Vitamin K and Equine Osteocalcin: an Enigma Explored

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Vitamin K and Equine Osteocalcin: An Enigma Explored

Jazmine Elizabeth Skinner

BApp.Sc Equine (Hons)

A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2020 School of Agriculture and Food Sciences

Abstract

Vitamin K has received considerably less attention over the past 50 years compared to other fat soluble vitamins. Intakes of vitamin K beyond that required for normal blood coagulation were believed to confer no additional benefits, and therefore rarely investigated. The requirements and physiological role of vitamin K appeared to be secure. This view of vitamin K has changed recently following the delineation of its cofactor role in protein carboxylation, and the growing awareness of its interactions with other essential metabolic functions, beyond that of blood coagulation; there is much to be discovered. At present, seventeen vitamin K- dependent proteins (VKDPs) have been characterised, but the functionality of some still remains to be elucidated. Vitamin K has recently been implicated in osteoarthritis and accumulating evidence from human studies supports a protective role for vitamin K. To date however, little research into the role vitamin K may play in equine bone development exists.

Developmental orthopaedic disease (DOD) is a term that encompasses a number of bone related conditions in the horse. It is a significant cause of lameness and wastage in a number of breeds, in particular Thoroughbreds and Warmbloods. It has clinical features in common with that of osteoporosis and osteoarthritis in the elderly. The VKDP osteocalcin is one of the few that has been extensively studied in humans and rodents. Located within bone, this protein plays a vital role in bone metabolism, facilitating the binding of bone minerals with protein. Decreased functionality of this protein has been found to be associated with increased fracture risk and osteoporosis in humans.

The studies in this program were initiated to establish the relationship between vitamin K status and the development of DOD in foals. As bone development begins in utero, the first step was to examine placental transfer of the vitamin and then determine availability to the foal through milk. It was found in the initial experiments (Chapter 3), that foals at birth, had negligible concentrations of vitamin K1 (<0.01ng/mL) in both umbilical cord and foal plasma. The results clearly demonstrated that there is limited trans-placental transfer of vitamin K in the horse. It was also shown that milk can be enriched with vitamin K by supplementation of the mare with the vitamin. These studies confirmed that plasma concentrations were not a reliable measure of overall vitamin K status.

Circulating osteocalcin and, its carboxylation status, has been proposed as one of the indicators of vitamin K status and a ‘biomarker’ of bone metabolism. Current assays available to measure

i osteocalcin, however, do not provide information about the degree of osteocalcin carboxylation. The aim of the subsequent studies, was to exploit the recent advances in the use of mass spectrometry (MS) based techniques and investigate the feasibility of a method to accurately measure the carboxylation status of circulating osteocalcin in equine plasma, after vitamin K supplementation.

The development of a MS-based method to quantify circulating carboxylated osteocalcin in plasma was investigated in Chapter 5. In this study, equine osteocalcin was in-silico digested into peptides and synthetically synthesised. A targeted MS-analysis, by LC-MS/MS MRM method, was developed based on these osteocalcin specific peptides. The method was not sensitive enough to detect native osteocalcin in plasma samples collected in Chapter 3. Studies conducted in Chapter’s 6 and 7 aimed to validate the use of a proteomics based enrichment strategy, to increase the sensitivity of this assay for detection of osteocalcin in plasma and other carboxylated VKDPs.

The use of hydroxyapatite was investigated in Chapter 6 as an as an enrichment strategy to detect osteocalcin in plasma. A method was successfully developed and validated with the osteocalcin synthetic standards; Carboxylated Analysis by Ceramic Hydroxyapatite Enrichment (CACHE). The studies conducted in Chapter 7 characterised and investigated the applicability of the CACHE protocol on biological samples with known elevation in vitamin K using foal plasma. Plasma samples taken from the foals at day 14 (Chapter 3) that showed a statistically significant difference between the control group and the supplemented group (P<0.05) were chosen for this study. In this study a number of unique proteins were identified in the enriched elute plasma fraction however, no carboxylated VKDPs, other than prothrombin, were detected. It is anticipated that further optimisation of the CACHE method will facilitate the detection other carboxylated VKDPs.

When the findings of these Chapters are considered, further examination of the quantification and role of osteocalcin is warranted. A key outcome of this thesis was the development of a novel method: CACHE. With further developments in the fields of proteomics explored in this thesis, it is anticipated that new insights into the metabolism and mechanisms of vitamin K, and its intrinsic relationship to osteocalcin and other VKDPs will be uncovered. It should then be possible to delineate the relationship of vitamin K status to DOD development in the foal.

ii Declaration by author

This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis.

I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my research higher degree candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award.

I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School.

I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis.

iii Publications included in this thesis

“No publications included”.

iv Submitted manuscripts included in this thesis

“No manuscripts submitted for publication”.

Other publications during candidature

Book chapter

Skinner, JE., Hilly, L.J., Li, X., Cawdell-Smith, A.J. and Bryden, W.L. (2019) Equine production systems and the changing role of horses in society. In Squires, V.R. and Bryden, W.L. Livestock: Production, Management Strategies and Challenges. Nova Science Publishers, NY. pp. 389-433. Invited papers

Skinner, J.E., Sadowski, P, Regtop, H, Biffin, R, Talbot, A, Cawdell-Smith, AJ, Bryden, WL (2018) Quantification of vitamin K and osteocalcin in horses. Australasian Equine Science Symposium, 7: 11.

Skinner, JE., Cawdell-Smith, AJ, Regtop, HL, Biffin, JR, Bryden, WL (2020) Equine vitamin K nutrition and metabolism – Review paper. Animal Production Science. In progress Journal articles

Skinner, JE., Fischer, T, Cawdell-Smith, AJ, Regtop, HL, Biffin, JR, Bryden, WL (2019) Trans-placental transfer of vitamin K in the horse. Equine Veterinary Journal. In progress

Skinner, JE., Regtop, HL, Talbot, AM, Biffin, JR, Cawdell-Smith, AJ, Bryden, WL (2019) Absorption of different vitamin K compounds in the horse. Equine Veterinary Journal. In progress

Skinner, JE., Sadowski, P, Satake, N, Cawdell-Smith, AJ, Bryden, WL (2019) Proteomic profiling of gamma-carboxylated proteins in horse plasma upon vitamin K supplementation. Journal of Proteomics. In progress Conference abstracts

Skinner, JE., Sadowski, P, Regtop, HL, Biffin, JR, Cawdell-Smith, AJ and Bryden, WL (2018) Equine vitamin K and osteocalcin: Application of LC-ICP-MS. 5th International Vitamin Conference, Sydney, Australia.

Skinner, JE., Regtop, HL, Talbot, AM, Biffin, JR, Cawdell-Smith, AJ and Bryden, WL (2018). Absorption of different vitamin K compounds in the horse. 5th International Vitamin Conference, Sydney, Australia.

Fischer, TJ., Regtop, HL, Talbot, AM, Biffin, JR, Skinner, JE, Cawdell-Smith, AJ and Bryden, WL (2018). Placental transfer of vitamin K in the horse. 5th International Vitamin Conference, Sydney, Australia.

v Munjizun, A., Cawdell-Smith, AJ, Skinner, JE, Regtop, HL, Biffin, JR and Bryden, WL (2018) The effect of vitamin K administration to lactating mares on plasma Vitamin K concentrations in foals. Australasian Equine Science Symposium, 7: 39.

Skinner, J.E., Cawdell-Smith, AJ, Biffin, JR, Talbot, AM, Regtop, HL and Bryden, WL (2016) Is vitamin K3 converted to MK4 in the horse? Australasian Equine Science Symposium, 6: 36.

vi Contributions by others to the thesis

“No contributions by others”.

vii Statement of parts of the thesis submitted to qualify for the award of another degree

“No works submitted towards another degree have been included in this thesis”.

Research involving human or animal subjects

The horses used in this study were located at the UQ Equine Research Unit, Gatton. The experimental procedures were approved by the University of Queensland Animal Ethics Committee (SAFS/421/16; Appendix 1).

viii Acknowledgements

Where do I start, my deepest gratitude goes to Dr Judy-Cawdell-Smith and Professor Wayne Bryden for their continuous encouragement and support, not only with this research but over the entire course of my studies, thank-you for your encouragement and mentorship during this journey.

I am truly grateful to Dr Pawel Sadowski, for all the time and patience you dedicated to teaching me the ropes of mass spectrometry and proteomics. Your passion and excitement for your field was so inspiring. I am a much better research scientist equipped with the skills and knowledge you have imparted. Special mention also goes to Dr Raj Gupta and the rest of the CARF team for their assistance.

My sincere thanks goes to my dear friend and supervisor Dr Nana Satake. I will be forever grateful for all your help. Thank-you for taking me on under your wing! I am especially grateful for the support you gave me particularly over the final few months, when I really needed it most.

I would also like to acknowledge Ray Biffin and Hub Regtop and their company, Agricure Pty Ltd for the funding that has made this research possible; thank-you Ray, Hub and Andrea Talbot for your ongoing support and assistance with sample analysis.

To all my friends who provided me with some very much needed ‘downtime’ and all the equine unit crew, thanks for the coffee dates and wine nights that helped keep me sane! Sincere thanks to Mitch Coyle and students that have helped me over the period of this project. To my best friend and fellow PhD student in crime Halley; we have been through a lot together!

To my beautiful parents who have supported my every decision, I realise how fortunate I am to have such a loving and supportive family. Thank-you for keeping me grounded and raising me to be the person I have become. Finally to my fiancé Pete. You have been a part of my university journey from the very beginning, thank-you for supporting me through the trials and triumphs of this PhD.

ix Financial support

This research was supported by an Australian Government Research Training Program Scholarship.

Agricure Pty Ltd donated funds and analytical support for the research program.

x Keywords

Equine, mass spectrometry, multiple reaction monitoring (MRM), osteocalcin, proteomics, vitamin K.

Australian and New Zealand Standard Research Classifications (ANZSRC)

ANZSRC code: 060101, Analytical Biochemistry, 60%

ANZSRC code: 060102, Bioinformatics, 15%

ANZSRC code: 070204, Animal Nutrition, 25%

Fields of Research (FoR) Classification

FoR code: 0702, Animal Production, 60%

FoR code: 0699, Other Biological Sciences, 40%

xi Table of Contents

Abstract ...... i

Declaration by author ...... iii

Other publications during candidature ...... v

Acknowledgements ...... ix

Keywords ...... xi

Australian and New Zealand Standard Research Classifications (ANZSRC) ...... xi

Fields of Research (FoR) Classification ...... xi

CHAPTER 1: Introduction and Objectives of the Study ...... 1

CHAPTER 2: Review of the Literature...... 7

Trans-placental Transfer and Milk Deposition of vitamin K1 in the Mare. .... 67

General Materials and Methods, Method Development and Optimisation..... 93

CHAPTER 5: Osteocalcin Peptide Assay Method Development ...... 110

: Carboxylated Analysis by Ceramic Hydroxyapatite Enrichment (CACHE): Development of a novel method ...... 152

CHAPTER 7: Application of the Carboxylated Analysis by Ceramic Hydroxyapatite Enrichment (CACHE) method to the plasma of vitamin K supplemented foals...... 170

General discussion and future directions ...... 196

Bibliography ...... 209

Appendices ...... 236

xii List of Abbreviations

α Alpha

AA Amino acid

ACN Acetonitrile

ANOVA Analysis of variance

APCI Atmospheric pressure chemical ionisation

β Beta

BCA Bicinchoninic acid

BLAST Basic Local Alignment Search Tool

BSA Bovine Serum Albumin

CACHE Carboxylated Ceramic Hydroxyapatite Enrichment

CARF Central Analytical Research Facility cOC Carboxylated osteocalcin

CO2 Carbon dioxide

CPS Counts per second

CR Chylomicron remnants

C-terminus Carboxy terminus

CID Collision-induced dissociation

DDA Data-dependent acquisition

°C Degrees Celsius

Da Daltons

DIA Data-independent acquisition

DNA Deoxyribonucleic acid

DTT DL-Dithiothreitol

EDTA Ethylenediaminetetraacetate

xiii ELISA Enzyme-linked immunosorbent assay

ESI Electro-spray ionization

FA Formic acid

FASP Filter-aided sample preparation technology

FASTA Text-based format for representing peptide/protein sequences

FDR False Discovery Rate

γ Gamma

GGCX Gamma-glutamyl carboxylase

Gla Gamma-carboxyglutamic acid

Glu Glutamic acid

HAP Hydroxyapatite

HDL High-density lipoproteins

HPLC High-performance liquid chromatography

IAM Iodoacetamide

ICP-MS Inductively-coupled plasma mass spectrometry

ID Identification

IgG Immunoglobulin iST In-stage tip iTRAPQ Isobaric tags for relative and absolute quantitation

K1 Vitamin K1

K2 Vitamin K2

K3 Vitamin K3 ka Kilo annum kg Kilogram

xiv KH2 Reduced vitamin K

KO Vitamin K epoxide

KQ Quinaquinone™

LA-ICP-MS Laser-ablation inductively coupled plasma mass spectrometry

LC Liquid chromatography

LC-MS/MS Liquid chromatography tandem mass spectrometry

LDL Low density lipoproteins

LDLR Low density lipoprotein receptors

LRP LDL receptor-related proteins m/z Mass-to-charge ratio

MALDI-TOF-MS Matrix assisted laser desorption/ionization time-of-flight mass spectrometry

µg Microgram

µL Microliter

µm Micrometer mg Milligram

MGP Matrix Gla protein mgf Mascot generic format

MK-4 Menaquinone-4

MK-7 - MK-13 Long chain menaquinones mL Millilitre mM Millimolar

MRM Multiple Reaction Monitoring mRNA Messenger ribonucleic acid

MS Mass spectrometry

xv MS/MS Tandem mass spectrometry

nanoLC-ESI-MS/MS Nano-liquid chromatography electrospray ionization tandem mass spectrometry

NCBI National Centre for Biotechnology Information

N-terminus Amino terminus

1D SDS-PAGE One dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis

OCD Osteochondritis dessicans

ω Omega

PCR Polymerase chain reaction

pH Acidity/alkalinity

pmol Picomole

ppb Parts per billion

PTM Post-translational modification

QC Quality control

QH Quarter horse

QTOF Quadrupole time-of-flight

RT Retention time

SD Standard deviation

SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis

SAFS School of Agriculture and Food Sciences

SILAC Stable isotope labelling with amino acids in cell culture

SRM Selected Reaction Monitoring

SWATH Sequential window acquisition of all theoretical fragment ion mass spectra

TB Thoroughbred

xvi TD Tibial dyschondroplasia

TFA Trifluroacetic acid

TG Triglycerides

TIC Total ion chromatogram

TOF Time-of-flight

TripleTOF 5600+ AB SCIEX mass spectrometer

Tris Trisaminomethane

TRL Triglyceride rich lipoproteins ucOC Undercarboxylated osteocalcin

UniProtKB Universal Protein Resource Consortium Knowledgebase

VKDPs Vitamin K-dependent proteins

VKDB Vitamin K deficiency bleeding

VKOR Vitamin K epoxide reductase

VKR Vitamin K reductase

VLDL Very low-density lipoprotein wiff SCIEX instrument raw data file output wiff.scan SCIEX instrument raw data file scan output

XIC Extracted ion chromatogram

xvii

CHAPTER 1 Introduction and Objectives of the Study

1.1 Preface ...... 1 1.2 Context of this research ...... 3 1.2.1 Horse wastage ...... 3 1.2.2 Emerging role of vitamin K in bone development...... 4 1.3 Objectives of the research program ...... 5

1.1 Preface Vitamin K is a group of fat-soluble compounds formed from a naphthoquinone ring attached to a poly-isoprenyl side chain of variable length and saturation. As described by van Oostende

et al. (2011) the main natural forms are phylloquinone (vitamin K1) that is found in plants, green algae, and some cyanobacteria, and menaquinones (vitamin K2) that is found in most archaea and bacteria, some cyanobacterium, red algae and diatoms. Vitamin K-synthesizing organisms appear to contain either vitamin K1 or vitamin K2 but not both (van Oostende et al. 2011). Biologically, vitamin K has a pivotal role in vertebrate blood coagulation and photosynthesis in plants and other organisms.

Vitamin K was discovered independently in 1935 by two research scientists; Henrich Dam working in Denmark (Dam 1935a, 1935b) and Herman Almquist in the USA (Almquist & Stokstad 1935a, 1935b); two references are cited for both Dam and Almquist here, as both references are cited interchangeably for each author when the discovery of vitamin K is reviewed. The first reference to the anti-haemorrhagic factor in chickens as the discovery of a new vitamin by both authors is a letter to Nature in 1935 (Almquist & Stokstad 1935a; Dam 1935b). Interestingly, the letter from Dam (1935b) is published under the general heading “The Antihaemorrhagic Vitamin of the Chick” with a subheading “Occurrence and chemical nature” along with another letter under the same general heading, but with the subheading “Measurement of biological action” (Schonheyder 1935). In this letter, Schonheyder was the first to describe a role for vitamin K in blood coagulation. Ham and Schonheyder were from the same Institute, published together, and submitted their letters on the same day. In his letter, Dam (1935b) named the factor vitamin K, as it was the next letter in alphabet that had not been 1 used to designate a vitamin and coincidently the first letter of the Danish word “koagulation”. Edward Doisy’s group in St Louis, USA, subsequently described the chemical structure of both vitamin K1 and K2 (Doisy et al. 1941). In 1943, the Nobel Prize in Physiology or Medicine was co-awarded to Henrik Dam ‘for his discovery of vitamin K’ and to Edward Doisy ‘for his discovery of the chemical nature of vitamin K’. Dam and Almquist have both given accounts of the discovery of vitamin K (Almquist 1941; Dam 1942; Almquist 1975). It is interesting to compare these accounts, with those prepared by Jukes (1980) and van Oostende et al. (2011).

Since the discovery of vitamin K in the middle of last century it has received considerably less attention over the intervening period than other fat soluble vitamin, despite its importance to both animal and plant metabolism. In animals, the general consensus has been that intakes beyond that required for normal blood coagulation confer no additional benefits and therefore were rarely investigated (Shearer 1995); the role of vitamin K appeared to have been delineated (Shearer 1995). However, research into the physiological role of vitamin K over the past few decades has revealed its intrinsic involvement in other metabolic functions, beyond that of coagulation (Booth 2009; Pizzorno 2011). Warfarin therapy was found to not only inhibit blood coagulation, but also detrimentally effect bone density (Gundberg et al. 2012). A number of age related diseases in humans have also been related to vitamin K inadequacy (Pizzorno 2011). In particular bone related diseases such as osteoporosis have been linked to inadequate vitamin K intake in humans, rats and laying hens (Fleming et al. 2003; Lanham-New 2008). Furthermore, it has been found that a higher requirement for vitamin K is necessary to avoid bone diseases, in comparison to that required for normal blood coagulation in humans (Booth 2009; Terachi et al. 2011). This finding lead to the development of the triage theory by McCann and Ames (2009) who proposed that;

“When the availability of a micronutrient is inadequate, nature ensures that micronutrient- dependent functions required for short-term survival are protected at the expense of functions whose lack has only longer-term consequences, such as the diseases associated with aging”

Interestingly, none of the major reference authorities on the nutrition of the horse in the North America (NRC 2007), France (INRA 2015), or Germany (GEH 2013), list requirements for vitamin K. This reflects a lack of data on which to predict requirements. In many respects vitamin K has been neglected in equine nutrition research, due in part to very few instances of bleeding disorders being reported in the horse. There is also a widely held belief that horses

2 receive an adequate intake of vitamin K from grass and forage, along with microbial synthesis in the gut. However, to date there is no or very limited data to substantiate these assertions.

1.2 Context of this research The equine industry, in particular the Thoroughbred breeding and racing sectors, contribute significantly to the Australian economy. It is estimated that these sectors contribute 12 million GDP annually (Hardy & Limoli 2019 ). The Upper Hunter Valley region of NSW ranks alongside Kentucky in the USA and Newmarket in England as one of the most significant Thoroughbred breeding nurseries globally (Robinson 1996). This is not surprising, as Australia has the greatest number of racetracks per capita in the world and is second to the USA in the number of Thoroughbred brood mares (Skinner et al. 2019).

1.2.1 Horse wastage A major concern for the horse industry, is the loss or wastage of horses from racing or other equestrian pursuits and this has been examined in Australia by a number of authors (Bourke 1995; Bailey et al. 1997; Dyer 1998; More 1999; Thomson et al. 2014). In excess of 35% of horses are lost to the racing industry annually for a variety of reasons (Thomson et al. 2014), with the major cause being lameness or other bone related or musculoskeletal conditions (Rogers et al. 2020). It should not be forgotten that horses are also lost to the industry prior to sale as yearlings due to anomalies in bone development. Pre-sale x-rays are now a requirement for all Thoroughbred sales. However, lesions visible on x-ray are always of pathological concern (Bourebaba et al. 2019).

DOD are most commonly observed in young growing animals. Aldred (1998) estimated that 10% of Thoroughbred foals born in the Hunter Valley each year, could not be sold as yearlings due to DOD, costing the industry about $10 million annually. It is evident that measures to reduce the incidence of DOD lesions occurring are necessary. In order to do this, an increased understanding of the underlying mechanism involved in the pathogenesis of these diseases is imperative (Desjardin et al. 2014; Bourebaba et al. 2019).

3 Osteochondrosis is an important DOD in young horses (Desjardin et al. 2014). The primary lesion is caused by a defect in the growing cartilage during endochondral ossification (Bourebaba et al. 2019). It may lead to the development of osteochondritis dissecans (OCD) in which the primary lesion separates from the underlying bone. While the aetiology of the disease is complex the general consensus is that it involves abnormal chondrocyte maturation and/or failure of differentiation (Olstad et al. 2011). This can lead to subchondral fractures, cysts, cartilage flaps and synovitis (Olstad et al. 2011). Much research has studied possible causative factors including genetics, and diet, the relative contribution of which appears to vary (Coskun et al. 2016; Kemper et al. 2019). Osteoporosis and osteoarthritis in humans, resemble OCDs in horses with loss of articular cartilage and degeneration. Perhaps similar mechanisms are behind the development of the human and equine conditions.

1.2.2 Emerging role of vitamin K in bone development In recent years, with the delineation of the cofactor role of vitamin K in protein carboxylation (Berkner 2005), a growing awareness of the interactions between vitamin K and other essential metabolic functions, has been investigated in humans, poultry and rodents (Booth 2009). This lead to the discovery of the intrinsic role vitamin K plays in many other functions including; glucose homeostasis, calcification, sphingolipid metabolism and bone metabolism (McCann & Ames 2009). Vitamin K deficiency has recently been implicated in osteoarthritis and there is accumulating evidence in human studies supporting a protective role of vitamin K (Shea & Booth 2017b). To date however, little research has been conducted into the role of vitamin K in equine nutrition and health.

The emerging roles of vitamin K beyond that of blood coagulation has been discovered through rodent and human studies, which have uncovered a plethora of vitamin K dependent proteins (VKDPs) (Booth 2009). The major role of vitamin K is to facilitate the carboxylation of these proteins and in so doing, determine their functionality. At present, seventeen VKDPs have been characterised, but the exact underlying functionality of some still remains to be elucidated (Booth 2009). The VKDP osteocalcin is one of the few that has been extensively studied in humans and rodents. Located within bone, this protein plays a vital role in bone metabolism, facilitating the binding of bone minerals with protein (Booth 2009) and is the most abundant non-collagenous protein of the bone matrix and an important component of bone formation (Prats-Puig et al. 2010; Entenmann et al. 2017). Decreased functionality of this protein has

4 been found to be associated with increased fracture risk and osteoporosis in humans (Shea & Booth 2008).

Although extensive research on vitamin K has been carried out in humans and rats on bone related disorders, little research has been conducted in horses. This raises the question, can knowledge gained in other species be extrapolated to horses? Preliminary findings by Biffin et al. (2010) reveal an association between increased occurrences of osteochondrosis in horses with lower serum vitamin K concentrations. The equine industry incurs considerable economic losses annually as a result of these potentially debilitating skeletal disorders such as osteochondritis dissecans (OCD); a developmental orthopaedic disease (DOD) that can affect horses early in life (Lepage et al. 1998).

An understanding of the relationship between vitamin K and bone metabolism will permit the role of the vitamin in normal equine bone development and skeletal disorders such as DOD to be defined. This will facilitate the determination of specific dietary requirements of vitamin K for horses, which will aid in determining supplementary levels that may limit the occurrence of bone diseases. This will improve equine welfare and reduce lameness and wastage in the equine industries.

1.3 Objectives of the research program The studies in this program were initiated to establish the relationship between vitamin K status and the development of DOD in foals. If a relationship was established, it would allow the development of appropriate strategies for vitamin K supplementation of equine diets. The overall objective of this study was therefore to investigate the role of vitamin K in equine bone development. As bone development begins in utero, the first step was to examine placental transfer of the vitamin and then determine availability to the foal through milk. These objectives were examined with the hypotheses; that vitamin K does not cross the equine placenta, and that the concentration of the vitamin in mare’s milk can be modulated by oral supplementation.

Experiments were therefore designed to answer the following research questions;

• Is there placental transfer of vitamin K in the mare? • Does vitamin K supplementation increase the concentration of the vitamin in milk? • Are circulating concentrations of vitamin K a reliable indicator of vitamin K status?

5 The initial experiments (Chapter 3) demonstrated that there was limited placental transfer of vitamin K, milk concentrations could be increased by supplementation, and plasma concentrations were not a good biomarker of vitamin K status. It had been intended to conduct more horse studies but the lack of a reliable marker of vitamin K status was a significant impediment to further equine work. The search for a biomarker began and osteocalcin became the molecule of choice. Osteocalcin is considered to be a more sensitive measure of osteoblastic vitamin K status and potential ‘biomarker’ to detect bone disease (see Chapter 2). The question then arose of how to measure osteocalcin?

As described in Chapter 2, there are numerous pitfalls with the current osteocalcin analysis methods. There are immunoassays available however, they do not differentiate the degree of under carboxylation of osteocalcin, which is key to vitamin K status. This finding prompted a series of studies using mass spectrometry, in an attempt to quantify circulating concentrations of osteocalcin in different carboxylation states as a marker of vitamin K status.

It should be noted that in conjunction with these studies, some of the data contained within Chapter 3 of this thesis was also presented by Tracey Fischer and Ahmad Munjizun as part of their Honours theses.

6 CHAPTER 2 Review of the Literature 2.1 Introduction and scope of the review ...... 7 2.2 Physiology of vitamin K ...... 8 2.2.1 Chemical structures and molecular form...... 8 2.2.2 Metabolism of vitamin K ...... 10 2.2.3 The vitamin K cycle and γ-Carboxylation...... 15 2.2.4 Vitamin K–dependent proteins ...... 18 2.3 Vitamin K status: challenges in determining requirements & deficiency ...... 20 2.3.1 Vitamin K deficiency bleeding (VKDB)...... 25 2.3.2 Skeletal health ...... 28 2.3.3 Determining requirements ...... 28 2.4 Bone development and pathophysiology ...... 30 2.4.1 Endochondral Ossification ...... 30 2.4.2 Equine vitamin K and bone metabolism ...... 35 2.5 Osteocalcin ...... 39 2.5.1 Structure & biochemistry of osteocalcin ...... 39 2.5.2 Osteocalcin and bone metabolism and disease ...... 42 2.5.3 Methods currently used to measure osteocalcin ...... 47 2.6 Modern proteomics approaches and analytical techniques ...... 53 2.6.1 An introduction to liquid-chromatography mass spectrometry (LC- MS) ...... 55 2.6.2 Mass Spectrometry (MS) for routine detection of proteins ...... 56 2.6.3 Targeted and untargeted proteomics approaches ...... 57 2.6.4 Development of MS based techniques to analyse osteocalcin: current status ...... 61 2.7 Conclusion ...... 66

2.1 Introduction and scope of the review Within the last 20 years our understanding of vitamin K and its interactions has received considerably more attention, with the discovery of its involvement in many other functions beyond that of blood coagulation (Booth 2009). In 1974, Suttie and colleagues discovered the vitamin K dependent (VKD) carboxylation of γ-carboxyglutamic acid (Gla) residues in the liver microsomes of rats, paving the way for research to uncover various vitamin K- dependent proteins (VKDPs) throughout the body (Shah & Suttie 1974). Several VKDPs have now been discovered with distinct and vital roles in an array of functions including haemostasis, calcium homeostasis, growth and energy metabolism, signal transduction and apoptosis (Hallgren et al.

7 2013). Involvement of these proteins extends beyond that of the liver, to metabolism in a range of areas, including bone, sphingolipids, glucose and even reproduction (Manna & Kalita 2016).

This review will explore current understanding of the physiological role and metabolism of vitamin K and, explore its role in other metabolic processes beyond that of coagulation, especially bone metabolism. Where data exists, the role of vitamin K and VKDPs in the horse will be discussed. The intrinsic relationship between vitamin K and osteocalcin has informed this review. The focus is on the function of vitamin K and osteocalcin across species, but more particularly the horse. The structure and function of osteocalcin will be extensively reviewed along with current methods of analysis and the potential of ‘omics methodologies.

2.2 Physiology of vitamin K Vitamin K occurs naturally in a number of chemical forms that dictate the metabolism of the molecule and ultimately functionality.

2.2.1 Chemical structures and molecular form Vitamin K is a fat soluble vitamin, found in 3 structurally similar molecular forms (McDonald et al. 2019). Each of the forms (Figure 2.1) share a common 2-methyl-1,4naphthoquinone ring with individual differences denoted by the number of isoprenoid units in the alkyl side chains

(Booth & Suttie 1998; McDonald et al. 2019). The three forms are; Phylloquinones (K1) are

found in green leafy plants; Menaquinones (K2) are produced by the intestinal bacteria of mammals, and are also found in vegetable oils and fermented foods such as natto and cheese;

Menadione (K3), is a synthetically derived form, predominately added to stock feed premixes as it is not approved for human use (Schurgers & Vermeer 2000; Booth 2009); it can also have toxic side-effects (Rebhun et al. 1984; Thijssen et al. 2006). To complicate matters, MK-4

appears to be produced in the tissues of some mammals via conversion form both K1 and K3

after either injection or oral supplementation (Al Rajabi et al. 2012). In the intestine, K3 may

be produced as an intermediary from the conversion of K1 within the gut endothelium, where it is converted to MK-4 by the enzyme UBIAD1 (Thijssen et al. 2006; Kimie et al. 2010).

Figure 2.1: The different forms of vitamin K; (A) K3, (B) K1, (C) MK-4, (D) K2 (adapted from Booth 2009).

Surveys (Bolton-Smith et al. 2000; Booth 2012; Harshman et al. 2014a; Centi et al. 2015; Fu et al. 2016; Harshman et al. 2017; Vermeer et al. 2018) show that vitamin K is provided in the human diet by leafy green vegetables, vegetable oils, natto, cheese and other animal products.

There is no consensus on the importance vitamin K2 to the vitamin economy of a human or animal host (McCann et al. 2019)(McCann et al. 2015), and this is discussed further in the next section. Moreover, little attention has been given to vitamin K concentrations of animal feedstuffs, because of the lack of bleeding conditions in livestock and the availability of menadione as an animal feed additive. The concentration of the vitamin in cereal grains is very low (0.2-0.4mg/kg DM) but much higher (3-22 mg/kg DM) in forages (Siciliano et al. 2000a). However, Biffin et al. (2008b) reported that the vitamin K content was rapidly reduced in forage as it dried during hay production. They concluded that stabled horses fed hay and a grain-based complete feed should be supplemented with vitamin K.

2.2.2 Metabolism of vitamin K The metabolism (absorption, distribution, excretion) of vitamin K is complex due to the different molecular forms of the vitamin. Some aspects of metabolism are poorly defined and species differences seem to exist but require elucidation.

Absorption transport and tissue distribution Each of the different forms of vitamin K varies in absorption efficiency, and reflects different chemical structures and the rate of release from the food or bacterial matrix in which it is encased (Schurgers & Vermeer 2000). As a fat-soluble substance, vitamin K is absorbed in a manner similar to other dietary fats, and there is evidence that absorption of the vitamin is enhanced by dietary fat (Goncalves et al. 2015). The absorption of vitamin K was first described by Hollander (1973), who identified maximal uptake of K1 in the duodenum and jejunum in rats. In contrast, Goncalves et al. (2015) found greater uptake in the ileum. Vitamin

K1 is tightly bound within the membrane of chloroplasts in plants (Harshman et al. 2016). On

the other hand, vitamin K2 is bound within bacterial cells and occurs in various forms with different side chain lengths (MK-n) (Shearer & Newman 2008). There are short and long chain menaquinones from MK-4 to MK-14 (Shearer & Newman 2014). The length of the side chain

influences absorption (Thane et al. 2002). Vitamin K3, unlike the other forms, lacks a side chain and prevents this form from activating extra-hepatic VKDPs, other than those involved in the blood clotting cascade (Al Rajabi et al. 2012).

A schematic diagram showing the absorption and cellular uptake of vitamin K1 and MK-7 (an example of K2) is depicted in Figure 2.2. In the intestinal lumen, K1 and MK-7 are incorporated into mixed micelles and then taken up by enterocytes (Shearer et al. 2012). Micelles are a combination of bile salts, products of pancreatic lipolysis and other lipids (Shearer et al. 2012).

After being absorbed into the enterocytes of the small intestine, K1 is thought to be transported by a carrier protein, but specific carrier proteins for intra-cellular transport across the enterocyte have not been identified (Reboul & Borel 2011). In the enterocyte, the vitamin becomes bound to chylomicrons (Schurgers & Vermeer 2002) and in this configuration it is released into circulation via the lymphatic system to be recruited by the liver (Shea & Booth 2007). The release into circulation of these particles is achieved via the thoracic duct (Shearer & Newman 2008). Receptor mediated uptake of the chylomicron remnants (CR) into the liver is then facilitated by hepatic parenchyma cells (Schurgers & Vermeer 2002).

Figure 2.2: Schematic representation of the transport and uptake of K1 and K2 (MK-7). Post digestion, dietary vitamin K and the products of pancreatic hydrolysis of triglycerides are emulsified by bile salts to form mixed micelles. These are taken up by the enterocytes of the intestinal epithelium, and processed into nascent chylomicrons which contain apoA and apoB- 48. Chylomicrons are then secreted into the lacteals within the intestinal villi. The lacteals drain into larger lymphatic vessels eventually emptying into the blood circulation via the thoracic duct. Once in circulation, chylomicrons acquire apoC and apoE and HDL. Within the capillaries of muscles and adipose tissue, chylomicrons are stripped of their TG by the action of lipoprotein lipase (LPL). The resultant smaller CR re-enter the circulation having lost much apoA and apoC but retaining vitamin K in the lipophilic core. Uptake by the liver: In the liver chylomicrons enter hepatocytes by binding to LDLR and LRP followed by receptor mediated endocytosis. Their lipids are repackaged into VLDL (containing apoB-100) and return to the circulation where they acquire apoC and apoE. Further TG is removed by LPL in the capillaries resulting in VLDL remnants. Subsequent metabolism and loss of apoC and apoE from these remnants gives rise to smaller LDL particles containing almost exclusively apoB- 100. Vitamin K is presumed to be still located in the lipophilic core. Uptake by bone: Circulating lipoproteins such as chylomicrons and LDL can deliver lipids to osteoblasts which are attached to the surfaces of bone matrix. Osteoblasts express lipoprotein receptor such as LDLR which can interact with chylomicrons and LDL, allowing receptor mediated endocytosis of the particles and their cargoes of vitamin K. Evidence suggests that osteoblasts obtain most of their K1 via the chylomicrons pathway and most of their MK-7 via the LDL pathway (adapted from Shearer & Newman 2008).

Transport of vitamin K, like that of vitamin E appears to be facilitated by lipoproteins however, unlike vitamin E, vitamin K has no specific plasma carrier protein (Shearer 1995). The primary

transporters of K1 are believed to be triglyceride rich lipoproteins (TRL) and very low-density

lipoproteins (VLDL) (Shea & Booth 2007). After intestinal absorption, both K1 and K2 are transported by these lipoproteins to the liver (Shearer et al. 2012). Binding of the chylomicrons to these lipoproteins allows them to enter the hepatocytes via receptor mediated endocytosis as depicted in Figure 2.2.

The liver contains the largest concentrations of vitamin K, and is the primary site for synthesis of vitamin K-dependent coagulation factors (Shearer 1995; Shearer & Newman 2008). It is well documented in humans and rats that K1 preferentially accumulates in the liver (McCann

& Ames 2009). There is now good evidence that the hepatic turnover of long-chain K2 is very

much slower than that of K1 in both rats and humans, resulting in much greater hepatic stores

of K2 than K1 in many species; in particular MK-7 to MK-13 (Shearer & Newman 2008). It

has been suggested that the intake of K2 may be of greater importance than intake of K1 in alleviating risk of age-related diseases such as osteoporosis (Inoue et al. 2009; Pizzorno 2011).

This may be attributable to their different absorption and transport pathways with K2 found to

be readily transported to extra-hepatic tissues in humans, whereas most of K1 is excreted (Falcone et al. 2011).

Inoue et al. (2009) suggested that K2 may not be as nutritional significant in horses as often proposed. They found significantly lower plasma concentrations of bacterial synthesised MK-

7 in comparison to K1. This supports the findings of Hirauchi et al. (1989) who found that K1

concentrations were much higher in almost all horse tissues compared to values for K2. These results suggest that absorption and distribution of vitamin K in horse may be different to humans and rodents and requires further research. It also raises the question of vitamin K availability, which is a function of digestion, absorption and utilisation.

For most nutrients, digestion and absorption are the rate limiting steps in availability and for vitamin K there is no information on the digestion and release of K1 from plant cells or K2 from

bacterial cells. Once released, K1 will be readily absorbed in the small intestine, but the situation

is less clear in the large intestine or hindgut. In the caecum and colon of the horse, K2 will be

tightly bound to cytoplasmic membranes of bacteria and if released may not be absorbed. For absorption, bile salts are required for micelle formation but are lacking in the hindgut.

Placental transfer Often the discussion of nutrient metabolism neglects what happens during pregnancy. While research has been conducted on the placental transfer of vitamin K in humans and rodents, to

date no research investigated placental transfer of vitamin K in the horse. In humans, K1 concentrations in umbilical cord blood have been found to be extremely low and challenging to measure, implying that placental transfer of vitamin K is poor (Shearer et al. 2012).

The supplementation of vitamin K orally before delivery in humans, has been found to

significantly increase maternal plasma vitamin K1 concentrations (Kazzi et al. 1990). Similar findings were also reported in umbilical cord plasma, where the treated group had a

significantly higher concentration of vitamin K1 compared to the untreated group (Kazzi et al. 1990). In humans it appears that vitamin K traverses the placenta poorly, as a result, infants who do not receive a prophylactic dose of vitamin K at birth are at risk of haemorrhagic disease (Dror & Allen 2018).

Uptake by bone Vitamin K is taken up by extra-hepatic tissues such as bone (Newman et al. 2002) and this was discovered with the isolation of VKDPs from the bone matrix (Shearer et al. 2012). The exact molecular basis of vitamin K transport and uptake by extra-hepatic tissues however, has not been as extensively studied. There are recent studies that attempt to elucidate the underlying mechanisms of the uptake of vitamin K into bone (Shearer et al. 2012).

The primary delivery vesicles of vitamin K into bone are believed to be chylomicrons, with low density lipoproteins (LDL) being predominate lipoprotein transporters, as depicted in Figure 2.2. This was established by Newman et al. (2002) who found that the most efficient uptake of K1 into osteoblasts was achieved via LDL. While TRL are believed to be the primary transporters, vitamin K2, has been detected in both LDL and high density lipoproteins (HDL) suggesting differential transport and uptake routes, perhaps in different tissues (Shea & Booth 2007). This is supported by known differences in human tissue specificity between different

isomers, with K2 the major form in circulation and bone, and K1 stored at higher concentrations

in the liver (Shea & Booth 2007). LDL are the primary transporters of K1 to osteoblasts, however uptake can also occur via TRL and HDLs (Shearer et al. 2012)

Synthesis of osteocalcin by osteoblasts is dependent on vitamin K (Zoch et al. 2016). This permits γ-glutamyl carboxylation of osteocalcin, which will be discussed later. The presence of LDL receptors (LDLR) and LDL receptor-related proteins (LRP) on the surface of osteoblasts implies that vitamin K can be delivered directly to them via the vascular system

(Shearer et al. 2012). This supports findings where dietary K1 supplementation was found to decrease the percentage of circulating undercarboxylated osteocalcin (%ucOC) in healthy subjects (Binkley & Suttie 1995).

Vitamin K transformation After absorption and tissue uptake, many nutrients are transformed for storage or function but little is known about what occurs to vitamin K in horses; for all species the situation is unclear.

Most effort has been directed at K1 and the following putative biosynthetic pathway (Figure

2.3) showing the conversion K1 to MK-4, with MK-4 being the “active” metabolite/vitamer

(Shearer & Okano 2018). In this pathway menadione or K3 is an intermediary, and this appears to provide an explanation of why K3 has proved successful as an animal feed supplement.

Figure 2.3: Metabolism of dietary phylloquinone to menaquinone-4 (MK-4). During intestinal absorption, a fraction of dietary phylloquinone is cleaved by an unknown enzyme(s) to release menadione. Menadione enters the blood circulation via the thoracic lymphatic system, is delivered to target tissues, and is reduced to menadiol by a redox enzyme(s). Finally, menadiol undergoes a prenylation reaction with geranylgeranyl diphosphate catalyzed by the enzyme UBIAD1 (UbiA prenyltransferase domain-containing protein 1) to produce menaquinone-4 (adapted from Shearer & Okano 2018)

If a similar conversion occurs for the series of K2 vitamers (MKn) to MK-4, it is possible to explain how the different K vitamers are able to maintain vitamin K status and biological function of an individual or animal. This scenario as described by Shearer & Okano (2018) is shown in Figure 2.4.

Figure 2.4: Overview of synthesis and cellular functions of MK-4. The enzyme UBIAD1 mediates the in vivo tissue conversion of vitamin K forms to MK-4 in extrahepatic tissues. Besides its canonical role as a cofactor for GGCX-mediated synthesis of VKDPs, MK-4 has been shown to modulate gene expression and signal transduction. Examples shown are those initiated by binding to nuclear receptors (SXR/PXR) or through activation of PKA. Abbreviations: GGCX, γ-carboxyglutamyl carboxylase; MK-4, menaquinone-4; PKA, protein kinase A; PXR, pregnane X receptor; SXR, steroid and xenobiotic receptor; UBIAD1, UbiA prenyltransferase domain-containing protein 1; (adapted from Shearer & Okano 2018).

2.2.3 The vitamin K cycle and γ-Carboxylation The Vitamin K cycle as depicted and detailed in Figure 2.5; This cycle facilitates the conversion of Glutamate (Glu) to gamma-carboxyglutamate (Gla) by gamma-glutamyl carboxylase

(GGCX) using a reduced form of vitamin K (KH2) (Stafford 2005). In so doing, vitamin K

undergoes oxidation and reduction in three stages and is regenerated. In the cycle, KH2 is oxidised to vitamin K epoxide (KO). KO is reduced to vitamin K by vitamin K epoxide reductase (VKOR) (Li et al. 2004). The reduction of vitamin K to KH2 is carried out primarily

by this enzyme (Hammed et al. 2013). The oxidation of KH2 to vitamin K 2,3–epoxide (KO)

is a two–step reduction process, enabling KO to then be converted back to KH2 via the enzymes VKOR and VKR (Tie et al. 2016). Two primary enzymes involved in the carboxylation process; are gamma-glutamyl carboxylase (GGCX) and Vitamin K epoxide reductase (VKOR) (Tie et al. 2016). While less is currently understood about the enzyme VKR, evidence depicts 15

its importance in the vitamin K cycle however, to date its exact identity remains unknown (Caspers et al. 2015). In summary the vitamin K cycle is coupled to the carboxylation of proteins.

Figure 2.5: The Vitamin K cycle. During vitamin K-dependent carboxylation, glutamate (Glu) is converted to gamma-carboxyglutamte (Gla) by gamma-glutamyl carboxylase (GGCX) using a reduced form of vitamin K (KH2), carbon dioxide, and oxygen as cofactors. KH2 is oxidized to vitamin K epoxide (KO). KO is reduced to vitamin K by vitamin K epoxide reductase (VKOR). The reduction of vitamin K to KH2 is carried out by VKOR and an as-yet-unidentified VKR (adapted from Tie & Stafford 2016).

Γ-Carboxylation reactions The carboxylation of vitamin K dependent proteins is a vital post-translational modification (PTM) process that coverts specific glutamate residues (Glu) to γ-carboxyglutamate (Gla) residues in VKDPs (Berkner 2005; Lacombe & Ferron 2015). It is required for the biological function of numerous VKDPs including the clotting factors (Wallin & Martin 1985). Gla residues adopt a calcium-dependent conformation that promotes clotting factors binding to a membrane surface (Tie & Stafford 2016). The resulting Gla residues produced show an increased affinity for calcium ions (Malashkevich et al. 2013). Binding then elicits a conformational change rendering the VKDPs biologically active (Shearer & Newman 2014). The γ-Carboxylation of glutamate residues present in VKDPs is depicted in Figure 2.6. The 16

extent to which these proteins become carboxylated influences their activity. It is believed that vitamin K is preferentially taken-up by the liver to facilitate optimal blood coagulation (Booth 2009). The remaining vitamin K is then made available to facilitate carboxylation of extra- hepatic VKDPs such as osteocalcin (McCann & Ames 2009). It is for this reason that inadequate vitamin K intake may result in underlying diseases such as osteoporosis (Shiraki et al. 2000; Misra et al. 2013). For instance, although osteocalcin is not specifically regulated by vitamin K, vitamin K influences the extent to which osteocalcin becomes carboxylated (Gundberg et al. 2012) and therefore functional.

Figure 2.6: γ–Carboxylation of vitamin K dependent proteins (VKDPs) (adapted from Tie & Stafford 2016).

Vitamin K metabolites

As introduced earlier, K1 and MK-4 in their reduced forms are essential cofactors for the enzyme, γ-glutamyl carboxylase (GGCX) in the carboxylation of glutamate (Glu) residues in proteins (Edson et al. 2013; McDonald et al. 2019). During this process there are a number of metabolites produced.

Both K1 and MK-4 are removed from this homeostatic cycle primarily through initial cytochrome P450(CYP)4F2-mediated metabolism (Edson et al. 2013). CYP4F2 oxidises the terminus of the side chain, converting the ω-methyl group first to a primary alcohol (Edson et al. 2013). This is then oxidised to form the resulting vitamin K catabolites; Acid I and acid II (Edson et al. 2013). These catabolites are similar in structure to fatty acids as they are derived from the β-oxidation pathway (Tadano et al. 1989). Tadano et al. (1989) was one of the first to isolate and identify these metabolites in rat urine.

These urinary metabolites may prove to be useful measures of overall vitamin K status, but it

is not possible to determine if they originate from K1 or MK-4 (Shearer & Barkhan 1973; Landes et al. 2003). There are only a few methods available to measure these metabolites, none of which have been validated for use in horses (Harrington et al. 2005; Harrington et al. 2007). McDonald et al. (2019) recently published an LC-MS/MS method for the quantitation of these metabolites and identified another urinary metabolite; MK1 ω-COOH (McDonald et al. 2019).

This is believed to be formed from either K1 or MK-4 via P450- mediated ω-oxidation reactions (McDonald et al. 2019). These reactions initially generate the terminal carboxylic acids, followed by 5 rounds of β-oxidation side-chain truncation that ultimately produce the K acid I (McDonald et al. 2019). K acid I then undergoes further rounds of β-oxidation to produce MK1 ω-COOH which is then reduced to generate K acid II. (Gentili et al. 2014; Ferland et al. 2016)

2.2.4 Vitamin K–dependent proteins For many years only the VKDPs involved in the blood coagulation cascade were recognised. However, it is now appreciated that that vitamin K functions in the posttranslational modification of at least seventeen VKDPs (Table 2.1) and the search continues. The location and action of many of these proteins is fairly well described, but some have not been well characterised with the location and functionality of the transmembrane Gla-proteins 1, 2, 3 and 4 still unknown (Cancela et al. 2012; Wen et al. 2018).

Table 2.1: Vitamin K- dependent proteins (adapted from Cancela et al. 2012; McCann & Ames 2009; Rishavy & Berkner 2012 and Wen et al. 2018).

Proteins (VKDP) Location Function

Hepatic proteins:

Prothrombin, Factor VII, IX, Liver Coagulation & X

Proteins C, S & Z Unknown Anti-coagulation

Extra-hepatic proteins:

Cartilage, vascular Matrix Gla-protein (MGP) Inhibition of calcification tissue

Osteocalcin Osteoblasts Bone turnover and glucose metabolism

Gla-rich protein (GRP) CNS, spinal cord, Modulation of Ca thyroid

Regulation of vascular Gas6 Smooth muscle, homeostasis and sphingolipid endothelium metabolism

Periosteum, Fibrilogenesis and wound healing Periostin osteoblasts, remodelling

tissues

Keratoepithelin Most extra-hepatic Microtubule stability and positive tissues regulation of mutations.

Transmembrane Gla- Unknown Unknown proteins (TMG) 1, 2, 3, 4

2.3 Vitamin K status: challenges in determining requirements & deficiency At about the time of the discovery of vitamin K by Dam and Almquist (see Chapter 1), there was interest in North America in a haemorrhagic condition that occurred predominantly in cattle consuming mouldy sweet clover (Melilotus alba and Melilotus officinalis) hay (Cheeke 1998). Horses are reported to have suffered from sweet clover poisoning/toxicity, but the only recorded case is of a Percheron mare in Canada (McDonald 1980). Link (1959) provides a fascinating description of the discovery of dicumarol, the compound responsible for sweet clover poisoning/toxicity. Sweet clover contains a glycoside that is converted to dicumarol by fungal metabolism. The isolation of dicumarol and its derivatives, which were shown to be potent vitamin K antagonists, was conducted at the University of Wisconsin. The most widely used dicumarol derivative is warfarin (Figure 2.7). Warfarin is named for the Wisconsin Alumni Research Foundation and it is used globally both in medicine and as a rat poison.

Figure 2.7: The structural relationship of warfarin to dicumarol (adapted from Link 1959).

Figure 2.8 provides a detailed explanation of how warfarin inhibits vitamin K activity and in so doing, disrupts γ-carboxylation of proteins. In effect, vitamin K deficiency irrespective of whether it is derived from inadequate dietary intake or the presence of an antagonist in the diet, it is the resulting failure of γ-carboxylation that gives rise to the clinical signs of deficiency. It was the side effects of long term warfarin therapy that alerted researchers to the impact of vitamin K beyond coagulation (Booth & Mayer 2000).

Studies that report the metabolism, status and deficiency of vitamin K in the horse are listed in Table 2.2. This a stark reminder of how little we know about vitamin K in the horse.

Figure 2.8: Enzyme activities of the vitamin K (VK) cycle in the (a) absence and (b) presence of warfarin. (a). The enzyme γ-carboxyglutamyl carboxylase (GGCX) (activity 1), with the cofactor VKH2, facilitates the transformation of peptide-bound glutamate (Glu) to γ- carboxyglutamate (Gla) residues and the subsequent synthesis and secretion of carboxylated VKDPs. The γ-carboxylation reaction results in the generation of the epoxide metabolite VK>O, which is reduced to VK quinone by the enzyme VK epoxide reductase (VKOR) (activity 2). VK quinone is then reduced to the VKH2 cofactor by an unidentified nicotinamide adenine dinucleotide phosphate, reduced [NAD(P)H]-dependent reductase(s) (activity 3), or possibly by VKOR itself (activity 2), to complete the cycle. (b) In the presence of a VK antagonist, such as warfarin, VKOR (activity 2) is inhibited, resulting in the cellular synthesis and secretion of inactive species of undercarboxylated VKDPs called PIVKAs (proteins induced by vitamin K absence or antagonism). Given sufficient input of VK into the cycle, an alternative quinone reductase pathway (activity 3) can bypass the VKOR to provide the VKH2 substrate for GGCX and hence overcome the inhibitory action of warfarin, even under extreme blockade (adapted from Shearer & Okano 2018).

A vitamin K deficiency results in hypoprothrombinemia and prolonged blood clotting time and haemorrhages. With low vitamin K intake, blood clotting may be normal but there is increased risks of osteoporosis, articular cartilage damage, and osteoarthritis (Misra et al. 2013; Shearer & Newman 2014). The following sections will briefly review the two major aspects of inadequate vitamin K consumption; haemorrhagic diseases and skeletal health.

Table 2.2: Summary of equine studies investigating vitamin K.

Study cohort/samples Response Form of vitamin K and intake Outcome

Duello and Matschiner Horse liver Tissue K1 No vitamin K administered. Identification of K1 in horse liver. (1970) - Tissue analysis of K1

n = 8 horses Coagulation time, No vitamin K administered, 1g Synthesis of extrahepatic VKDPs is reduced in parallel with Vermeer and Ulrich growth, tissue K1 anticoagulant administered. the synthesis of the coagulation factors II, VII, IX and 9. (1982) - Effect of warfarin (4 warfarin treated, 4 controls)

Rebhun et al. (1984) - n = 6 horses Plasma K3 Parenteral administration of Vitamin Vitamin K3 induced renal toxicity in 5 of the 6 horses. Effect of K3 K3 (intake unknown).

n = 4 horses Coagulation time, K1 (100mg/dose), 300mg of vitamin K1 After discontinuation of warfarin administration there was a Byars et al. (1986) - Serum vitamin K1 IV, and 2 horses were given 300mg of prothrombin time (PT) reversal time of approximately 5 Therapeutic response of vitamin K1 subcutaneously. days from the last dose of warfarin. The 100mg dose of K1 vitamin K1 shortened the therapeutic response time to 12 hours and the PT reversal time to 24 hours.

n = 12 horses Total OC (HAP Both groups were allowed free access No significant correlation was present between the serum Siciliano et al. (2000a) - (QH,18-24 months) binding capacity), to brome grass hay (273mg of measures, osteocalcin and hydroxyapatite binding capacity Effect of exercise on serum vitamin K1 K1/100g). Additionally, the exercise of serum osteocalcin, and the bone measures. vitamin K status group received 40mg of K1/100g on the days they were exercised.

n = 15 horses (QH Total OC (HAP No vitamin K administered. In conclusion, vitamin K status increased and serum Siciliano et al. (2000b) - and TB foals) binding capacity), osteocalcin decreased with age. In addition, vitamin K status Effect of age at weaning serum vitamin K1 tended to increase at a greater rate in foals weaned early as on vitamin K status. compared to those weaned late.

Table 2.2: Summary of equine studies investigating vitamin K continued.

Study cohort/samples Response Form of vitamin K and intake Outcome

Piccione et al. (2005) - n = 5 horses (TB Serum vitamin K1 No vitamin K administered. Serum vitamin K concentration displayed an acrophase Diurnal monitoring of mares, 8 years) which occurred during the evening. vitamin K

Biffin et al. (2008a) - n = 69 horses (TB Total % OC 3mg K1 in proprietary soluble form In those horses administered vitamin K1, %cOC increased. Effect of vitamin K on yearlings) administered to 3 of these horses for 9 Results suggest that pasture source and vitamin form can bone density and OCD days. markedly change the vitamin K status of horses.

TB (young horses Plasma vitamin 9 mg/day of supplemental vitamin K3. Plasma MK-7 concentration was extremely low in horses, Inoue et al. (2009) - and mares). K1, MK-4, and the nutritional importance of vitamin K2, which is Circulating vitamin K MK-7 synthesized in the intestinal tract, may not be significant in status and absorption (n = unknown) horses.

SB colt (n = 1) Coagulation No vitamin K administered. Although vitamin K administration resolved colt A's VKDB, McGorum et al. (2009) - factors and coagulation indices were normal 5 days after cessation Case study: VKDB of vitamin K administration, colt A was euthanized at 9 weeks of age because of prolonged failure to thrive.

Biffin et al. (2010) - Effect n = 26 horses (TB, Growth, bone 7mg QAQ/day and the control group, a Horses may receive suboptimal intakes of vitamin K and of vitamin K 2 years) density, plasma blank powder. further research is required to determine equine vitamin K supplementation on bone vitamin K1 requirements in different management systems and the density efficacy of different forms of the vitamin.

n = 16 horses Plasma K1, MK-4, Each group was given K1, MK-4, or K3 The K1 concentration increased (P < 0.001) after feeding in Terachi et al. (2011) - K3 at 58µmol/d, or no vitamin K the K1 group but no changes in the plasma K1 concentration Circulating vitamin K (8 mares and 8 supplement for 7 d. were observed after feeding in the other groups. Plasma status and absorption geldings) MK-4 concentration was greater (P < 0.001) in the K3 group.

Table 2.2: Summary of equine studies investigating vitamin K continued.

Study cohort/samples Response Form of vitamin K and intake Outcome

n = 12 horses Plasma was There were six treatments (200mg); The soluble form of the vitamin, KQ was the most Skinner et al. (2014) - (geldings) analysed for K1, control, K1, K2 (in the form of MK-4), efficiently absorbed. There is no specific conversion of K1 to Absorption of vitamin K MK-4 and K3. K3 and KQ (Quinaquanone™, a soluble K3 or K3 to MK-4 in plasma. form of K1 and K2 in the ratio of 10:1).

n = 4 horses Plasma K1, MK-4 200mg oral of either (1) KQ (2) KQ There was no further uptake of K1 from the spheres in the Skinner et al. (2015) - (geldings) and K3 coated with 1.5% calcium alginate or hind gut, suggesting that the hind gut does not facilitate Hindgut absorption of K (3) K1 oil. vitamin K absorption in the horse. vitamers

n = 18 horses Plasma and milk Treatment groups (n = 6): no vitamin K Vitamin K1 concentrations in the foal pre-suck plasma and Fischer et al. (2017) - (mares and foals) sampled for K1 supplementation (carrier paste only); umbilical cord samples were below the detection limit of the Maternal transfer of and K3 vitamin K3 (15mg); vitamin KQ a analysis (<0.01 ng/mL) across all groups. vitamin K soluble form of K1 and K2 (15mg).

2.3.1 Vitamin K deficiency bleeding (VKDB) Bleeding disorders occur in the new born and neonate and are rarely reported in adults.

The human neonate Limited placental transfer of vitamin K in humans, can lead to a syndrome known as vitamin K deficiency bleeding (VKDB), where haemorrhagic events occur and can be fatal in the newborn (Shearer et al. 2012). A few studies have observed a link between warfarin based anticoagulants, administered to treat thrombosis during pregnancy, with an increased risk of bleeding, cartilage calcification and abnormal bone development in the neonate (McCann & Ames 2009; Fujioka et al. 2017).

Aside from maternal exposure to warfarin during early pregnancy, there are other contributing factors to the incidence of VKDB in the human neonate. Studies have shown that human breast milk contains low concentrations of vitamin K, with the concentrations of clotting factors measured at birth low. Deficiency of vitamin K is evidenced by the low plasma concentrations of K vitamers within liver tissue of neonates (Shearer 2009a). To reduce the risk of VKDB, vitamin K is therefore routinely administered to the infant at birth, by either an oral dose or an intramuscular injection (Greer et al. 1997; Shearer 2009a). Prophylaxis programs throughout the world aim to reduce the incidence of VKDB within the population, however, early infancy deficiency is still prevalent in developed countries (Shearer 2009b).

The human neonate is most at risk during the first six months of life (Shahrook et al. 2018). Infants presenting with intracranial bleeding between three to eight weeks of age, may suffer permanent neurological damage, or may die (Shearer et al. 2012). Shearer et al (2012) found that there is no predisposing factor to VKDB currently identified, although it is possible that some pathologies which cause malabsorption could be a contributing factor.

Beulens et al. (2013) suggests that the deficiency of vitamin K in the new born stems from a number of sources, beginning with a low placental transfer of vitamin K to the foetus. Breast milk has also been found to be low in vitamin K, along with a sterile gut at birth, therefore no bacterial production of vitamin K. These factors reduce the neonate’s potential intake of this essential vitamin within the first few months of life, resulting in low clotting factors (produced from VKDPs) and the increased risk of haemorrhage (Beulens et al. 2013). Interestingly, the

vitamin K1 content of formula based feeds have been found to be 50 fold greater, than breast milk, resulting in a much reduced risk of VKDB occurring (Shearer et al. 2012).

Incidence of VKDB within the horse VKDB, has only been reported in a few case studies in horses. McGorum et al. (2009) reported an incidence of VKDB in a Standardbred colt. The administration of vitamin K successfully stopped the bleeding however, the horse was subsequently euthanised due to ill thrift (McGorum et al. 2009). There are no other cases of VKDB that have been reported in foals, possibly due to the presenting clinical signs (McGorum et al 2009).

There are a few early studies that investigated the effect of Warfarin on vitamin K and blood parameters however, the prognosis was not good for the long term health of the horses and ponies used in these studies (Scott et al. 1978, 1979; Scott et al. 1980). In one of the studies vitamin K1, was administered intravenously to reverse anticoagulation however, it was only successfully reversed for 2 of the 4 horses in the study (Scott et al. 1980).

Composition of milk in mares An important aspect of reducing the incidence of VKDB is the provision of vitamin K to the neonate by milk. Most research investigating the composition and quality of mares’ milk has been conducted within Western Europe (Krešimir et al. 2011), where there is an increased focus on nutrition and health status of the newborn foal. In some areas, mares are milked specifically for the production of products for human consumption. Mares milk has a different composition compared to other domestic species however, it is relatively similar in composition to breast milk, and is frequently utilised as a substitute for infants intolerant to cow’s milk in some countries (Doreau & Boulot 1989; Pieszka et al. 2016; Barreto et al. 2019).

Similarities to human milk include low casein to whey protein ratio, low nitrogen content, high lactose content, and low cholesterol content (Krešimir et al. 2011; Pieszka et al. 2016). The main difference in composition is in fat levels, where mare milk is 12.1gm/kg, compared to 36.4gm/kg in human breast milk (Table 2.3)

Table 2.3: Gross composition of mare milk in comparison to human milk (g/kg) (adapted from (Krešimir et al. 2011)). Mare Human Cow Fat 12.1 (5-20) 36.4 (35-40) 36.6 (33-54) Crude protein 21.4 (15-28) 14.2 (9-17) 32.5 (31-39) Lactose 63.7 (58-70) 67 (63-70) 48.8 (44-49) Ash 4.2 (3-5) 2.2 (2-3) 7.6 (7-8) Gross energy * 480 (390-550) 677 (650-700) 674 (650-712) * measured as kcal/kg mean value and between brackets, ranges are varied between literature.

In the newborn foal the ingestion of colostrum during the first 36 hours of life is crucial to their health. During that period the small intestine is able to absorb antibodies and large proteins with Immunoglobulin G (IgG) being the main antibody that is provided to the foal via colostrum (Mateja et al. 2014; Dror & Allen 2018).

The crude protein content in milk is found to drop rapidly within 12 hours post-parturition (19.1% at birth to 3.8% 12 hours later). This likely attributable to a decline in IgG concentration (Krešimir et al. 2011).

There are some differences in the fat composition of mare milk between that of colostrum, and the remaining milk produced during lactation. Mare milk has higher concentration of phospholipids with the composition of these being different from that of human and cow’s milk (Csapó et al. 1995; Krešimir et al. 2011).

Colostrum is a more concentrated source of vitamins for foals than milk produced throughout lactation (Csapó et al. 1995). As depicted in Table 2.4, vitamin K1 concentrations were 1.48% higher in colostrum, than milk produced between 8 and 45 days post-parturition.

Table 2.4: Vitamin content of mare colostrum and milk (mg/kg) (adapted from (Csapó et al. 1995)).

Postpartum days Vitamin 0-0.5 8-45 5-270 A 0.8800 0.3400 0.3520 D3 0.0054 0.0032 0.0029 E 1.3420 1.1280 1.1350 K1 0.0430 0.0290 0.0320 C 23.800 17.200 15.320

It is postulated that the mare is very inefficient at transferring ingested nutrients into milk, and as such the mares requirements for dietary nutrients are relatively high prior to and during lactation (Mateja et al. 2014). In direct contrast to the cow, the mare will decrease its milk protein content when fed high-concentrate diets, attributable to a higher milk yield causing a dilution effect (Barreto et al. 2019). Likewise, feeding a high concentrate diet with a low amount of roughage reduces the milk fat content of mare’s milk, which is attributed to lower de novo synthesis of acetate and butyrate (Barreto et al. 2019).

2.3.2 Skeletal health With the isolation of vitamin K dependent proteins located in bone, especially osteocalcin (see Section 2.5), there has been much interest in delineating the role of vitamin K in skeletal health, especially osteoporosis and osteoarthritis (Misra et al. 2013; Shearer & Newman 2014). All of the research into the relationship of vitamin K to bone accretion and turnover has centred on these human maladies that usually occur in older adults; similar conditions have not been reported in older animals. However there are a number of developmental bone diseases in horses, pigs, chickens, turkeys and dogs (Ytrehus et al. 2007) that have many similarities with osteoporosis. As yet research into the animal conditions has not been undertaken. The possible role of vitamin K in the pathogenesis of equine osteochondrosis is examined in Section 2.4.2.

2.3.3 Determining requirements Traditionally, assessment of vitamin K status has relied on the general observation of optimal blood coagulation, for which vitamin K is an essential cofactor and is therefore indicative of adequate vitamin K status (Suttie & Booth 2011). In humans circulating levels of vitamin K have been found to have large inter- and intra-individual variation (Booth 2010; Shea et al. 2012). Such findings make determining species and gender specific dietary requirements challenging.

In addition, the relative absorption efficiencies of each of the isomers of vitamin K vary among species. This has ramifications for assessing dietary vitamin K requirements in horses, as most studies have been conducted predominately with rodents and humans (Shearer et al. 2012). It

has been well documented however, that K1 is predominate form in herbivores and K2 in omnivores and carnivores (Thijssen et al. 1996). The horse almost exclusively utilises and

stores K1 and therefore their vitamin K profile is different to that of rodents; the species in which most research has been conducted. Will et al. (1992) found that poultry have a higher dietary vitamin K requirement than rodents. This is presumably due to their higher need for vitamins and minerals for egg deposition and to aid in egg shell development.

The triage theory proposed by McCann & Ames (2009) highlights the importance of vitamin K, its different forms and their individual roles in modulating the activity of VKDPs. While vitamin K is essential for blood clotting, current recommendations of vitamin K intake have been found to be suboptimal to maintain the functioning of extra-hepatic VKDPs (Harshman et al. 2014b). The difficulty however, as mentioned previously is that vitamin K absorption can vary considerably depending on several factors. For instance, as it is a fat-soluble vitamin, its 28

absorption is, improved when accompanied by fat within a meal and is also influenced by the degree and method of cooking (Harshman et al. 2016). With regards to animals particularly grazing species, the vitamin K status of pastures is also known to vary considerably (Booth 2012). It is largely dependent on environmental factors such as the season, weather and time of day. Likewise, the vitamin K content of hay varies and is sensitive to drying and exposure to ultraviolet radiation (Erkkilä et al. 2004). Therefore, in some cases the vitamin K content of hay can be very minimal (Biffin et al. 2008b). This has implications for animals with restricted access to pasture, where the vitamin K status of their diet may be insufficient to ensure optimal functioning of extra hepatic VKDPs (Biffin et al. 2008a).

With the various challenges presented in determining vitamin K status, there is an urgent need to explore other possible measures. Whilst utilisation of prothrombin time appears to be the preferred method of determining status of vitamin K. The indirect measurement of osteocalcin has been proposed to be a more suitable measure of subclinical vitamin K deficiency (Gundberg et al. 1998). However, recent research suggests the degree of carboxylation of VKDPs, especially serum osteocalcin concentrations, is a more sensitive indicator of vitamin K status than coagulation (Fusaro et al. 2016a)

To date, there has been only a limited number of studies that have measured and investigated vitamin K concentrations and functioning in horses. An overview of these studies can be found in Table 2.2. According to National Research Council (2007) the dietary requirement for vitamin K in the horse is yet to be confirmed. Contrary to this, Siciliano et al. (2000b) states that microbial synthesis of K2 within the gut, as well as the supply of K1 via green pasture is enough to meet a horse’s daily requirements. Unfortunately, this only refers to coagulation, as

the degree of carboxylation was not determined. Furthermore, Will et al. (1992) found that K2 of microbial synthesis made a minimal contribution to the overall vitamin K status in both poultry and rodents. More recently, McCann et al. (2019) found that while some evidence does

exist that vitamin K2 is produced by gut bacteria, the mechanisms by which they are absorbed

from the colon in humans are unknown (Karl et al. 2017). This questions the contribution K2 to vitamin K status in horses.

In summary, there are two biomarkers that have been routinely used to assess vitamin K status;

plasma K1 and prothrombin time. However, there is a growing consensus that circulating concentrations of undercarboxylated osteocalcin maybe a more appropriate measure of vitamin K status. 29

2.4 Bone development and pathophysiology Over the duration of an animal’s life bone undergoes a process known as remodelling whereby it is continuously resorbed and formed (Lepage et al. 1998). This process is regulated by several key proteins osteoblasts and osteoclasts (Lepage et al. 1998). Osteoclasts are responsible for bone resorption, which entails the breakdown of bone and its integral collagen matrix (Lepage et al. 1998). Osteoblasts are responsible for bone formation, synthesising proteins to form the new bone matrix (Lepage et al. 1998). Osteoclasts also play a role in calcium homeostasis, allowing the calcium within bones to be withdrawn and released into circulation when it is needed by the body (Colville & Bassert 2008). This continuous process of remodelling yields specific molecules which can be used as markers to assess bone metabolism. Osteocalcin for instance is secreted by osteoblasts and incorporated into the bone matrix, before being released into circulation during the process of bone resorption (Donabedian et al. 2008). Serum levels can therefore be used as an indicator of the extent of bone turnover, potentiating its use as a non-invasive measure of bone metabolism in horses (Donabedian et al. 2008; Lepage et al. 1998).

The process of bone development is relatively similar among species, with only a few minor modifications despite their diverse array in morphology (Kronenberg 2003). While first initiated during foetal development, it occurs primarily after birth (Dibner et al. 2007). Osteoblasts form the extracellular matrix of bone (ECM) which is initially composed of a network of collagen fibres in particular type 1 collagen fibres (Dibner et al. 2007).

The bone matrix is comprised of both inorganic and organic substances; with crystalline hydroxyapatite [3Ca3 (PO4)2] (OH2)] making up most of the inorganic component. The organic constituent of bone is comprised mostly of type I collagen fibres which make up over 90% of this component (Feng & Teitelbaum 2013). This provides the structural support for which hydroxyapatite crystals, responsible for bone hardening can bind, a process known as endochondral ossification which occurs in long bones (appendicular skeleton) (Colville & Bassert 2008; Dibner et al. 2007).

2.4.1 Endochondral Ossification Endochondral ossification depends largely on trace mineral availability, calcium and phosphorous (Dibner et al. 2007). Calcium and phosphorous are both deposited in the cellular bone matrix in the form of hydroxyapatite crystals. The main minerals found to play a crucial role in bone development include manganese, copper, zinc, calcium, phosphorous as well as 30 vitamin D (Dibner et al. 2007). Mineralisation is essential for bone development, with most taking place within the epiphyseal plates. Excessive or inadequate mineralisation within the epiphyseal plate is implicated in the pathogenesis of several diseases including osteoporosis (Dibner et al. 2007).

The first step in endochondral ossification is that cartilage cells known as chondrocytes form cartilaginous rods, which will be gradually replaced by bone (Kronenberg 2003; Dibner et al. 2007). As can be seen in Figure 2.9, this process of cartilage removal and subsequent bone development begins in the central area of long bones known as the diaphysis (Kronenberg 2003). As depicted in Figure 2.9, growth cartilage consists of three primary zones; resting (RZ), proliferative (PZ) and hypertrophic zones (HZ) (Wongdee et al. 2012). In the proliferative zone chondrocytes are responsible for the production of the extracellular matrix (ECM) (Wongdee et al. 2012). These cells then differentiate into hypertrophic chondrocytes which is followed by a subsequent increase in the secretion of non-peptides (C2C) and C-terminal (CPII) pro- peptides of type II collagen (Hoogen et al. 1999; Kronenberg 2003). These are thought to act as nucleation sites for mineral deposition to occur, and are the principle cells responsible for the growth and regulation of bone (Kronenberg 2003). Within the hypertrophic zone matrix vesicles appear which are responsible for the transportation of alkaline phosphatase, calcium and phosphate, which are essential for the development of hydroxyapatite; the main matrix component of bone (Hoogen et al. 1999).

Figure 2.9: Structure of a long bone (adapted from Wongdee et al. 2012). 31

As this is taking place, osteoblasts are delivered by the vascular system to these sites of new bone formation and become embedded in the matrix forming osteocytes (bone cells) (Kronenberg 2003). The hypertrophic chondrocytes then undergo cell death allowing the invading osteoblasts and blood vessels to transform the cartilage into bone; primary spongiosa (Semevolos 2017). This takes place predominately in the calcified septae of the lower hypertrophic zone, depicted as the growth plate in Figure 2.10. Cartilage canals facilitate the blood supply to this area and are not only responsible for the delivery of bone cells but also the delivery of essential nutrients to the area (Olstad et al. 2008). Epiphyseal cartilage is distinguishable histologically from the overlying articular cartilage, by the presence of these cartilage canals (Semevolos 2017).

As new bone is laid down and expands, a few of these hypertrophic chondrocytes will remain to allow additional bone development to occur (Ytrehus et al. 2007). This takes place in just two areas, known as the secondary centres of ossification located in the proximal and distal epiphyses (Kronenberg 2003; Wongdee et al. 2012). These sites are known as the growth plates or epiphyseal plates, and they are located between the diaphysis and epiphysis of each end of the bone (Kronenberg 2003). These areas allow additional lengthening of the bone as the animal grows with epiphyseal plate closure occurring once the animal has reached

Figure 2.10: Schematic diagram of Endochondral Ossification (adapted from Mackie et al. 2008).

a certain growth stage milestone (Ytrehus et al. 2007; Mackie et al. 2008). Radiographic studies on horses have shown that maximum bone mineral content across all breeds is not achieved until the horse is approximately 6 years old (Lawrence 2005). This is characterised by the ossification of the epiphyseal plates when all cartilage is replaced with bone (Semevolos 2017). Subsequently, the regression of cartilage canals from the epiphyseal cartilage occurs, a process referred to as chondrification (Ytrehus et al. 2007). In humans’ closure of the growth plates occurs at the onset of adolescence (puberty) which can vary between individuals (Kronenberg 2003). Most bones in the body develop via this process of endochondral ossification as depicted in Figure 2.10 (Mackie et al. 2008; Bourebaba et al. 2019). It is therefore the primary mechanism that determines an animal’s underlying skeletal morphology, and can be influenced by several factors, in particular physiological conditions and nutritional status (Lepeule et al. 2013).

Developmental orthopaedic disease (DOD) & Osteochondritis dessicans (OCD) Developmental orthopaedic disease (DOD) is a term that encompasses an array of growth related disturbances in horses (Jeffcott 1991). Osteochondrosis falls under this banner and can be defined a group of conditions that result from a disturbance to the cartilage growth, generally observed as a primary lesion in the articular and/or epiphyseal cartilage and growth plates which can result in osteochondritis dissecans (OCD) (Bruggeman et al. 2010; Jeffcott 1991).

Figure 2.11: Diagrammatic representation of the development of osteochondritis dissecans (OCD) and subchondral cystic lesions (adapted from Baxter 2011). OCD is a multifactorial disease with a very complex aetiology that has been identified in a number of mammals including pigs, dogs, cattle, cats, horses, rodents and humans (Ytrehus et al. 2007). It is a significant cause of limb weakness in pigs and lameness in horses, significantly lowering their sales prices (Robert 2013). An overview of the development of osteochondrosis and the lesions it can manifest is provided in Figure 2.11 (Wright & Minshall 2005; Baxter 2011). It develops due to impairment in the endochondral ossification, generally as a result of

failure of calcification and/or vascular invasion to occur (Olstad et al. 2011; Ytrehus et al. 2007).

There are a few manifestations of the disease depending on the site of the endochondral ossification defect (Lykkjen et al. 2012). The clinical significance of osteochondritis dissecans (OCD) also varies (Frantz et al. 2010). Radiographic identification of OCD may or may not be accompanied by clinical signs. It has also been shown that some lesions have a higher potential to self-correct than others (Mendoza et al. 2016). Those often causing the most significant clinical problems are lesions and subchondral bone cysts as depicted in Figure 2.11 (Lykkjen et al. 2012).

OCD represents one of the main challenges in equine orthopaedics. This disease can compromise horse performance causing career–limiting or career–ending lameness (Chiaradia et al. 2012). The cause of OCD remains uncertain but various factors have been implicated such as skeletal growth rate, nutrition, genetics and physical activity (Lepeule et al. 2013). Delayed diagnosis precludes preventative or timely therapeutic actions. Proposed diagnostic markers were suggestive of an inflammatory and cartilage turnover condition (Chiaradia et al. 2012). There is an urgent need to identify individuals at earlier stages of the disease process because conservative methods of treatment can help affected animals resolve the condition when identified early (Billinghurst et al. 2004). Radiography and magnetic resonance imaging (MRI) are some of the imaging modalities currently used for detecting established lesions, and can also be used to monitor disease progression; however radiography is insensitive for detecting early changes, especially in cartilage and can be a laborious and time–consuming process (Fontaine et al. 2013).

2.4.2 Equine vitamin K and bone metabolism Such evidence supporting a possible linkage between vitamin K and bone development has led to the question of the role played by vitamin K in equine bone development, especially the pathogenesis of bone related diseases.

It is evident that a number of key nutrients are intimately linked to the processes involved in bone development. Some have a direct affect including calcium and phosphorous, while others, such as vitamin K act indirectly (Wongdee et al. 2012). As previously discussed, vitamin K is necessary for the optimal functioning of VKDPs, in particular osteocalcin and matrix Gla protein (MGP) in bone (Wongdee et al. 2012). Osteocalcin and MGP are essential proteins

involved in bone development with each possessing the ability to bind calcium and therefore provide a framework for calcification of the cartilaginous matrix to occur (Wongdee et al. 2012). A disease known as ‘Keutel syndrome’ is a result of a mutation in the MGP gene and is characterised by abnormal cartilage development and calcification (Wongdee et al. 2012). It bears some resemblance to OCD seen in horses, which is generally characterised by retention and thickening of the cartilage as a result of impaired bone development (Hoogen et al. 1999).

The initial lesions of OCD arise in the growth cartilage disturbing the development of cartilage cells and the surrounding matrix (Jeffcott 1991). This detrimentally affects bone development in this area. The development of cartilage canal vessels is particularly prone to failure within this area, with necrosis of these vessels implicated as an underlying cause in its pathogenesis (Ytrehus et al. 2007). This is supported by the findings of Olstad et al. (2011) in which early lesions of OCD in foals were consistently found in regions where early ossification and vascular invasion were occurring. Likewise experimental lesions, identical to those of OCD in pigs can be induced by arresting the supply of blood to epiphyseal cartilage (Carlson et al. 1995). This implies that areas in which cartilage canal vessels are required to traverse tissue junctions; such as that between growth cartilage and bone, increases their vulnerability to failure therefore, also increasing the risk of OCD lesions developing (Olstad et al. 2011). Such findings also suggest that only a narrow window of opportunity exists during which OCD develops and could be treated. This is because as the individual matures, vascular tissue regresses and articular cartilage becomes avascular, deriving nutrition from synovial fluid instead (Olstad et al. 2011).

Mineral and nutrient imbalances have been confirmed as a significant contributing factor in the pathogenesis of the disease (Jeffcott 1991). Deficiencies and/or imbalances of vitamin D, calcium, phosphorous, copper and zinc have all been identified in its underlying pathology, adding further weight to the potential role vitamin K may indirectly play in its pathogenesis.

Lesions of OCD can be detected before 9 months of age in horses, suggesting that initiation of the lesion can occur at a very young age (Wright & Minshall 2005). Evidence in support of this link has also been identified in humans, pigs and dogs (Jeffcott 1991). Although there are some distinct differences in the pathology of the disease between species the underlying pathological development is relatively uniform (Jeffcott 1991). An example of this can be seen in Figures 2.12 and 2.13, showing a comparison between OCD lesion seen in horses and a similar disease found in poultry, known as tibial dyschondroplasia (TD) (Dibner et al. 2007). 36

Initial lesions of OCD are very similar in both foals and pigs, with its histopathology also found to resemble that of TD in birds (Olstad et al. 2008). TD is characterised by the arrested development of chondrocytes in their pre-hypertrophic state (Dibner et al. 2007). This is accompanied by distinct biochemical changes in the growth plate cartilage, with an observed reduction in osteocalcin and growth related factors; collagen type X and growth factor-β (Dibner et al. 2007).

A causal relationship between abnormalities in cartilage canal vessels and lesions of OCD has been suggested in horses, pigs and dogs. In both horses and dogs the incidence of OCD appears to be associated with early bone development, before closure of the growth plate occurs (Kane 2013). High metabolic activity during this period of early growth, makes the skeleton most susceptible to nutritional and metabolic insults, increasing the risk of such diseases (Kane 2013). The large performance horse breeds such as Warmbloods and Thoroughbreds have the highest incidence of OCD (van Weeren & Jeffcott 2013). A similar phenotypic and genetic predisposition has also been identified in dogs with larger breeds such as Great Danes and Doberman’s the most susceptible (Kane 2013). The bird condition TD also serves as an example of a leg problem that accompanies rapid growth (Dibner et al. 2007). This suggests a link between the development of bone diseases and the genetic potential for rapid growth.

Figure 2.12: Normal growth plate cartilage in birds (A) and that of a TD lesion (B) (adapted from Dibner et al. 2007).

Figure 2.13: Cartilage development in the horse: (A1) Healthy distal tibial articular cartilage from a 2 year old horse, and (B1) the site of an OCD lesion on the femoral condyle articular cartilage of a weanling (adapted from Olstad et al. 2008).

Osteocalcin as outlined above; of which vitamin K is an essential cofactor plays a vital role in cartilage development and therefore, this poses the question of whether under-carboxylated osteocalcin contributes to the development of OCD in horses? A preliminary study by Biffin et al. (2010) found that OCD lesions in a group of affected 2 year old Thoroughbreds appeared to regress after supplementation with an oral proprietary formulation of vitamin K (Figure 2.14). The preliminary results of this study must be interpreted with caution however, as some OCD lesions have been found to be developmentally sensitive and regress overtime without any intervention (Mendoza et al. 2016). These results therefore warrant further investigation in a larger, controlled cohort of horses. 38

Figure 2.14: A) OCD lesion of the stifle, B) Regression of the same lesion after supplementation with vitamin K (adapted from Biffin et al. 2010).

2.5 Osteocalcin In 1975 osteocalcin; a VKDP produced by osteoblasts in bone was characterised (Hauschka et al. 1975; Camarda et al. 1987). Formerly, it was referred to as Bone Gla Protein (BGP) in the early literature (Price et al. 1976; Price et al. 1981)

2.5.1 Structure & biochemistry of osteocalcin Osteocalcin is the most abundant non-collagenous protein found in bone and is comprised of 49 amino acids (AA) (Zoch et al. 2016). Initially discovered in bone by Hauschka et al. (1975) it has a highly conserved sequence among vertebrates. As depicted in Figure 2.15, it is a vital constituent of the bone matrix, binding with calcium to form hydroxyapatite crystals (Hoang et al. 2003). Vitamin K increases the carboxylation of glutamic acid (Glu) residues in osteocalcin which is crucial to facilitate its binding to hydroxyapatite in bone (Krueger et al. 2009). However, a small fraction of the osteocalcin is released into the bloodstream where it is metabolised by the kidney and liver and excreted (Price et al. 1981). Insufficient vitamin K can result in under-carboxylated osteocalcin (ucOC) which cannot bind hydroxyapatite and is

released into circulation in nanomolar concentrations (Price & Nishimoto 1980; Gundberg & Weinstein 1986).

Figure 2.15: Diagrammatic representation of the tertiary crystal structure of osteocalcin and its coordinated binding of Gla residues to calcium (Ca2+) to form hydroxyapatite in bone (adapted from Hoang et al. 2003).

Osteocalcin contains three glutamic acid (Glu) residues (Karsenty 2012). In order to form hydroxyapatite in bone it must bind to calcium ions (Hoang et al. 2003). A lack of binding to calcium results in ucOC. If all three sites are carboxylated this results in carboxylated osteocalcin (cOC) (Hoang et al. 2003). When only one or zero sites are carboxylated this results in under or ucOC respectively (Levinger et al. 2014). Although osteocalcin binds tightly to hydroxyapatite, a fraction of the newly synthesised molecule will be released into the blood (Fusaro et al. 2016a). In humans the carboxylation of osteocalcin has also been identified to be an ordered process (from Glu-24, Glu-21 to Glu-17). At any one instance in time depending on the vitamin K status of the body osteocalcin can be found circulating in a number of different truncated forms (Rehder et al. 2015).

The ability to determine the carboxylation status of osteocalcin may therefore be an important marker to assist in determining vitamin K status of the body (McKeown et al. 2002b). However, to date a universal analysis method has not been developed to be able to accurately determine this. Furthermore, its circulating forms have not been well defined (Rehder et al. 2015). Several assays have already been developed for measuring the circulating osteocalcin concentrations

in various species (Patterson-Allen et al. 1982; Garnero et al. 1994). However, an assay which can discriminate between the different circulating forms; ucOC and cOC remains elusive.

Circulating kinetics Garnero et al. (1994) reported the presence of low-molecular weight osteocalcin fragments in serum that were not related to bone resorption. In humans they detected four key fragments; N terminal, mid, N-terminal mid and mic-C terminal. These were detected in the serum of healthy patients and patients with metabolic bone disease (Garnero et al. 1994). They were unable to detect the c terminal figment (43-49) suggesting that it is rapidly degraded and excreted (Garnero et al. 1994). Findings in the literature suggest that in human’s osteocalcin bonds involving arginine residues are the most susceptible to proteolytic cleavage; 19-20 and 43-44 (Price et al. 1976; Gundberg & Weinstein 1986).

Osteocalcin has also been documented to be “glycated” whereby glucose reacts non- enzymatically with certain groups on a protein (Gundberg et al. 1986). For instance glycation is known to be responsible for inducing conformational change in albumin, altering the binding capability of ligands (Gundberg et al. 1986). Studies by Gundberg et al. (1986) revealed that the extent of glycation of osteocalcin in human bone is age dependent and may influence its affinity for hydroxyapatite. However, the recent findings of Thomas et al. (2017) suggest that glycation may in fact not interfere with osteocalcin affinity for hydroxyapatite. It may however, interfere with its interaction with other bone proteins; collagen I and osteopoitin (Thomas et al. 2017). Either way, glycated fragments released from bone during bone resorption could be used to assess bone quality at least in the N-terminal region (which is the fragment where glycation has been identified) (Thomas et al. 2017).

The N-terminal mid-fragment were detected at the highest concentration compared to other fragments (Garnero et al. 1994). Furthermore the concentration of this peptide in serum was not altered when patients were treated with an inhibitor of bone resorption. This indicates that this large peptide is not released into circulation during osteoclastic degradation (Garnero et al. 1994). Pooled serum sample results also found this peptide to be the most abundant, representing about 30% of the total osteocalcin concentration in health adults (Garnero et al. 1994). Their study also highlighted the importance of maintaining samples at 4°C immediately after sampling and freezing them within 1 hour to prevent further degradation (Cleland et al. 2016).

In this study, serum creatinine concentrations were also analysed using a commercially available test kit (Garnero et al. 1994). Creatinine values were found to be in the normal range of 90-140µmol/L (Garnero et al. 1994). Serum/plasma creatinine concentration is indicative of renal function (Lepage et al. 1990). Impaired renal function has been found to affect osteocalcin concentration, so determination of creatinine is essential when looking at osteocalcin (Price et al. 1981; Delmas et al. 1983b).

Diurnal variations in osteocalcin concentrations has also been reported in the literature (Markowitz et al. 1987; Lepage et al. 1990) with highest osteocalcin concentrations during the morning and night (Black et al. 1999). A negative logarithmic regression was reported between age and serum osteocalcin in healthy subjects (Carstanjen et al. 2004). Higher osteocalcin concentrations in weanlings when compared to adult horses has been reported (Mäenpää et al. 1988; Black et al. 1999).

While there is conflicting findings amongst the literature with regards to the relationship between osteocalcin and other proposed roles such as energy metabolism (Centi et al. 2015), there is evidence linking increased plasma ucOC concentrations to bone diseases in both animal and human models (Poser et al. 1980; Urano et al. 2015). This warrants further investigation in horses.

2.5.2 Osteocalcin and bone metabolism and disease Equine skeletal development is significantly influenced by nutritional related factors (Staniar 2010). Hauschka et al. (1983) were one of the first research groups to speculate the importance of osteocalcin to bone metabolism. Using the chicken model they identified the presence of Gla at the onset of longitudinal growth in the developing chick embryo around day 7-8, which coincided with the onset of mineralisation and calcium deposition (Hauschka et al. 1983). It may therefore be used as a biomarker in which to study diseases or conditions associated with bone diseases, exercise, different therapy forms and/or dietary changes (Carstanjen et al. 2003).

Bone has long been known to be influenced by hormonal status of the body, with the well documented literature regarding the relationship between pre and post–menopausal, oestrogen and bone loss (Iwamoto et al. 1999; Booth et al. 2004a; Parazzini 2014). Certain diseased states have also been well revised with regards to the actions they exert on bone (McCabe et al. 2013). These changes to bone however, were previously thought to be a side–effect of all the changes rather than a pivotal mediator in the cause of diseases such as osteoporosis and osteoarthritis.

Supplementation with vitamin K is used to treat and prevent osteoporosis and osteoarthritis in humans (Neogi et al. 2006; Oka et al. 2009; Misra et al. 2013; Shea et al. 2017). In Japan MK- 4 is frequently used to aid in the prevention and treatment of osteoporosis with high doses of 45mg/day prescribed (Fu et al. 2012). However, for the wider population such a dosage is unachievable from diet alone. Only a few studies have looked at the effects of supplementing vitamin K in healthy individuals with levels that are achievable through the diet (Booth et al. 1999; Bonjour et al. 2009; van Summeren et al. 2009). However, no definitive causal relationship was found linking vitamin K to an increase in BMD or bone health (Bonjour et al. 2009). This may be attributable to an overall healthy diet and therefore general state of health in the subjects studied. In the majority of observational studies undertaken however, K1 intake was found to be associated with a decreased risk of hip fracture and decreased percentage of under-carboxylated osteocalcin (Shea & Booth 2008; van Summeren et al. 2009). Similarly, studies examining the effects of vitamin K supplementation in rats have consistently demonstrated a protective influence of vitamin K supplementation on bone loss. Sogabe et al.

(2011) found an increased BMD of the femur in rats supplemented with K1 as opposed to controls. This supports the findings of Iwamoto et al. (2005) who administered vitamin K to rats fed a normal or low calcium diet. They found increased cancellous bone mass and cortical bone mass respectively (Iwamoto et al. 2005).

Is there a relationship between osteocalcin and equine bone diseases? Currently research has been focused on links to age related diseases including; dementia and osteoporosis in humans (Noori et al. 2014; Grimm et al. 2016). As such there is an abundance of research in human and rodent studies, with limited research conducted in other species (Desjardin et al. 2012). With the increasing research implicating vitamin K in extra-hepatic roles vital to health and longevity in humans, it is only appropriate that increased research pertaining to animals particularly production animals be carried out. In the long term this could increase production capability by ensuring optimal health and welfare. With studies implicating osteocalcin as a marker of bone metabolism in states of metabolic bone disease (Poser et al. 1980; Delmas et al. 1983a) it is only appropriate that its possible relationship and role in the aetiology of equine bone disease be explored. The correlations observed between serum/plasma concentrations and histomorphometric findings related to aging and disease in human studies warrants further investigation in horses (Delmas et al. 1983a; Anderson et al. 2020).

Mäenpää et al. (1988) was the first to document that serum osteocalcin concentrations were highest in young horses compared to mature horses. Since this study in Finn horses, other studies have supported these findings (Black et al. 1999; Fletcher et al. 2000). Fletcher et al. (2000) conducted several studies to investigate variables affecting osteocalcin concentration; sex, exercise, weaning and circadian rhythm. They found sex related differences in circulating osteocalcin concentrations of exercising horses, with fillies depicting higher circulating osteocalcin concentrations (Fletcher et al. 2000). It must be mentioned that this however was in comparison to geldings rather than colts. In weanling’s, circadian rhythm didn’t have a significant impact on osteocalcin concentrations which is also supported by the findings of Black et al. (1999). However, circadian rhythm induced changes have been reported in adult horses (Lepage et al. 1990; Black et al. 1999) with osteocalcin concentrations found to be higher during the night and early morning (Black et al. 1999). Black et al. (1999) also investigated the effect of cortisol on osteocalcin concentrations. They found no significant effect of cortisol on osteocalcin concentrations in adult and weanling horses (Grafenau et al. 2000).

In the horse, osteocalcin concentrations have been reported to vary from 3.68 – 17.31 ppb (Carstanjen et al. 2003). These low circulating concentrations make its accurate detection challenging. Aside from increased circulating concentrations found to be associated in human studies with bone disease (Gundberg & Weinstein 1986), circulating osteocalcin concentrations in young developing horses have been found to be significantly higher, attributable to higher bone turnover processes (Lepage et al. 1990; Black et al. 1999; Carstanjen et al. 2003). This makes detecting it in young horse plasma a key candidate for assay development. Furthermore, it has been found that bone developmental issues such as osteochondritis dissecans (OCD) can be detected in young foals (Billinghurst et al. 2004). This emphasises the importance of exploring the relationship between vitamin K and osteocalcin by using the young horse as a model.

Osteocalcin as a marker to detect bone diseases Biomarkers are continually being characterised for bone and cartilage. Alterations in the expression and distribution of components of the extracellular matrix of these tissues, and the enzymes that degrade them, have been reported for osteochondrosis lesions in cartilage from horses (Billinghurst et al. 2004). Biomarkers can be monitored through minimally invasive means in body fluids such as blood, synovial fluid and urine (Billinghurst et al. 2004). Some serum biomarkers of bone and cartilage metabolism have a role as putative indication of horses with severe OCD lesions that are unlikely to resolve. With this knowledge, early intervention and monitoring of treatment response may be possible by monitoring specific biomarkers of cartilage and radiographing horses with OCD (Billinghurst et al. 2004).

A Dutch study of 43 foals investigated the effect of exercise on OCD lesions. They identified that serum osteocalcin concentrations were significantly correlated with OCD severity and number of OCD lesions at 5 months of age. Osteocalcin concentrations were negatively correlated however, with OCD severity at 11 months of age (Billinghurst et al. 2004).

It is evident from these findings that the analysis of cartilage and bone by proteomic approaches will undoubtedly provide new insights into the biology of these important tissues (Desjardin et al. 2014) This will allow the molecular mechanisms involved in numerous osteoarticular pathologies such as osteoarthritis or osteoporosis to be probed (Desjardin et al. 2014). Likewise, DOD and especially OCD represents a major concern in the equine industry, such comparative proteomic approaches may prove useful to decipher molecular mechanisms involved in these disorders and refine entities. In addition it is likely that direct proteomic analysis of cartilage and bone will help identify new biomarkers (Desjardin et al. 2012).

Proteomic approaches applied to human rheumatoid investigations, have recently demonstrated that studying the expression pattern of synovial fluid proteins and its fluctuation during pathological states, may be helpful in clarifying the biology of these joint diseases (Chiaradia et al. 2012).

Roles of osteocalcin beyond bone metabolism As outlined in the previous sections, ucOC has been regarded as the inactive form however, a recent study in rodents suggests that this form may in fact have a role in the regulation of glucose metabolism (Brennan-Speranza & Conigrave 2015). While such a conclusion is not entirely unwarranted with Poser et al. (1980) back in the 80’s noting that osteocalcins small size is comparable to that of other hormones. It also possess some sequence features which are 45

characteristic of hormones such as paired basic residues, which in proinsulin are the sites of proteolytic cleavage and activation (Poser et al. 1980) . To date however, studies investigating its relationship to energy metabolism have been inconclusive in humans (Centi et al. 2015). This is further supported by the fact that much of our knowledge regarding vitamin K insufficiency and linkage to diseases has come about because of the widespread use of warfarin (Wallin & Martin 1985; Hilton & Van Horn 2017). Warfarin blocks the action of vitamin K, consequently causing ucOC and detrimentally affecting other VKDPs within the body (Danziger 2008). In recent years bone has now been implicated as an endocrine organ in its own right, with findings emerging revealing its intrinsic role in the maintenance of energy metabolism (Mathieu et al. 2008). With the discovery of insulin receptors in osteoblast cells and the finding that they appear to release osteocalcin into circulation, this is further evidence in support of its role in the regulation of glucose homeostasis (Razzaque 2011). Studies in mice have found that those lacking insulin receptors in osteoblasts have less β cell receptors in the pancreas (Fulzele et al. 2010). The Esp gene encoding the osteo-testicular protein tyrosine phosphatise (OST– PTP) has also been implicated in the carboxylation process (Ferron et al. 2010a). This is further supported by evidence of Esp null mice in which they exhibit a phenotype characterised by increased circulating ucOC and hypoglycaemia (Mizokami et al. 2013). Chronic Kidney disease (CKD) patients while highly susceptible to fractures and extra skeletal calcification (Fusaro et al. 2016b), have also been noted to be more likely to develop insulin resistance (McCabe et al. 2016).

Adjunct to the hypothalamic–pituitary–gonadal axis, another pathway has been proposed specifically involving osteocalcin (De Toni et al. 2016). Osteocalcin has been postulated to act on the testis with the characterisation of a membrane receptor protein responsible for mediating the activity of ucOC, identified to be expressed by the leydig cells of the testis (De Toni et al. 2016).

It is of interest to mention that in a study which supplemented five times more than the current US dietary recommendation, complete carboxylation of osteocalcin was not observed, even at this level (Shea et al. 2016). This begs the question whether complete carboxylation is necessary for optimal functioning? It is evident from these research findings that there is still much to be learned and understood about osteocalcin, and its interplay between nutrition and diseased states.

2.5.3 Methods currently used to measure osteocalcin Many of the findings in human studies remain inconclusive as to the protective role of vitamin K and osteocalcin in preventing age related bone loss and disease (Binkley et al. 2009; Koitaya et al. 2014; Hong et al. 2015). These studies however, relied on methods which approximated the degree of carboxylation therefore, determination of the amount of vitamin K required to carboxylate osteocalcin couldn’t be reliably evaluated.

An important difference in the molecular structure of osteocalcin has been noted in humans and other close relatives (primates) (Christina et al. 2005). They lack hydroxyproline, which has been observed on osteocalcin in other mammals such as Bos Taurus (Nielsen-Marsh et al. 2005).

While there has been some initial research conducted in the horse, it has mostly been conducted to determine the sequence of osteocalcin rather than an accurate means of quantifying it (Ostrom et al. 2006). Nevertheless, these studies have proved useful in deciphering and determining what approaches are best to employ. The finding that osteocalcin sequence is relatively conserved between species is of key significance (Carr et al. 1981; Tie et al. 2014). It’s homogeneity between species and conservation of its three key Gla binding residues allows inference of its key biological roles. Human osteocalcin for instance, bears 98% homogeneity to bovine (Colombo et al. 1993; Prigodich & Vesely 1997). Horse osteocalcin likewise, bears about 96% homogeneity to bovine (Grafenau et al. 2000; Ostrom et al. 2006). While the conservation of the Gla residue binding sites is of key significance to assay development, there is some contention amongst the literature to the exact sequence of equine osteocalcin (Carstanjen et al. 2003; Ostrom et al. 2006). There are two key studies which have looked at equine osteocalcin specifically. Carstanjen et al. (2002) reported the sequence to differ, with residues 48 and 49 being the amino acids proline (P) and valine (V). Ostrom et al. (2006) however, found there to be a discrepancy in this, with the mass reported in this study not corresponding to their findings. While the spectra generated by MS/MS of the asp-n enzyme peptide residues 34-49 of osteocalcin, derived from prehistoric horse bone, confirmed that residues 34-46 were the same as the previously published sequence, residues 47-49 were unable to be assigned (Ostrom et al. 2006).

Figure 2.16: Osteocalcin sequence (adapted from Ostrom et al. 2006).

Edman sequencing was therefore carried out by the authors. This revealed a 24Da decrease in the m/z for osteocalcin in the C-terminus of the protein, relative to the previously published sequence (this is reflective of the fully decarboxylated molecule, as the observed matrix used during MALDI/MS results in decarboxylation of γ-carboxyglutamic acid (Gla) (Ostrom et al. 2006). They sustained that this mass difference in charge could be accounted for by the last two residues, which they reported to be the amino acids threonine (T) and Alanine (A) (Figure 2.16). This was further confirmed by comparing the sequences of 42ka horse, modern horse, zebra and donkey by MS/MS (Ostrom et al. 2006). This has significant implications for assay development. Establishing the correct sequence is key in being able to accurately identify and quantify. It is also of interest to note that in the genus Equus, there has been no changes to the osteocalcin sequence over 1.2Ma, further highlighting it’s highly conserved nature (Hauschka et al. 1989).

Human osteocalcin is initially synthesized by the osteoblast as an 11kD molecule that consist of a 23-residue hydrophobic signal peptide, a 26-residue pro-peptide, and the 49-residue mature protein (Hauschka & Wians 1989; Hosoda et al. 1993). The hydrophobic region presumably targets the protein for secretion and the pro-region. This is homologous for corresponding regions in the vitamin K-dependent blood coagulation factors, which contains the γ- carboxylation recognition site (Hosoda et al. 1993). After the hydrophobic region is cleaved by a signal peptidase, the pro-osteocalcin is γ-carboxylated (Hauschka & Wians 1989). Subsequently the pro-peptide is removed and the mature protein is secreted (Hauschka & Wians 1989). However, nothing is known about the fate of this peptide.

To test the usefulness of the pro-peptide as a potential marker of bone metabolism, Gundberg and Clough (1992) investigated whether it could be measured in human serum. However, they were unable to detect it from any of their subjects, including those with a high rate of bone turnover. Likewise, it was also unable to be detected in culture media under numerous conditions. The authors concluded that the absence of detectable pro-peptide in human serum was probably due to intracellular cleavage and catabolism, which was confirmed to occur in- vitro. This is contrary to other markers of bone metabolism, such as human procollagen type I and II which are cleaved extracellularly (Jung & Lein 2014).

A disulphide bridge is also a key characteristic of osteocalcin and has been found to be conserved between species, with Ostrom et al. (2006) confirming its presence in a prehistoric horse osteocalcin extract. A disulphide bridge is responsible for the proteins tertiary structure; stabilising the III turn structure that is responsible for connecting the two N-terminal α-helices (Hoang et al. 2003). Reduction and alkylation has confirmed the presence of a disulphide bridge (Ostrom et al. 2006). Hoang et al. (2003) also proposed the presence of three α-helices within the hydrophobic core.

The conservation of this small protein through species divergence makes it a useful candidate through which to further develop protein sequencing approaches. The three glutamic acid residues responsible for binding to hydroxyapatite are found to be analogous in all mammals, birds, amphibians and bone fish (Carstanjen et al. 2003). Only in humans and the kangaroo is residue 17 found to be incompletely carboxylated (Huq et al. 1985). Osteocalcin is an ordered process with Glu-24 carboxylated first followed by Glu21 then Glu17 in humans (Cairns & Price 1994). Important to make note that in most species osteocalcin is completely carboxylated; incomplete carboxylation observed in humans has been postulated to be due to inadequate vitamin K intake (Cairns & Price 1994).

Research is starting to make headway in the human field, with the discovery that deciphering intact osteocalcin from its fragments, is key in understanding the underlying role and functioning of this elusive protein. Until now, osteocalcin has been reported as a percentage ratio or as total intact osteocalcin, neither of which provide information of much physiological significance (Cleland et al. 2016). With the emerging evidence of ucOC role in bone and now glucose metabolism, it is of benefit to further develop our understanding of this intriguing protein and develop more accurate means of measuring it. The following sections will outline

the assays currently available to measure osteocalcin and those proposed, with attention given to the underlying biology and structure of osteocalcin.

Immunoassays The use of immunoassay-based methods to detect specific protein targets is a widely used scientific method (Lisitsyn et al. 2014). Immunoassay methodologies are based on the non– covalent antigen–antibody binding capacity of proteins, producing a highly stable and distinct complex for targeting, enabling the detection of a protein or protein fragment (Lisitsyn et al. 2014). Most immunological assays require three key components; the protein to be detected (antigen), the antibody and the label attached to the antibody, which can be detected using a highly sensitive chemical, biological or physical method (Terenghi et al. 2009; Lisitsyn et al. 2014). The most commonly used label types are; Radioactive labels (radioimmunoassay), Luminescent labels (fluorescent antibodies), Enzyme labels (Enzyme linked immunoassay; ELISA) and DNA fragment (immune–PCR). The sensitivity and applicability of each of these techniques varies considerably (Lisitsyn et al. 2014). Immunoassay-based methods have been developed to detect osteocalcin with varying sensitivity and reproducibility results.

As mentioned previously, equine osteocalcin shows a strong homology with bovine (98%) sheep (96%) and human (94%) osteocalcin (Carstanjen et al. 2002). The bovine osteocalcin dosing kits therefore make it possible to measure concentrations of serum osteocalcin in horses (Carstanjen et al. 2003).

Radioimmunoassay (RIA) Total osteocalcin concentration can be determined by radioimmunoassay following the method of Gundberg et al. (1998) (Figure 2.17). However, these antibody-based methods provide little or no information on the posttranslational modifications of the protein (γ-carboxylation status) (Rehder et al. 2015). While some other complementary methods have been developed to determine undercarboxylated status like hydroxyapatite binding assay, this method is unable to decipher between that which has 0, 1 or 2 Gla γ-carboxyl groups (Ferron et al. 2010b).

Currently the percentage carboxylation of osteocalcin is used however, this can be quite inaccurate in determining overall vitamin K status, as it only depicts the status of the blood in that instance of time (Rehder et al. 2015). Immunological methods have been employed for analysis (Rehder et al. 2015). At present the major circulating form is thought to be the intact polypeptide, and large N-terminal-mid molecular fragment (Ivaska et al. 2003). However, as

of yet this has not been confirmed. Cross-reactivity has been reported between rabbit anti- bovine osteocalcin sera and equine and human serum osteocalcin (Patterson-Allen et al. 1982). The use of bovine specific osteocalcin assay has therefore been utilised (Mäenpää et al. 1988; Black et al. 1999). The human specific immunoradiometric assay (IRMA) did not recognise equine serum osteocalcin (Lepage et al. 1997).

Carstanjen et al. (2003) developed an equine specific radioimmunoassay (RIA) for osteocalcin quantification in equine plasma and serum. RIA is a technique for determining antibody concentrations in a sample by incorporating an antigen labelled radioisotope (Price & Nishimoto 1980; Bouillon et al. 1992). The radioactivity of the antibody is then determined (Zuckerman & Howard 1979). To enable accurate detection, osteocalcin was extracted and purified from the long bones of a foal to act as the standard (Carstanjen et al. 2003). Extraction and purification of osteocalcin from bone is a common method employed to provide a reliable standard (Ostrom et al. 2006). This is a common experimental procedure in order to verify the presence of the molecule in blood (Choppin et al. 2009; Bruggeman et al. 2010) In this study, the researchers performed Edman sequencing to obtain the complete amino acid profile (Colombo et al. 1993). Enzyme cleavages were performed using trypsin and chymotrypsin, with the peptide fractions then subjected to reverse-phase high-performance liquid chromatography (RP-HPLC) analysis (Colombo et al. 1993).

To obtain the antibody, Carstanjen et al. (2004) immunised rabbits against equine osteocalcin. It is important to mention that different antisera will bind to different regions of osteocalcin (Carstanjen et al. 2003). It has been reported that the antigenic site of osteocalcin is often located within its carboxy-terminal end (Price et al. 1980; Gundberg & Weinstein 1986) however, amino-terminal and mid-molecular epitopes have been proposed (Taylor et al. 1988).

There is disparity reported in the literature between the different fragments that various antibodies can detect. It is clear from these findings that different antibodies have different affinities for the various peptide fragments of osteocalcin (Garnero et al. 1992; Garnero et al. 1994).

Figure 2.17: Schematic diagram of osteocalcin (adapted from Gundberg & Weinstein 1986).

Hydroxyapatite binding assay Methods have been developed, with the recognition that affinity of osteocalcin for hydroxyapatite, depends on the number of Gla residues in the protein. The portion of osteocalcin that does not bind to hydroxyapatite has been taken to be ucOC. Studies using this method have found a number of significant results, with one study reporting that non-bound (ucOC) was higher in postmenopausal women with osteoporosis, than premenopausal women (Vergnaud et al. 1997). However, as stated by Gundberg et al. (1998) none of these studies controlled for the total amount of osteocalcin in the samples.

There are several problems posed using hydroxyapatite preparations, including differences in the way in which they are prepared, and differences in the binding characteristics. This makes accuracy and reproducibility of this method difficult. Gundberg et al. (1998) suggested that if using hydroxyapatite preparations to measure ucOC, the preparations must be standardized before use, and the precent binding corrected for basal osteocalcin concentrations reported. Furthermore, the authors made several key observations. A major catabolic fragment present in circulation is suggested to be that of residues 1–43 (Gundberg et al. 1998). This fragment is homologous to the intact molecule and will bind to hydroxyapatite. Therefore, they suggested that assays with known specificity to the intact molecule, or the intact plus the 1–43 fragment, should be used when assessing the carboxylation status of osteocalcin (Gundberg et al. 1998). Likewise, because the helical structure facilitates the binding of osteocalcin to hydroxyapatite, partially carboxylated osteocalcin may have reduced binding to hydroxyapatite. 52

2.6 Modern proteomics approaches and analytical techniques The field of proteomics and use of mass spectrometry (MS) has made significant headway in the study of proteins in the last few years (Soares et al. 2012). MS is one of the most powerful tools available in chemical analysis, due to its ability to provide the molecular mass of analytes (Vähätalo et al. 1999). The field of proteomics refers to the science that studies the proteome (Soares et al. 2012). These new advanced approaches in MS have enabled the accurate quantitation of the whole proteome, permitting the robust and simultaneous examination of thousands of proteins (Soares et al. 2012).

Detection and characterisation, relies on extensive purification methodologies before analysis. This includes; protein separation, identification, characterisation and quantification. The flow chart depicted in Figure 2.18, shows the processes involved in the use of such techniques. As can be observed, the first step relies on the use of extraction techniques (Sabidó et al. 2012). This is proceeded by the separation and digestion of the extracted proteins, using either gel or gel–free based methods (Soares et al. 2012). Protein identification and characterisation is then undertaken by the use of MS (Sabidó et al. 2012). The peptide digest is separated most commonly by liquid chromatography based methods, coupled with mass spectrometry (LC- MS) (Soares et al. 2012). The peptide digest is analysed by MS, and its mass and fragmentation information is then submitted to protein sequence databases (Sabidó et al. 2012). The database search marries up the information available regarding proteins, with that from the experimental data analysed. This allows the accurate identification of proteins in the sample provided (Soares et al. 2012). The determination of protein and peptide mass is a complex process, and is dictated by the capabilities of these protein sequence databases. The digestion and fragmentation of each protein will provide a set of specific peaks, which should be very similar to the one’s predicted in silico, using the known parameters of each enzyme, and the theoretical masses of the generated peptides (Soares et al. 2012). For each spectrum, the search algorithm determines which peptide sequence provides the best match.

Protein Protein Peptide

Plasma and tissue separation digestion purification and collection separation

Figure 2.18: Schematic representation of the mass–spectrometry (MS) workflow (adapted from Soares et al. 2012).

There are several parameters which can be changed; mass tolerance, maximum number of proteases, missed cleavages allowed and to which database the experimental data is compared to. This is however reliant on extensive sequencing of genomes, of which does not yet exist for some species (Soares et al. 2012).

There are a number of bioinformatics tools that enable unknown spectra to be analysed and matched against known databases. (Dunn et al. 2011) (Table 2.5 and Figure 2.19). These software tools each have their own advantages and disadvantages, and searches that they are more adeptly suited for. After protein identification, quantification is the next phase. There are also a number of bioinformatics software tools available many of which are instrumentation specific however, Skyline daily is a licence free software which is gaining in popularity for these purposes (MacLean et al. 2010). It has been applied in many studies to assist in the quantification of peptides and proteins.

The development of proteomic approaches has proven useful to unravel molecular and cellular mechanisms that contribute to disease aetiology and progression, and to identify biomarkers in human diseases such as osteoarthritis (Desjardin et al. 2012). With DOD’s representing a major concern to the horse industry, such proteomic approaches may prove useful to decipher molecular mechanism involved in these disorders (Desjardin et al. 2012). MS based proteomic assays are emerging as a promising approach that have the advantage of specificity and high throughput of protein analysis.

2.6.1 An introduction to liquid-chromatography mass spectrometry (LC-MS) Chromatography is used to separate peptides from a protein digest to reduce the complexity of the sample. High performance liquid chromatography (HPLC) involves elution of peptides through a pressurised capillary column (Nováková et al. 2017). Ion exchange chromatography is also another common method (Ly & Wasinger 2011). Peptides are separated based on the salt content or pH of the mobile phase by way of anion or cation exchange, and is often used in combination with other fractionation strategies (Tang et al. 2008). These methods may combine strong cation exchange (SCX) and this is one of the most commonly used methods for peptide separation in bottom-up proteomics (Tang et al. 2008). The different forms of chromatography are drawn from the same principle as they all have a stationary phase and a mobile phase (Tang et al. 2008). The most common column packing material for reversed phase chromatography is C18 (Ly & Wasinger 2011). The absorption of peptides on these as they traverse the column depends on a number of factors; hydrogen bonds, Van der waals forces

and solubility (Ly & Wasinger 2011). Peptide absorption can also be varied by changing the solvent or the temperature or pH of the solvent (Tang et al. 2008).

Optimum and reproducible LC-MS performance is determined by a number of factors. The time taken for a particular peptide to travel through an HPLC column to the detector is known as retention time (RT) (Wang et al. 2006). This is unique for each peptide and is measured from when a sample is injected into a column to the point at which it reaches maximal detection. The narrower the peaks, the better the chromatographic resolution. Peak area can also be used to determine the quantity of peptide present in the sample, with the area under the peak being proportional to the quantity of protein present (Wang et al. 2006). When this is coupled with an MS output this corresponds to when the detector is showing a peak, some ions that are passing through the detector at the time can be diverted (mass selected) and converted into either a mass spectrum, a chromatographic display or both. This gives a fragmentation pattern that can be compared against bioinformatics database of known patterns. Ideally, the peaks of interest in an MS/MS spectrum are those represented by β and y ions, corresponding to the prefix of N-terminal (b-ion) and the suffix of C-terminal (γ-ions) fragments (Gillet et al. 2012).

2.6.2 Mass Spectrometry (MS) for routine detection of proteins As introduced, mass spectrometer is an instrument that separates compounds with different mass to charge ratio (m/z) and determines the amounts of each peptide in a mixture (Gundry et al. 2009). Proteins are digested into peptides dissolved in a polar solvent, ionised, separated and then conveyed through a detector that quantifies the ions by displaying their peptide spectra (Diez Fernández et al. 2012). The most common types of ion generation for protein analysis are matrix-assisted laser desorption/ionisation (MALDI) and electrospray ionisation (ESI) (Gundry et al. 2009). These two forms of ionisation are capable of ionising proteins and peptides while preserving their chemical structure. Other forms include atmospheric pressure chemical ionisation (APCI) (Soares et al. 2012).

A quadrupole mass analyser comprises of a set of conducting rods arranged in parallel. Ions are separated based on the stability of their flight paths through an oscillating electric field in the quadrupole (Diez Fernández et al. 2012). Only ions of a certain mass to charge (m/z) value will have a stable flight path through the quadruple whilst all others will not reach the detector. A triple quadrupole MS consists of two quadrupole mass analysers with a collision cell between them (Diez Fernández et al. 2012). The first quadrupole mass analyser selects the precursor ions. These precursor ions are then fragmented inside by a process known as collision-induced

56 dissociation (CID) to obtain tandem MS spectra (MS/MS) (Gundry et al. 2009). The product ions are analysed or selected by the second quadrupole mass analyser and then passed on to the detector. The precursor and product ion pairs are called mass transitions (Anderson & Hunter 2006).

Time of flight (TOF) ion detectors are designed to have large areas, rapid response times to provide good timing resolution with correspondingly accurate m/z determinations and high sensitivity (Jannetto & Fitzerald 2016). Peptides form molecular ions in the ionisation chamber of the MS that can be mass selected and detected.

2.6.3 Targeted and untargeted proteomics approaches

Multiple-reaction-monitoring mass spectrometry (MRM-MS) Multiple-reaction-monitoring; MRM (or SRM) is a highly selective and sensitive technique (Table 2.5). It allows the detection of specific analyses of interest with known fragmentation properties (Anderson & Hunter 2006). A specified number of analyte transitions are monitored at once during the chromatographic run, and only those that have the specified mass transition (precursor/product ion pair) are able to be detected. This technology is a useful avenue for detecting and verifying assays for low-abundance proteins (Corthals et al. 2000). It has been widely used for both small molecule and protein analysis (Faktor et al. 2017). This targeted analysis permits the quantification of low abundance proteins in complex matrices such as blood. MRM-MS is performed using triple quadrupole instruments. As discussed in the previous section, during analysis precursor and product ions are selected to form an MRM transition. The selected transitions are sequentially analysed (cycle time), resulting in the collection of chromatographic data. A minimum of three transitions per peptide is considered the industry standard, with some studies monitoring up to eleven (Anderson & Hunter 2006). MRM-MS assay development and optimisation can be laborious and expensive, especially if stable-isotope peptide standards are used (Anderson & Hunter 2006). It is also relatively low throughput however, will quantify low abundance proteins with high accuracy and reproducibility once developed.

57 Sequential Windowed Acquisition of All Theoretical Fragment Ion Mass Spectra mass spectrometry (SWATH–MS) The operation of SWATH-MS as depicted in Figure 2.19, is similar to MRM but with much wider isolation window (Gillet et al. 2012) (Table 2.5). These isolation windows are cycled at a sufficient speed to provide MS/MS fragmentation of all analytes present in the sample, inside a given mass range (Schubert et al. 2015). While it is an untargeted form of data acquisition, it relies on targeted data extraction and analysis (Faktor et al. 2017). SWATH-MS is a form of data independent acquisition (DIA), and unlike MRM relies on spectral libraries generated from data-dependent acquisition (DDA) experiments, to identify proteins (Schubert et al. 2015). SWATH-MS quantifies all peptides by DIA but also allows the choosing of protein targets during subsequent data analysis (Jakob et al. 2014). it is best suited to target high and medium abundance proteins, and is known for providing comprehensive sample coverage with negligible reduction in quantitative precision (Krisp & Molloy 2017). The downside of this discovery proteomics workflows though is the reduced ability to identify proteins of low abundance, and the complex bioinformatics processes required to analyse the data (Facility 2010). For these reasons, it would be reliant on osteocalcin concentration being high enough in the plasma to be detected accurately.

Innovations in label free methods for absolute quantitation of peptides and proteins are continuously been developed and improved. SWATH-MS based protein quantification has been used routinely in human medicine to search for protein biomarkers (Rosenberger et al. 2017) however, its use in animal science is in its infancy. The success of the technique requires comprehensive and well curated reference DDA peptide spectral libraries for protein identification and quantitation (Soares et al. 2012). However, due to the relatively young nature of the SWATH-MS methodology, the expansion of spectral libraries across a variety of organisms and biological sample matrices is lacking (Rosenberger et al. 2017). Only four such repositories are currently publicly available (human, mouse, yeast and bacteria). In domestic production and companion animals, blood plasma is a fairly non-invasive and routinely obtained source of proteins and, potentially, a large source of biomarkers (Soares et al. 2012). However, it is one of the most challenging samples for analyses by advanced MS instrumentation, due to its wide dynamic range of protein concentrations which spans several orders of magnitude.

Table 2.5: Different modes of data acquisition in MS.

MRM/SRM–MS

Multiple reaction monitoring (MRM) or Selected A highly selective method that allows for reaction monitoring (SRM) the targeted quantitation of specific compounds within a sample (Vincent et Software: Skyline daily al. 2009; Barbas et al. 2015). Scan: MS1 (MS) and MS2 (MS/MS)

DDA–MS

Automated data dependent acquisition (DDA) or From one scan the most abundant ion species are selectively collected and Information dependent acquisition (IDA) picked (Mullard et al. 2015). Software: ProteinPilot – Only certain ions are fragmented.

Scan: MS2 (MS/MS) – Bias towards most intense peptides.

–Untargeted and not quantitative.

SWATH–MS

Sequential Windowed Acquisition of All A method that allows the quantitation of Theoretical Fragment Ion Mass Spectra every detectable compound within the (SWATH) or sample. It is a form of untargeted data acquisition (Gillet et al. 2012; Egertson et Data independent acquisition (DIA) al. 2013). Software: PeakView –Every ion is fragmented.

Scan: MS2 (MS/MS) – Complete digital fingerprint of sample

–Not as sensitive as MRM/SRM.

Figure 2.19: A comparison of the steps involved in MRM-MS and SWATH-MS data acquisition workflows (Vincent et al. 2009; Facility 2010).

2.6.4 Development of MS based techniques to analyse osteocalcin: current status

Application of Matrix–Assisted Laser Desorption Ionisation (MALDI) & Electrospray Ionisation (ESI) mass spectrometry. Mass spectrometry can provide a direct means with which to measure the post-translational modifications of osteocalcin (Rehder et al. 2015) MALDI-MSIA and ESI-MSIA were employed to assess measures of osteocalcin carboxylation in patients receiving vitamin K supplementation (Rehder et al. 2015). These assays are more useful in that they are able to combine the use of two different approaches; antibody and mass spectrometry technologies (Soares et al. 2012). The antibody is used to bind to a key fragment/peptide of interest in osteocalcin sequence (Gundberg & Weinstein 1986). In this case a peptide that is homologous to the species in question is key, particularly if using a standard (Carstanjen et al. 2003). Standards can be synthetic in nature or acquired via extraction methods (Hoofnagle et al. 2016). Synthetic standards are usually heavy labelled to increase the confidence and accuracy of detection. Bone extraction is the most common means with which to extract osteocalcin (Price et al. 1976; Cleland et al. 2012; Cleland & Vashishth 2015). This method is usefully in that it allows the native form of the protein to be extracted however, it is laborious and time consuming (Price et al. 1976). There doesn’t appear to be a best place to derive the bone for osteocalcin extraction, with reports utilising numerous locations in a multitude of species (Colombo et al. 1993; Cleland et al. 2012).

Based on findings from immunological assays, the primary forms of osteocalcin in circulation have been found to be the intact molecule and the N-terminal mid molecule fragment (Liu et al. 2011). This large fragment is postulated to be the result of trypsin-like activity or a by- product of sample handling (Garnero et al. 1992). Enzymatic digestion provides a means with which to obtain structural information (Baumgrass et al. 1997). Trypsin is a common enzyme used. Tryptic digestion of human osteocalcin is known to yield the resulting osteocalcin fragments; 1-19, 20-43 and 45-49 (Vähätalo et al. 1999).

Circulating variants of carboxylated osteocalcin are also known to occur in humans and human bone (Booth et al. 2004b; Booth & Al Rajabi 2008). Preliminary observational studies have found an association between vitamin K status and bone health, with decreased vitamin K intake found to be associated with a higher proportion of ucOC and subsequent bone loss (Binkley et al. 2009; Rehder et al. 2015).

Current assays available to measure total osteocalcin are however, indifferent to carboxylation status; methods are unable to decipher between that which is fully, partially or ucOC in circulation (Gundberg et al. 1998; Rehder et al. 2015). Therefore, even though the amount of ucOC relative to the total in circulation (%ucOC) is a relative biomarker of vitamin K status in bone, there is no consensus on the precise amount in circulation, or how many of the three potential Gla residues are carboxylated (Rehder et al. 2015). Deciphering between these different forms is vital, especially considering research in rodent models has implicated ucOC as the key active hormone involved in glucose metabolism (Lee et al. 2007; Ferron et al. 2010a). While a few human studies have reported a relationship between total osteocalcin concentrations and measures of energy metabolism, such as insulin and glucose. There are few studies that have directly considered the carboxylation status of osteocalcin, or taken into account its reported daily fluctuations, which are sensitive to circadian rhythm and vitamin K consumption (Markowitz et al. 1987; Shearer et al. 2012).

Recently Rehder et al. (2015) were able to report new qualitative and semi-qualitative information on osteocalcin molecular fragments in human blood plasma and serum samples. To further complicate its determination however, over twelve different truncated forms of osteocalcin were identified to circulate in humans (Cleland et al. 2016). This work is fundamental to determining the functional significance of different carboxylated fragments of osteocalcin and their relationship to health and disease (Rehder et al. 2015).

MALDI-MS was able to identify some low abundance osteocalcin fragments that hadn’t been previously accounted for in the literature (Rehder et al. 2015). However, a disadvantage of this technique is that it is unable to detect γ-carboxylation because the γ-carboxyl groups are instantly lost as CO2 when the laser meets the sample (Kalume et al. 2000; Ivaska et al. 2003). On the contrary γ-carboxy groups are retained during ESI-MSIA so the use of this technique was able to relatively determine osteocalcin with 0, 1, 2 or 3 γ-carboxyl groups (Rehder et al. 2015).

Human osteocalcin was found to circulate in over twelve N and/or C-terminally truncated forms (Rehder et al. 2015). Interestingly, vitamin K supplementation in this study was not found to have a significant effect on the relative abundance of these truncated forms however, it did significantly increase the fractional abundance of osteocalcin with 3 Gla residues compared to that with 0 in the control group (Rehder et al. 2015).

Cleland et al. (2016) also analysed osteocalcin from purified bovine using ESI-MS. For full- length osteocalcin they were also able to detect forms possessing between 0-3 Gla residues (Gundberg et al. 1984). They also detected chromium adducted (+511.9) forms with the 0-3 carboxylation’s (Niiranen et al. 2002). Chromiun-adducted forms were found to be more consistency carboxylated, suggestive of a stabilising property of chromium (Cleland et al. 2016). This finding signifies that some of the variation in carboxylation found may be the result of decarboxylation during or before liquid chromatography. This is an important consideration to take into account in future MS carboxylation quantitation experiments (Cleland et al. 2016). As reported by Rehder et al. (2015) Cleland et al. (2016) also emphasised the importance of taking into account CO2 neutral losses, with the fragmentation of carboxylated osteocalcin depicting the distinct neutral losses characterised by carboxylated glutamic acids. Detection of

Gla by mass spectrometry will be improved if neutral loss of CO2 during peptide fragmentation is eliminated (Hallgren et al. 2013).

It is also well documented that the stability of proteins is affected by a number of degradation pathways, of which the most prominent occur via oxidation of methione (Met), deamidation of asparagine (Asn) and cleavage of peptide bonds at aspartic acid (Asp) (Nabuchi et al. 1997; Vähätalo et al. 1999). This has implications specific to osteocalcin which must be considered, as osteocalcin has four Asp residues susceptible to acid catalysed cleavage (Vähätalo et al. 1999). Vähätalo et al. (1999) conducted an experiment to test the stability of osteocalcin. They conducted a degradation assay on a synthetic osteocalcin derivative. The products of degradation under acidic conditions (pH 2) were noted to be the fragments 1-14 and 15-46 formed via the cleavage between Asp-14 and Pro-15 (Vähätalo et al. 1999). The N-terminal glutamine also formed pyroglutamic acid; a well-documented phenomenon in the literature whereby glycine and glutamine at the N-terminal can, under the right physiological conditions convert to pyroglutamate (Vähätalo et al. 1999; Liu et al. 2011). They concluded from this experiment that osteocalcin is much more stable under neutral and alkaline conditions (Vähätalo et al. 1999).

Incomplete carboxylation was also noted on the N-terminal peptide and middle peptide following trypsin digestion. Trypsin is the enzyme of choice for mass spectrometry (Hallgren et al. 2013) Chromium adduction was not found to be associated with the tryptic peptides generated (Cleland et al. 2016). In this study however, they were unable to detect tryptic peptide fragments encompassing the carboxylation, instead only b-ion fragments of the N-terminal

peptide or y-ion fragments of the middle peptide were detected (Cleland et al. 2016). This emphasises the difficulty in detecting fragments due to carboxylation (Hallgren et al. 2013).

The authors in this study therefore concluded that detection of osteocalcin peptides using MS based techniques is hindered by two main factors; γ-carboxylation of one or more glutamic acids, and the acidity of osteocalcin which may impact its detectability (Hallgren et al. 2013). Taking this into consideration a technique which may prove of more value in detecting this is the use of HPLC coupled with ICP-MS (Dowd et al. 2001b).

Inductively coupled plasma mass spectrometry (ICP-MS) This is a type of mass spectrometry capable of detecting elements (Yu et al. 2013). It works by converting the atoms of the elements in the sample to ions, which are then detected by the mass spectrometer (Chudzinska & Baralkiewicz 2011). As with any mass spectrometry technique the ions are then separated by their mass to charge ratio (m/z). It has numerous applications and is widely used to detect trace elements in foods (Khan et al. 2014). It is a sensitive and accurate technique, offering very low detection limits of major and trace elements in different matrices (Salomon et al. 2002). As a stand–alone technique however, ICP–MS is unable to provide much information of value concerning the structural and chemical characteristics of the sample (Meermann et al. 2012). Paired with chromatography techniques it can be used as a valuable detector, allowing specific separation of the molecule of interest by HPLC and then detection by ICP–MS (Meermann et al. 2012). As depicted by Rehder et al. (2015) during MALDI–MS and other mass spectrometry techniques, calcium is lost during ionization as the sample undergoes decarboxylation. Therefore, calcium bound osteocalcin peptides were unable to be detected (Rehder et al. 2015). The use of ICP–MS may therefore prove to be valuable in detecting the number of calcium bound ions in the osteocalcin sample, particularly if paired to the separation capabilities of HPLC to allow specific detection.

Sample handling Proteolysis of osteocalcin has been reported to occur during sample handling and preparation (Hoofnagle et al. 2016). The researchers noted an increase in the fragmentation of osteocalcin when samples were left out at room temperature overnight (Novak et al. 1997; Hoofnagle et al. 2016). They suggested that sequential exopeptidase activity may also be responsible for the production of other fragments observed (Rehder et al. 2015; Hoofnagle et al. 2016). Larger fragments detected (residues 8-42), were postulated to be the result of proteolysis of the intact osteocalcin in vivo (Rehder et al. 2015). All of these could contain 0-3 Gla residues therefore,

64 as state by the authors, an assay that measures the mid-molecular forms along with the intact would be of the most clinical relevance (Rehder et al. 2015). Due care must however be taken to ensure the stability of samples to avoid ex vivo proteolysis, and ensure accurate determination of intact osteocalcin (Cleland et al. 2016).

Other antibodies specific for the C- or N- terminus, and capable of providing enough mass spectral signal, may provide additional insights into the nature of circulating osteocalcin (Rehder et al. 2015). They also didn’t observe a cleavage that might be expected from trypsin or plasmin activity, suggesting smaller fragments encompassing the first twenty-three residues is unlikely (Novak et al. 1997). The antibody utilised by Ivaska et al. (2003) was also able to recognise larger fragments.

Contamination is avoided as osteocalcin is only found within vertebrates and not common contaminants such as bacteria and plants (Oldenburg et al. 2015). While this is useful for analysis purposes, there are some inherent issues and challenges which need to be taken into consideration. Interpretation of complex mass spectra can be challenging. Peaks as a result of the self-digestion of enzymes and/or the plasma matrix can be easily confused with those of an enzymatic digest (Ostrom et al. 2006). This is also why it is of key importance to cross-validate findings where possible.

2.7 Conclusion The role of vitamin K in blood coagulation has been known for many decades, but in recent years it has been shown to be a factor in many other physiological processes. This review of the literature highlights the role of vitamin K, and its dependent proteins, in bone metabolism.

Placental transfer of vitamin K is limited in the human, and there has been limited research conducted in horses on this aspect of vitamin K metabolism, or transfer of the vitamin into mare milk. In light of the link between vitamin K and bone health identified in human and rodent studies, it would seem important to investigate transfer and uptake of vitamin K in the foal. This would aid in establishing requirements for vitamin K in the horse.

Interest is developing around understanding the role and importance of osteocalcin in bone development, with current research emphasis on elucidating its role in osteoporosis and osteoarthritis of the elderly. Currently, limited research investigating its role in the bone metabolism of rapidly growing young animals exists. This review of the literature also emphasises the possible role it may play in the development of bone diseases such as DOD and OCD in horses. This warrants further investigation in the horse.

Furthermore, the carboxylated status of osteocalcin is considered a more valuable measure of assessing overall vitamin K status. To date however, there is no consensus on the best method to analyse it in circulation. As a result, there is disparity in the literature with regards to the circulating concentrations of osteocalcin reported in blood for many species, including the horse. It is evident that further research is necessary to investigate and clarify this.

CHAPTER 3

Trans-placental Transfer and Milk Deposition of vitamin K1 in the Mare. 3.1 Introduction ...... 67 3.2 Materials and Methods ...... 69 3.2.1 Mares and Husbandry...... 69 3.2.2 Experimental Design and Protocol ...... 71 3.2.4 Statistical Analysis ...... 75 3.3 Results ...... 76 3.3.1 Experiment 3.1 - Pre-parturition study ...... 76 3.3.2 Experiment 3.2 - Post-parturition study ...... 82 3.4 Discussion ...... 88 3.5 Conclusion ...... 91

3.1 Introduction Vitamin K plays an intrinsic role in blood coagulation, and in recent years has been shown to be a key cofactor in many other physiological processes through the function of vitamin K dependent proteins (VKDPs; see Chapter 2). Studies in both humans and rodents have highlighted the role of vitamin K, and its dependent proteins, in bone metabolism, energy utilisation, fertility, sphingolipid metabolism and immunity. Osteocalcin is a VKDP that has an essential role in bone metabolism (Booth 2009) and McCann and Ames (2009) found that the increased risk of fractures in humans with osteoporosis, can be attributed to decreased functionality of osteocalcin. Studies in humans have shown that there is a higher requirement for vitamin K to prevent bone disease, than for the maintenance of normal blood coagulation (Booth 2009; Terachi et al. 2011).

Osteoporosis, a disease reported predominantly in aging human populations, is the main driver for the investigation of the role of vitamin K in bone metabolism. There has been much less research into vitamin K and bone abnormalities of the young of many species, including humans, horses, dogs, pigs and poultry (Ytrehus et al. 2007). Developmental orthopaedic disease (DOD) is a term encompassing different skeletal disorders, such as osteochondritis dessicans (OCD), which affect horses early in life (Lepage et al. 1998). Nutrition is regarded as an important factor in the aetiology of OCD, especially mineral and energy intake (Richardson & Zentek 1998). Lower circulating concentrations of vitamin K in yearlings, have

67 been implicated in the occurrence of OCD (Biffin et al. 2010) but there are no other equine publications on vitamin K and bone health. Importantly, lesions associated with OCD may be present at birth or shortly thereafter (Rejno & Stromberg 1978; Henson et al. 1997; Olstad et al. 2008) suggesting that some skeletal disorders may be initiated in utero, where the placenta is key to foetal development.

Trans-placental transfer of vitamin K in human pregnancies has been shown to be limited, with negligible transport of the vitamin (Kazzi et al. 1990). This lack of vitamin K transfer to the foetus can result in haemorrhagic episodes and be fatal for the human neonate (Shearer 2009a). Maternal supplementation with vitamin K before delivery, or a vitamin K injection of the baby shortly after birth have been recommended to prevent coagulation complications in babies with inadequate vitamin K status (Y de Vries et al. 2018). There is very little information on placental transfer of Vitamin K in other species. No published research has investigated the maternal transfer of vitamin K from the mare to the foetus pre-partum, or to the foal following milk consumption postpartum. These questions were examined in these studies with the hypotheses; vitamin K does not cross the equine placenta, and the concentration of the vitamin in mare’s milk can be modulated by oral supplementation.

68 3.2 Materials and Methods

The experimental studies described in this Chapter were conducted in the Equine Research Unit on the Gatton Campus of the University of Queensland, in south-eastern Queensland, Australia, during the months of July 2015 - August 2015 and July 2016 – March 2017. The experiments were approved by the University of Queensland Animal Ethics Committee (Production and Companion Animal AEC; Approval numbers SAFS/147/13 and SAFS/421/16), in accordance with the Australian Code for the Care and Use of Animals for Scientific Purposes (NHMRC, 2013).

3.2.1 Mares and Husbandry Twelve pregnant mares with known breeding history from the UQ Gatton Campus teaching herd were used in these studies. All mares had been maintained on pasture in the teaching herd for at least twenty-four months prior to experimentation. Mares were aged between six and sixteen years, with foaling dates estimated from breeding records and confirmed by palpation per rectum and/or trans-rectal ultrasonography. The mares were allocated to treatment groups in Experiment 3.1 based on gestational stage, breed, and parity (Table 3.1). Experiment 3.2 was undertaken in the following breeding season, and the same cohort of mares was used (see Appendix 2). In both breeding seasons all mares were mated to an Australian Stock Horse stallion.

Mares were brought in from the pasture and maintained in foaling yards approximately a week before their expected foaling date. A calcium test kit (CHEMetrics FoalWatch®, Galgo, UK) to monitor calcium concentrations in the mare’s milk was used to predict foaling within 48 hours (a high calcium reading was indicative of imminent parturition). Care was taken when collecting samples at foaling, due to the nature of live births and the importance of the formation of a bond between the mare and her foal. The health of the mares and foals was monitored, daily throughout both study periods.

The mares had ad libitum access to pasture for the duration of the experimental periods and offered a commercial balanced ration (Pryde’s EasiFeed®; BioMare Cubes®; see Appendix 3 for details) while in foaling yards both pre and post-parturition. This was to ensure that there vitamin and mineral requirements of the mares were being met and to mimic industry practice (NRC 2007). Pasture availability was scarce during winter and grass hay was offered. Pasture intake increased during the lactation period, following rainfall and increased pasture growth during spring (Figure 3.1).

69 a b

Figure 3.1: Pasture over the late winter period-August (a), and pasture after rainfall over the spring-September/October (b). Table 3.1: Description of Mares used in Experiments 3.1 and 3.2.

Mare ID Age Breed* Parity Treatment

1 14 ASH MP Control

2 6 ASH MP Control

3 12 SB MP Control

4 8 SB MP Control

5 16 ASH MP Control

6 10 SB MP Control

7 7 SB MP KQ**

8 12 ASH MP KQ

9 12 ASH MP KQ

10 15 SB MP KQ

11 13 ASH MP KQ

12 9 SB MP KQ

* ASH, Australian stock horse; SB, Standardbred; MP, multiparous mare. **KQ, Oral vitamin K treatment.

3.2.2 Experimental Design and Protocol The twelve mares used in both studies were allocated to a control or treatment groups as shown

in Table 3.1. The treated group received 15mg K1, orally in 4g of a carrier paste. The vitamin

was supplied in a soluble form of K1 and K2 in a ratio of 10:1 (KQ, Quinaquone™). The control mares were treated with the paste devoid of vitamin K. Both treatments were supplied by Agricure Pty Ltd., in ready to use syringes of a single dose (4g) of paste intended for oral administration. Information about the components contained within the formulations was not formally disclosed, due to proprietary ownership of the information by Agricure Pty Ltd.

Experiment 3.1- Pre-parturition Study Treatment of both control and KQ pregnant mares began four weeks prior to their expected foaling date in Experiment 3.1. All mares were dosed three times per week (Monday, Wednesday, Friday), with 4g of prepared paste orally. Prior to foaling, blood samples were collected from mares in the paddock (Figure 3.1), weekly on Monday, and prior to dosing. At the time of foaling, pre-suck samples were collected from the mare (blood and colostrum), the foal (blood), and the placenta (umbilical cord blood). Blood samples from mares and foals were collected from the jugular vein, into lithium heparin tubes (BD vacutainer) maintained on ice, centrifuged (10 minutes, 4ºC, 3500rpm), decanted into storage vials and stored at -80ºC for later analysis. A colostrum sample was obtained from the mare prior to her suckling the foal. Colostrum was tested using a Brix refractometer (ARS Equine Colostrum Refractometer) to determine the quality of immunoglobulins available for the foal, and stored at -80ºC. Milk samples were collected at birth and 12 hours, 24 hours and 7 days after parturition.

Experiment 3.2- Post-parturition Study The same cohort of mares was inducted into Experiment 3.2, and the study commenced at parturition and continued till the foals were about 3 months of age. The treatments (Table 3.1) used the same doses as in Experiment 3.1, were administered at parturition and then three times per week (Monday, Wednesday and Friday) for the entire study period. At foaling, pre-suck samples were collected from the mare (blood and colostrum) and the foal (blood). These samples were collected and processed as described in Experiment 3.1. Mares and foals were maintained in yards for a few days post-parturition before being turned out onto pasture for the remainder of the study.

Plasma and milk samples were collected from mares and foals at birth, 12 hours and 24 hours after foaling and then on day 7, 14, 21, 28, 35, 42, 49, 56, 70, 84 and 98. Mares and foals were

restrained in a crush for sample collection. Blood samples were collected from the jugular vein of the foal and mare, using a 21G needle, transferred into lithium heparin vials, centrifuged, decanted into vials and stored at -80ºC for later analysis. Milk was stripped from the mare into sterile screw top containers, and then distributed into storage containers and stored at -80ºC for further analysis.

3.2.3 Chemical Analysis

Pasture and feed analysis Representative samples of pasture, hay and BioMare Cubes® were collected throughout both experiments and sent to Equi-Analytical Laboratories (DairyOne, USA) for analysis of nutrient content (see Appendix 3 and 4).

Plasma Vitamin K

Plasma vitamin K1 (phylloquinone) concentrations were determined using HPLC (High performance liquid chromatography). The analysis was based on the methodologies of Wang et al. (2004) with modifications to both the extraction and quantification processes from other publications (Griminger & Brubacher 1966; Booth et al. 1994; Zhang et al. 2003; Damon et al. 2004).

Reagents and standards The mobile phase consisted of methanol and isopropanol (90:10) and contained a reducing agent. The deproteinising-agent was a mix of methanol, dichloromethane (70:30) and reducing agent. The reducing agent was prepared from zinc chloride 2M, sodium acetate 1M and acetic

acid 1M. The internal standard of synthetic vitamin K [IS-K (25)], reference standards for K1 and the deproteinising and reducing agents were all purchased from Sigma.

Instrumentation and Chromatographic conditions An analysis was performed using a HPLC system (Agilent 1100 series) with a Waters 474 scanning fluorescence detector (16μL flow cell) and was controlled by Agilent Chemstation

software. The column was a Kinetex (Phenomonex) 5μ C18 (150 x 4.6mm) with a C18 (4 x 3.0mm) security guard (Phenomonex) cartridge filter. Post-column reduction was achieved using a stainless steel (100 x 4.6 mm) column packed with zinc dust (Sigma).Vitamin K was eluted isocratically with the detection wavelengths; 244nm (excitation) and 430 (emission). An LC-MS system (LCMS-2010EV, Shimadzu, Kyoto) was used to confirm the structure of vitamin K1.

Standard preparation

Stock solutions of K1, and the internal standard IS-K (25) were prepared by addition of 20mg of each standard into 1L isopropanol. These standards were kept at -20° C in the dark until

required. Working solutions of each standard were prepared by further dilution of the stock standards with mobile phase, in the range 5- 2000ng/ml.

Sample preparation and quantification Plasma samples were thawed under reduced light conditions. Samples (200μL) were pipetted into siliconised tubes with 20μL of IS-K (25) (20ng/mL) and 780μL of deproteinising agent added. The samples were vortexed gently for 1 minute then centrifuged at 3000rpm for 3 minutes. The samples were then injected onto the HPLC. The detection limit of the analysis was 1ng.

Milk Vitamin K Milk vitamin K concentrations were determined using the HPLC method, described above for plasma. The milk samples were thawed in a water bath at 37ºC, and placed on a heated rotary mixer prior to analysis. An aliquot (0.5 mL) was pipetted into a centrifuge tube, and 1.0ml of methanol (Sigma) added, and the tube was vortexed for 30 seconds. Ethyl acetate (1.0ml) 99% (Sigma) was then added to the tube and vortexed for a period of 2 minutes, to ensure a thorough mixing of the sample. Tubes were then centrifuged for 5 minutes at 3000rpm. After centrifugation, the supernatant was decanted to a HPLC vial for analysis as described for plasma.

3.2.4 Statistical Analysis

All data analysis was conducted by using the SAS/STAT® software. Visualisation and data tabulation was carried out using GraphPad Prism version 8.2.1 for Windows (GraphPad® Software).

Changes in the plasma concentration of vitamin K1 over time were analysed by a repeated measures analysis of variance using the SAS MIXED procedure. The mean group values were

calculated for each time point and the data was log10 transformed before analysis for each treatment response. Pair-wise comparisons were generated to determine the significance of

differences at the indicated time points. Data for measuring vitamin K1 in milk fat were log10 transformed before analysis. The mean ± standard deviation (SD) and mean ± standard error (SEM) group values for each time-point were considered to be different at P<0.05.

The experimental design can be expressed by the following model:

, , = μ + Treatment + , + Time + Treatment x Time + , ,

𝑌𝑌𝑖𝑖 𝑗𝑗 𝑡𝑡 𝑖𝑖 𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑖𝑖 𝑗𝑗 𝑡𝑡 𝑖𝑖 𝑡𝑡 𝑒𝑒𝑖𝑖 𝑗𝑗 𝑡𝑡 (The italicised terms represent variances or random effects).

where: , , is the plasma concentration of vitamin K1, μ is the overall mean, Treatment is

the fixed𝑌𝑌𝑖𝑖 effect𝑗𝑗 𝑡𝑡 of treatment (i = 1 to 2), Time is the fixed effect of sampling time (t = 1 to𝑖𝑖 4; 1 to 14), the interaction between treatment and𝑡𝑡 time (Treatment x Time ), the random effect

of the horse is , , representing variation between horses 𝑖𝑖within treatments𝑡𝑡 and , , is

the residual error𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆. Pair𝑖𝑖-𝑗𝑗wise comparisons were generated to determine the significance𝑒𝑒𝑖𝑖 𝑗𝑗 𝑡𝑡of differences between treatments at indicated time points.

Mare and foal plasma was analysed using area under the curve (AUC), following the trapezoidal rule and assuming equally spaced observations.

= + + + + 𝑦𝑦0 𝑦𝑦4 𝐴𝐴𝐴𝐴𝐴𝐴 2 𝑦𝑦1 𝑦𝑦2 𝑦𝑦3 2 These data were analysed using a simple one-way ANOVA model with the SAS GLM procedure. Data for cord plasma included a large amount of data below the detection limit of 0.1ng/ml. All horses, did not have detectable amounts of . Data were initially analysed using

a one-way ANOVA (as for AUC). As a further check data𝐾𝐾1 for the KQ group were analysed

using the SAS LIFEREG procedure, assuming a log-normal distribution with zero readings regarded as being between zero and the detection limit of 0.1 ng/ml.

3.3 Results The results of the pasture, hay and BioMare Cubes®, indicate that the mares would have met their nutrient requirements (NRC, 2007). Interestingly, all samples tested were below the detection limit for vitamin K. Throughout the results section all values for vitamin K refer to

vitamin K1. Vitamin K1 was the only form measured in plasma and milk, as it is predominate circulating form.

3.3.1 Experiment 3.1 - Pre-parturition study All mares appeared clinically normal during the experimental period and the outcome of the study is shown in Table 3.2. However, two mares from the control group were removed from the trial; mare five did not produce a live foal, and mare six was removed due to misadventure. The remaining 10 mares delivered normal foals with an average body weight of 47.71kg ± 5.43kg. Mares foaled at an average of 1.38 days beyond their expected due date, therefore increasing the number of treatment days (31 days) and amount of concentrate ingested (16 days on feed) (Table 3.2).

Table 3.2: Experimental outcome for mares in Experiment 3.1

AFD ͣ No. Days No. days in Horse Foal Birth Treatment relative to Fed Treatment ID Weight (kg) EFD ᵇ Concentrate ⁿ Period 1 Control 6 20 31 50.5 2 Control 1 15 31 47.0 3 Control 3 17 32 49.0 4 Control -5 9 25 52.0 7 KQ -6 8 26 41.0 8 KQ 14 28 44 47.5 9 KQ -2 12 26 55.0 10 KQ 3 17 32 49.0 11 KQ -14 7 21 37.0 12 KQ 1 15 28 42.5 ªAFD, actual foaling date; ᵇEFD, estimated foaling date = day 0 ⁿBiomare cubes fed at 1kg/500kg bodyweight

Circulating concentrations of vitamin K1 in maternal plasma pre-parturition

Concentrations of vitamin K1 in plasma of mares prior to foaling varied greatly between animals. To ascertain if there was a cumulative treatment effect, data was transformed and the area under the curve (AUC) calculated for the period of KQ administration (Table 3.3), but no significant differences in circulating concentrations of vitamin K were found, irrespective of supplementation.

Table 3.3: Circulating concentrations of vitamin K1 in maternal plasma prior to foaling

Control KQ Horse 1 2 3 4 7 8 9 10 11 12 ID AUC* 1.3 8.4 9.2 10.3 3.0 9.6 1.8 4.7 9.0 7.9

*AUC area under the curve.

Vitamin K1 concentration at parturition

The concentration of vitamin K1 in maternal plasma, colostrum, umbilical cord (UC) and foal plasma at parturition are shown in Table 3.4. Concentrations of vitamin K1 in maternal plasma

and colostrum were measured immediately after parturition. Vitamin K1 in colostrum does not appear to reflect the amount determined in maternal plasma. Compared to plasma concentrations, colostrum concentrations in mares supplemented with KQ were four fold and significantly (P<0.05) higher than colostrum produced by mares in the control group. Plasma

K1 concentrations in UC plasma and foal plasma at birth (pre-suckle) were either very low or below the detection limit of the analysis (<0.01ng/ml); demonstrating that placental transfer of vitamin K in the mare is limited (Table 3.4).

Table 3.4: Mean ±SE concentration (ng/ml) of vitamin K1 at parturition in maternal plasma, umbilical cord (UC) plasma, pre-suck foal plasma and colostrum.

Pre-suck Foal Mare Plasma UC Plasma Colostrum Plasma C 1.90 ±0.35 a 0ˣ 0.12 ±0.04 (3) 1.79 ±1.26 a KQ 1.49 ±0.29 a 0.32 ±0.22 (4)* 0.48 ±0.35 (3) 6.16 ±1.03 b ˣall observations were below the detection limit (0.01ng/mL) of the analysis ²a,b values in the same column with different superscript are significantly different (P<0.05) * numbers in brackets refer to the number of samples above the detection limit

Circulating concentrations of vitamin K in mares post-parturition There was no significant difference between treatment groups in circulating concentrations of vitamin K1 in mares at parturition (Table 3.5). However, samples taken at one week post foaling showed a significant (P< 0.05) difference between the control and treated group.

Table 3.5: Mean (±SE) concentration (ng/ml) of vitamin K1 in maternal plasma²

Time (day) C KQ 0 1.90 ±0.35 a 1.49 ±0.29 a 7 2.52 ±0.29 a 0.99 ±0.24 b ²a,b values in the same row with different superscript are significantly different (P<0.05).

Figure 3.2 shows graphically that circulating vitamin K1 in maternal plasma of treated group declined following cessation of supplementation. In contrast, concentrations of vitamin K1 in control mares increased during this period.

2.5

2 C 1.5 KQ

0.5

Vitamin K1 concentration (ng/mL) concentration K1 Vitamin 0 0 7

Days after birth

Figure 3.2: Mean concentration (ng/ml) of vitamin K1 in maternal plasma. There was no significant difference (P >0.05) in mean plasma vitamin K1 concentration between the control (n = 4) and treatment group (KQ) (n = 6) except at 7 days post-parturition.

Circulating concentrations of vitamin K1 in foals

Foal plasma K1 concentrations at birth (Table 3.6) were similar for both groups, with most samples being below the detection limit of the analysis (<0.01ng/ml). Half a day after birth and suckling, plasma values increased in the control and KQ foal groups, presumably reflecting uptake from milk. There was however, no significant difference between the groups, as depicted graphically in Figure 3.3.

Table 3.6: Mean (±SE) log transformed concentration (ng/ml) of vitamin K1 in foal plasma²

Time (day) C KQ 0 0.05 ±0.06 0.13 ±0.05 0.5 0.24 ±0.07 0.24 ±0.06 1 0.34 ±0.11 0.47 ±0.09 7 0.34 ±0.13 0.51 ±0.11 ²a,b values in the same row with different superscript are significantly different (P<0.05).

0.6

0.4 C KQ 0.2

Vitamin K1concentration (ng/ml) K1concentration Vitamin 0.0 0 2 4 6 8 Days after birth

Figure 3.3: Mean concentration (ng/ml) of vitamin K1 in foal plasma. There was no significant difference in mean plasma vitamin K1 concentration between the control and treatment group.

79 Vitamin K1 concentration in mare’s colostrum and milk

Milk concentrations in vitamin K1 at parturition (Table 3.7) showed that supplementation with KQ, resulted in significantly (P<0.05) higher concentrations in colostrum. Higher concentrations for the KQ group were evident in the following 24 hours but had returned to control concentrations within 7 days of cessation of supplementation (Figure 3.4).

Table 3.7: Mean (±SE) concentration (ng/ml) of vitamin K1 in milk²

Time (day) C KQ 0 1.79 ±1.26 a 6.16 ±1.03 b 0.5 2.05 ±1.51 a 8.04 ±1.23 b 1 2.86 ±2.23 a 8.15 ±1.82 a 7 1.47 ±0.67 a 2.89 ±0.54 a ²a,b values in the same row with different superscript are significantly different (P<0.05).

6 C 4 KQ

Vitamin K1 concentration ng/ml) concentration K1 Vitamin 0 0 0.5 1 7

Days after birth

Figure 3.4: Mean concentration (ng/ml) of vitamin K1 in colostrum and milk. There was a significant difference (P<0.05) in mean plasma vitamin K1 concentration between the control (n = 4) and treatment groups (KQ) (n = 6) up to 12 hours (0.5 days) post-parturition.

The milk concentrations for vitamin K1 corrected for milk fat content are shown in Table 3.8.

These corrected values more closely reflect the values for vitamin K1 in milk. This is also apparent when the data is graphed (Figure 3.5), and the dynamics of vitamin K concentrations for 7 days post-parturition exhibit a similar pattern to that observed in Figure 3.4. At parturition 80

concentrations of vitamin K1 (ng/g) in milk corrected for milk fat show that the KQ group had the highest concentration (1.26 ± 0.16; Table 3.8). Over the first 12 hours postpartum concentrations of vitamin K1 in the KQ treatment group were higher than in control mares, although this appeared to plateau at around 24 hours with concentrations decreasing between 1 and 7 days postpartum (Figure 3.5).

Table 3.8: Mean (±SE) log transformed vitamin K1 concentration (ng/g) corrected for milk fat²

Time (day) C KQ 0 0.92 ±0.20 a 1.26 ±0.16 a 0.5 0.73 ±0.16 a 1.36 ±0.13 b 1 0.89 ±0.20 a 1.49 ±0.16 b 7 0.85 ±0.18 a 1.07 ±0.15 a ² a,b values in the same row with different superscript are significantly different (P<0.05).

1.5

1 C

0.5 Vitamin K1 concentration (ng/mL) milk fat milk (ng/mL) concentration K1 Vitamin 0 0 0.5 1 7

Days after birth

Figure 3.5: Mean concentration of vitamin K1 (ng/g) corrected for milk fat. Over the first 12 hours (0.5 days) postpartum concentrations of vitamin K1 in the treatment group were significantly higher than in control (P<0.05).

3.3.2 Experiment 3.2 - Post-parturition study The mares remained clinically normal throughout the experimental period. At the start of the study the mares had a mean body weight of 485 ± 14.2kg and a mean body condition score of 5.6 ± 0.6 (Henneke et al. 1983)At the end of the study the mares had a mean body weight of 496 ± 15.6kg and a mean body condition score of 5.4 ± 0.8. Individual body weights fluctuated over the study period. This is attributable to normal post-parturition body weight changes in mares during lactation and reflects, in part, changes in pasture availability.

The foaling dates of the mares are shown in Table 3.9. A control and KQ mare where matched for foaling date to reduce the impact of environmental factors during the experimental period. Matched control and KQ mares foaled within a few days of each, except for one pair, which foaled 15 days apart. Four mares (control and KQ treatment) foaled in mid- to late September while the other eight mares foaled throughout October.

Table 3.9: Foaling dates of control and KQ treatment mares in Experiment 3.2

Mare ID Control Mare ID KQ Interval (days)

2 17/09/2016 8 30/09/2016 15

1 24/09/2016 7 24/09/2016 0

3 6/10/2016 9 5/10/2016 1

4 8/10/2016 10 6/10/2016 2

6 16/10/2016 12 12/10/2016 4

5 20/10/2016 11 17/10/2016 3

The average body weight of the foals at birth was 50.29kg. The foals remained clinically normal throughout the study and had a similar weight gain trajectory as shown in Figure 3.6. There was no difference (P>0.05) in mean body weight between the control and KQ group during the study.

250 Control 200 KQ Treatment

150

100

Foal weight (kg) 50

1 7 14 21 28 35 42 49 56 70 84 98 0.5 112 140 Days after birth

Figure 3.6: The mean ± standard error of foal body weight over the duration of the experimental period. There was no significant difference (P >0.05) in mean body weight between the control (n = 6) and treatment group (KQ; n = 6).

Circulating Vitamin K1 concentrations in foal plasma

There was no significant difference in foal plasma vitamin K1 concentrations between the control and KQ treatment group except at day 14 (Table 3.10). The concentration of vitamin

K1 in foal plasma prior to suckling was below the detection limit (<0.05 ng/ml) for both groups

(Table 3.10). There was a significant increase (P<0.05) in the plasma vitamin K1 concentrations during the first 24 hours but no further increase in circulating concentrations of the vitamin was apparent in either group, for the next 27 days (Figure 3.7). A significant increase (P<0.05) occurred between days 28 and 56 in both groups and this increase was still evident at day 98 (Figure 3.7).

Table 3.10: Mean (±SE) log transformed concentration (ng/ml) of vitamin K1 in foal plasma.

Days Control KQ

0 <0.05a <0.05a

0.5 0.73±0.43a 0.90±0.37a

1 2.57±4.23a 4.89±4.40a

7 2.79±1.36a 3.53±1.25a

14 1.88±1.10b 4.47±1.5a

21 2.92±0.98a 3.64±1.08a

28 3.95±1.19a 4.40±1.55a

56 13.55±7.89a 11.82±5.38a

98 12.50±6.16a 11.28±7.27a

a,b values in the same row with different superscripts differ significantly (P<0.05)

Control

20 KQ Treatment

* 5 Vitamin K1 concentrationVitamin (ng/ml) K1 0 0 1 7 0.5 14 21 28 56 98

Days after birth

Figure 3.7: Mean vitamin K1 concentrations (ng/ml) in foal plasma. There was no significant difference (P >0.05) in mean plasma vitamin K1 concentrations between the control (N = 6) and treatment groups (KQ) (N = 6) except at 14 days post-parturition. * represents a significant difference between the two treatments in (P<0.05).

Vitamin K1 concentration (ng/ml) in mare’s colostrum and milk

The values for vitamin K1 reported here have been corrected for milk lipid concentration. Vitamin K or KQ supplementation significantly increased (P<0.05) milk vitamin concentration during the immediate post -partum period (Table 3.11) but from day 7, concentrations in milk from the control mares was essentially the same for the next 6 weeks (day 49). During the

following 7 weeks (day 98) the milk concentration of vitamin K1 doubled in both groups (Figure 3. 8). Plasma vitamin K concentrations were not analysed in Experiment 2, due to the failure to detect a difference in circulating concentrations following supplementation in Experiment 1.

Table 3.11: Mean (±SE) concentration (ng/ml) of vitamin K1 in colostrum and milk.

Days Control KQ

0 2.05±0.61a 9.61±4.65a

0.5 9.21±1.84b 24.50±6.14a

1 11.45±2.12b 21.00±5.02a

7 5.14±2.93a 7.85±1.05a

14 3.20±1.13a 6.44±1.60a

21 4.31±1.35a 5.76±2.21a

28 4.34±1.83a 6.18±1.47a

35 3.11±1.19a 3.99±1.13a

42 4.45±1.90a 1.34±0.37a

49 5.51±2.37a 6.11±2.67a

56 10.89±5.05a 10.37±3.45a

70 8.80±2.18a 13.85±1.06a

84 14.88±5.23a 12.80±1.49a

98 9.97±1.55a 11.47±1.77a a,b values in the same row with different superscripts differ significantly (P<0.05)

40 Control KQ Treatment 35 *

30 * 25 20 15 10 5 Vitamin K1concentration (ng/ml) 0 0 1 7 0.5 14 21 28 35 42 49 56 70 84 98 Days after birth

Figure 3.8: Mean vitamin K1 colostrum and milk concentrations (ng/ml). There was no significant difference (P >0.05) in mean milk vitamin K1 concentrations between the control (n = 6) and KQ group (n = 6) * represents a significant difference between the two treatments in (P<0.05).

3.4 Discussion There is still debate as to which source of vitamin K is most relevant to metabolism studies

(Halder et al. 2019). Most studies use vitamin K1, and a soluble form of the vitamin; vitamin KQ was used in these studies. It has been shown to be more readily absorbed than other forms of the vitamin in the horse, following oral supplementation (Skinner et al. 2014). Moreover, human studies have shown that vitamin K1 is the form predominantly used by the foetus

(Shearer 1992) and demonstrated that vitamin K1 is deposited in milk to a much greater extent

that vitamin K2 (Shearer 2009b).

Vitamin K in neonatal foals The transfer of nutrients across the placenta and deposition in milk is important for foetal and neonatal development. It appears, that as in humans, the horse has limited placental transfer of vitamin K. The concentration of the vitamin was below the detection limit (<0.01ng/ml) in both umbilical cord and pre-suck plasma samples of foals from mares not receiving vitamin K supplementation. A systematic review by Shahrook et al. (2018) found that cord concentrations

of vitamin K1 in humans were also below the limit of detection in most instances. Of the fat soluble vitamins, vitamin K is the least transferred by the placenta (Shearer 2009b) and little is known about the mechanisms responsible for membrane transfer of vitamin K (Sânzio Gurgel et al. 2017).

The dynamics of circulating vitamin K in human neonates can be summarised as follows. In babies, circulating levels of vitamin K are often below the detection limit at birth, especially when mothers have not been supplemented with the vitamin. Moreover as bleeding is rare in newborn babies, placental transfer must be sufficient for normal haemostasis during foetal development. However, vitamin K is usually detected after breast feeding and reaches adult levels during the first week post-partum (Greer et al. 1991; Shearer 2009a). The results with foals mirrored the dynamics in babies. Foals born to mares that were supplemented with vitamin K tended to have a higher concentration of vitamin K in plasma and cord blood at birth; this has also be shown in babies (Kazzi et al. 1990; Greer et al. 1991). The rapid increase in circulating concentrations of vitamin concentrations in the foals in the first day after birth reflects colostrum intake.

Colostrum produced by supplemented mares had a significantly higher concentration of the vitamin, and this was reflected in the plasma concentrations of their foals. Studies in woman

found that supplementation with vitamin K1 significantly increased the vitamin K1

concentration in breast milk (Thijssen et al. 2002; Shahrook et al. 2018). Mares were supplemented throughout Experiment 2 but with the exception of day 14, when foal plasma from the supplemented group was significantly higher than the control, there were no significant difference detected at any other time point. However, there was a tendency for the

circulating plasma concentrations of vitamin K1 to be higher in the supplemented group over the course of the study. This lag in the rise of vitamin K levels in the foal’s plasma may be attributable to the sequestering of the vitamin in the liver. As the foals are born with little or no store of vitamin K in the liver, it may take several weeks before a rise in plasma level is observed (Siciliano et al. 2000b).

Plasma vitamin K concentrations also fluctuate as a result of daily intake and supplementation (Y de Vries et al. 2018). The increase in both groups between days 28 to 56 probably reflects pasture intake (see Figure 3.1b) as green grass has high photosynthetic capacity, significant

concentrations of vitamin K1 would be expected within the grass (Biffin et al. 2008b). By this

stage of the foal’s development there is likely to be synthesis of vitamin K2 in the gastrointestinal tract, but this source of the vitamin is unlikely to impact on the vitamin K economy of the foal (see Chapter 2).

Vitamin K1 in the mare

Milk concentrations of vitamin K1 showed that oral supplementation was able to modulate the concentration of the vitamin in milk especially in the first few days post-partum. Both studies showed the same trend in milk vitamin K concentration between control and supplemented

horses however, concentrations of vitamin K1 in mare’s milk reached significantly higher concentrations in Experiment 2 at 12 and 24 hours post-parturition. This is likely due to the timing of supplementation. The mares in Experiment 2 received their first dose at the time of parturition whereas the mares in Experiment 1 were only dosed prior to parturition. As expected in Experiment 1, the vitamin K1 concentration in the milk of supplemented mares declined to control values during the week after parturition, following the cessation of supplementation. For the six to eight week period after parturition, milk concentrations remained at about 10

ng/ml with a tendency for higher values in the supplemented group. Milk vitamin K1 concentrations during lactation have also been shown to either remain stable or increase (Thijssen et al. 2002). After this period there was noticeable increase in milk vitamin concentration, presumably reflecting seasonal fluctuations in pasture concentrations (Biffin et al. 2008b; Peugnet et al. 2015). As noted above, a similar pattern of increase was observed in

foal plasma concentrations of vitamin K1.

There was however, as shown in Experiment 1, no correlation between milk and plasma vitamin K1 concentrations. This agrees with the findings of Thijssen et al. (2002) in which vitamin K1 concentrations in plasma did not correlate with milk concentrations in nursing

mothers. Suggesting that plasma vitamin K1 is not a good indicator of vitamin K requirements as it reflects only a small fraction of the total body pool (Shearer 1995; Gundberg et al 2012). In woman, mobilisation of vitamin K into milk for the purpose of maintaining vitamin K status in the infant has precedence over maintaining vitamin K stores (Greer et al. 1997). This may

explain, in part, the failure to detect any difference in the circulating vitamin K1 concentrations in mares but it may also be a function of the time of blood sample collection. A previous study, in which horses were supplemented with a bolus of vitamin K per os, found that the half-life of vitamin KQ in plasma is short, and an optimal plasma sampling time is about two hours after oral administration (Skinner et al. 2014). In the current study a bolus dose was also given, but mares were sampled prior to dosing and some three days after the previous dose; determined values would therefore reflect baseline values. Moreover, in a study with nursing mothers, a similar scenario was described. Haroon et al. (1982) found that circulating concentrations of vitamin K1 increased sharply and then rapidly declined after mothers were given a single oral

dose of vitamin K1.

Determination of the dose of vitamin K1 for supplementation of lactating mares is difficult as there is no recommended intake of vitamin K for horses (NRC 2007; GEH 2013; INRA 2015).

However, from these experiments it is possible to estimate the effect of vitamin K1 supplementation of mares on the intake of foals with the following assumptions. Daily milk consumption of a 50kg foal ranges from 10% to 25% of its body weight (NRC 2007) and milk production by mares of 500kg body weight range between 15 and 30kg daily (Doreau & Boulot

1989). According to the vitamin K1 concentration of milk in plasma of KQ supplemented mares (8.15 ng/ml), 12.5 litres of milk will contain 102μg, which translates to be 2μg/kg body weight per foal daily. This value is twice what is recognised as an adequate intake for humans (1μg/kg;

(Turck et al. 2017)). Meanwhile, foal vitamin K1 intake from milk of non-supplemented mares is estimated to be 36μg, which is equal to 0.72μg/kg body weight, less than the adequate intake recommended for humans. Based on this estimate, foals of unsupplemented mares require an increased intake of vitamin K1, especially in the first week of life. However, vitamin K deficiency bleeding disorder and prophylaxis (Shearer 2009a) is not commonly seen in foals

(McGorum et al. 2009). This indicates that vitamin K1 intake is adequate for blood clotting but requirements may be greater for other VKDPs.

90 Pregnancy and lactation are periods of significant influence on bone metabolism (Filipovic et al. 2010). The intensive foetal growth, and the mineralisation of the foetal skeleton is substantial. Bone has also recently been identified to act as an endocrine organ in its own right, with osteocalcin implicated as one of the key players in bone development (Ducy 2011; Karsenty 2012; Ferron & Lacombe 2014). In young animals, modelling of the skeleton is accompanied by increased concentration of bone remodelling markers in the blood (Pastoret et al. 2007). Could vitamin K status be better assessed by the use of an alternative candidate marker, such as osteocalcin?

3.5 Conclusion To our knowledge, this is the first controlled study to investigate the effects of vitamin K

supplementation on vitamin K1 status in mares and foals. While these studies failed to demonstrate any significant effect of routine oral supplementation on circulating vitamin K status, milk concentrations showed that supplementation was able to significantly modulate the

concentration of vitamin K1 in milk, especially in the first few days following parturition. This suggests that colostrum is a critical source of vitamin K for foals in the first few days, and also before their gut is developed enough for them to obtain requirements from pasture and potential microbial derived synthesis of vitamin K (Heidler et al. 2003). The hypothesis that vitamin K has only limited transport across the equine placenta, was supported by the negligible

concentrations of vitamin K1 found in both umbilical cord and foal plasma at birth.

The results suggests that mares and foals maintained on quality grass and legume pasture, probably do not require additional vitamin K supplementation to maintain vitamin K status during lactation and growth. However, in order to make informed decisions regarding diet formulation and supplementation in horses, the effects of vitamin K supplementation during lactation and growth on vitamin K status needs to be further investigated, specifically the effect on vitamin K dependent markers of bone metabolism.

While supplementation of vitamin K using Quinaquanone (KQ) did not affect vitamin K1 plasma concentrations in mares, this implies that circulating plasma concentrations of vitamin K may not be a reliable indicator of overall vitamin K status. Osteocalcin has been implicated as a more reliable indicator of overall vitamin K status. Further research should examine the association between vitamin K status and osteocalcin, to better assess the effect of supplementation on bone metabolism in the horse, as the results suggest that in most instances the intake of vitamin K is sufficient for normal haemostasis.

91 92 General Materials and Methods, Method Development and Optimisation 4.1 Introduction ...... 93 4.2 Animals ...... 94 4.3 Standards, reagents and solvents ...... 94 4.4 Equipment ...... 94 4.5 Consumables ...... 95 4.6 Sample preparation & optimisation for proteomics-based mass spectrometry ...... 95 4.6.1 Equine plasma pre-treatment ...... 95 4.7 Optimisation of digestion and desalting ...... 95 4.7.1 Preparation of bovine serum albumin (BSA) ...... 96 4.7.2 Preparation of equine plasma...... 96 4.8 Preparation of osteocalcin synthetic standards ...... 97 4.8.1 Osteocalcin peptide standards...... 97 4.9 Digestion methods for protein analysis ...... 97 4.9.1 In solution digestion ...... 97 4.9.2 Filter Aided Sample Preparation (FASP) ...... 98 4.10 Desalting ...... 98 4.11 Mass Spectrometry Analysis ...... 99 4.11.1 Liquid Chromatograph Mass Spectrometer (LC-MS) Instrumentation Discovery platform ...... 99 4.12 Data Acquisition ...... 100 4.12.1 Data-dependent acquisition (DDA) ...... 100 4.12.2 Data independent acquisition (DIA) ...... 100 4.12.3 Targeted data acquisition ...... 101 4.13 Data Analysis and processing ...... 102 4.13.1 Skyline daily ...... 102 4.13.2 Peakview...... 104 4.13.3 Protein Pilot ...... 104 4.13.4 Mascot ...... 105 4.14 Statistical analysis of the processed data ...... 105

4.1 Introduction Research over the last decade has uncovered a plethora of proteins that bind to calcium upon vitamin K-dependent carboxylation (VKDPs) (Chapter 2). The VKDP, osteocalcin, plays a vital role in bone metabolism. In human studies, osteocalcin defects have been associated with increased fracture risk and osteoporosis. Circulating osteocalcin and, its carboxylation status, has been proposed as one of the indicators of vitamin K status and a ‘biomarker’ of bone metabolism (Chapter 2).

93 Current assays available to measure osteocalcin, however, do not provide information about the degree of osteocalcin carboxylation. The aim of the studies presented in the following chapters, was to exploit the recent advances in the use of mass spectrometry (MS) based techniques and investigate the feasibility of a method to accurately measure the carboxylation status of circulating osteocalcin in equine plasma, after vitamin K supplementation (Chapter 3).

This chapter describes the general material and methods used throughout the subsequent studies presented in this thesis (Chapters 5 – 7). Variations to these methods, and those methods specific to further experiments are described in the subsequent chapters.

4.2 Animals The equine plasma samples used in the proceeding chapters of this thesis were collected with the required endorsement of the University of Queensland Animal Ethics Committee (AEC Approval number SAFS/421/16). The samples were collected as part of a study (see Chapter 3) that was conducted at the Equine Research Unit at the University of Queensland, Gatton campus in South-Eastern Queensland, Australia. For full blood collection and sampling procedures refer to section 3.2.2 in Chapter 3.

4.3 Standards, reagents and solvents Standards were purchased from Mimtopes (VIC, Australia). The solvents and reagents used throughout this thesis were predominately obtained from Thermo Fisher Scientific Pty Ltd (VIC, Australia) and Bio-Rad (NSW, Australia). All solvents used were of LC-MS grade.

4.4 Equipment Tandem LCMS/MS (Triple quadrupole mass spectrometer LCMS 8050, Shimadzu Pty Ltd, Japan), Quadrupole time-of-flight mass spectrometer (TripleTOF® 5600+ System, SCIEX) with Nanospray III ion source (SCIEX Pty Ltd, USA), Nano-HPLC (Eksigent Ultra 2D, Eksigent Technologies, Dublin, CA), Triple quadrupole Inductively Coupled Plasma Mass Spectrometer (ICP-MS 8800 System, Agilent Technologies, Inc. USA), Laser Ablation Inductively Coupled Plasma Mass Spectrometer (LA-ICP-MS 8800 System, Agilent Technologies, Inc. USA), Speedvac Concentrator (Christ® cat. No. RVC 2-33 IR).

94 4.5 Consumables Desalting pipette tips (Millipore® Zip Tips SCX, Sigma-Aldrich, cat no. ZTSCXS096), MS auto sampler vials (12 x 32mm Snap Neck Vial, Waters, cat. No. 186005224), CHT Ceramic Hydroxyapatite (Type 1, 20µm particle size, BIO-RAD, cat. No. 1582000), Hydroxyapatite (Calbiochem cat no. 391947-100gm, Merck).

4.6 Sample preparation & optimisation for proteomics-based mass spectrometry Plasma is a source of proteins that can provide insight into the nutritional status of the animal, at the instance of sampling. The range of proteins present in the plasma however, can be up to 12 orders of magnitude. Highly abundant proteins such as albumin need to be depleted before analysis can occur otherwise, they may mask the presence of lower abundant proteins. Sample collection and preparation of plasma is therefore crucial. The quality and consistency of sample preparation influences the time and cost of mass spectrometry analysis and the reliability of the results. For mass spectrometry-based proteomics to reach its full potential, variability associated with the sample preparation steps that precede mass spectrometry analysis must be eliminated where possible. For this reason, optimisation of sample preparation is imperative before further sample processing is conducted. It is vital to run quality control (QC) samples and check the reproducibility of sample preparation.

4.6.1 Equine plasma pre-treatment Plasma must undergo pre-treatment to remove insoluble materials and lipids before analysis. To precipitate the insoluble fraction, cold acetone (-20°C) was added to the sample and incubated overnight at 4°C. After incubation the samples were then centrifuged, supernatant removed and then the pellet was washed with acetone. After washing the pellet was then resuspended in urea/ammonium bicarbonate (AMBIC) buffer. Finally, it was centrifuged for 10 minutes at 14,000 x g. The supernatant was removed and kept for further protein analysis. The protein concentration of the supernatant was determined using the BCA Protein Assay kit (Bicinchoninic acid kit, Pierce™, cat. No. 23225) and spectrophotometer (NanoDrop 2000, Thermo Scientific) following the manufacturer’s instructions.

4.7 Optimisation of digestion and desalting Prior to conducting the experiments, it was necessary to assess how reproducible sample preparation and data acquisition were from in-solution digests of BSA standards for in-house

95 quality control of the instrument. This was done in order to assess the satisfactory reproducibility of repeat injections of individual samples.

Sample preparation has involved utilising both Filter Aided Sample Preparation (FASP) (Wisniewski et al. 2009) and in-solution (Kulak et al. 2014) digestion methods. To assess the reproducibility of these methods, QC samples of BSA were digested. The FASP digestion technique has consistently proved to be the most accurate and reproducible (Figure 4.5).

4.7.1 Preparation of bovine serum albumin (BSA) In order to optimise procedures, BSA was prepared at a concentration of 10µg/µl for digestion and analysed with equine plasma for QC. BSA replicates were in-solution digested and desalted using strong cation exchange (SCX) membrane to test reproducibility of samples.

4.7.2 Preparation of equine plasma Equine plasma was initially loaded onto a pre-cast SDS-PAGE gel to check the protein concentration (Figure 4.1). This was conducted to confirm there was an adequate amount of protein before performing sample digestion and submitting for further analysis. For this QC test, 20µg and 50µg of equine plasma was prepared for loading onto the pre-cast gel. The samples were prepared in 1X buffer with 20mM DDT, incubated for 3 minutes at 60°C before loading onto the gel. Once the run was completed the gel was stained with aqua stain and left on the agitator for 10 minutes. De-staining was then carried out in water on the agitator.

96 Figure 4.1: SDS-PAGE image of 50µg and 20µg of equine plasma (EP) compared to 10µg of bovine serum albumin (BSA) and a protein reference ladder.

To test the reproducibility of equine plasma preparation, five technical replicates of 20µg/µl were digested separately with the endoproteinase asp-n (which selectively cleaves protein and peptide bonds N-terminal to aspartic acid residues) and trypsin to compare the digestion with both enzymes (refer to Chapter 5 for full details).

4.8 Preparation of osteocalcin synthetic standards 4.8.1 Osteocalcin peptide standards The exact amount of each pure peptide was calculated based on percent purity provided by Mimtopes Pty Ltd. (eg. 1.1mg at 69% purity = 0.759mg of pure peptide). The standards were then individually solubilised in 50% acetonitrile/0.1% formic acid at a concentration of 1pmol/µl. All peptides containing cysteine were then reduced and alkylated.

4.9 Digestion methods for protein analysis 4.9.1 In solution digestion In the following experiments, 10-20µg of acetone precipitated plasma protein was processed (10µg of BSA). Disulphide bridges were reduced by 5mM of dithiothreitol (DTT) and incubated at room temperature (27°C) for 60 minutes. Alkylation was then carried out by 14mM of iodoacetamide (IAM). The sample was then incubated in the dark for 20 minutes.

97 The reaction was quenched by 5mM DTT and 10mM of calcium chloride (CaCl2). Finally, the samples were enzymatically digested using trypsin and left in the incubator overnight at 37°C with gentle agitation. The next day the samples were dried (Christ® cat. No. RVC 2-33 IR) and then reconstituted in 10% acetonitrile (ACN) in 0.1% trifluoroacetic acid (TFA). The samples were desalted in preparation for mass spectrometry (MS). This method, while simpler than the steps required for FASP digestion, was less reproducible.

4.9.2 Filter Aided Sample Preparation (FASP) In the following experiments, 10-20µg of acetone precipitated plasma protein was processed (10µg of BSA). For this protocol the protein was firstly solubilised in SDS-Tris lysis buffer. This was then combined with DTT-Urea buffer within a 30kDa Microcon YM-30 centrifugal filter device (Millipore®, Merk, cat no: MRCF0R030) and incubated at room temperature for 60 minutes on an agitator. After incubation, a series of centrifugation steps was then carried out. The filters were centrifuged at 14,000 x g at 21°C for 15 minutes and flow through discarded, before being washed again with Urea-Tris buffer and then centrifuged. Alkylation was then carried out via the addition of 50mM of iodoacetamide in Urea-Buffer and incubation at room temperature (27°C) for 20 minutes on the agitator. The samples were then repeatedly washed with Urea-Tris buffer to carboxyamidomethylate thiols and remove and remaining detergent. The filters were then equilibrated with two final washes with 100mM AMBIC. Trypsin was then added to the filters and the samples were left overnight for digestion in the incubator at 37°C. The next day, the filters were transferred to clean Eppendorf tubes and the peptides collected by centrifugation at 14,000 x g at 21°C for 15 minutes, with elution carried out by the addition of 100mM AMBIC. The samples were then speedvac and reconstituted in 10% acetonitrile, 0.1% trifluoroacetic acid (TFA). The samples were finally desalted in preparation for mass spectrometry analysis.

4.10 Desalting This protocol is for cleaning, desalting and concentrating of peptide sample prior to mass spectrometry analysis. It is carried out to improve signal quality and remove salts and particulate matter to avoid clogging of the column and extend analytical life of the column (Kulak et al. 2014). Clean up was conducted using a strong cation exchange (SCX) membrane (Empore®, Sigma Aldrich Co-LLC, Sydney, NSW, Australia).

98 Figure 4.2: Stage tip preparation; a small disk of the SCX membrane was excised in a petri dish using a cutting tip. The membrane was then released into a pipette tip (200µl microtip).

To prepare the stage tip, the empore extraction membrane was placed in a clean petri dish. A needle tip was then used to cut a small piece of membrane disk (see Figure 4.2). This was then ejected into a pipette tip (200µl) microtip. The stage tip was then activated by passing 100% acetonitrile through the tip using positive pressure. Conditioning was then carried out by adding 5% ammonium hydroxide/80% acetonitrile and passing it through the tip before the last remaining liquid had left the tip to avoid the tip drying completely. It was then equilibrated by adding 0.2% TFA. The sample was then loaded onto the tip and passed through, binding the peptide material to the tip. Three final washes of 0.2% TFA were then carried out. The peptides were then eluted by passing 5% ammonium hydroxide/ 80% acetonitrile through the tip into a clean Eppendorf tube. The sample was then speedvaced and reconstituted in 2% acetonitrile/0.1% formic acid (or irt buffer for mass spectrometry analysis). Peptide concentration was then normalised across samples by using the peptide assay kit (Quantitative Colorimetric Peptide Assay, Pierce™, cat no. 23275) following the manufacturer’s instructions. Finally, the samples were then transferred to a plastic autosampler vial for mass spectrometry analysis.

4.11 Mass Spectrometry Analysis 4.11.1 Liquid Chromatograph Mass Spectrometer (LC-MS) Instrumentation Discovery platform A NanoLC-MS/MS (TripleTOF® 5600+ System, SCIEX) was used to generate all peptide data. Peptides were separated by performing reversed-phase chromatography using an Eksigent ekspert™ nanoLC 400 system coupled to MS/MS instrument. The mobile phases were; mobile

99 phase A: water/0.1% FA; mobile phase B: ACN/0.1% FA; and mobile phase C: water/2% ACN/0.1% FA. The LC was configured as a trap and elute platform with a 10 mm x 0.3 mm trap cartridge packed with ChromXP C18CL 5 µm 120Å material and an analytical column (150mm x 75µm packed with ChromXP C18 3µm 120Å). Trapping was performed in mobile phase C for 5 minutes at 5µl/minute, followed by an elution configuration across a 90 minute gradient using mobile phases A and B at a conserved flowrate of 300nL/minute. To minimise retention time (RT) drift, the analytical column was maintained at 40°C.

4.12 Data Acquisition 4.12.1 Data-dependent acquisition (DDA) All samples were analysed using DDA acquisition mode. BSA samples were analysed using 25 minute DDA. Osteocalcin peptide standards and equine plasma samples were analysed using 95 minute DDA. The DDA mode of the instrument was set to obtain high resolution (30,000) TOF-MS scans over a mass range of 350-1350 m/z followed by up to 40 (in the 95 minute method) and up to 20 (in the 25 minute method). High sensitivity MS/MS scans of the most abundant peptide ions per cycle were collected and the fragmentation spectra stored in .wiff format (SCIEX).

Equine NCBI database processing: The custom database was assembled in FASTA format downloaded on the 5th of April 2017 from a repository of non-redundant and predicted protein sequences of equine species sourced from UniprotKB (Universal Protein Resource Knowledgebase – http://www.uniprot.org/). Added contaminates from the common Repository of Adventitious Proteins (cRAP), was also assembled in FASTA format and iRT peptides were also incorporated to facilitate RT calibration. Protein lists from DDA experiments were presented in spreadsheet and charts and then exported for analysis.

4.12.2 Data independent acquisition (DIA) Eluted peptides were subjected to cyclic data-independent acquisition (DIA) using a generic SWATH-MS™ acquisition mode. The instrument was operated using a mass range of 100 msec for the survey scan (MS), followed by performing MS/MS on all precursors in a cyclic manner using an accumulation time of 0.1 seconds per SWATH-MS window (36 windows total, each 26 m/z units wide) for a total cycle time of 3.75 seconds. The above parameters allowed for collection of at least 6 data points for each chromatographic peak of a peptide to ensure reasonably accurate quantitation.

100 4.12.3 Targeted data acquisition A triple quadrupole liquid chromatograph mass spectrometer (LC-MS 8050, Shimadzu Pty Ltd, Japan) was used to analyse all QC samples (BSA and equine albumin) and for MRM method development and characterisation of standards. MRM–MS is a highly sensitive and targeted method of data acquisition allowing the specific detection of peptide or small proteins of interest in complex biological samples (Vincent et al. 2009).

Multiple-Reaction-Monitoring (MRM) MRM method development was carried out on a Shimadzu LC-MS TQ-8050 (Shimadzu Corporation, Tokyo, Japan) using a KINETEX® 2.6µm EVO C18 100Å, 100x2.1mm (Phenomenex cat no. 00D-4725-AN) maintained at 40°C. For a given protein, selection of a signature peptide and optimum transitions for the peptide in MRM analysis is crucial. Quantitation of a signature peptide target in a complex digest preferably requires 2 to 3 transitions, although the use of a spiked reference peptide can reduce this requirement. Quantitation of the precursor protein also requires comparable results from 1 or 2 additional signature peptides. Selection of precursor-to-fragment transitions has been facilitated by in- silico methods based on algorithms that use theoretical and empirical data generated from proteomic analysis worldwide. There are accessible repositories of MS/MS spectral libraries of peptides, as well as a wide array of computational tools such as Skyline.

Osteocalcin (OC) peptide LC-MS-MRM method The final LC-MS conditions of the instrument used to develop the osteocalcin peptide MRM method were: uHPLC conditions; Mobile phase A: 0.1% (v/v) formic acid in water, Mobile phase B: 0.1% (v/v) formic acid in acetonitrile with a total flow rate of 500µl/min between both pumps maintained. Peptide elution was achieved by a binary gradient. 10% of mobile phase B was increased to 30% over 4 minutes then increasing to 95% at 4 minutes. This gradient was maintained for 3 minutes before then rapidly reducing back down to 10% at 7.01 minutes and maintained at 10% until pump pressures were returned to stable initial column pressure. The total chromatographic separation was achieved over 10 minutes and was conducted in positive ion mode. The Electrospray Ionisation (ESI) source parameters were; nebulising gas: 2.0L/minute, heating gas flow: 15.0L/minute, interface temperature: 350°C, DL temperature: 200°C, heat block temperature: 400°C, drying gas flow: 3.0L/minute, collision gas pressure at 350kPa.

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4.13 Data Analysis and processing 4.13.1 Skyline daily Skyline is a vendor-neutral and licence free software package that facilitates quantitative analysis of mass spectrometry data (MacLean et al. 2010). Skyline provides a number of ways to build and edit MRM methods (Henderson et al. 2018). For osteocalcin analysis, a Skyline template was established by in-silico predicting asp-n and trypsin digested peptide sequences of equine osteocalcin. An example Skyline annotated chromatogram can be found in figure 4.3.

The MS raw data was analysed using Skyline software. Proteins were extracted and annotated with amino acid sequence from a custom built database using the Paragon algorithm. A background proteome was built from FASTA formatted equine sequences. This forms the basis of the expected experimental protein matrix. Skyline digests proteins and fragments peptides in-silico to facilitate peptide precursor and transition picking as well as automated picking using in-built filters (MacLean et al. 2010).

Data were collected and concentrations were calculated by importing the data into Skyline- daily (version 19.1.1.309). Identification and quantification of osteocalcin peptides was carried out using the calibration feature of the software package.

102

Transition Ions

Peak RT

Peak Transitions

Figure 4.3: Annotated example MRM chromatogram in Skyline depicting the peak RT, Intensity, transition ions and peak transitions of an osteocalcin peptide standard.

In Skyline, CV% for peak area of peptides (Table 4.1) and retention time (RT) across replicates was monitored. DDA data acquired for plasma samples were subjected to ProteinPilot (ProteinPilot™ Software 5.0 Revision Number: 4769, SCIEX) analysis.

Skyline analysis of BSA sample displayed extracted ion chromatogram, RT and peak area CV for several BSA peptides (Figure 4.4 and Figure 4.5). Overlaid extracted ion chromatograms for all BSA peptides were used to demonstrate the digestion efficiency and RT, and peak area CV were used to demonstrate reproducibility of sample processing. The peak area is the integration of the mass per unit volume (concentration) of the eluted peptide. Peak area CV for peptides indicated accuracy and robustness of the whole sample preparation. The black line on the peak area CV graph indicates 20% CV cut-off. Skyline profile for FASP digestion technique was found similarly acceptable for both desalting methods since peak area CV was less than 20% for most of the peptides and chromatogram and RT pattern were also similar for each replicate.

103 Each of these Skyline document templates includes a library to facilitate targeted data extraction. Before importing experimental SWATH-MS, technical replicates of PBQC SWATH files were imported (PBQC was the mixture of all experimental sample in a single vial and this sample run on LC-MS/MS for four times). After importing the PBQC files, all detected peaks were refined using Skyline tools and peptides under 20% CV were only considered for further quantification of experimental files. This somewhat overly stringent cut off was applied to ensure statistical significance of the result in the case of limited number of biological replicates.

Skyline was also used to assess the digestion reproducibility of asp-n and trypsin digested equine plasma by comparing equine albumin. Separate templates were set-up for asp-n and trypsin generated equine albumin peptides to allow SWATH import. Background proteome: Horse; .DAT files from mascot searches imported as the spectral library.

4.13.2 Peakview Peakview software was used for an initial exam of consistency and quality of collected data. The summed peptide signals (total ion chromatograms, TIC) of samples as displayed in this software.

4.13.3 Protein Pilot MS/MS spectra were searched against the equine database using ProteinPilot (ProteinPilot™ Software 5.0 Revision Number: 4769, SCIEX) search engine. This software evaluated an amino acid (AA) sequence of the fragmented peptides during the database search. Once sequences of all fragmented peptides have been determined, the protein sequence were built additional computational analysis after applying statistical filtering where all proteins were identified at 1% FDR. The software combined the Paragon™ Algorithm for in-depth sample interrogation the Pro Group™ Algorithm for confident protein assignment. This was also used to annotate the MS/MS spectra from the imported osteocalcin database.

The following settings were used for the searches: Sample type: Identification; Cys Alkylation: Iodoacetamide; Digestion: Trypsin/Asp-N; Instrument: TripleTOF5600; Special Factors: None; Search effort: Thorough ID; ID Focus: Amino acid substitution; Results Quality: Unused ProtScore ≥ 0.05 with 10% false discovery rate (FDR). The instrument .wiff files were individually analysed by the software. ProteinPilot™ then automatically generated a .group, .mgf output file for each and excel report.

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4.13.4 Mascot The group file data in ProteinPilot™ was exported as calibrated generic format (.mgf) to Mascot search engine (Matrix Science, London, UK; version 2.5.1). Mascot was set-up to search the same custom database that was used in ProteinPilot with the following search parameters; Type of search: MS/MS Ion Search; Enzyme: Trypsin; Fixed modifications: 0; Variable modifications: Carbadmiomethyl (C), Carboxy (E); Mass values: Monoisotopic; Protein mass: Unrestricted; Peptide mass tolerance: ± 20ppm; Fragment mass tolerance: ± 20 Da; Max missed cleavages: 0; Instrument type: ESI-QUAD-TOF. Identification search was manually inspected and Peak list data from the search was exported in a DAT format for further processing in Skyline.

Search parameters used for the full-length ucOC and cOC standards: Variable modification: carbamidomethylation (C), carboxy (E), Label: 13C(5) (P), Oxidation (P); Enzyme: Asp-n and/or trypsin; 2 missed cleavages; Peptide tolerance ± 50ppm; Fragment mass tolerance ± 0.02 Daltons; Peptide charge: 2+, 3+ and 4+; Monoisotopic mass values; top 100 hits reported.

The following parameters were used in Mascot to search the peptides;

Search parameters for osteocalcin peptide standards: Variable modification: carbamidomethylation (C), carboxy (E), oxidation (P), Cation: Ca[II] (DE) ; Enzyme: Asp-n and/or trypsin; 2 missed cleavages; Peptide mass tolerance ± 100ppm; Fragment mass tolerance ± 0.02 Daltons; Peptide charge: 2+, 3+ and 4+; Protein mass: unrestricted; Monoisotopic mass values; top 100 hits reported.

Peptide and protein lists in the excel file were manually curated by applying the followig criteria; removal of contaminants; such as keratins, immunoglobulins were omitted, 1% FDR cut-off (by applying the Global FDR recorded in the report), removal of proteins with a unique score < 2

4.14 Statistical analysis of the processed data Protein lists from DDA experiments were presented in spreadsheet and charts (Microsoft® Excel™ 2010, Microsoft Corporation) and then exported for visual analysis to BioVenn software version 2007-2017.

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The proteins identified by Mascot searches were subjected to gene ontology (GO) analysis using Protein Analysis Through Evolutionary Relationships (PANTHER) classification tool. The gene entries were analysed by aligning them to Equus Caballus. The results of this analysis were displayed in Excel™ charts.

STRING v10.5 is a database of known and predicted protein-protein interactions. The interactions include direct (physical) and indirect (functional) associations; they stem from computational prediction and from interactions aggregated from other (primary) databases. In this experiment, this tool used to see the interaction between the proteins which showed significant expression and distinguish the biological process of those proteins.

Statistical analysis was conducted using Statistica ™ for windows with R integration (version 13.2, StatSoftInc Pty. Ltd., Tulsa, OK, USA). Heat maps for peptide identification were generated. The peptide concentrations were normalised against the different fractions and a z- score reported for each.

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Table 4.1: Calculated CV of individual QC-BSA peptides following in-solution digestion protocol.

Test Run CV% Date (non KQTALVEL LGEYGFQN HLVDEPQN HPYFYAPELL LSQKFPK AEFVEVTK LVTDLTK YIYEIAR QTALVELLK LVNELTEFAK ATEEQLK DAFLGSFLYEYSR DLGEEHFK TVMENFVAFVDK alkylated) LK ALIVR LIK YYANK 26-Oct > 100% <20% <20% <20% <20% <20% <20% <20% >100% <20% <20% >100% <20% <20% 1-Nov <40% <20% <20% <20% <30% <30% <20% <20% <30% <20% <30% <70% <80% <40% 2-Nov <20% <20% <20% <20% <20% <20% <20% <20% <30% <20% <20% <30% <20% <30% 7-Nov <40% <20% <20% <20% <20% <20% <20% <20% >100% <30% <20% <30% <20% <20% 9-Nov <30% <20% <20% <20% <30% <20% <20% <20% <30% <20% <80% <20% <20% <20% 14-Nov <20% <30% <30% <30% <40% <60% <20% <20% <70% <20% <90% <30% <50% <20% 16-Nov <20% <20% <20% <20% <20% <20% <20% <20% <20% <20% <20% <20% <20% <20% 21-Nov <50% <20% <20% <20% <20% <20% <20% <20% <50% <30% <20% <20% <20% <20%

(alkylated) YICDNQDTISSK QNCDQFEK LCVLHEK RPCFSALTPDETYVPK SLHTLFGDELCK DDPHACYSTVFDK ETYGDMADCCEK EYEATLEECCAK GLVLIAFSQYLQQCPF MPCTEDYLSLILNR 26-Oct <30% < 20% <50% <30% <50% <70% <30% <30% <30% <30% 1-Nov < 30% <20% <40% < 30% < 40% < 100% < 80% > 100% < 40% < 70% 2-Nov <20% <20% <20% <20% <20% <30% <20% <30% <20% <20% 7-Nov <20% <50% < 30% <20% <20% <20% <20% <20% <20% <20% 9-Nov < 30% < 30% <50% <20% <50% 80% <70% <80% <20% <20% 14-Nov < 30% < 30% <20% < 30% <20% <30% <20% <20% <20% <20% 16-Nov <20% <20% <20% <20% <20% <30% <20% <30% <20% <20% 21-Nov <20% <60% <20% <20% <20% <20% <20% <20% <20% <20%

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Figure 4.4: Reproducibility of QC-BSA in-solution digestion, desalting with SCX membrane. The black line displays the 20% CV threshold for the peptides with most peptides below the cut-off.

Figure 4.5: Reproducibility of QC-BSA FASP digestion, desalting with SCX membrane. The black line displays the 20% CV threshold for the peptides with all peptides below the cut-off.

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109 CHAPTER 5 Osteocalcin Peptide Assay Method Development 5.1 Introduction ...... 110 5.2 Materials and Methods ...... 113 5.2.1 Animal Samples ...... 113 5.2.2 Solvents and reagents ...... 113 5.2.3 Osteocalcin standards ...... 113 5.2.4 Experimental design ...... 114 5.2.5 Instrumentation and data acquisition ...... 116 5.2.6 Data Processing ...... 122 5.3 Results ...... 123 5.3.1 Equine osteocalcin synthesis ...... 123 5.3.2 OC characterisation ...... 130 5.3.3 Detection of carboxylation ...... 130 5.3.4 MRM method development ...... 133 5.3.5 Offline liquid chromatography inductively coupled plasma mass spectrometry ...... 143 5.3.6 Laser-ablation inductively coupled plasma mass spectrometry (LA- ICP-MS) ...... 144 5.3.7 Glu/Gla amino acid initial screening results...... 145 5.4 Discussion ...... 146 5.5 Conclusion ...... 151

5.1 Introduction Osteocalcin is a non-collagenous polypeptide (49 amino acid long side chain protein, molecular weight (MW) 5.8kDa). Its primary function is to bind calcium (Ca) and that is dependent on the number of γ-carboxyglutamic acid (Gla) residues in its sequence (Hauschka 1986). When fully carboxylated with three glutamate (Glu) residues converted to Gla residues, osteocalcin is bound to bone hydroxyapatite (HAP) (Figure 5.1) (Hauschka & Wians 1989). The partially carboxylated (two or one Glu residue converted to Gla residue) or undercarboxylated (ucOC) form (no Gla residues presence in the sequence) of osteocalcin, is believed to circulate in plasma (Ivaska et al. 2004). Due to its vitamin-K-dependent biosynthesis, it serves as an indicator of osteoblastic vitamin-K status, thus osteocalcin is a useful marker of bone formation (Lian et al. 1978).

110 Figure 5.1: Schematic diagram of the vitamin-k-dependent post-translational modification of glutumate (Glu) to γ-carboxyglutamic acid (Gla) in osteocalcin.

Emerging evidence for novel and distinct roles of osteocalcin in health and disease necessitates sensitive and selective methods for quantifying osteocalcin in the plasma matrix. Plasma osteocalcin concentrations are currently only routinely monitored using immunoassays (Ferron et al. 2010b; Cleland et al. 2016). Immunoassays available for osteocalcin include; Immuno- radiometric assay (IRMA), enzyme-immunoassays (EIA and ELISA), and Enzyme Amplified Sensitivity Immunoassays (ELISA) using either monoclonal or polyclonal antibodies. The protein sequence of osteocalcin is highly conserved between species, with bovine osteocalcin sharing 90% homology with human osteocalcin. Despite the homologous sequence, minor species differences in the N-terminal region of the protein mean that antibodies raised against bovine osteocalcin may react differently with that of osteocalcin from other species (Hoang et al. 2003). This yields artefacts (Pesce & Michael 1992) and has contributed to contradictions in circulating plasma concentrations of osteocalcin.

Concentrations of osteocalcin in equine plasma are low and very variable, ranging from 3.68 – 127.31ng/ml (Lepage et al. 1997). The variability is attributable to a number of factors including age, gender and breed (Mäenpää et al. 1988; Grafenau et al. 2000; Carstanjen et al. 2002; Pastoret et al. 2007; Greiner et al. 2012). Discrepancy in results however, may be due to the lack of a reliable method to measure osteocalcin in blood (Power et al. 1991). Furthermore immunoassays can be limited by their availability and high failure rates (Hoofnagle & Wener 2009; Beck & Lock 2015). An attractive alternative to immunoassays is the development of targeted MS-based approaches (Hoofnagle & Wener 2009).

111 The use of Mass Spectrometry (MS) to study osteocalcin Mass spectrometry(MS)-based assays have the advantage of specificity and high throughput analysis, but these assays, especially for animals are still limited (Angel et al. 2012). The difficulty in developing these assays, lays in the lack of relevant standards and, in the case of incompletely sequenced organisms; protein sequence information (Vincent et al. 2009). Gla

resides undergo neutral loss of carbon dioxide (CO2) from the γ-carboxy carbon during collision-induced dissociation (CID). This renders it undetectable (Hallgren et al. 2013) and hence analysis of Gla containing proteins such as osteocalcin has so far alluded MS (Cleland et al. 2016). MRM/SRM is a targeted approach that is commonly used for protein quantification in human medicine and traditionally has been used for small molecule analysis (Camerini & Mauri 2015). It is also used for verification of results from discovery (shotgun) proteomic analysis (Camerini & Mauri 2015). Discovery proteomics is acquired in data-dependent acquisition (DDA) and can detect hundreds to thousands of proteins in biological samples (Aebersold & Mann 2016). It is however, biased towards the most abundant precursor ions and therefore not sensitive enough to detect osteocalcin in plasma without extensive fractionation or prior depletion of plasma. SWATH-MS is a data-independent acquisition (DIA) method. It fragments all peptides that enter the MS within a predetermined mass window regardless of abundance (Aebersold & Mann 2016). It does however, rely on well curated spectral libraries acquired by DDA. If the target peptide is not present in the spectral library it will not be detected in SWATH-MS (Aebersold & Mann 2016).

Figure 5.2: The generic experimental workflow depicting: sample preparation of proteins, mass-spectrometry detection, identification and analysis.

112 The aim of this study was therefore to exploit the recent advances in the use of MS, and develop an assay capable of measuring the carboxylation status of circulating osteocalcin. As with any new method development a lengthy exploration phase must be undertaken to determine feasibility, suitability and sensitivity of the method. Figure 5.2 illustrates the method development workflow, processes involved and general techniques investigated in this chapter.

5.2 Materials and Methods

5.2.1 Animal Samples The equine blood plasma samples used in this Chapter were collected from mares and foals in Chapter 3. The samples were stored at -80°C till analysed at the Proteomics and Small Molecule Mass Spectrometry Laboratory at CARF, QUT.

5.2.2 Solvents and reagents The LC/MS grade chemicals and solvents used throughout method development were purchased from Sigma Aldrich Co. LLC (Sydney NSW, Australia) and Thermo Scientific (VIC, Australia) unless otherwise stated. Asp-n endproteinase was purchased from Roche® and prepared in 50mM AMBIC at a concentration of 0.01µg/µ1. Refer to Chapter 4, section 4.3 for a detailed description of further solvents and reagents.

5.2.3 Osteocalcin standards Initial method development was started with the full-length protein. The carboxylated (cOC) and undercarboxylated (ucOC) full-length sequences of equine osteocalcin were synthesised. The standards were heavy proline labelled (hydroxyproline modified residues).

Osteocalcin peptide standards were synthesised and purchased from Mimtopes Pty. Ltd. (Melbourne, Australia). Based on in-silico prediction and BLAST search results, 11 candidate peptides of predicted asp-n and trypsin digestion were synthesised (Table 5.1). The peptides were synthesised by an automated synthesiser using the mild Fmoc chemistry method. The exact masses of the molecular ions recorded on the certificates of analysis were compared against the predicted masses in the Skyline peptide template (see Table 5.1). Carbamidomethylation accounts for some of the inconsistency observed between the masses. This occurs when cysteine residues react with IAM.

113 Table 5.1: Peptide sequences and their respective information for synthesis of peptide standards by Mimtopes Pty Ltd. (Melbourne, Australia). The precursor ion masses extracted from Skyline were compared to the theoretical molecular weight.

Theoretical Carboxylated/ Molecular Skyline Enzyme Peptide modified sequence Weight (daltons) Precursor Uncarboxylated (m+h)

Trypsin YLDHWLGAPAPYPDPLEPR Uncarboxylated 2223.48 2223.09

Trypsin EVCELNPDCDELADHIGFQEAYR Uncarboxylated 2666.88 2780.19

Trypsin EVCELNPDCDELADHIGFQEAYR Carboxylated 2710.89 2824.18

Trypsin EVCELNPDCDELADHIGFQEAYR Carboxylated 2754.90 2868.17

Asp-N YLDHWLGAPAPYP Uncarboxylated 1515.68 1515.73

Asp-N DHWLGAPAPYP Uncarboxylated 1239.35 1239.58

Asp-N DPLEPRREVCELNP Uncarboxylated 1666.88 1723.84

Asp-N DPLEPRREVCELNP Carboxylated 1710.89 1767.83

Asp-N DPLEPRREVCELNP Carboxylated 1754.90 1811.82

Asp-N DPLEPRREVCELNP Carboxylated 1798.91 1855.81

Asp-N DHIGFQEAYRRFYGPV Uncarboxylated 1955.17 _

5.2.4 Experimental design

5.2.4.1 Equine plasma sample preparation For method development, a select number of equine plasma samples were used. Replicates of plasma were asp-n (n = 5) and trypsin (n = 5) in-solution digested (SCX desalted) to compare the reproducibility and efficiency of digestion between the two enzymes. For this experiment 20µg of foal plasma was in-solution digested as per the protocol outlined in Chapter 4, section 4.9. The samples were then dried and re-suspended in 20µl of iRT buffer before being submitted to ABSciex for DDA and SWATH analysis. Digestion reproducibility was then assess by comparing the CV% of equine albumin peptides using the equine albumin LC-MS

114 multiple reaction monitoring (MRM) method developed between the replicates for both asp-n and trypsin individually.

NB: As with any method development a lengthy phase must be undertaken to determine what the most feasible approach is. It was outside the scope of this thesis to detail every equine plasma sample used during this process. It must be noted that over the duration of this PhD numerous mare and foal plasma samples were digested using a number of different methods including iST (in-stage tips), in-solution digestion and FASP digestion for screening and method development optimisation procedures.

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5.2.4.2 Preparation of osteocalcin synthetic standards

Full-length osteocalcin standards (cOC and ucOC) MRM development was initially tested on the full-length synthetic standards (both with and without alkylation). The standards were prepared in 10% acetonitrile/0.1% formic acid at a concentration of 1pmol/µl. The standards were also simultaneously FASP digested with trypsin.

Osteocalcin peptide standards The amount of pure peptide was calculated for each and solubilised in 50% acetonitrile/0.1% formic acid at a concentration of 1pmol/µl. All peptides containing cysteine were then reduced and alkylated (Chapter 4, Section 4.7). For construction of the calibration curve, samples were prepared as a serial dilution of osteocalcin peptide stock: 0ppb, 3.9ppb, 7.8ppb, 15.6ppb, 31.1ppb, 62.5ppb, 125ppb, 250ppb, 500ppb, and 1000ppb. The calibration curve samples were prepared in triplicate and resuspended in an appropriate amount of 10% acetonitrile/0.1% formic acid.

5.2.5 Instrumentation and data acquisition

5.2.5.1 nanoLC MS/MS analysis Peptide spectral data was generated using nanoLC-MS/MS on an ABSciex Triple TOF® 5600+ instrument. Refer to Chapter 4, Section 4.11 for details.

Data-dependent acquisition (DDA) The DDA acquisition mode of the instrument was set to obtain high-resolution TOF-MS scans (30,000). Osteocalcin peptide standards were analysed using 95 minute DDA. Refer to Chapter 4, Section 4.12 for details.

Data-independent acquisition (DIA) Data-intendent acquisition (DIA) was performed using a SWATH-MS™ acquisition method; refer to Chapter 4, Section 4.12. While SWATH-MS is not as sensitive as MRM it can collect thousands of proteins and therefore be used to create a spectral library for osteocalcin.

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5.2.5.2 LC-MS TQ-8050

Multiple reaction monitoring (MRM) The MRM platform was used to investigate the development of an MRM assay targeting full length equine-specific ucOC and cOC and the development of an MRM assay targeting equine- specific peptides derived from enzyme digested ucOC and cOC. Refer to Chapter 4, Section 4.11 for instrument method details.

Table 5.2: The MRM was developed manually and the following precursor, product ions, dwell time and collision energy were selected and optimised for each peptide.

Peptide modified sequence Precursor Product m/z Dwell Time Collision m/z (msec) Energy (CE) YLDHWLGAPAPYPDPLEPR 741.7000 823.4500, 611.3500 21.0 -26.6

EVCELNPDCDELADHIGFQEAYR 927.4000 1120.5500, 870.4000 21.0 -33.5

EVCELNPDCDELADHIGFQEAYR 942.0500 1120.5500, 870.4000 21.0 -34.0

EVCELNPDCDELADHIGFQEAYR 956.7500 1120.5500, 870.4000 21.0 -34.6

YLDHWLGAPAPYP 758.3500 956.4500, 1140.5500 21.0 -29.8

DHWLGAPAPYP 620.3000 864.4000, 680.3000 21.0 -24.3

DPLEPRREVCELNP 575.3000 804.9000, 536.9500 21.0 -20.4

DPLEPRREVCELNP 589.9500 826.900, 804.900 21.0 -21.0

DPLEPRREVCELNP 604.6000 826.9000, 804.9000 21.0 -21.5

DPLEPRREVCELNP 619.3000 826.9000, 804.9000 21.0 -22.1

DHIGFQEAYRRFYGPV - - - -

* DHIGFQEAYRRFYGPV was discounted from the MRM method development as it was unable to be detected

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5.2.5.3 Alternative method development approaches A number of other different technologies and methodologies were evaluated. These were: ICP- MS, LA-ICP-MS and amino acid analysis. It was however, outside the scope of this thesis to pursue these approaches further due to the extra time and funding that was required. Preliminary data collected however, and methods used is detailed below, to emphasise the scope of the exploratory phase undertaken during this PhD.

Inductively coupled plasma mass spectrometry (ICP-MS) Offline LC-ICP-MS was tested to ascertain the feasibility of developing an assay to assess the carboxylation status of osteocalcin, via detection of calcium (Ca) bound peptides. The developed MRM osteocalcin peptide method was used to collect offline fractions of each of the synthetic osteocalcin peptides (Figure 5.3). These were then submitted for ICP-MS analysis of Ca and Sulphur (S) (for comparative purposes to determine the ratio of Ca to S). The fractions collected are shown below as well as their expected results and calibration curves (Table 5.3 and Figure 5.4).

• Spotting on aluminium plate

DPLEPRREVCELNP - 3 Ca, 1 S

Figure 5.3: Delineation of how the fractions were manually collected. The dotted lines are the time point across the run in which the fractions were collected. The black arrow points to the peak on the chromatogram that depicts the DPLEPRREVCELNP peptide (retention time of 1.65mins and fragmentation of m/z 657) that contains three carboxylation sites [and therefore three calcium binding sites (3 Ca) and one suspected site of sulphur binding (1 S)]. A peptide assay was conducted on each of the peptides and 100ppb of each was run on the LC- MS to confirm their presence. Individual peptides were then run at the same volume and ‘pure’ HPLC fractions were then collected (to minimise Ca contamination).

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Table 5.3: Peptide fractions collected from HPLC and submitted for ICP-MS analysis. Their expected results for the number of bound calcium (Ca) and sulphur (S) ions are shown for each peptide.

Peptide modified sequence Expected results YLDHWLGAPAPYPDPLEPR 0 Ca, 0 S YLDHWLGAPAPYP 0 Ca, 0 S DHWLGAPAPYP 0 Ca, 0 S DPLEPRREVCELNP 0 Ca, 1 S EVCELNPDCDELADHIGFQEAYR 0 Ca, 2 S EVCELNPDCDELADHIGFQEAYR 1 Ca, 2 S EVCELNPDCDELADHIGFQEAYR 2 Ca, 2 S DPLEPRREVCELNP 1 Ca, 1 S DPLEPRREVCELNP 2 Ca, 1 S DPLEPRREVCELNP 3 Ca, 1 S

Figure 5.4: Calibration curve of S and Ca optimised in 02/H2 (left to right) mode respectively. Standards were within the linear range for both sulphur (S) and calcium (Ca) (S, r2 = 0.9971 and Ca, r2 = 0.9982). Detection is calibration curve dependent and is reported as counts per second % (CPS).

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Laser-ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) This technique uses a laser to scan and detect elements of interest. As a proof-of-concept to test this method, CaCl2 and cysteine samples were prepared in solution (ACN/FA). CaCl2 was mixed in equimolar proportions with cysteine (1:1) and at a 3:1 ratio, as well as individual

serial dilutions of CaCl2 and cysteine (4.5-1mM). The fractions were then spotted onto a plate and dried down (Figure 5.5). Aluminium (Al) was tested as a background matrix on one of the plates. The plates were then submitted for Ca and S analysis.

Figure 5.5: Aluminium covered plate and wells (left) and plate without aluminium (right). The samples were spotted onto each plate and then dried down before analysis.

Amino acid analysis of Glu/Gla ratio by alkaline hydrolysis The aim of this method was to hydrolyse the plasma sample and determine the global Gla/Glu ratio of the sample. This method will not ascertain specific carboxylation concentrations but it may provide a global picture of the effect of vitamin K supplementation on carboxylation via analysis of the Gla residue in comparison to Glu.

Alkaline hydrolysis was first performed on BSA with (positive control) and without (negative control) spiked Gla residue. The conditions used were modified from the protocols of Price et al. (1976). The samples were prepared in 5M NaOH and were heated to 110°C for 24 hours. After cooling, 2.5ml of 98% formic acid was added and the pH adjusted to 3.5.

A previously developed Glu/Gla amino acid MRM method was used to analyse the hydrolysed samples. As observed in Figure 5.6, the amino acids eluted early in the void volume, indicating that resolution of these amino acids could be further optimised in future experiments.

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Figure 5.6: Glutumate and γ-carboxyglutate (Glu/Gla) amino acid MRM method. As depicted both the Glu (green trace; [Glu+H]+ 147.9000[M+H]) and Gla (pink trace; [Gla+H]+ 192.0503[M+H]) synthetic standards eluted close to each other, and early during the chromatographic run (within 0.4-0.54 mins). The Mobile phases consisted of; A: water and 0.1% formic acid; B: acetonitrile and 0.1% formic acid. The column used was: Kinetex EVO.

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5.2.6 Data Processing

5.2.6.1 Database To generate a theoretical spectral library for osteocalcin a custom database was assembled from UniprotKB (Universal Protein Resource Knowledgebase – http://www.uniprot.org/) and the equine osteocalcin sequence was downloaded in FASTA format on the 5th of April 2017. The database was assembled using the osteocalcin sequence and added contaminants from the common Repository of Adventitious Proteins (cRAP), (http://www.thegpm.org/crap/).

5.2.6.2 Peakview The MS/MS spectra collected from DDA and SWATH was visually appraised for reproducibility by assessing the total ion chromatogram (TIC) traces for replicates in PeakView® (ver. 2.2) software (SCIEX).

5.2.6.3 Protein Pilot ProteinPilot™ was used to analysis the acquired DDA data (Refer to Chapter 4, Section 4.13.3 for search parameters).

5.2.6.4 Mascot The .mgf file output from ProteinPilot™ search was uploaded to the Mascot search engine to conduct an MS/MS ion search. Mascot was used, as unlike ProteinPilot™, it has the specific capability to select carboxylation as a modification to be searched. Mascot search results returned a protein/peptide list and .DAT files. These .DAT files were utilised to create a library in Skyline.

5.2.6.5 Skyline The data was imported into Skyline-daily (ver. 19.1.1.309) as a library. DDA and SWATH was collected on the full-length, digested osteocalcin standards and this was compared in Skyline to the results from the Mascot reports. This was to confirm if peptides of interest were being detected, in what samples, and where. For the osteocalcin peptide standards, precursor ion masses were extracted from the DDA data for each peptide and the most dominate charged state was chosen. The SWATH data was then imported into Skyline to see if it matched the DDA. This skyline template was used as the basis for MRM method development.

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5.3 Results

5.3.1 Equine osteocalcin synthesis The development of assays targeting both full–length and peptide MRM were investigated. The MRM platform by default was optimised to target equine-specific ucOC standards for uc0C and cOC, therefore, development was initiated on the full-length protein. While the full-length ucOC standard could be detected, there was some inherent problems. Peptide MRM is renowned for being more sensitive, reproducible and stable, therefore although full-length protein analysis was attempted, it was decided to develop a peptide MRM assay.

5.3.1.1 Full-length osteocalcin synthetic standards (ucOC and cOC) The standards were synthesised with heavy-isotope labels and reagent characterisation was conducted on the discovery MS platform (Figure 5.7). Reagent characterisation included polypeptide stability (exposure to several freeze/thaw cycles) as fragments were reported in the literature, unexpected modifications and trial trypsin digestion.

Figure 5.7: Synthesised heavy labelled cOC sequence. The proline residues were heavy labelled to differentiate the standard from the native protein. The sequence differs in the literature with Ostrom et al. (2006) reporting the last residues as Threonine (T) and alanine (A). NH2 depicts the N-terminal of the protein and COOH the C-terminal. The sites of possible carboxylation in the sequence are numbered and shown by the red glutamate residues (E).

The characterisation of the standards is crucial, as it is vital to ensure synthesis has been carried out correctly. The ucOC tryptic peptide and full-length ucOC standards were run on the MRM and the full-length ucOC standard was identified.

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5.3.1.2 MRM method set–up to target total osteocalcin (tOC) peptides The LCMS/MS MRM method was then set up to target those peptides to be used to monitor tOC that were identified via the MASCOT® software search; DHWLGAPAPYP. The MASCOT® peptide data generated for the ucOC asp–n digested standard sample and the three most abundant transitions for each peptide were scrutinised, identified and selected to increase the sensitivity of the method. The instrument method was exported from Skyline daily into the lab solutions software. The ucOC asp–n standard sample was submitted and run using the method developed to target the tOC peptides. As depicted in Figure 5.8 the peptides were successfully extracted and identified in the standard sample. Once extracted, the most abundant transition states were identified. The extracted peptides were identified to be very hydrophobic, eluting late in the acetonitrile wash. The plasma digested samples were then submitted and run however, the peptides were not successfully extracted.

Figure 5.8: ucOC full-length synthetic standard, digested with asp-n (tOC peptide; DHWLGAPAPYP). As depicted in the key above (A) each of the coloured traces depicts a transition of the synthetic standard (it also shows the ion and their mass (m/z) and charged state). The chromatogram (B) depicts the retention time of the protein standard and the intensity of each of the transitions shown in the key. The black arrow points to the retention time (9.0 mins) and intensity of the highest transition (b8 – 956.4625+ (heavy)).

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5.3.1.3 Enzyme selection and in silico prediction of synthetic peptides It was established that while trypsin is the best enzyme for proteomics it may not generate the most ideal fragments for osteocalcin analysis. A thorough search of the literature revealed that asp-n may be a more suitable enzyme then trypsin for osteocalcin based on its predicted peptide fragments as observed in Figure 5.9. A NCBI BLAST® search revealed that this may also be more suitable for developing an assay targeted for multiple species, not just the horse (Ostrom et al. 2006). Asp-n was purchased (Roche®) and the standards were digested using in-solution digestion protocol and submitted for SWATH analysis.

Osteocalcin sequence retrieved from NCBI database was imported into Skyline and it was used to predict trypsin and asp-n cleavage sites. A BLAST® search was performed to confirm homology of predicted sequences across numerous species (Altschul et al. 1990).

Figure 5.9: Synthesised cOC sequence with the predicted cleave sites of the asp-n endoprotease annotated in black and blue. The black annotation depicts a peptide that encompasses all the carboxylation sites in the sequence.

The MS/MS spectra was then submitted to MASCOT® software and several peptides of interest were identified (Figure 5.9). The peptides highlighted may be useful for monitoring osteocalcin in samples as it contains the sites of γ–carboxylation in the protein sequence.

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5.3.1.4 Optimisation of asp-n and trypsin digestion in equine plasma Digestion optimisation was necessary before conducting further experiments to ensure reproducible sample preparation.

A B

Intensity Intensity Intensity Intensity

Time Time

Figure 5.10: Total ion chromatogram (TIC) traces of trypsin digested equine plasma samples (A) and asp-n digested equine plasma samples (B). The traces for each enzyme exhibit good reproducibility between the samples (shown as the different coloured traces overlapping) (n=5). However, the gauchan curve of the asp-n digested equine plasma samples (B) does not follow the same pattern as (A). This likely attributable to the different peptides generated by asp-n as opposed to trypsin. The general reproducibility of the SWATH samples was observed in PeakView by overlaying extracted ion chromatograms with both sets exhibiting the characteristic gauchan curve. As can be observed (Figure 5.10) the total ion chromatogram (TIC), traces of each of the samples follow the same gauchan curve therefore sample preparation could be deemed reproducible.

Skyline analysis of the extracted ion chromatograms for asp-n digested equine albumin and trypsin digested equine albumin was used to further assess digestion reproducibility. Peak area CV was used to demonstrate the reproducibility of sample preparation with a CV < 20% considered optimal. Peak area can be defined as the concentration of the eluted peptide. The black line on the peak area CV graph demarks a CV of ≤ 20%. Sample preparation was deemed reproducible for both trypsin in-solution digested equine albumin and asp-n in-solution digested equine albumin (Figure 5.11 and Figure 5.13) with most peptides below the 20% CV

126 threshold. The concentration of each peptide across the replicates can also be observed in Figures 5.12 and 5.14, with the concentration of most of the peptides being relatively uniform between the samples.

C D

Figure 5.11: Reproducibility of in-solution trypsin digestion of equine albumin, desalted with SCX membrane. (A) Depicts a panel of chromatograms (5 replicates) showing the intensity and retention time of each of the equine albumin trypsin peptides, listed in the panel (B). (C) The retention time of each of the peptides across the replicates. (D) The peak area CV (%) of each of the peptides. The black line indicates the 20% cut-off CV, with most of the peptides below this.

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Figure 5.12: Heatmap showing the reproducibility of the trypsin digested equine albumin peptides between the replicates. Colour key represents the difference in concentration (intensities) of each of the peptides across the sample replicates. Dark blue = peptides of higher concentration, and dark red = peptides of lower concentration. A uniform colour across the replicates indicates the same peptide concentration.

C D

Figure 5.13: Reproducibility of in-solution asp-n digestion of equine albumin, desalted with SCX membrane. (A) Depicts a panel of chromatograms (5 replicates) showing the intensity and retention time of each of the equine albumin asp-n peptides, listed in the panel (B). (C) The retention time of each of the peptides across the replicates. (D) The peak area CV (%) of each of the peptides. The black line indicates the 20% cut-off CV, with most of the peptides above this.

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Figure 5.14: Heatmap showing the reproducibility of the asp-n digested equine albumin peptides between the replicates. Colour key represents the difference in concentration (intensities) of each of the peptides across the sample replicates. Dark blue = peptides of higher concentration, and dark red = peptides of lower concentration. A uniform colour across the replicates indicates the same peptide concentration. It is evident that most of the peptides are of low concentration (red) with one peptide (DEKLFTFHA) dominating in concentration.

To assess digestion efficiency the samples were run through a digestion efficiency calculator (m/z density histogram). The figures below depict the target peptide fragments over the calculated windows. The blue line represents the proteome of interest and the red line represents the isolation window width. Figure 5.15, depicts a normal m/z distribution and isolation window width. As can be observed below, trypsin digestion was more efficient in comparison to asp-n.

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A B C

Figure 5.15: (A) This plot depicts the default digestion efficiency plot. The smooth blue line represents the peptides of interest and optimal digestion efficiency and the red line represents the isolation scheme window width (B) This plot illustrates the digestion efficiency of the trypsin digested equine plasma samples. The blue line suggests that digestion was suboptimal (C) This plot illustrates the digestion efficiency of the asp-n digested equine plasma samples. Digestion was also deemed suboptimal for asp-n.

5.3.2 Osteocalcin characterisation DDA data was collected for the synthetic peptides (ucOC and cOC trypsin and asp-n enzymatically cleaved peptides). This data acted as the spectral library in Skyline for SWATH data analysis. Firstly, the .wiff files were searched in Protein pilot software and then the .mgf MS/MS spectra was submitted to Mascot as this search engine allows for carboxylation detection.

Once the Mascot searches were completed the data was then saved as .DAT files and combined to make a spectral library in Skyline. This library was then used to search the acquired DDA data for the peptides. Precursor ion masses were extracted for each peptide and the most dominate charged state (+++) was chosen for each peptide.

5.3.3 Detection of carboxylation Full-scan MS in positive and negative ion mode was then conducted to see if any calcium (Ca) bound precursors could be observed (Figures 5.16 to 5.19). 100mM CaCl2/0.05% FA was added to each peptide and the extracted massed were then analysed and compared to that what was observed in Skyline for each peptide; Ca, Al, Mg, Cr and Pb have all been documented in the literature to bind to osteocalcin (Dowd et al. 1994; Dowd et al. 2001a; Nousiainen et al. 2002). Using this low resolution instrument, it was postulated that the masses observed

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corresponded to the following annotated elements. The literature also suggests that osteocalcin has a higher affinity for Al and Pb than for Ca, making the displacement of Ca in-solution, a possible explanation for the observations in Figures 5.16 to 5.19.

A +0 CO2

Figure 5.16: (A) Full-scan MS in positive and negative ion mode of DPLEPRREVCELNP peptide. Shows the positive ion mode scan. Annotated peak without Ca (575) was the most intense with the source conditions used. (B) Shows the negative ion mode.

+1 CO2

Figure 5.17: (A) Full-scan MS in positive and negative ion mode of DPLEPRREVCELNP peptide. Shows the positive ion mode scan. Annotated peak with 1 Ca (590) was the most intense with the source conditions used. (B) Shows the negative ion mode.

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+Mn/2 Cr/+0+(Ca-2H) = 623m/z +Al/Mg +2 CO2 K +2 Ca = 630m/z A

Figure 5.18: (A) Full-scan MS in positive and negative ion mode of DPLEPRREVCELNP peptide. Shows the positive ion mode scan. Annotated peak with 2 Ca (605) was the most intense with the source conditions used. Masses potentially corresponding to other elements have also been annotated; Al/Mg, K, Mn/Cr, Ca. (B) Shows the negative ion mode.

+3 CO2 A

Figure 5.19: (A) Full-scan MS in positive and negative ion mode of DPLEPRREVCELNP peptide. Shows the positive ion mode scan. Annotated peak with 3 Ca (620) was the most intense with the source conditions used. (B) Shows the negative ion mode.

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5.3.4 MRM method development The development of the MRM method used the transition/spectra from the acquired DDA and SWATH data. For each peptide the 5 to 6 most intense product ions were selected. The transition list was exported to the instrument and conditions were further optimised for each peptide. Intensity in Skyline was peptide dependent with the peptide without calcium observed to be the most intense using these source conditions (Figure 5.20).

A product ion scan was also conducted to try and optimise the collision energy conditions; this did work. Method development was achieved, however, by theoretical predictions in Skyline.

A B A

Figure 5.20: (A) Osteocalcin peptide MRM method showing the summed intensity for the optimised transitions and retention time of each of the peptides. (B) Depicts the C02 loss of each of the Gla (Ca) containing peptides (indicative of their reduced signal intensity) (Left to right); DPLEPRREVCELNP (0 carboxylation), DPLEPRREVCELNP (1 carboxylation), DPLEPRREVCELNP (2 carboxylation), DPLEPRREVCELNP (3 carboxylation).

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5.3.4.1 Sensitivity of MRM on synthetic peptides The sensitivity of the developed MRM method was tested both with and without plasma background and in spiked asp-n and trypsin digested equine plasma, separately. Asp-n synthetic osteocalcin peptides consistently yielded greater signal intensity at the equivalent concentration than trypsin digests (Figure 5.21).

A B

9000000 C 8000000 7000000 6000000 5000000 4000000 3000000

Peak Area Peak 2000000 1000000 0

Osteocalcin synthetic peptides

Figure 5.21: Comparison of synthetic osteocalcin trypsin (A) and asp-n (B) digested peptides. (C) Mean ± standard error of the mean (SEM) peak area comparison of synthetic peptide standards. (n = 3). Asp-n peptides (purple bars) show increased intensity of signal at the equivalent concentration compared to tryptic peptides (teal bars). The asp-n peptides with the highest intensities (DPLEPRREVCELNP and DHWLGAPAPYP) show no significant differences compared to the tryptic peptides, despite the high intensity of YLDHWLGAPAPYPDPLEPR. This is due to the higher variation in intensity exhibited between replicates for each of the tryptic peptides.

134 5.3.4.2 Peptide Calibration curves Calibration curves were established to assess the linearity of the osteocalcin synthetic peptides (Figures 5.22 to 5.31). Calibration curves in un-spiked plasma spanning 3.9 – 1000ppb demonstrated good agreement in standard-to-analyte ratios with r2 values ranging from 0.9916 – 0.9981. Each of the synthetic peptides were then spiked into 20ug of asp-n digested equine plasma. The results were calculated on the basis of the standard curve prepared in the same conditions. The limit of detection (LOD) and quantitation (LOQ) were determined by injecting a series of linear dilutions of known concentrations (Table 5.4). A linear response was not observed at all concentrations and endogenous peptides were not detected (Figures 5.22 to 5.31).

Table 5.4: Analytical parameters of LC-ESI-MS/MS quantitative method; data for calibration curves, limit of detection (LOD) and limit of quantification (LOQ) values for each of the synthetic osteocalcin peptides (extracted from 20µg of equine plasma).

Linear range Regression Peptide modified sequence LOD (ppb) LOQ (ppb) (ppb) coefficient (r2)

DPLEPRREVCELNP 31.3 - 250 0.9959 10.43 31.3

DPLEPRREVCELNP 3.9 - 500 0.9983 10.43 31.3

DPLEPRREVCELNP 3.9 - 1000 0.999 41.66 125

EVCELNPDCDELADHIGFQEAYR 62.5 - 500 0.999 20.83 62.5

EVCELNPDCDELADHIGFQEAYR 62.5 - 1000 0.9998 83.33 250

DPLEPRREVCELNP 3.9 - 1000 0.9999 166.66 500

DHWLGAPAPYP 7.8 - 500 0.9998 20.83 62.5

EVCELNPDCDELADHIGFQEAYR 3.9 - 1000 0.9993 20.83 62.5

YLDHWLGAPAPYP 3.9 - 1000 0.9999 1.3 3.9

YLDHWLGAPAPYPDPLEPR 3.9 - 1000 0.9996 5.2 15.6

135 A B

Figure 5.22: DPLEPRREVCELNP – Asp-n peptide (1 Gla carboxylation residue) neat standard serially diluted calibration curve (A) and equine plasma (20µg) matrix spiked calibration curve (B). The equation and r2 for the best line of fit are given for each.

B A

Figure 5.23: DPLEPRREVCELNP– Asp-n peptide (2 Gla carboxylation residues) neat standard serially diluted calibration curve (A) and equine plasma (20µg) matrix spiked calibration curve (B). The equation and r2 for the best line of fit are given for each.

136 A B

Figure 5.24: DPLEPRREVCELNP– Asp-n peptide (3 Gla carboxylation residues) neat standard serially diluted calibration curve (A) and equine plasma (20µg) matrix spiked calibration curve (B). The equation and r2 for the best line of fit are given for each.

B A

Figure 5.25: EVCELNPDCDELADHIGFQEAYR – Trypsin peptide (1 Gla carboxylation residue) neat standard serially diluted calibration curve (A) and equine plasma (20µg) matrix spiked calibration curve (B). The equation and r2 for the best line of fit are given for each.

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B A

Figure 5.26: EVCELNPDCDELADHIGFQEAYR– Trypsin peptide (2 Gla carboxylation residues) neat standard serially diluted calibration curve (A) and equine plasma (20µg) matrix spiked calibration curve (B). The equation and r2 for the best line of fit are given for each.

A B

Figure 5.27: DPLEPRREVCELNP neat standard serially diluted calibration curve (A) and equine plasma (20µg) matrix spiked calibration curve (B). The equation and r2 for the best line of fit are given for each.

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B A

Figure 5.28: DHWLGAPAPYP neat standard serially diluted calibration curve (A) and equine plasma (20µg) matrix spiked calibration curve (B). The equation and r2 for the best line of fit are given for each.

B A

Figure 5.29: EVCELNPDCDELADHIGFQEAYR neat standard serially diluted calibration curve (A) and equine plasma (20µg) matrix spiked calibration curve (B). The equation and r2 for the best line of fit are given for each.

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A B

Figure 5.30: YLDHWLGAPAPYP neat standard serially diluted calibration curve (A) and equine plasma (20µg) matrix spiked calibration curve (B). The equation and r2 for the best line of fit are given for each.

B A

Figure 5.31: YLDHWLGAPAPYPDPLEPR neat standard serially diluted calibration curve (A) and equine plasma (20µg) matrix spiked calibration curve (B). The equation and r2 for the best line of fit are given for each.

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5.3.4.3 Comparison of MRM osteocalcin spiked and un-spiked equine trypsin and asp- n digested plasma samples. Detection and validation of endogenous concentrations of the peptides in the plasma matrix was testing using the MRM method developed with both trypsin and asp-n digested plasma (spiked and un-spiked with synthetic peptides). The results indicated that none of the endogenous peptides were able to be extracted from either trypsin or asp-n digested equine plasma (Figures 5.32 and 5.33).

A B

C D

Figure 5.32: (A) Equine trypsin digested (20µg) plasma spiked with 100ppb of osteocalcin peptides and (B) equine un-spiked trypsin digested (20µg) plasma. (C) Equine asp-n digested (20µg) plasma spiked with 100ppb of osteocalcin peptides and (D) equine un-spiked asp-n digested (20µg) plasma.

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To ascertain if the failure to detect peptides in plasma was due to the small concentration of plasma digested and tested (20µg) pooled equine plasma samples were tested. 100µg of pooled plasma was digested with trypsin and asp-n respectively and then spiked and un-spiked corresponding samples were run on the MRM. As observed in Figure 5.33, only the spiked synthetic peptides were recovered from the samples; no endogenous peptides were detected.

A B

Figure 5.33: (A) 100ppb of osteocalcin peptides spiked into asp-n digested equine plasma (pooled 100ug of plasma from 5 mature mares and foals) and (B) 100ppb of osteocalcin peptides spiked into trypsin digested equine plasma (pooled 100ug from 5 mature mares and foals).

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5.3.5 Offline liquid chromatography inductively coupled plasma mass spectrometry The detected sulfur (S) and calcium (Ca) concentrations did not agree with the predicted results (Table 5.5). Ca was detected in the blank samples and the Ca concentrations were lower than expected for the quality control (QC) samples. A significant amount of Ca (24.2 ± 5.78) was also detected in the negative control samples where Ca was not expected to be present. Likewise, S was below the limit of detection except for DPLEPRREVCELNP and DPLEPRREVCELNP in which 0.4386ppb and 0.1414ppb were detected respectively. The predicted Ca/S ratio was not observed for any of the peptides tested as depicted in Table 5.5.

Table 5.5: Calcium (Ca) and Sulfur (S) concentrations (CPS) detected by offline ICP-MS.

Sulfur (S) Calcium(Ca) Sample Identification Expected results Concentration Concentration (ppb) (ppb) Blank 1 0 <0.000 0.003 Blank 2 0 <0.000 0.027 Blank 3 0 <0.000 0.002 Blank 4 0 <0.000 0.014 Quality Control (QC) 0.1 Ca <0.000 0.04 Quality Control (QC) 1 Ca <0.000 0.38 Negative Control 0 Ca, 0 S 0 29.67 Negative Control 0 Ca, 0 S 0 24.78 Negative Control 0 Ca, 0 S 0 18.15 DPLEPRREVCELNP 0 Ca, 1 S 0 37.34 EVCELNPDCDELADHIGFQEAYR 0 Ca, 2 S 0 27.21 DPLEPRREVCELNP 1 Ca, 1 S 0 54.17 DPLEPRREVCELNP 2 Ca, 1 S 0.4386 13.57 DPLEPRREVCELNP 3 Ca, 1 S 0.1414 24.3 EVCELNPDCDELADHIGFQEAYR 1 Ca, 2 S 0 43.9 EVCELNPDCDELADHIGFQEAYR 2 Ca, 2 S 0.0491 16.45

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5.3.6 Laser-ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) The detected calcium chloride (CaCl2) and cysteine (Cys) concentrations (CPS) did not match the serially diluted concentrations for either the aluminium or no aluminium sample plates tested in this experiment (Table 5.6). Furthermore, concentrations varied depending on the isotope measured (Ca 44 as opposed to Ca 43). This may be due to the presence of polyatomic interferences in which two or more different elements combine to produce the same mass as the element of interest (May 1998). Compared to the aluminium plate, the concentrations were higher for all samples and element isotopes tested for the plate that was not covered in aluminium.

Table 5.6: Calcium (Ca), Sulfur (S) and Aluminium (Al) concentrations (CPS) of LA-ICP-MS fractions.

Calcium (Ca 43) Calcium (Ca 44) Sulfur (S 34) Aluminium (Al 27) Sample Sample Concentrations Concentrations Concentrations Concentrations plate Identification (CPS) (CPS) (CPS) (CPS) Aluminium 1:1 CaCl2 : Cys 549.00 9720.00 599.00 48600.00 (w/w) Aluminium 3:1 CaCl2 : Cys 22.40 508.00 24.40 23180.00 (w/w) Aluminium 3:1 CaCl2 : Cys 566.00 14000.00 141.00 51200.00 (w/w) diluted Aluminium CaCl2 52.30 1420.00 12.70 29300.00

Aluminium CaCl2 diluted 87.30 2530.00 8.90 37400.00

Aluminium CaCl2 diluted 214.00 3820.00 15.60 53500.00 Aluminium Cys 1.71 19.00 466.31 30000.00 No 1:1 CaCl2 : Cys 9846.00 167920.00 14361.00 - Aluminium (w/w) No 3:1 CaCl2 : Cys 14250.00 244300.00 2940.00 - Aluminium (w/w) No CaCl2 12310.00 233000.00 71.90 - Aluminium No Cys 51.10 0.00 0.00 - Aluminium

144 5.3.7 Glu/Gla amino acid initial screening results. The initial screening using the BCA assay showed that no protein was detected in the alkaline hydrolysed samples (Table 5.7). This suggests that the samples were completely hydrolysed and that any remaining unhydrolysed protein would have been below the limit of detection (LOD). An expected concentration of ±2mg was detected for both control samples.

Table 5.7: BCA assay test results of protein concentrations before and after alkaline hydrolysis

Result reading Sample (mg) Blank (H20) 0 BSA std_2mg/ml 1.70 Control BSA + GLA std_2mg/ml 1.95 BSA std_2mg/ml 0 Alkaline hydrolysis BSA + GLA std_2mg/ml 0

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5.4 Discussion The current study investigated the development of a method to assess the carboxylation status of osteocalcin in plasma. The detection of osteocalcin carboxylation has so far alluded detection via mass spectrometry (Ostrom et al. 2006; Cleland et al. 2016). Current assays rely on a combination of immunoassay and hydroxyapatite based methods (Prakash et al. 2005). The downside of these assays is they are cumbersome and not high-throughput.

In this study equine osteocalcin was digested in-silico into peptides and synthetically synthesised. Similar concentrations to that reported in plasma were then analysed and characterised by LC-MS/MS. Osteocalcin in circulation is extensively fragmented and disparity in those fragments reported is highly convoluted (Rehder & Borges 2010). Furthermore, to pursue development on the full-length osteocalcin synthetic standard was obsolete as osteocalcin is found to circulate in many different fragments (Rehder et al. 2015). The protein was digested into peptides of interest and a targeted MS-analysis, by LCMS/MS MRM method, was developed based on these specific osteocalcin peptides. While quantitation with MS is achievable for proteins and peptides, quantification is more sensitive for peptides.

Optimisation of trypsin and asp-n in plasma In proteomic experiments trypsin is the most widely used protease because of its specificity and efficiency in protein cleavage (Hendriks, 2018). This study evaluated the digestion of samples with both asp-n and trypsin. Asp-n proved to be more useful in generating peptides to monitor cOC peptides and has been applied previously to investigate osteocalcin (Nousiainen et al. 2002). Asp-n cleaves osteocalcin into homologous fragments. For this study, Mimotopes Pty Ltd were commissioned to artificially synthesis equine osteocalcin sequence and performed the initial characterisation by MALDI-TOF-MS. Only minor variation was demonstrated (Table 5.1) in the masses observed between both Skyline theoretical in-silico peptides and MALDI-TOF-MS analysis. The precise sequence of equine osteocalcin has also not be confirmed. There is currently disparity regarding the last few amino acids in the sequence. The observed m/z for modern horse differs from the monoisotopic m/z 5743.7 predicted from the published sequence (Carstanjen et al. 2002) Furthermore, the last three amino acids in the sequence (GTA) differ from the published sequence at residues 48 and 49. Carbamylation of osteocalcin has also been proposed (Gundberg and Weinstein 1986) and could account for the 43 Da disparity reported.

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Deamidation, which can occur during sample handling, could also have caused some mass differences.

This study also investigated the two performance characteristics of the enzymes; digestion efficiency and reproducibility. Extracted ion chromatograms for equine albumin indicated that trypsin exhibited more efficient digestion and gave more reproducible data with a CV of <20% for most of the peptides. In contrast, most peptides from the asp-n extracted ion chromatograms were above the 20% CV cut-off. These results are not surprising as trypsin is widely regarded to be a superior enzyme for protein digestion (Hendriks et al. 2018). While both enzymes yielded different peptides of interest, based on these results trypsin was chosen for further sample processing.

Characterisation of osteocalcin standards The results observed in this study were consistent with those reported by Cleland et al. (2016). There was a distinct neutral loss of carboxylation observed with the intensity of the fully carboxylated peptide (three Gla residues) lowest compared to the corresponding peptide containing one and two Gla residues (Cleland et al. 2016). This may in part explain why MS/MS ion search using Mascot was not particularly successful in these experiments with many of the synthetic peptides only partially detected. The unmodified peptides were more successful however, the DPLEPRREVCELNP fragments containing the carboxylated Gla

residues were undetected. As observed in Figure 5.20 the characteristic C02 neutral loss of the Gla containing peptides has been annotated. This phenomenon makes identification of the carboxylated peptides more challenging (Nousiainen et al. 2002). As stated by Cleland et al. (2016) this further hinders detection using conventional software searches such as Mascot as these fragments can be easily mistaken for the unmodified forms. The methylation of Gla residues has been utilised as a method to successfully detect the carboxylase enzyme using LC- MS/MS (Hallgren et al. 2013). However, as mentioned by Cleland et al. (2016) this will introduce further modifications that will only further complicate the analysis and interpretation. Minor differences between the expected and observed values can be explained by the affinity of certain metal ions; sodium (Na 22.99 atomic mass), magnesium (Mg 24.31 atomic mass), chromium (Cr 52 atomic mass) and iron (Fe 55.85 atomic mass) to osteocalcin (Niiranen et al. 2002). This metal ion interference has been documented in a number of osteocalcin studies (Rehder et al. 2015; Cleland et al. 2016). While this hinders analysis these metal ions have a remarkable effect on the stability of the osteocalcin molecule and it is suspected that this may have biological implications (Niiranen et al. 2002). In a study by Niiranen et al. (2002) the

147 interfering effect of metal ions to MS spectra was eliminated with addition of EDTA to the osteocalcin sample. However, this was not investigated in this experiment as EDTA chelates Ca ions. Cleland et al. (2016) were able to detect osteocalcin peptides carrying zero to three Gla residues using ESI. They also identified a number of adducts including chromium-adducted (+51.9) fragments and reported that the chromium adducts appeared to stabilise the carboxylation. In the current study a number of possible adducts were identified with mass shifts possibly corresponding to adduct containing Mg and Al (Figure 5.18). These findings are consistent with the results reported by Nousiainen et al. (2002).

Assay characterisation experiments Validation and consistency of the method was evaluated by several criteria including; digestion efficiency, reproducibility and linearity of calibration curves (Bundgaard et al. 2014a). Calibration curves were established to assess the linearity of the osteocalcin synthetic peptides. An estimate of the method and LOD was performed by analysing osteocalcin synthetic peptides individually at different concentrations in both un-spiked and spiked equine plasma samples. Un-spiked plasma demonstrated good agreement in standard-to-analyte ratios with r2 values ranging 0.9916 – 0.9981. A linear response was however not observed when the peptides were spiked into 20µg of digested (trypsin and asp-n) equine plasma (Figures 5.22 – 5.31) and endogenous peptides were not detected (Figure 5.33 and 5.34).

The peptides chosen for MRM method development were not identified in the plasma tested as analyte signals were below the LOD and LOQ for all peptides. This could be due to a number of reasons. It is possible that osteocalcin fragments are too low in plasma/circulation to be detected. While this conflicts with the literature in horses (Lepage et al. 1990; Lepage et al. 1992; Lepage et al. 1997; Black et al. 1999; Fletcher et al. 2000; Pastoret et al. 2007; Filipovic et al. 2014; Anderson et al. 2018) it is possible. Only a small fraction of total osteocalcin is circulating as most is in bone (Weber 2001) and only released during bone resorption (Delmas et al. 1983a; Anderson et al. 2018). A process that will vary depending on nutritional status, physiological status and age.

The developed method appears to be sensitive for synthetic standards that may not behave in the same way as the native peptides. The synthetic standards also may not carry the same post- translational modifications (PTMs) and such variances can alter the kinetics of proteolysis and could compromise the anticipation of strict stoichiometry between them (Bundgaard et al. 2014a). It is also clear that the sensitivity of the method on the standards was peptide sequence

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dependent; each individual peptide behaved quite differently. While some calibration curves were also within the linear range this was not enough to detect the corresponding fragment.

To ascertain if this was attributable to low starting concentrations, pooled plasma samples (100µg) was digested with asp-n and trypsin separately, but only the spiked synthetic peptides were recovered (Figures 5.33). Peptides were spiked into pooled plasma samples in an attempt to increase recovery, but spiking recovery was not sensitive enough to detect endogenous concentrations. This implies one of two things either; osteocalcin peptides were not sufficiently high enough to be detected with this method or the peptide targeted are not found in circulation. Differences in assay performance for different peptides highlights the dependence of MRM assay performance in plasma on specific properties of the peptides selected as surrogates for targeted peptides (Gundry et al. 2009). The most frequent cause of poor peptide identification is interference from the background plasma digest matrix (Vincent et al. 2009). If a significant peptide is not detected in an MRM assay it is often unclear if this is because of a) losses from sample handling (fractionation, desalting) b) poor enzymatic digestion, concentration below LOD, PTM (glycosylation and phosphorylation) or artificial modification to reactive amino acids such as oxidation and carbamylation.

Alternative strategies investigated Offline ICP-MS & LA-ICP-MS In this experiment Ca and S did not behave as expected. The ratio of Ca/S did not exhibit the anticipated pattern for each of the peptides. This may be partially attributable to the pervasive nature of Ca (Salazar et al. 2011). It was not possible to decipher what Ca was being detected from the peptides and what was due to contamination during sample preparation (Table 5.5). This made it difficult to interpret the results as a significant amount of Ca was also detected in the negative controls. Furthermore, Ca can be replaced by Mg and different polyatomic (spectroscopic) interferences can play a role (Rehder et al. 2015). The presence of polyatomic interferences are probably the largest cause of interferences in ICP-MS and result from atomic or molecular ions that have the same mass-to-charge as the analyte of interest (May 1998; Machado et al. 2017). Moreover, the concentration (CPS) of Ca and S did not correlate with what was expected for each of the control samples in the LA-ICP-MS analysis (Table 5.6). The failure of this proof-of-concept experiment was further evidence that off-line ICP-MS analysis are not the right method, at least for detection of carboxylation sites. These systems may be appropriate to compare the crude Ca content between different biological samples however, it was not sensitive enough for the purposes of this experiment.

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It should be noted that on-line systems, where the element-selective detector is connected directly to the separation system, give faster results than off-line systems (Kesava Raju et al. 2013). Additionally, the risk for contamination or losses is reduced, as no fraction collection and storage is necessary (Machado et al. 2017). On the other hand, the collected fractions provide the possibility of several quality control checks.

Software can be developed to correct for most interferences however, it cannot correct for most polyatomic interferences that are caused by atoms formed from precursors having numerous sources, including the sample matrix, aqueous and acid solutions used for preparation, plasma gases and entrained atmospheric gases (Salazar et al. 2011; Kesava Raju et al. 2013). A prior knowledge of polyatomic interferences from the literature for a particular analyte mass may be helpful in future experiments to aid in selection of reagents and conditions that would preclude, or at least reduce the possibility of polyatomic formation (May 1998). Other possible spectral interferences include, double charged ions and isobaric interferences (Salazar et al. 2011). Future experiments should investigate the use of online LC-ICP-MS systems to try and eliminate or circumvent spectral and non-spectral interferences.

Glu/Gla AA alkaline hydrolysis Alkaline hydrolysis was also investigated as an alternative strategy as it has been used in previous studies. While not carboxylation status specific, it has the potential to provide an overall comparison of the amount of γ-carboxyglutmate (Glu) to glutamate (Gla) between samples. The initial screening using the BCA assay showed promising results with no protein detected in the alkaline hydrolyses samples (Table 5.7). This suggests that the samples were completely hydrolysed and that any remaining unhydrolysed protein was below the limit of detection (LOD). It is suggested that this method should be pursued in future experiments.

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5.5 Conclusion In summary, it is suggested that future experiments employ enrichment strategies to increase the sensitivity of such an assay for possible detection of osteocalcin in plasma. Biological samples vary considerably in complexity and in the relative abundances of the individual proteins. There is inherent limitations of molecular mass spectrometry techniques. Some of these include the narrow dynamic range for calibration and the different signal response factors of different peptides and are further discussed in Chapter 8. While this method proved unsuccessful in detecting osteocalcin in plasma, it is anticipated that this assay may prove useful in serving as a sensitive indicator of osteoblastic vitamin K status in bone, in which osteocalcin is known to be at a much higher concentration. This needs to be investigated and further validated in future experiments as outlined in Chapter 8.

151 Carboxylated Analysis by Ceramic Hydroxyapatite Enrichment (CACHE): Development of a novel method 6.1 Introduction ...... 152 6.2 Experimental Procedures ...... 156 6.2.1 Sample Preparation ...... 156 6.2.2 Mass Spectrometry Analysis ...... 157 6.3 Results ...... 158 6.3.1 OC synthetic peptides HAP optimisation and reproducibility ...... 158 6.3.2 Carboxylated BSA synthetic peptides HAP optimisation and reproducibility ...... 161 6.3.3 Peptide Calibration curves ...... 164 6.4 Discussion ...... 166 6.5 Conclusion ...... 168

6.1 Introduction One of the key findings of Chapter 3 was that supplementation of a mare post-parturition with vitamin K increases the amount of the vitamin available to the foal in the mare’s milk. However, from this study it became clear that measuring the concentrations of vitamin K in plasma is not informative regarding the effect of vitamin K supplementation on the animal. Vitamin K supplementation is known to affect many different proteins, via its action on the enzyme γ-carboxy glutamase (Shearer & Newman 2014). This enzyme reaction, known as γ- carboxylation, requires vitamin K as a cofactor. Γ-Carboxyglutamic acid is a unique amino acid that binds to calcium (Shearer & Newman 2014). In proteins, γ-carboxyglutamic acids form the calcium-binding sites that characterize this form of calcium-binding protein, the vitamin K-dependent proteins (VKDPs) (Li et al. 2004). Calcium (Ca) stabilises certain structural forms of the VKDPs, enabling these proteins to bind to cell membranes. In the absence of vitamin K, γ-carboxylation is inhibited and proteins are synthesized that are deficient in γ-carboxyglutamic acid. These proteins have no biological activity because they do not bind to Ca and do not interact with membrane surfaces (Macek et al. 2009).

Ƴ-Carboxylation is a post-translational modification (PTM). To become active all VKDPs must become carboxylated (at least to some degree). Like most PTMs however, the abundance of carboxylation is very low, and further complicated by the rapid turnover of the modification in vivo (Rehder et al. 2015). Furthermore, it has alluded mass spectrometry analysis due to the properties of its negatively charged residues (Gla) (Hallgren et al. 2013). To investigate the

152 effect of nutritional supplementation of vitamin K, it would be valuable to determine the global change in the degree of protein carboxylation. However currently, no such assay is available. In contrast, global phosphorylation (Giorgianni & Beranova-Giorgianni 2016), acetylation (Li et al. 2013), ubiquitynation (Qiu & Luo 2019) and methylation (Hallgren et al. 2013) assays are well established. It therefore would be valuable to establish a similar assay for carboxylation. Strikingly, most studies so far have only focused on osteocalcin and not considered any of the other VKDPs within this context. This will be critical for future nutritional studies, as well as for assessing the effect of carboxylation on other physiological processes. Given that there is currently no way to simultaneously monitor carboxylated plasma proteins (VKDPs), the possibility of a screening proteomics approach would be useful.

Proteomics of biological fluids presents specific challenges. A major issue stems from the wide dynamic range of protein levels and the presence of a small group of highly -abundant proteins such as albumin and immunoglobulins (IgG) that constitutes a large fraction of the total protein mass (Zhang et al. 2007; Lepczyński et al. 2018). These proteins tend to dominate proteomic analyses of plasma and other biological fluids and therefore present a barrier for detection of less-abundant proteins (Lepczyński et al. 2018). The very low plasma concentrations of VKDPs compared to highly abundant proteins, make an enrichment step absolutely crucial. The methods that have been developed for depleting high abundance proteins include protein- based approaches such as immunoaffinity depletion with antibodies (Sabidó et al. 2012). As discussed in Chapter 5 though, these methods are not specific to carboxylation.

The objective of this chapter was therefore to investigate the use of hydroxyapatite as an enrichment strategy. Hydroxyapatite (HAP) is comprised of a crystalline mineral structure of calcium and phosphate (Figure 6.1) and is the main component of bone and dentin (teeth) (Gorbunoff 1984). Proteins bind to HAP via interaction with these minerals. HAP chromatography is very well established and has been used for the purification of proteins and nucleotides since the 1950’s (Gorbunoff 1980; Oshida 2014). The binding conditions of different proteins (basic, acidic) have been well characterised (Gorbunoff 1980, 1984; Gorbunoff & Timasheff 1984).

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Functional groups of HAP consist of charged (+) pairs of crystal Ca ions (C-sites) and

negatively (-) charged oxygen (O2) atoms associated with triplets of crystal phosphates (P- sites) (Gorbunoff & Timasheff 1984). As depicted in Figure 6.1, eluting specific proteins depends on the pH, buffer composition and the surface properties of the protein (or solute) applied (Gorbunoff 1980). Binding of acidic proteins at acidic and neutral pH forms metal coordination complexes between C-sites and carboxyl clusters. Elution of acidic proteins relies on a displacer with a strong affinity for C-sites such as phosphate (Gorbunoff 1984). For this experiment the use of commercial HAP was investigated as an enrichment strategy for carboxylation (Figure 6.2).

Figure 6.1: HAP interactions with protein amino residues and carboxyl clusters. C-sites, P- sites (PO4) and hydroxyl (OH) groups are distributed on the crystal surface. This combination of active groups supports retention by at least 3 distinct mechanisms.

While enrichment may be performed at the protein level to isolate intact phosphoproteins, enrichment of phosphopeptides on the peptide level is the most widely applied strategy (von Stechow 2016). Every peptide-level phosphoproteomics assay is comprised of five main steps (Giorgianni & Beranova-Giorgianni 2016): Extraction of proteins from the matrix of interest, protein digestion, binding, washing, elution and identification. In this study the principles of phosphoproteomics were used to develop and test a protocol to enrich for peptide-level carboxylation (Figure 6.2). The efficiency and specificity of the HAP enrichment protocol was monitored using LC-MS to evaluate and optimise conditions on bovine serum albumin (BSA) and osteocalcin synthetic peptides.

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Figure 6.2: (A) The general proteomic workflow (B) schematic diagram of the method being employed to enrich for carboxylation (CACHE), based on the principles used in phosphoproteomics experiments to monitor phosphorylation.

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6.2 Experimental Procedures 6.2.1 Sample Preparation A 3mg/ml suspension of HAP in loading buffer; 1mM of unbuffered sodium chloride (NaCl) was prepared in a 1.5ml Eppendorf tube (Figure 6.3). The HAP suspension (100µl) was added to separate tubes containing 10µg of digested BSA peptides and 10µg each of the osteocalcin synthetic peptides; the peptides had been dried for 1hour at 30°C. The samples were vortexed thoroughly to suspend the peptides, briefly centrifuged and then placed on the incubator at room temperature (27°C) to agitate for 1 hour at 1000rpm. The following steps were then carried-out:

1. After 1 hour, each of the samples were centrifuged for 2 minutes at 10,000 x g. 2. While keeping the samples on ice, the flow through was then carefully pipetted off into a fresh labelled Eppendorf tube and the exact amount was recorded. 3. The equivalent amount to that which was recovered of washing buffer number 1 (5mM

magnesium chloride (MgCl2) was then added to the sample. 4. The samples were then briefly vortexed to resuspend the pellet and then centrifuged for 2 minutes at 10,000 x g. 5. Wash 1 was removed, the exact amount recorded, and kept aside on ice in a fresh labelled Eppendorf tube. 6. As with the flow through, the equivalent amount recovered was then added of washing buffer 2 (1M NaCl) to the hydroxyapatite and peptide pellet. 7. The sample was again vortexed thoroughly and then centrifuged for 2 minutes at 10,000 x g. 8. Washing buffer 2 was removed, the exact amount recorded, and kept aside on ice in a fresh labelled Eppendorf tube. 9. As with wash 1 and 2, the exact amount recovered was then added to the hydroxyapatite and peptide pellet, of the elution buffer; 300mM phosphate buffer (sodium phosphate monobasic). 10. The samples were then vortexed thoroughly and then centrifuged for 3 minutes at 10,000 x g. 11. The eluted fraction was then carefully pipetted off into a labelled tube on ice. The remaining pellet was set aside and kept in a -80°C freezer. 12. The flow-through, wash 1, wash 2 and elution fractions were then dried down for 90 minutes at 35°C.

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13. Once dried, the samples were then resuspended in acetonitrile and trifluoroacetic acid and then individually desalted using SCX membrane tips (see Chapter 4, section 4.10 for desalting protocol).

(NB. The exact amount was recorded and then added in steps 2-3, 5-6 and 8-9 respectively, to ensure the concentration of the peptide starting material was unchanged). After desalting the samples were then dried down again and re-suspended in an appropriate amount of 10% acetonitrile and 0.1% formic acid to be run and analysed on LC-MS 8050 (Osteocalcin and BSA combined MRM method) (Haney et al. 2011).

Figure 6.3: CACHE enrichment protocol. The biological samples was spilt into 3 technical replicates. These were equilibrated with HAP and incubated. The samples were then spun- down and the flow-through removed after two separate, consecutive washes; wash 1: 5mM MgCl2, wash 2: 1M NaCl. The peptides were then eluted with 0.3M of phosphate.

6.2.2 Mass Spectrometry Analysis The samples were run on LC-MS 8050 using a combined BSA and Osteocalcin method (See Chapter 4, section 4.12 for Osteocalcin method development).

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6.3 Results 6.3.1 Osteocalcin synthetic peptides HAP optimisation and reproducibility Skyline analysis of osteocalcin peptide synthetic standards displayed extracted ion chromatogram, retention time and peak area CV for several replicates. Retention time and peak area CV were used to demonstrate the reproducibility of sample optimisation. The skyline chromatographic profiles across each of the different fractions can be observed for all of the osteocalcin peptides in Figures 6.4 and 6.5. These figures depict the change in peak area and therefore retention of the different peptides across each of the fractions (Panel A: left to right); control, flow-through, wash 1, wash 2 and elution. It is obvious from these extracted ion chromatograms that there is some retention of the carboxylated osteocalcin (cOC) peptides in the elution fraction as opposed to the undercarboxylated osteocalcin (ucOC) peptides.

Figure 6.4: (A) Chromatographic profile of all osteocalcin peptides; each colour represents the peak area of an osteocalcin peptide (1 x replicate showing all fractions, 1ul injection) (B) Peak area of each of the osteocalcin peptides across the fractions (C) Peak area of all the undercarboxylated peptides (D) Peak area of all the carboxylated peptides.

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The red line on the peak area CV graph indicates a 20% CV cut-off. The chromatographic profile for both the control and elution (Figure 6.5) replicates was deemed acceptable with all osteocalcin peptides predominately below the 20% CV and chromatogram and retention time pattern similar for each replicate.

A B

Figure 6.5: Osteocalcin peptides (3 x replicates, 1ul injection). (A) Depicts a chromatogram of the control sample showing the intensity and retention time of each of the peptides and the peak area CV (%) of each of the peptides. (B) Depicts a chromatogram of the elute sample showing the intensity and retention time of each of the peptides and the peak area CV (%) of each of the peptides. Most of the peptides were below the 20% cut-off CV.

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The concentration of most of the ucOC peptides was significantly higher in the flow through compared to the other fractions (Figure 6.6). In contrast, the concentration of the carboxylated peptides and the uncarboxylated peptide EVCELNPDCDELADHIGF was significantly higher in the elute fraction. The concentration of the singly carboxylated peptide DPLEPRREVC(66)LNP was lowest in the elute fraction compared to the other carboxylated peptides, with increased concentration of this peptide also observed in the flow-through fraction.

Figure 6.6: Heatmap showing the concentration of each of the osteocalcin peptides; uncarboxylated and carboxylated synthetic peptides. FT: flow-through, W1: wash 1, W2: wash 2, E: elution. Colour key represents the difference in concentration (intensities) of each of the peptides across the sample replicates. Dark blue = peptides of higher concentration, and dark red = peptides of lower concentration. A uniform colour across the replicates indicates the same peptide concentration.

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6.3.2 Carboxylated BSA synthetic peptides HAP optimisation and reproducibility Skyline analysis of the synthetic carboxylated BSA peptide standards displayed extracted ion chromatogram, retention time and peak area CV for several replicates. As above, retention time and peak area CV were used to demonstrate the reproducibility of sample optimisation. The chromatographic profiles across each of the different fractions can be observed for all of the peptides in Figures 6.7 and 6.8. This depicts the change in peak area and therefore retention of the different peptides across each of the fractions (Panel: left to right); control, flow-through, wash 1, wash 2 and elution. It is clear from these extracted ion chromatograms that there is minimal retention of the carboxylated BSA peptides in the elution fraction as opposed to the carboxylated osteocalcin peptides.

Figure 6.7: (A) Carboxylated BSA synthetic peptides sample (1 x replicate, 1µl injection) (left to right) Control, flow-through, wash 1, wash 2 and elute fractions. (B) Depicts the loss in peptide concentration across the fractions, with very little retention of the peptides in the elute.

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The red line on the peak area CV graph indicates a 20% CV cut-off. The skyline profile for both the control and elution (Figure 6.8) replicates was deemed acceptable with all osteocalcin peptides predominately below the 20% CV and chromatogram and retention time pattern similar for each replicate. There was however, only two peptides retained in the elution fraction as opposed to the control samples.

A B

Figure 6.8: Carboxylated BSA peptides (3 x replicates, 1ul injection). (A) Depicts a chromatogram of the control sample showing the intensity and retention time of each of the peptides and the peak area CV (%) of each of the peptides. (B) Depicts a chromatogram of the elute sample showing the intensity and retention time of each of the peptides and the peak area CV (%) of each of the peptides. Most of the peptides were below the 20% cut-off CV.

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The peptide concentration in the flow through for both undercarboxylated and carboxylated BSA peptides is significantly higher than in all other fractions (Figure 6.9). The concentration of the uncarboxylated osteocalcin peptides and the carboxylated peptide (DPLEPRREVC(66)LNP) was significantly higher in the flow-through compared to the other fractions. In contrast, the concentration of the remaining carboxylated peptides was significantly higher in the elute fraction (Figure 6.9). This is in contrast to the initial experiment on the osteocalcin synthetic peptides in which EVCELNPDCDELADHIGF was significantly higher in the elute fraction and the concentration of the singly carboxylated peptide DPLEPRREVC(66)LNP was lowest in the elute fraction compared to the other carboxylated peptides (Figure 6.6).

A B

Figure 6.9: Heatmaps (A) BSA peptides; uncarboxylated and carboxylated synthetic peptides. FT: flow-through, W1: wash 1, W2: wash 2, E: elution. (B) Osteocalcin peptides; uncarboxylated and carboxylated synthetic peptides. FT: flow-through, W1: wash 1, W2: wash 2, E: elute.

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6.3.3 Peptide Calibration curves Calibration curves were established to assess the linearity and recovery of the osteocalcin synthetic peptides, when extracted from hydroxyapatite (CACHE protocol). These were compared to the spiked plasma calibration curves established for each of the synthetic osteocalcin peptides (Chapter 5) (Figure 6.10). The limit of detection (LOD) and quantitation (LOQ) were determined by injecting a series of linear dilutions of known concentrations (3.9- 1000ppb). A linear response was not observed at all concentrations and endogenous peptides were not detected (Table 6.1). However, for hydroxyapatite extraction of some of the carboxylated synthetic peptides (Figure 6.10) the LOD and LOQ were increased (Table 6.1).

Table 6.1: Data for calibration curves; limit of detection (LOD) and limit of quantification (LOQ) values for each of the synthetic osteocalcin peptides (extracted from hydroxyapatite (CACHE) protocol). Linear Regression LOD LOQ Peptide modified sequence range (ppb) coefficient (ppb) (ppb) (r2)

DPLEPRREVCELNP 3.9 - 500 0.9903 41.66 125

DPLEPRREVCELNP 3.9 – 500 0.9995 1.3 3.9 3.9 – 500 DPLEPRREVCELNP 0.9995 2.6 7.8

EVCELNPDCDELADHIGFQEAYR 15.6 - 250 0.998 10.43 31.3

EVCELNPDCDELADHIGFQEAYR 31.3 - 1000 0.9958 166.66 500

DPLEPRREVCELNP 15.6 - 500 0.9945 166.66 500

DHWLGAPAPYP 3.9 - 500 0.9916 41.66 125

EVCELNPDCDELADHIGFQEAYR 125 - 1000 0.9983 83.33 250

YLDHWLGAPAPYP 31.3 - 1000 0.9985 83.33 250

YLDHWLGAPAPYPDPLEPR 62.5 - 500 0.9974 83.33 250

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600000 1200000 y = 1914.8x 500000 R² = 0.9921 1000000 y = 1948.3x 400000 y = 575.61x 800000 R² = 0.9995 R² = 0.9903 300000 600000 y = 731.06x

Peak Area Area Peak 200000 R² = 0.9979

Peak Area Peak 400000 100000 200000 0 0 0 200 400 600 0 200 400 600 Analyte Concentration (ppb) Analyte Concentration (ppb) DPLEPRREVCELNP (1 carboxy) DPLEPRREVCELNP_HAP DPLEPRREVCELNP (2 carboxy) DPLEPRREVCELNP_HAP

400000 y = 733.18x 25000 R² = 0.9995 300000 y = 283.23x 20000 200000 R² = 0.9971 15000 y = 52.322x y = 47.07x R² = 0.998 R² = 0.9948 10000 Peak Area Peak 100000 Peak Area Area Peak 5000 0 0 0 500 1000 1500 0 200 400 600 Analyte Concentration (ppb) Analyte Concetration (ppb) DPLEPRREVCELNP (3 carboxy) DPLEPRREVCELNP_HAP EVCELNPDCDELADHIGFQEAYR (1 carboxy) EVCELNPDCDELADHIGFQEAYR_HAP

40000 3000000 y = 2401.4x y = 31.819x R² = 0.9999 30000 R² = 0.9958 2000000 20000 1000000 y = 75.504x Peak Area Area Peak y = 26.131x 10000 Area Peak R² = 0.993 R² = 0.9945 0 0 0 500 1000 1500 0 500 1000 1500 Analyte Concentration (ppb) Analyte Concentration (ppb) EVCELNPDCDELADHIGFQEAYR (2 carboxy) DPLEPRREVCELNP DPLEPRREVCELNP_HAP EVCELNPDCDELADHIGFQEAYR_HAP

4000000 60000 y = 55.872x y = 6244.6x 50000 R² = 0.9983 3000000 R² = 0.9996 40000 2000000 30000 y = 44.828x

Peak Area Peak y = 113.09x 20000

1000000 Area Peak R² = 0.9916 10000 R² = 0.9979 0 0 0 200 400 600 0 500 1000 1500 Analyte Concetration (ppb) Analyte Concentration (ppb) DHWLGAPAPYP DHWLGAPAPYP_HAP EVCELNPDCDELADHIGFQEAYR EVCELNPDCDELADHIGFQEAYR_HAP

5000000 y = 4184.6x 12000000 y = 11016x 4000000 R² = 0.9998 10000000 R² = 0.9989 3000000 8000000

Peak Area Peak 2000000 6000000 4000000 y = 91.366x 1000000 y = 36.421x Area Peak R² = 0.9985 2000000 R² = 0.9974 0 0 0 500 1000 1500 0 500 1000 1500 Analyte Concentration (ppb) Analyte Concentration (ppb) YLDHWLGAPAPYP YLDHWLGAPAPYP_HAP YLDHWLGAPAPYPDPLEPR YLDHWLGAPAPYPDPLEPR_HAP Figure 6.10: Comparison of plasma matrix spiked calibration curves (purple) hydroxyapatite enriched plasma matrix spiked and (teal) plasma matrix spiked calibration curve.

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6.4 Discussion The aim of this study was to evaluate the use of HAP as an option to enrich for carboxylation in equine plasma samples. The results proved promising on the initial optimisation experiment however, investigation of alternative synthetically carboxylated peptides from a different protein, in this case BSA was not as successful. There was limited binding of all the carboxylated BSA peptides, with highest concentration of each detected in the flow-through. This implies that HAP may not be as selective as was first anticipated, after the initial experiment on the osteocalcin synthetic peptides. This is not a new phenomenon, it has been well characterised and documented in the literature (Nimptsch et al. 2007). It is also already widely used in combination with other assays to measure circulating osteocalcin in humans (Vergnaud et al. 1997; Gundberg et al. 1998). Therefore, it makes sense both biologically and based on current evidence that HAP has a stronger affinity for osteocalcin than some other proteins, regardless of carboxylation, and that the current method may not be as specific to carboxylation as initially anticipated.

The carboxylated synthetic BSA peptides were also only singly carboxylated. A repeat HAP experiment on the osteocalcin peptides (Figure 6.9) revealed that the singly carboxylated DPLEPRREVCELNP peptide was not retained in the elution fraction, with highest concentration detected in the flow-through. Likewise, while the highest concentration of the same peptide was detected in the elution in the initial experiment (Figure 6.6) there was still a higher concentration of this peptide detected in the flow-through when compared to the other peptides. This suggests that singly carboxylated peptides do not bind as selectively or strongly to HAP as those containing two or three carboxylation sites. It is also plausible that decarboxylation of this peptide may be responsible for its lack of binding. HAP binding assay for instance is known to underestimate ucOC concentrations in human studies, as a significant proportion of decarboxylated osteocalcin does not bind specifically to HAP (Merle & Delmas 1990). This is interesting from a biological standpoint. It would be interesting to consider if singly carboxylated osteocalcin is higher in circulation than doubly carboxylated osteocalcin. Consideration of the composition and structure of HAP in bone suggests that this may be the case. Furthermore, it is likely that under biological conditions the state of circulating osteocalcin in plasma will influence its binding of osteocalcin to HAP. Plasma samples with a mix of two and three Gla residues may behave differently than mixtures of fully carboxylated and decarboxylated protein. However, circular dichroism measurements show that the Gla residue at position 17 in osteocalcin is essential for the conformational transition to an α-helix

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(Nakao et al. 1994). This is because the helical structure facilitates the selective binding of osteocalcin to HAP and therefore partially carboxylated osteocalcin may have reduced binding. Likewise, some circulating proteins also have the capacity to displace osteocalcin when concentrations of the binders are too low (Gundberg et al. 1998).

Reproducibility and homogeneity of different preparations of HAP may vary. Studies show that different preparations have different binding characteristics, and that each batch must be independently evaluated and characterised before use (Gundberg et al. 1998). Secondly, the optimal amount of HAP to be used in binding studies must also be investigated. Especially that which is able to discriminate between carboxylated and undercarboxylated osteocalcin. Gundberg et al. (1998) attempted to alleviate any of the variation from differences in the binding capacity of HAP by enacting a regression equation based on multiple binding curves for their specific experiment. This should be investigated in further experiments using the specific conditions and reagents used in this study.

This study documents the need to carefully standardise HAP binding assay. To our knowledge it has not been applied to optimise binding to carboxylated plasma proteins however, it has been applied as an enrichment strategy in some other studies (Arya et al. 2018). Arya et al. (2018) observed that 242 plasma proteins were able to readily bind HAP (either directly or in some cases via association with another HAP-binding protein).

HAP was able to increase the sensitivity of the synthetic osteocalcin carboxylated peptides for the MRM method developed in Chapter 5 of this thesis. An improved correlation coefficient (r2) was observed for a number of the synthetic peptides extracted from equine plasma including; DPLEPRREVCELNP (2Gla), DPLEPRREVCELNP (3Gla), EVCELNPDCDELADHIGFQEAYR (1 Gla) and EVCELNPDCDELADHIGFQEAYR (0 Gla). However, there was no significant difference observed for the DPLEPRREVCELNP (1 Gla) peptide. As previously mentioned this may be attributable to decarboxylation of this peptide due to prolonged or compromised storage of the standard (Gundberg et al. 1984).

This study focused on the optimisation of sample preparation however, sample storage and handling are also critical and were not evaluated here. Further studies should investigate the effect of sample storage and handling on the standards used as this is a plausible cause of some of the discrepancies in results observed. In the case of phosphoproteomics for instance, phosphatase inhibitors are added to the sample during preparation and acids are avoided (Macek et al. 2009). It is therefore necessary to investigate what effects these variables may

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have on osteocalcin, especially as it has been suggested that circulating osteocalcin fragments are generated by proteolysis in the circulation or during sample processing and storage (Garnero et al. 1994). In summary, if using HAP to measure differential binding of osteocalcin the method must be standardised before use. It must be emphasised that although these findings may have functional implications it is not possible yet to translate these observations directly to a clinical setting. Further experiments are needed to validate the results of this study.

6.5 Conclusion As the developed MRM assay (Chapter 5) was more sensitive at the peptide level, enrichment efforts were focused on the peptide level. However, the results of this Chapter suggest that the enrichment conditions were unable to enrich for singly carboxylated peptides. Future studies should investigate enrichment at the protein level. Longer osteocalcin sequence peptides or modification of buffer conditions may also improve enrichment of singly carboxylated peptides.

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169 CHAPTER 7 Application of the Carboxylated Analysis by Ceramic Hydroxyapatite Enrichment (CACHE) method to the plasma of vitamin K supplemented foals. 7.1 Introduction ...... 170 7.2 Materials and methods ...... 173 7.2.1 Sample collection and storage ...... 173 7.2.2 Experimental design ...... 173 7.2.3 Sample preparation ...... 174 7.2.4 Mass Spectrometry Analysis ...... 176 7.2.5 Data acquisition ...... 176 7.2.6 Data processing...... 176 7.3 Results ...... 179 7.3.1 Quality control (QC) checks – LC-MRM-MS...... 179 7.3.2 Outcome of sample comparison and protein data (PeakView ® DDA and SWATH (DIA) traces) ...... 181 7.3.3 SWATH (DIA) Quality control (QC) check ...... 182 7.3.4 Assay Characterisation ...... 183 7.4 Discussion ...... 189 7.5 Conclusion ...... 194

7.1 Introduction Vitamin K functions as a cofactor for the enzyme γ-carboxy glutamase to convert Glu residues to γ-carboxyglutamic acid (Gla) residues in proteins (Berkner 2005). These activated vitamin K dependent proteins (VKDP) are found in tissues and body fluids throughout the body (Booth 2009). Few studies have investigated the relationship between vitamin K intake and its effect on extra-hepatic VKDPs in horses. It is suspected that vitamin K supplementation of mares may change the VKDP plasma profile of their foals. There are however, no studies that have investigated the applicability of a proteomics-based approach to evaluate this relationship. Arya et al. (2018) applied a proteomics-based approach to analyse hydroxyapatite-binding plasma proteins in individuals with age-related macular degeneration. A similar approach may be feasible for VKDPs in plasma.

Plasma proteomics is a rapidly growing area of research as blood plasma is a rich source of circulating proteins and is easily accessible To date however, the equine proteome has not been well characterised in the horse there are still many unclassified proteins. This means that results of some of these studies should be interpreted with caution. Interrogation of the proteins

170 indentified in some of these studies as putative ‘biomarkers’ suggests that they may be affected by a number of physiological conditions. Furthermore, many of the same proteins have been idenitifed to be present under different physiological conditons. This includes the acute phase proteins (APP) which are commonly used as markers for inflammatory diseases (Bundgaard et al. 2014b). Haptoglobin is an APP that is considered a marker of acute and chronic inflammation in the horse (Bundgaard et al. 2014b). Likewise, fibrinogen is considered a diagnostic marker of inflammation. The determination of what conditions may up or down regulate their expression can therefore, be challenging. future progress in equine genome and proteome coverage will greatly improve the coverage of proteins and peptides.

There have been a few studies investigating the use of proteomics to detect potential biomarkers for a number of conditions in horses including; OCD (Chiaradia et al. 2012; Desjardin et al. 2012; Desjardin et al. 2014), laminitis (Steelman & Chowdhary 2012) and tying-up (Bouwman et al. 2010). More recently there has been a growing interest in characterising proteins in oviductal fluid (Smits et al. 2016), the equine embryo (Swegen et al. 2017), uterine and amniotic fluid (Isani et al. 2016; Bastos et al. 2018) and umbilical cord (Maia et al. 2017) in order to gain a better understanding of early pregnancy in the mare.

The blood plasma proteome is comprised predominately of albumins and globulins as well as the following proteins; fibrinogen, immunoglobulins, α1-antytrypsine, transferrin, α-2 macroglobulin, haptoglobin and complement C3 (Leeman et al. 2018; Lepczyński et al. 2018). These highly abundant proteins make up to 90% of the protein concentration in plasma and are responsible for maintaining normal blood architecture and homeostasis: osmolality, pH and ion concentrations (Leeman et al. 2018). The remaining 10% is comprised of medium and low abundant proteins, most likely to be of key interest as targets for biomarkers. Proteins secreted by different tissues into blood are the most promising group of potential biomarkers, as they are reflective of the state of the cells in the tissue under given conditions.

Scoppetta et al. (2012) attempted to evaluate plasma proteome changes induced by prolonged exercise in horses, however these results are considered with caution as there was no abundant protein depletions conducted prior to analysis, and albumin and immunoglobulins were identified as the predominate markers associated with changes in exercise. It is probable that in this study these proteins masked lower abundant proteins that better reflected exercise induced changes in the blood.

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During the past two decades mass spectrometry (MS) has emerged as a method of choice to study post-translational modification (PTM) of proteins and is considered an ideal detector of phosphorylation events (Giorgianni & Beranova-Giorgianni 2016). Phosphorylation is a common PTM that involves the reversible attachment of phosphate groups to the side chains of specific amino acids (Macek et al. 2009). In humans one-third of proteins are phosphorylated, often at multiple sites and in a transient manner (Macek et al. 2009). Unlike phosphorylation, little is known about the amount that carboxylation contributes to the plasma proteome. This is partially attributable to the fact that requirements for vitamin K have not been well defined in humans and are even less defined in horses.

MS can in principle identify each phosphopeptide and localise the phosphoryation groups in the peptide sequence. This principle also applies to carboxylation. However, MS characterisation of phosphopeptides is not a trivial task (Macek et al. 2009). Phosphopeptides in complex protein digests often escape detection and idenitification by standard MS analaysis because of their low abundance, and inadequate fragmentation patterns (Macek et al. 2009). This may pose an even greater challenge for the detection of carboxylation as it is expected to represent an even smaller proportion of all peptides present in the plasma proteome. The aim of this study was therefore to increase the chance of detecting carboxylated VKDPs. This was investigated by applying the principles of phosphoproteomics and the enrichment startegy developed in Chapter 6; CACHE. In any method development workflow the first step is to characterise the method. Therefore, the first stage in this workflow centres on qualitative discovery (Giorgianni & Beranova-Giorgianni 2016). This is a crucial phase to characterise, assign and catalogue possible carboxylation sites, to test the feasibility of the chosen workflow and ascertain if further optimisation is necessary (Macek et al. 2009).

In Chapter 6, the use of HAP as a method to enrich for equine plasma carboxylation was developed using osteocalcin standards. The next step is to test this optimised method on a cohort of biological samples and in this Chapter, the method is applied to a subset of experimental samples from Chapter 3. While HAP enrichment is well established for osteocalcin, an attempt to enrich for other VKDPs has not been investigated. Initial characterisation was undertaken in this study with the objective to apply the developed method in a future quantitative study.

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7.2 Materials and methods 7.2.1 Sample collection and storage Plasma samples were taken from foals (see Chapter 3, section 3.2.2. for full details). Blood samples were collected in lithium heparin vacutainers and centrifuged at 10,000 x g for 10mins. Plasma was then decanted and stored at -80°C until proteomic analysis was performed.

Experimental design This study was applied to plasma samples collected at the 14 days post-parturition time-point that a statistically significant difference was observed for compared to the controls (refer to Chapter 3). For this experiment three biological replicates were taken from the control group and three biological replicates from the vitamin K supplemented group. These samples were all acetone precipitated and randomised, before being spilt into three technical replicates of each (Figure 7.1). This pull-down method can be done at either the peptide or protein level. This means that the sample is either digested before or after pull-down. For this experiment, CACHE was conducted at the peptide level, as this is what the protocol had been optimised for (refer to Chapter 6). The samples were then all in-solution digested and after digestion, the equine albumin MRM method was run on each as quality control (QC) to check digestion efficiency. Each digested sample was then spiked with osteocalcin synthetic peptides before proceeding with the CACHE protocol as an added QC to check pull-down efficiency.

Figure 7.1: Peptide CACHE experimental workflow. Plasma was collected from each of the experimental groups (three biological replicates of each). These were then split into technical replicates, digested and analysed via LC-MS/MS.

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7.2.2 Sample preparation Equine plasma samples To prepare the samples for peptide CACHE, the equine foal plasma samples (20µg) were digested using the in-solution digestion protocol (Chapter 4, Section 4.9). After digestion the samples were dried down for 90 minutes at 35°C. Samples were set aside after digestion to be run on LC-MS 8050 for quality control (QC) using the developed equine albumin MRM method (refer to Chapter 5). Three digestion replicates were then generated for each of the samples. The CV’s were then checked and any samples that had CV ≥ 20% were immediately rejected. The samples were then prepared for peptide CACHE. As a further QC measure, 10µl of synthetic asp-n OC peptides were spiked into each of the samples (this concentration was taken from the spiked calibration curve in Chapter 5). The samples were then dried and resuspended in 5µl of loading buffer (1mM NaCl). The tubes (1.5ml) were then prepared for each of the samples containing a 3mg/ml suspension of hydroxyapatite. The 5µl of the resuspended peptide sample was then added to 95µl of the equilibrated suspension of hydroxyapatite in loading buffer. The mixture was thoroughly vortexed and then placed on the incubator at 27°C to agitate (1000 rpm) for 1 hour. When the samples had finished agitating, they were then centrifuged for 2 minutes at 10,000 x g. The samples were then placed on ice and the flow through was carefully pipetted into a fresh, labelled tube and the amount removed was recorded. The pellet was then washed with washing buffer 1 (5mM MgCl2). The sample was vortexed thoroughly to resuspend the pellet and then centrifuged for 2 minutes at 10,000 x g. This was then collected into a fresh tube and placed on ice. The equivalent amount of washing buffer 2 (1M NaCl) was then added to the pellet. The sample was vortexed thoroughly and then centrifuged for 2 minutes at 10,000 x g. The wash 2 was then collected and set aside in a new tube on ice. The peptides were then eluted by adding an equivalent amount of elution buffer (300mM Phosphate buffer – sodium phosphate monobasic monohydrate) to the pellet. The sample was vortexed thoroughly and then centrifuged for 3 minutes at 10,000 x g. The eluted fraction was then carefully removed and pipetted into a fresh, labelled tube. The pellet was placed in the -80°C freezer for safe keeping. The clean-up of all samples was done by desalting each of the fractions with SCX membrane (Chapter 4, section 4.10). Before submitting for LS-MS/MS analysis, each of the elution fractions was QC tested using the osteocalcin MRM method to check the asp-n QC peptides and peptide concentrations were normalised (Chapter 4, Section 4.12). When the peptide concentration was normalised, each of the samples was resuspended in iRT buffer and submitted for MS analysis.

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Equine tissue samples To increase the likelihood of detecting more vitamin K-dependent carboxylated proteins of interest, this exact same protocol was applied to a pooled vitamin K supplemented plasma sample (using 200µg as opposed to 20µg) and samples of equine liver and muscle. The reasoning behind this was that if this approach was successful, the equine liver sample should serve as a library of carboxylated peptides. Most VKDP are produced and sequestered in the liver, while muscle would act as a negative control as none of the vitamin K-dependent peptides of interest would be expected to be present in this tissue. Equine tissue samples were also collected and prepared for peptide CACHE in an attempt to enrich the spectral library for carboxylated proteins of interest. Equine liver and muscle samples were prepared as depicted in the figure below (Figure 7.2). The samples were collected opportunistically from a mature gelding that underwent post-mortem at the School of Veterinary Science (SVS). Collection was covered under the veterinary proteomics biomarker assay development project (Animal Ethics approval number; ANRFA/SVS/541/18). The horse was cleared of any health conditions that may have impeded analysis and the samples were deemed healthy for the purposes. The samples (25mg) were homogenised with 200µl of lysis buffer and then placed on a shaker and beaten with glass beads in order to lyse the sample. The lysed samples were centrifuged at 16,000 x g for 10 minutes to clarify the samples. The clarified protein extract was solubilised in Urea-Tris buffer. The protein concentration of the samples was determined by BCA assay (Chapter 4, Section 4.6) before proceeding with FASP digestion protocol (Chapter 4, Section 4.9).

Figure 7.2: Experimental protocol for tissue protein extraction. Equine liver and muscle samples were homogenised, clarified and solubilised. Protein concentration was then ascertained by Bradford Assay (BCA assay). The samples were then FASP digested and analysed via LC-MS/MS.

175 7.2.3 Mass Spectrometry Analysis The elute fractions were analysed by LC-MRM-MS using the Osteocalcin method developed (refer to Chapter 5 for full details).

7.2.4 Data acquisition Protein identification after CACHE- Data acquisition in data-dependent mode (DDA) All the wash and elute fractions were pooled and submitted separately for DDA to act as respective libraries for carboxylated and non-carboxylated peptides for future SWATH analysis and to assess if any carboxylated peptides/proteins were indeed detected and enriched for in the elute fractions. DDA analysis was conducted on a quadrupole time-of-flight instrument (TripleTOF® 5600+ System, SCIEX). Mobile phase A was composed of 0.1% (v/v) formic acid (FA) in water, while mobile phase B was composed of 0.1% (v/v) FA in ACN.

Data acquisition in data independent SWATH mode Sequential window acquisition of all theoretical fragment-ion spectra mass spectrometry (SWATH-MS). For SWATH-MS data acquisition, the same mass spectrometer and LC-MS/MS setup as described above were used but operated in SWATH mode. For quantitation a sample specific reference spectral library was generated by DDA analysis of the CACHE generated plasma proteins. To generate a SWATH assay library, a pooled mixture of all digested protein samples was measured in a positive data dependent acquisition (DDA) mode in three technical replicates (injections). The results were saved for future Skyline analysis.

7.2.5 Data processing. Equine NCBI database processing To identify peptides, the experimentally-generated mass spectra were matched against protein sequence databases, generating a qualitative list of potential matches, ranked by confidence, of proteins that may be present in the sample. An equine database was assembled in FASTA format; HORSE_NCBI containing 133,454 sequences (downloaded on the 5th of September 2019). This was used in both ProteinPilot and Mascot search engines. To increase the possibility of detecting a known carboxylated protein, a customised FASTA file was manually created of known VKDP (refer to Appendix 5) in the horse and elute pooled fraction was searched against this.

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ProteinPilot analysis DDA data acquired from the above instrument was subjected to ProteinPilot (ProteinPilot™ Software 5.0, Revision Number: 4769, SCIEX) analysis. The acquired MS/MS data were annotated with amino acid sequences from the imported equine database using the Paragon™ Algorithm provided in the software (refer to Appendix 6). For the ProteinPilot™ searches the following parameters were applied: Sample Type: Identification; Cys Alkylation: Iodoacetamide; Digestion: Trypsin; Instrument: TripleTOF5600; Special Factors: Urea Denaturation; Species: None; Search effort: Thorough ID; ID Focus: Amino acid substitution; Results Quality: Detected protein threshold [Unused ProtScore (Conf)] ≥ 0.05 with 10% false discovery rate (FDR) selected. The automatically generated excel file was manually curated to filter out contaminates and false positive results. The highest confidence number of proteins identified at 1% critical FDR was obtained from the Global FDR fit in the excel report and applied. Proteins with a unique score < 2.0 were discounted and only proteins identified at FDR ≤1% with ≥ 2.0 peptides from the remaining list were retained for further comparatives analysis.

Mascot The .mgf file exported from the .group output file from the ProteinPilot search was submitted to Mascot. Mascot employs an algorithm that will attempt to predict the presence of certain modifications based on sequence patterns (Matthias & Ole 2003). This search engine was used to see if carboxylation could be detected in the acquired MS/MS data (refer to Appendix 7). The mass to charge ratios of possible peptide fragments (b- and y- ions) are calculated and matched against the experimental spectra. In an MS/MS experimental search the modified peptide variant is distinguished from the unmodified variant by comparing the mass shift (m/z value) of the modified peptide spectrum to the unmodified peptide spectrum.

The wash and elute files were searched independently with both the Equine NCBI database and the assembled VKDP database. The protein sequences are digested into peptides in silico by the chosen protease utilised in the experimental workflow (in this case trypsin). A theoretical spectrum is then generated and compared to the experimental spectrum with all possible candidate theoretical spectra calculated.

To initiate the search, a list of search parameters must be specified. The files were submitted for a MS/MS Ion Search and the following parameters were applied for each: Enzyme: Trypsin; Variable Modifications: Carbamidomethyl (C), Carboxy (E); Mass values:

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Monoisotopic; Protein mass: Unrestricted; Peptide mass tolerance: ± 20ppm; Fragment mass tolerance: ± 20ppm; Max missed cleavages: 0, Instrument type: ESI-QUAD-TOF. The same Mascot parameters were used to search DDA data for both wash and elute fractions initially. This was done using the VKDP database and allowed for a more meaningful search to be conducted by reducing the list of peptides/proteins to the most likely candidates. The wash data was then searched against the entire horse NCBI database. This was done so that the MS/MS spectra from the wash data could potentially be used to validate carboxylated hits from the elute fractions, if any were identified. This is common practice in phosphoproteomics experiments to identify phosphorylated sites.

STRING analysis STRING (version 10.5) is an online database tool to predict and associate known protein- protein interactions. In this experiment the tool was used to compare the protein-protein interactions between the wash and elute fractions. (Downloaded on the 8th of October 2019, www.string-db.org).

PANTHER analysis The proteins identified by ProteinPilot™ for the elution tissue sample searches were analysed by the gene ontology software using Protein Analysis Through Evolutionary Relationships (PANTHER) tool (version 15.0). In this tool the gene entries were analysed by associating them with the Equus Caballus species. (Downloaded on the 9th of December 2019, www.pantherdb.org) (refer to Appendix 8).

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7.3 Results 7.3.1 Quality control (QC) checks – LC-MRM-MS Skyline analysis of equine albumin peptides displayed extracted ion chromatogram, retention time and peak area CV for each of the six biological replicates (Figure 7.3). Retention time and peak area CV were used to demonstrate the reproducibility of sample optimisation. The peak area CV was ≤ 20% for each of the peptides. However, some variability is expected to be present between individual biological samples from different animals.

B C

Figure 7.3: (A) Equine albumin peptide extracted ion chromatograms, retention time and peak area 20% cut-off CV for each of the 6 biological replicates (B) Example of extracted ion chromatogram of a control sample and (C) sample from the vitamin K supplemented group, depicting retention time and intensity of each of the eluted trypsin digested equine albumin peptides.

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Skyline analysis of spiked in synthetic asp-n peptides displayed extracted ion chromatogram, retention time and peak area CV for each of the technical replicates (18 samples) (Figure 7.4). Retention time and peak area CV were used to demonstrate the reproducibility of sample processing. The peak area CV was ≤ 20% for each of the peptides. This was not unsurprising as some variability is expected as not all of these peptides elute at the same concentration. Extraction of each of the peptides to some extent was achieved indicating that the CACHE protocol had been achieved successfully and the carboxylated osteocalcin synthetic peptides were indeed enriched. This confirmed that the samples were able to be submitted to LC-MS/MS for DDA and DIA analysis. A

B C

Figure 7.4: (A) Spiked-in standard asp-n osteocalcin peptides extracted ion chromatograms, retention time and peak area 20% cut-off CV for each of the 18 technical replicates. (B) Example of extracted ion chromatogram of a control sample and (C) sample from the vitamin K supplemented group, depicting retention time and intensity of each of the eluted spiked-in asp-n osteocalcin standard peptides. The fully carboxylated DPLEPRREVCELNP peptide eluting at 2 minutes exhibited the highest concentration indicating that the CACHE protocol successfully enriched for carboxylation in the elute fractions.

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7.3.2 Outcome of sample comparison and protein data (PeakView ® DDA and SWATH (DIA) traces) The collected DDA and DIA data was intially examined in PeakView® software. The total ion chromatogram traces (TIC) which represent the summed peptide signals were observed (Figure 7.5). The distinct different traces observed between the wash and elute fractions are indicative of differing peptide profiles. These results were promising as they suggest that the CACHE protocol was potentially enriching for a different subset of peptides. Likewise the TIC traces from the collected DIA (SWATH) data exhibited a similar pattern. It was evident that the peptide traces were distinctly different between the pooled vitamin K treated samples and that of the control samples. Based on these observations, further processing of the data was undertaken.

Figure 7.5: Total ion chromatogram (TICs) of DDA collected peptides representing the wash fraction (pink) and elute fraction (blue) of pooled equine plasma samples (left panel); and (right panel) SWATH collected traces; blank (blue trace), PBQC of vitamin K supplemented samples elute fraction (pink trace) and PBQC of control samples elute fraction (red trace).

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7.3.3 SWATH (DIA) Quality control (QC) check Signals for the spiked in asp-n synthetic osteocalcin peptides were extracted from each of the technical replicates. This was done as a further QC measure to check if the CACHE protocol had worked and had successfully extracted the spiked in peptides from the collected DIA data. While the reproducibility varied for each of the peptides between the technical replicates, each of the peptides was able to be successfully extracted to some degree. An example of the DPLEPRREVCELNP is depicted in Figure 7.6 (refer to Appendix 9 for all other extracted peptide chromatograms).

B C

Figure 7.6: (A) Skyline SWATH extracted ion chromatograms showing the precursor and product ion transitions for the spiked in standard asp-n osteocalcin peptide; DPLEPRREVCELNP from each of the 18 technical replicates. Retention time, peak area CV and replicate comparison of this peptide is also depicted. (B) Example of extracted ion chromatogram of a control sample and (C) sample from the vitamin K supplemented group, depicting the retention time and intensity of the spiked-in asp-n osteocalcin standard peptide DPLEPRREVCELNP which was successfully extracted from the SWATH analysis. The precursor and product ion transitions are annotated.

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7.3.4 Assay Characterisation Analysis of DDA data - ProteinPilot™ search results of plasma CACHE experiment A spectral library contains LC-MS/MS spectra that is used as a reference library for peptide identification. It relies on the collection of data-dependent acquisition (DDA), allowing the identification of proteins analysed using SWATH-MS approach (Schubert et al. 2015). After acetone precipitation both wash and elute fractions were in-solution digested and analysed on the mass spectrometer. The total number of proteins identified in the wash fraction was 94 and 72 were detected in elute (refer to Appendix 10). A higher number of proteins were detected in the wash as compared to the elute fraction as displayed in the venn diagrams below (Figure 7.7). This was expected as CACHE should increase the number of higher abundant proteins in the wash fraction and enrich for lower abundant proteins. However, there were also 41 commonly shared proteins identified in each of the fractions. Considering the overlap between the different fractions, no significant difference was found.

Figure 7.7: (left) peptides, (middle) distinct peptides, (right) proteins. Venn diagram depicting the number of proteins identified in elute and wash fractions following in-solution digestion and CACHE protocol with SCX membrane for desalting. The total number of proteins was also evaluated in the equine liver and muscle tissue samples. A total of 748 proteins were identified in the wash fraction and 91 were detected in the elute (Figure 7.8). A higher number of proteins were detected in the wash as compared to the elute fraction and of these 77 proteins were commonly shared between each of the fractions. There was only 14 unique proteins identified in the elute fraction.

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Figure 7.8: (left) peptides, (middle) distinct peptides, (right) proteins. Venn diagram depicting the number of proteins identified in elute and wash fractions following FASP digestion and CACHE protocol of tissue samples with SCX membrane for desalting.

The curated number of acidic and basic peptides in the wash and elute plasma fractions was also compared. There was a higher number of acidic peptides identified in the elute fraction, however, there was also a number of acidic peptides identified in the wash fraction (Figure 7.9). This indicates that there may have been a loss of key peptides of interest in the wash fraction and also that the conditions used may not have been as selective for acidic peptides as anticipated.

100 90 80 70 60 50 40 30 20 10

Number of curated peptides peptides curated of Number 0 Wash Elute Wash Elute BASIC ACIDIC

Figure 7.9: Number of peptides in the wash fraction (113) and number of peptides in the elute (83) fraction of the plasma.

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Gene ontology analysis All proteins identified in the wash and elute fractions (Figure 7.10) were assessed in the STRING gene annotation tool. The majority of the protein – protein interactions expressed in the proteins were related to the regulation of biological functions. The lines between the nodes represent known interactions derived from curated databases and those predicted experimentally. Small nodes represent proteins of unknown structure and large nodes represent proteins with some known structure. All proteins displayed have a SAINT score > 0.75 and STRING analysis was performed at the high confidence interval. Highlighted nodes are those possibly related to VKDP functioning. While no key VKDPs of interest were able to be identified, it was of interest to see what biological functions the proteins that were identified were involved in. In the wash fraction most of the identified proteins were involved in functions not as closely related to VKDP functioning.

A B

Figure 7.10: String analysis of expressed proteins in the wash and elute plasma fractions as determined by mass spectrometry analysis (DDA). This demonstrates that some of the proteins identified in elute and wash fraction are functionally associated with each other. The coloured nodes, are involved in pathways which could be directly associated with vitamin K-dependent protein functioning.

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Gene ontology analysis of tissue samples The proteins identified in the tissue samples were subjected to GO-term analysis using the PANTHER classification tool. In the PANTHER tool, the gene entries were analysed by aligning them with Equus caballus. The PANTHER analysis resulted in only a small number of equine aligned genes entries as listed in Appendix 8. Observation of the molecular function domain of the proteins identified in the elute fraction shows that binding proteins account for nearly half of those identified.

3% (A) Molecular function

44% 38%

binding

structural molecule activity

molecular function regulator

catalytic activity 3% 12% transporter activity

Figure 7.11: Molecular functioning partitioning (A) Gene Ontology (GO) analysis of 44 ProteinPilot IDs from the elute fraction of the pooled tissue samples. 44% of the protein entries identified were binding proteins, 38% catalytic activity, 12% structural molecule activity, 3% molecule function regulators and 3% transport activity.

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Mascot data analysis In the elute fraction no significant carboxylation result was identified by a Mascot search of the MS/MS ion search generated by LC-MS/MS. Prothrombin was the only VKDP that was identified (Figure 7.12). There was no other peptides identified to be related to VKDPs and no other carboxylation hits occurred in either elute or wash fractions (refer to Appendix 11).

Figure 7.12: Probability based protein score of Prothrombin (scores >13 = p<0.05).

A protein match, observed in Figure 7.12, was identified as a significant hit with a protein score >13. The ion score is a measure of how well the observed MS/MS spectrum matches the identified peptide; prothrombin. As observed in Table 7.1, all the listed m/z ions were statistically significant (score>13). The expect value indicates the probability that the observed match between MS/MS spectra and peptide sequence would be identified by chance. High confidence matches should exhibit an expect value <0.1. In this case all peptides were identified to be significant prothrombin matches however, none of the peptides identified had carboxylation as a detected modification (Table 7.1). As shown in Table 7.1 there was also no carboxylation modification detected by the search engine on any of the prothrombin peptides identified in the wash fraction. Likewise, no significant carboxylation hits were detected in any of the peptides identified in the wash fraction (refer to Appendix 11).

187 Table 7.1: Peptide summary report of the MS/MS ion search of the VKDP database by MASCOT (www.matrixscience.com).

m/z M(expt) M(calc) Peptide Score Expect Hits

397.1991 1188.5755 1188.5716 K. YGFYTHVFR.L 32 0.00063 Prothrombin

787.3743 1572.7340 1572.7348 R. TTDEDFPLFFDVK.T (34) 0.00044

787.3743 1572.7340 1572.7348 R. TTDEDFPLFFDVK.T 44 4.1e-05

188 7.4 Discussion This study applied the CACHE protocol developed in Chapter 6 to plasma samples from foals, with known elevation in vitamin K, whose dams had been supplemented with a high dose of vitamin K. As outlined in Chapter 3, the supplementation of mares resulted in significant transfer of vitamin K into the colostrum and subsequently showed significant increased circulating vitamin K concentrations in the foals. Plasma samples taken from the foals at day 14 that showed a statistically significant difference between the control group and the supplemented group were chosen for this study (Chapter 3, Day 14: Figure 3.7). It was anticipated that supplementation may increase carboxylation in the plasma proteome, facilitating detection by CACHE.

Validation of the CACHE protocol in plasma (Quality control (QC) checks) Reproducibility of the equine albumin peptides confirmed that digestion was successful in the biological replicate samples (Figure 7.3). The next step was to evaluate the CACHE protocol in plasma and this was done by spiking-in and then assessing the recovery of osteocalcin synthetic peptide standards from the samples. The CACHE protocol was validated using known carboxylated standards where clear enrichment of carboxylation was evident (as observed in Chapter 6, Figure 6.6). Initial quality control checks confirmed that the protocol was working in this experiment, with the extraction of the fully carboxylated DPLEPRREVCELNP peptide exhibiting the highest level of recovery in the samples, compared to the other spiked-in carboxylated peptides (Figure 7.4). The signals for these peptide standards were also able to be detected by SWATH-DIA (Figure 7.6). This was further validation that the CACHE protocol and experimental workflow had worked as expected on the osteocalcin standards.

Characterisation of wash and elute fractions (DDA) The wash and elute fractions were pooled and analysed via DDA to act as the spectral library for future SWATH-DIA. The TIC traces for DDA and SWATH-DIA depicted a clear difference between the wash and elute fractions (Figure 7.5). This was evidence to suggest that the wash and elute fractions were enriching for different subsets of proteins and peptides. The proteins and peptides were then characterised by assessing the DDA collected data to determine what the assay had specifically enriched for in plasma. After the curation of the proteins (Figure 7.7), 94 proteins were identified in the wash and 72 in the elute fractions including some albumin and immunoglobulin fragments (Appendix 10). There were 31 proteins identified to be unique to the elute fraction.

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It must be noted that tissue samples were also digested in this experiment (Figure 7.2) however, it was evident from interrogation of the DDA data that further optimisation was necessary as digestion was suboptimal for these samples. Furthermore, while there was evidence of some enrichment between the different fractions (Figure 7.8) no proteins or peptides of interest were able to be detected in the elute fraction and as depicted in Figure 7.11, and of those proteins detected in the elute fraction most of them were binding proteins. It is recommended that future experiments optimise this protocol for tissue samples, in particular bone where known carboxylated proteins are present such as osteocalcin. This will aid in enriching the library to increase the chance of potentially detecting VKDPs of interest in the blood plasma proteome.

While the overall number and quantity of proteins may be stable, at the peptide level, it was necessary to investigate if there were any differences in the peptides being eluted in comparison to the wash. A comparison of the profile of peptides in the wash and elute plasma fractions identified that acidic peptides contributed relatively more to the profile of peptides in the elute fraction than basic (Figure 7.9). This to be expected as the CACHE protocol was developed to target acidic proteins and peptides such as osteocalcin however, there was still a proportion of acidic peptides lost in the wash fraction. As portrayed in Chapter 6 (Figure 6.1), the mechanisms of HAP binding are influenced by a number of factors, in particular pH and isoelectric point. There are also a number of different interactions a peptide or protein may have with HAP depending on its specific sequence and attached groups (Gorbunoff 1984). For this reason HAP binding of some proteins and peptides is not always uniform or easily predicted.

The gene ontology tool STRING, demonstrated that a higher number of the blood plasma proteins in the elute fraction were involved in the coagulation cascade as well as a number of other biological processes (Figure 7.10). The complement system is integral to the immune system response (Parente et al. 2017). A number of those proteins were identified to be unique to the elute fraction; complement-C3 and, complement factor H-like. An isoform of the serine protease, α-2 macroglobulin was also identified. α-2 macroglobulin is responsible for the inactivation of plasmin and is therefore key to blood coagulation cascade (Bundgaard et al. 2016). Many of these proteins can be found in the plasma of healthy horses, especially those that are highly abundant therefore, deciphering what proteins are expressed due to a stimulus is one of the challenges of proteomics (Lepczyński et al. 2018). As expected, prothrombin was detected and is a key protein involved in the clotting cascade (McKeown et al. 2002a). Extra-hepatic VKDPs of interest, like osteocalcin or MGP, were

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undetected. As albumin can mask lower abundant proteins, peptides from the higher abundant coagulation factors, such as prothrombin, may mask some of the lower abundant extra-hepatic VKDPs that may be present.

There was only low quality carboxylation hits detected on peptides in elute and wash fractions including that of prothrombin (Figure 7.12 and Table 7.1). This may be the result of an intensity issue (Krisp & Molloy 2017). Vitamin K catalyses many other coagulation related factors in the blood (Booth 2009) and therefore, despite no increase in carboxylated proteins being observed in this experiment, it does not mean that it was not necessarily absent.

Analysis of a biological fluids such as blood plasma, presents a major challenge for proteomics due to the highly complex nature of its proteins and biomolecules (Aebersold & Mann 2016). The very dynamic nature of all these biomolecules with many at trace levels makes detecting them difficult as although they are no less active or critical, signal intensities cannot be amplified to allow their detection. Proteins are additionally challenging for analytical techniques due to the post-translational modifications (PTMs). PTMs are also very dynamic, and like carboxylation, when present are only at very low concentrations. Therefore the peptides with these PTMs, may be overlooked for fragmentation in the mass spectrometer (Corthals et al. 2000; Leitner et al. 2007). Fragmentation patterns of certain modified peptides can be difficult to predict, especially in the case of carboxylation, which is a PTM that has received considerably less attention than phosphorylation or glycosylation (Scott et al. 2002; Ghesquière et al. 2006). Fragmentation patterns in published literature are therefore lacking. A better understanding of the effect of carboxylation on peptide fragmentation should improve identification in future studies.

Carboxylation is responsible for the activation of VKDPs (Figure 7.13). As depicted in Figure 7.13 these VKDPs such as osteocalcin are postulated to be present in circulation with a varying number of γ-carboxyl residues attached (Rishavy & Berkner 2012). Furthermore the carboxylation reaction depends on not only vitamin K as a cofactor but also the carboxylase enzyme to be present. The carboxylase is only capable of recognising this family of VKDPs as it exhibits no homology to any other known enzymes (Rishavy & Berkner 2012). The reaction catalysed by the carboxylase is also unique in biochemistry making an understanding of it crucial. Compared to other PTM’s it has also not been well characterised and the mechanisms that regulate it in circulation are not well understood. The literature suggest that most of these VKDPs can be identified in circulation with varying numbers of these γ-carboxyglutamic acid

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residues (Gla) attached. Multiple Gla residues are present in VKDPs, ranging from 3 to 16 residues within individual proteins (Hallgren et al. 2013). However, the extent and distribution of Gla residues and their specific function for each of the protiens are unknown.

Figure 7.13: Process of carboxylation of the VKDP osteocalcin. Uncarboxylated (0 γ- carboxyglutumate residues) and undercarboxylated (1 or 2 γ-carboxyglutumate residues). Carboxylation of the glutamate (Glu) residues to form γ-carboxyglutamic acid residues (Gla) occurs via the carboxylase enzyme and the co-factor vitamin K.

While MS-methods in proteomics and phoshoproteomics are becoming the methods of choice for discovery-driven protein analysis at a system level (Humphrey et al. 2015; Giorgianni & Beranova-Giorgianni 2016; von Stechow 2016). To this day large-scale analysis of complex phosphoproteomes is a highly challenging endeavour and even in validated experiments modifications can complicate interpretation. As carboxylation has received comparably less attention in the literature it is clear that an increased understanding of the carboxylation status of these VKDPs in circulation is needed. Furthermore the findings of this experiment suggest that the presence of these VKDPs in blood plasma may be lower than has been previously reported and this may also account for osteocalcin lack of detection by MS to-date (Cleland et al. 2016).

Further inspection of the data revealed no other PTMs of interest in the wash or elute fractions. However, this doesn’t rule out any changes in peptides which may be indicative of PTM’s (Eng et al. 2011). Noisy spectra or impurities, unusual enzymatic cleavage and unexpected modifications not considered in the search create difficulty in biological samples (Nørregaard

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Jensen 2004; Nielsen et al. 2006). Carboxylation may have been masked by modifications induced by sample preparation; adducts, carbamidomethyl, oxidation and/or deamidation (Nørregaard Jensen 2004; Nielsen et al. 2006).

The presence of isobaric peptides (Motorykin et al. 2014), can frequently form when nearby PTM’s co-localise on the same peptide (Ren et al. 2018). This is an inherent issue in MS/MS spectra database search for spectra matches due to the mass shift caused by an unknown modification (Motorykin et al. 2014). Discrimination of such peptides by DDA is insufficient as the precursor masses can be the same (Ren et al. 2018). The same precursor mass can be hundreds of isobaric forms and these modifications can hide fundamental information about biological systems and PTM crosstalk (Ren et al. 2018). Independently of quantitation method, the profile of the fragment ions is required to discriminate isobaric forms (Ren et al. 2018). Likewise the presence of any isomers may imply PTM enrichment (Rentsch et al. 2015). The isoforms detected in the elution fractions may therefore be the result of a PTM (Appendix 11). The SWATH data collected in this experiment would need to be further examined to investigate if isobaric peptides were present in these samples.

Future studies to improve the CACHE workflow As introduced in Chapter 6, there are numerous enrichment strategies available. While these have been extensively studied and applied for phosphopeptide enrichment, to date, similar strategies have not been investigated for carboxylation enrichment of the plasma proteome. After digestion, it was anticipated that the peptides would be non-carboxylated compared to their carboxylated counterparts. Therefore enrichment of carboxylation at the peptide level is necessary and is the most common approach applied in phosphoprotemics workflows (Nilsson 2012). It is evident from these findings that further studies should evaluate the binding capacity of HAP, to determine the optimal ratio of protein and/or peptide to the amount of HAP being loaded. It would also be advisable to optimise the CACHE protocol on the protein level to investigate if proteins bind to HAP in the same manner as peptides. Based on the principles of HAP chromatography it is probable that a different profile of proteins may be enriched (Oshida 2014) however, this is speculative and would need to be investigated in future experiments. The possibility that HAP carboxylation enrichment only pertains to osteocalcin also can’t be disregarded. However, the completely different chromatographic profiles (Figure 7.5) insinuate that a certain subset of peptides are being enriched.

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Detection problems can be partly improved by various sample preparation techniques, such as the incorporation of the use of antibodies to the workflow and other affinity-based enrichment strategies. However, as discussed in previous chapters this was outside the scope of this study to pursue. As natural carboxylation in blood plasma is expected to be very low, an in-vitro method of inducing and verifying carboxylation would be ideal. It is therefore suggested that to improve the current workflow future in-vitro experiments investigate the feasibility of inducing carboxylation within the samples of interest by the carboxylase enzyme. This experiment is suggested to be carried out after the CACHE protocol, to investigate if a difference can be detected between the samples prior to MS-analysis being carried-out.

7.5 Conclusion To the author’s knowledge, this is the first study to investigate a proteomics-based method to decipher VKDPs in equine plasma. The potential of the CACHE method developed in Chapter 6 was evaluated. However, this method did not enrich VKDPs of interest. While further optimisation of this method in plasma is necessary, the results provide a protocol that has laid the groundwork for future studies investigating the function of VKDPs in the horse. The importance of vitamin K and extra-hepatic VKDPs continues to be a key focus in human research and equally relevant to other species. Bioinformatics for PTM discovery is a very active research area and tools are always improving. This study has paved the way for the development of an alternative approach to the vitamin K conundrum, in the hope that it may lead to unravelling the complexity of the vitamin’s functions.

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195 Implications and future directions

8.1 Introduction ...... 196 8.2 Outcomes of the research ...... 196 8.2.1 Vitamin K nutrition of the mare and foal ...... 197 8.2.2 Investigation of osteocalcin with mass spectrometry ...... 198 8.3 General considerations ...... 198 8.4 Equine vitamin K biology ...... 200 8.4.1 Vitamin K metabolism ...... 200 8.4.2 Vitamin K requirements ...... 201 8.4.3 Which biomarker should be used? ...... 201 8.5 Future directions...... 202 8.5.1 Vitamin K in metabolism and bone development ...... 202 8.5.2 Vitamin K and bone health ...... 203 8.5.3 The possible role of other VKDPs in bone development ...... 204 8.5.4 Vitamin K and interactions with other fat-soluble vitamins ...... 204 8.5.5 Role of new technologies for studying vitamin K ...... 205 8.6 The final word ...... 207

8.1 Introduction Vitamin K has been shown to play a vital role in bone development (Shea et al. 2017). This role stems from its relationship to the bone protein, osteocalcin (Shea & Booth 2008). While the relationship to osteocalcin and the mechanism of uptake and absorption of vitamin K in humans and the human neonate have been widely studied (Shearer et al. 2012; Harshman et al. 2014b), similar investigations have not occurred in the horse. In humans vitamin K status has been implicated in osteoporosis, which has similarities with equine OCD. The aim of this thesis was not to study OCD per se, but rather to define the role of vitamin K and osteocalcin in mares and their foals. It was hoped that these studies would aid in elucidating the role that vitamin K plays in equine bone development, and thus bone related diseases in the horse. The stumbling block with the research was determining vitamin K status, especially the measurement of osteocalcin.

8.2 Outcomes of the research While the initial objective was to investigate the role of vitamin K in the mare and foal, this evolved into a quest to quantitate osteocalcin. In undertaking the research on these two complimentary aspects of equine vitamin K metabolism, the first reported controlled

experiments investigating the effects of vitamin K supplementation on vitamin K1 status in

196 mares and foals were completed. Likewise, the experiments that investigated carboxylation as it pertains to osteocalcin and other VKDPs are possibly the first proteomics-based studies to attempt this.

8.2.1 Vitamin K nutrition of the mare and foal The studies undertaken in Chapter 3 were designed to reflect field conditions and establish the role of vitamin K in early pregnancy and lactation. In Experiment 1, maternal transfer of vitamin K and the effect of pre-parturition supplementation on circulating vitamin K concentrations in mares and foals were assessed. In Experiment 2, mares were supplemented from parturition for 3 months. During this period, vitamin K concentrations in milk and in foal plasma were measured.

The main outcomes of these studies was that supplementation of mares modulated the concentration of vitamin K in milk, particularly in the first 24 hours post-parturition. It was

also discovered, that as in human infants, negligible concentrations of vitamin K1 are found in both umbilical cord and neonatal plasma of the foal at birth. Thus the hypothesis that there is limited placental transfer of vitamin K to the equine neonate was substantiated.

While these studies failed to demonstrate any significant effect of routine oral supplementation on circulating vitamin K status of mares, in part due to study design, modulation of colostrum

concentration of vitamin K1 was achieved. This suggests that colostrum is a critical source of vitamin K for foals in the first few days after birth, before the gut develops enough for them to obtain requirements from pasture, and potential microbial derived synthesis of vitamin K.

A key outcome of these studies was that plasma concentrations of vitamin K may not be a reliable indicator of vitamin K status. Osteocalcin has been suggested as a more reliable indicator of vitamin K status (refer to Chapter 2). However, methods to analyse osteocalcin have relied on immunoassay based methods, which do not permit the differentiation of the degree of carboxylation; a key indicator of vitamin K status. This difficulty sparked the investigation into osteocalcin undertaken in Chapter 5, and the exploration of a possible mass spectrometry based method to analyse it.

197 8.2.2 Investigation of osteocalcin with mass spectrometry The outcome of Chapter 3 prompted investigations of alternative approaches to measure osteocalcin. The studies were undertaken in collaboration with researchers at QUT and the proceeding chapters evolved from that collaboration.

In Chapter 5 a number of MS-based approaches were considered. An MRM method was developed but osteocalcin was not detected in plasma using this approach. This may indicate that osteocalcin is not in the circulation or circulates in another form. In light of the outcome, it was decided to investigate the use of an enrichment method. Whilst this was initially undertaken in the hope of enriching specifically osteocalcin it was anticipated that all circulating VKDPs would be enriched. This was attempted in Chapters 6 and 7 by the application of a novel proteomics based method.

In Chapter 6 an enrichment strategy for osteocalcin using hydroxyapatite (HAP) was developed; CACHE. It was found that HAP increased the sensitivity of synthetic osteocalcin peptides using the MRM method developed in Chapter 5. In Chapter 7 the CACHE method was extended and evaluated with horse samples. To do this an experiment using foal plasma samples collected in Chapter 3 was carried-out. Although it was found that this method did not enrich the VKDPs of interest, it is the first study to investigate the use of such an application in equine plasma.

8.3 General considerations In this research an effort was made to investigate a number of complex research questions with limited resources. The University of Queensland Equine Unit herd was used for this research, but only a limited number of animals were available for the experiments. Due to the high cost of undertaking research involving horses, the number of mares in the experimental groups was restricted. While this allowed for accurate determination of the date of ovulation, and a reasonably tight foaling period of three months, it also meant that when mares had to be withdrawn from the study it was not possible to replace them, resulting in two less mares in the control group in Experiment 1 (Chapter 3). Statistical power was therefore reduced and statistical significance could not be assigned in aspects of the initial experiment.

Limitations of this study include the fact that these horses were not inherently deficient in vitamin K1 and would have received vitamin K1 from pasture. It would have been ideal to record individual feed intakes of the horses to estimate vitamin K intake, but one constraint of

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the studies was that the experimental procedures had to complement the current management practices of the horses in the equine science teaching program. However, management practices were typical of current industry practices. Pasture analysis over the course of an experiment is recommended for future studies. This will allow the determination of the contribution of the pasture to overall vitamin K status.

Vitamin K1 is transported on triglyceride-rich lipoproteins. As such, circulating serum/plasma are generally obtained in a fasted state and/or corrected for triglycerides in order for circulating

K1 to be reflective of vitamin K nutritional status. While milk vitamin K1 concentrations were corrected for milk fat, plasma concentrations were not corrected for triglycerides in this study. It would be prudent to do this in future studies examining requirements.

The method development and optimisation was a significant component of this thesis. While the aim was to investigate an alternative method to analyse osteocalcin, the optimisation of sample preparation is a crucial phase in an MS-based workflow. While this was a lengthy process, it took much longer than initially anticipated.

In all analytical work authentic standards are essential and in this work the synthesis of the osteocalcin standards was led by the informed understanding that their concentrations would elevate following vitamin K supplementation. However, the findings of Chapter 5 imply that the osteocalcin peptides that were emulated by the synthetic peptides, exist at concentrations in plasma well below this. It is true that the most important and specific changes generally occur in proteins of low abundance, this therefore warrants their enrichment to enable detection (Lepczyński et al. 2018). This is why an enrichment strategy was explored, to try and increase detection in plasma. Since plasma has a wide and dynamic range of protein concentrations, depletion of high abundance protein can simplify the matrix and facilitate the depletion of several medium and low abundance proteins (Lepczyński et al. 2018). However, immuno- depletion kits are lacking for animal plasma and identification of low abundance proteins in animal plasma is challenging (Ghodasara et al. 2017).

In Chapter 7, SWATH data was collected and initially it was intended to include this in overall analysis. However, this did not occur as the VKDPs of interest were not detected in the spectral library DDA data on which SWATH relies. Whilst anecdotal evidence suggests that further optimisation of the CACHE protocol will enable detection of these VKDPs, such a goal was not within the timeframe of this thesis.

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The difficulty of embarking on method development using MS, is that the available MS run time is often limited by demand for equipment, as well as by the cost of access to it. In these experiments, the relatively small-scale size may have reduced the reliability and validity of results. Alternate strategies were also briefly investigated in this project, however, due to financial and time constraints these were not pursued.

8.4 Equine vitamin K biology

In contrast to other nutrients, there is not an established dietary concentration of vitamin K that has been recommended for the horse; the vitamin K requirement for the horse is yet to be confirmed (NRC, 2007). This makes dietary supplementation challenging, and raises a number of questions about the biology of the vitamin in the horse.

8.4.1 Vitamin K metabolism As discussed in Chapter 2, there are species differences in the metabolism of vitamin K and a clear definition of what occurs in the horse had not been described. Like other species, horses

receive vitamin K from two major sources; diet, especially pasture (vitamin K1) and microbial

synthesis (vitamin K2) in the gut. In addition, compounded feeds may be supplemented with

menadione (vitamin K3), a synthetic form of the vitamin. As a grazing species, horse depend on the vitamin K content of pastures, that can vary considerably (Booth 2012). It is largely dependent on environmental factors such as the season, weather and time of day. Likewise, the vitamin K content of hay varies and is sensitive to drying and exposure to ultraviolet radiation (Erkkilä et al. 2004). Therefore, in some cases the vitamin K content of hay can be very minimal (Biffin et al. 2008b). This has implications for animals with restricted access to pasture, where the vitamin K status of their diet may be insufficient to ensure optimal functioning of extra hepatic VKDPs (Biffin et al. 2008a).

It is generally considered that the microbial synthesis of K2 in the equine hindgut is an available source of the vitamin but evidence to the significance of this source is lacking. There is increasing evidence to suggest that microbial synthesis may not be as important to overall vitamin K status as previously thought (Skinner et al. 2015). McCann et al. (2019) found that

while vitamin K2 is produced by gut bacteria, the mechanisms by which absorption occurs from the colon in humans are unknown (Karl et al. 2017). Similar questions need to be addressed to determine the contribution the K2 of microbial origin to the vitamin K economy of horses.

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In addition, the relative absorption efficiencies of each of the homologs of vitamin K vary among species. This has ramifications for assessing dietary vitamin K requirements in horses.

It has been well documented however, that K1 is predominate form in herbivores and K2 in omnivores and carnivores (Thijssen et al. 1996). The horse almost exclusively utilises and stores K1 and therefore their vitamin K profile is vastly different to that of rodents; the species in which most research has been conducted (Biffin et al. 2008b). The underlying mechanism of vitamin K1 utilisation by tissues also needs further exploration in the horse as do the metabolites excreted.

8.4.2 Vitamin K requirements While vitamin K is essential for blood clotting, current recommendations of vitamin K intake, based on coagulation, have been found to be suboptimal to maintain the functioning of extra- hepatic VKDPs (Harshman et al. 2014b). Current equine feeding practices would suggest that vitamin K intake is adequate for blood coagulation in horses but the adequacy or otherwise for the other functions of the vitamin is an open question.

Late pregnancy and lactation significantly influence the extent of bone remodelling with associated changes in vitamin K requirements. Vitamin K supplementation was suggested as a way to improve maternal osteocalcin carboxylation, including the newborn (Sânzio Gurgel et al. 2017). Greater maternal liver storage of vitamin K would increase the amount of the vitamin available for transfer to breast milk and consequently improve newborn vitamin K status (Dror & Allen 2018). The newborn carboxylase system is immature and has less capacity to maintain the creation of complete carboxylated osteocalcin (Lanham et al. 2015). This further supports the need for increased maternal vitamin K supplementation for foetal and neonatal development.

8.4.3 Which biomarker should be used? Conventional methods to assess vitamin K status have relied on coagulation assays such as prothrombin time (PT) and measurement of serum vitamin K1 concentration. Undercarboxylated VKDPs may provide better insight into the utilisation of vitamin K by extra-hepatic tissues. Recently urinary Gla has been used as a marker of vitamin K status as it is reflective of VKDP turnover and degradation. However, there is no consensus on Gla levels in urine that indicate adequate vitamin K status; further studies are required. Recently, the alterations in VKDPs induced by vitamin K presence or absence have proven to be suitable

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biomarkers for detecting vitamin K deficiency. The indirect measurement of osteocalcin has been proposed as a more sensitive measure of subclinical vitamin K deficiency.

While there is no single biomarker that is considered a robust measure of vitamin K status (Shea & Booth 2016), there is an urgent need to identify a biomarker or panel of biomarkers for the determination of vitamin K status. To date, there has only been a limited number of studies that have investigated vitamin K status and metabolism in horses.

8.5 Future directions In the sections of this Chapter above, the implications of the research outcomes have been outlined and in this section, possible future studies are further defined. Much of the present work has been exploratory in nature. It will be necessary for future experiments to be designed to repeat and validate what has been observed. While initial attempts to use a combination of methods to assess osteocalcin were unsuccessful, these studies were not in vain. They can be used to inform further work in this area. A novel outcome of the research was the development of a method which may prove to be a useful tool. Whilst this method did not prove sensitive enough to ascertain changes in these initial experiments it is anticipated that with further optimisation it will do so.

8.5.1 Vitamin K in metabolism and bone development Pregnancy and lactation are periods of significant influence on bone metabolism (Filipovic et al. 2010). The intensive foetal growth, and the mineralisation of the foetal skeleton is substantial. Moreover, bone is not inert and has also recently been identified to act as an endocrine organ in its own right with osteocalcin implicated as one of the key players in bone development (Ducy 2011; Karsenty 2012; Ferron & Lacombe 2014). In young animals, modelling of the skeleton is accompanied by increased concentration of bone remodelling markers in the blood (Bourebaba et al. 2019). Is osteocalcin an appropriate marker of vitamin K status and bone turnover? An answer to this question is important for defining equine vitamin K requirements. The results in Chapter 3 suggest that mares and foals maintained on quality grass and legume pasture, probably do not require additional vitamin K supplementation to maintain vitamin K status during lactation and growth. However, in order to make informed decisions regarding diet formulation and supplementation in horses, the effects of vitamin K supplementation during lactation and growth on vitamin K status needs to be further investigated. Specifically the effect of vitamin K on vitamin K-dependent markers of bone metabolism needs to be defined.

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An important aspect of defining the role of osteocalcin is determining degree of carboxylation. The posttranslational modification (PTM) of osteocalcin by vitamin K is complex. PTM’s of proteins is an increasingly studied area of research. To our knowledge, this is one of the first proteomics-based studies to attempt to investigate carboxylation as it pertains to osteocalcin and other VKDPs. It is evident from these findings that this is a monumental task; the groundwork laid in Chapters 6 and 7 should assist this undertaking.

The relevance of circulating concentrations of undercarboxylated VKDPs is still uncertain. It remains controversial in humans, whether normal vitamin K status implies that circulating levels of VKDPs in particular, MGP and osteocalcin, would be fully carboxylated in all tissues. There is discrepancy among research findings regarding the presence of circulating undercarboxylated (ucOC) osteocalcin and whether or not it is indicative of vitamin K deficiency; are concentrations of ucOC of physiological significance? Is another question which needs clarification in the horse. While studies in mice and humans suggests a putative role of ucOC in energy metabolism, further evidence in the horse is needed.

8.5.2 Vitamin K and bone health A role for vitamin K in osteoporosis has been established and there is accumulating evidence to support a protective role for vitamin K in osteoarthritis (Chapter 2). However, it is premature to make recommendations regarding vitamin K’s efficacy in reducing the progression of bone pathology, especially during development. Further studies examining the effect of vitamin K on bone are needed, particularly given the uncertainty surrounding the reliability of current biomarkers to capture the putative mechanisms by which the vitamin may protect joint health.

The association of vitamin K nutritional status with joint health has been evaluated primarily in observational studies in humans. Oka et al. (2009) found that an increase in vitamin K intake was associated with a lower risk of osteoarthritis of the knee. Misra et al. (2013) proposed a threshold of 1.0nmol/L plasma vitamin K1 as potentially beneficial to joint health. Likewise, a health, aging, and composition study found that participants with bone issues such as

subarticular cysts were more likely to have plasma vitamin K1 concentrations <0.2nmol/L (Shea et al. 2015).

Numerous nutrients have been related to the metabolism of equine cartilage particularly, energy, protein, calcium, phosphorous, copper and zinc. While caution must be taken when extrapolating, in light of the recently discovered roles of VKDPs; matrix gla-protein (MGP) and osteocalcin in osteoarthritis in humans, it is most likely that vitamin K also has a role in

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equine bone disease, but proof requires much further investigation. The complexity of investigating osteocalcin in foals, demonstrates the range of data required to delineate the pathogenesis of bone disease.

8.5.3 The possible role of other VKDPs in bone development Research in this thesis concentrated on osteocalcin but the role of other VKDPs in bone metabolism is becoming increasingly apparent. Recent findings in humans have found that subclinical vitamin K deficiency may contribute to an increased risk of radiographic knee osteoarthritis (Misra et al. 2013). Misra et al. (2013) compared cartilage cells derived from both normal and osteoarthritic conditions and found that carboxylated MGP was only produced under normal conditions. MGP and osteocalcin share some common functionalities (Viegas et al. 2013) and in studies with knockout mice clearly show that both play integral roles in bone mineralisation. MGP has been found to prevent vascular calcification and ectopic mineralisation and osteocalcin has been found to ensure calcium deposition in bone (Wallin et al. 2010). While it remains to be determined whether both osteocalcin and MGP work in an antagonistic or synergistic manner, a protective role of MGP in bone development is possible (Wallin et al. 2010). Likewise, increased expression of periostin, which derives its name from its location in the cortical periosteum of bone, has been found to be associated with increased bone strength in mice (Rani et al. 2009).

Gla-rich protein (GRP) is one of the most recently identified members of the VKDP family and has been implicated in the crosstalk between inflammation and the calcification of articular tissues in osteoarthritis (Rafael et al. 2014). Additional VKDPs; Growth-arrest-specific 6 protein (GAS-6) and transforming growth factor B-inducible protein have also been found to promote chondrocyte survival and differentiation (Loeser et al. 1997). However, since antibodies for the carboxylation status of these proteins have not yet been developed, the role of these VKDPs in bone disease is unknown (Shea & Booth 2017a) . Nonetheless, the presence of multiple VKDPs in bone and joint tissue (Loeser et al. 1997; Wallin et al. 2010; Rafael et al. 2014) emphasises the important role of vitamin K in bone health.

8.5.4 Vitamin K and interactions with other fat-soluble vitamins Vitamin K and the other fat soluble vitamins A, D and E are presumably absorbed by similar mechanisms. Recent research suggests that vitamin E and vitamin K may compete for transporter mechanisms (Shea & Booth 2019). In vitro competition has been observed for both vitamin K, D and E (Goncalves et al. 2015). However, a better understanding of these

204 interactions and mechanisms of absorption of fat-soluble vitamins is needed in the horse. It is unknown if vitamin E affected the results of this study, but there is a trend in the animal feed industry to increase dietary vitamin E concentrations to improve antioxidant status. What effect this practice is having on the metabolism of other nutrients, including vitamin K should be investigated.

The synthesis of osteocalcin depends on both vitamin K and vitamin D. While vitamin K is necessary for the γ-carboxylation of osteocalcin, vitamin D is an essential cofactor involved in the transcription of osteocalcin. It is therefore evident that a nutritional interaction between these fat-soluble vitamins exists, but the interaction is likely to be more complex when calcium and phosphorus are also considered. These two minerals are essentially what bone is composed of and their metabolism is inextricably linked to vitamin D. Osteocalcin may play a role in calcium regulation via the removal of calcium ions form blood for bone matrix synthesis On the other hand, the deposition of calcium in vascular tissues and the increased the risk of cardiovascular disease is also mediated by VKDPs. The relationship of vitamins D and K and their roles in these events awaits detailed investigation.

8.5.5 Role of new technologies for studying vitamin K During the last decade, tandem mass spectrometry (MS/MS) has emerged as the principal technology for global-scale qualitative and quantitative examinations of protein PTM analysis (Giorgianni & Beranova-Giorgianni 2016). In the past few years MS-based protein research has evolved from producing mere lists of proteins identified, to producng highly sophisticated strategies for the analysis of complex biological systems (Macek et al. 2009).

When the results of Chapters 5, 6 and 7 are considered together, further examination of the role of osteocalcin is warranted. A key outcome of this thesis was the development of a novel method: CACHE. This method requires further optimisation and validation that was outside the scope of this thesis.

With the exciting developments in the fields of proteomics, this methodology was touched on in the last chapter of this thesis. A new proteomics-based method was outlined that could potentially quantify the systemic effect of vitamin K on the carboxylated proteins of the plasma proteome of the horse. Vitamin K like all vitamins, has effects on many different proteins therefore, it made sense to develop a method that employed cutting-edge proteomics technology to enrich for carboxylation. It is anticipated that this approach will provide new

205 insights into the metabolism of vitamin K and its intrinsic relationship to osteocalcin and other VKDPs.

Initial method development and optimisation of an alkaline hydrolysis method was undertaken, but due to time constraints and scope of the PhD project this could not be pursued. This technique however, is very promising and should be developed in future experiments. A global amino acid (alkaline hydrolysis) assay may be useful. This method however, is not protein specific and no genomic information will be gleaned from results. It would provide a global picture of the carboxylated amino acids in a tissue but not identify the specific proteins.

The applicability of ICP-MS was also explored. Initial planning sought to harness both LC and ICP-MS capabilities however, due to the significant financial investment this required in order for the instrumentation to be operational this had to be abandoned. The alternative was to collect offline fractions and analyse them via ICP-MS. Due to contamination issues this approach did not produce quality data. If possible, future studies should investigate the use of LC-ICP-MS as it is anticipated that this approach should yield better quality results.

Any future studies should include NMR experiments to analyse the structure of native equine osteocalcin. Equine bone samples have also been collected and stored to extract osteocalcin (native form) in future experiments. This would ensure that osteocalcin peptides of biological significance to the horse can be monitored.

206

8.6 The final word The pioneering work of Dam (1935a) and Almquist and Stokstad (1935a) established the key role of vitamin K in coagulation but who would have imagined the plethora of roles now attributed to this “forgotten” vitamin. It is through the actions of VKDPs that vitamin K exerts it many roles. While osteocalcin has been the forefront of research to delineate the role of vitamin K in bone metabolism, other VKDPs have recently come to light, in particular MGP and GRP. It appears that vitamin K exerts dietary cues to cartilage and bone via VKDPs and presumably through this mechanism is involved in the development of bone diseases in horses. While the aetiology of bone diseases is multifactorial and undoubtedly caused by a number of cumulative issues, unravelling the role(s) of vitamin K will be difficult. When this occurs, it will join vitamin D, calcium and phosphorus in being central to the maintenance of this very important tissue.

Biological systems are immensely complex. The complexity and the interdependence of certain functions is reflected in many aspects of this thesis. The results of the current studies pose as may questions as they answer. However, the findings do provide new insights into the possible relationship between maternal nutrition and skeletal abnormalities in growing horses. Although there is still much to be discovered, it is evident that there may be many interactions between the VKDPs to maintain homeostasis. While early evidence points to this in the horse, much research is needed in order to better understand these complex interactions. Only then can the vitamin K and osteocalcin enigma be unmasked

207

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Appendices

Appendix 1: Animal ethics approval certificate……………………………………………..237 Appendix 2: Table A.2.1 Mare and foal data…………………………………………….…..239 Appendix 3: BioMare Cubes® nutritional information……………………………….…….243 Appendix 4: Analysis of pasture samples……………………………………………….…...245 Appendix 5: VKDP FASTA file search………………………………………………….….247 Appendix 6: Example of protein search summary report in ProteinPilot™ software…….….251 Appendix 7: Example of mascot search summary report…………………………………....252 Appendix 8: List of 25 equine aligned gene entries (elute tissue fraction) from 44 protein entries submitted to the PANTHER classification tool………………...……..253 Appendix 9: Swath (DIA) extracted synthetic asp-n peptides…………………………….…254 Appendix 10: List of equine plasma proteins identified from wash and elute fractions CACHE……………………………………………………..………………..256 Appendix 11: Elute and wash fraction Mascot VKDP searches………………………...…...263

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Appendix 1: Animal ethics approval certificate

237

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Appendix 2: Table A.2.1 Mare and foal data

MARE ID FOAL ID SEX SIRE ID TREATMENT FOALING DATE Baseline 12hr 24hr 7days 14days

1 1’16 C A Control 17/09/2016 17/09/2016 17/09/2016 18/09/2016 23/09/2016 30/09/2016

0:45 12:00 0:05 9:25 11:15

2 2’16 F S Control 24/09/2016 24/09/2016 24/09/2016 25/09/2016 30/09/2016 7/10/2016

5:40 16:50 5:55 10:50 10:45

8 8’16 C A KQ 24/09/2016 24/09/2016 24/09/2016 25/09/2016 30/09/2016 7/10/2016

8:25 20:55 8:40 12:00 11:10

10 10’16 F S KQ 30/09/2016 30/09/2016 30/09/2016 1/10/2016 7/10/2016 14/10/2016

4:20 16:45 5:05 9:00 9:15

7 7’16 C S KQ 5/10/2016 5/10/2016 5/10/2016 6/10/2016 12/10/2016 19/10/2016

3:25 3:20 3:15 10:50 10:30

6 6’16 F S Control 6/10/2016 6/10/2016 6/10/2016 7/10/2016 12/10/2016 19/10/2016

0:08 12:20 0:00 10:45 11:05

9 9’16 F A KQ 6/10/2016 6/10/2016 6/10/2016 7/10/2016 12/10/2016 19/10/2016

8:05 20:20 8:15 9:25 11:25

3 3’16 C S Control 8/10/2016 8/10/2016 8/10/2016 9/10/2016 14/10/2016 21/10/2016

1:30 13:30 12:30 10:40 12:00

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11 11’16 C A KQ 12/10/2016 12/10/2016 12/10/2016 13/10/2016 19/10/2016 26/10/2016

2:30 14:30 2:00 11:30 9:50

5 5’16 C A Control 16/10/2016 16/10/2016 17/10/2016 17/10/2016 24/10/2016 31/10/2016

23:40 12:15 23:25 10:00 10:26

12 12’16 F S KQ 17/10/2016 17/10/2016 17/10/2016 18/10/2016 24/10/2016 31/10/2016

4:45 17:05 4:40 9:30 10:00

4 4’16 F A Control 20/10/2016 20/10/2016 21/10/2016 21/10/2016 28/10/2016 4/11/2016

21:30 10:00 21:15 14:30 9:55

240

21days 28days 35days 42days 49days 56days 70days 84days 98days 112days 140days

7/10/2016 14/10/2016 21/10/2016 28/10/2016 4/11/2016 11/11/2016 25/11/2016 9/12/2016 23/12/2016 6/01/2017 3/02/2017

11:45 10:15 11:25 12:50 10:55 9:51 8:55 9:15 9:03 8:51 8:15

14/10/2016 21/10/2016 28/10/2016 4/11/2016 11/11/2016 18/11/2016 2/12/2016 16/12/2016 30/12/2016 13/01/2017 10/02/2017

9:35 9:45 12:40 9:40 9:36 10:00 7:30 8:51 7:01 8:05 8:29

14/10/2016 21/10/2016 28/10/2016 4/11/2016 11/11/2016 18/11/2016 2/12/2016 16/12/2016 30/12/2016 13/01/2017 10/02/2017

11:00 10:30 13:05 10:25 7:12 9:45 7:41 8:39 6:50 7:40 8:27

21/10/2016 28/10/2016 4/11/2016 11/11/2016 18/11/2016 25/11/2016 9/12/2016 23/12/2016 6/01/2017 20/01/2017 17/02/2017

9:10 14:00 10:45 9:36 10:07 8:45 8:50 8:51 8:42 8:28 8:35

26/10/2016 2/11/2016 9/11/2016 16/11/2016 23/11/2016 30/11/2016 14/12/2016 28/12/2016 11/01/2017 25/12/2017 22/02/2017

11:10 11:00 9:55 10:05 8:32 8:49 7:22 8:30 7:40 8:06 9:00

26/10/2016 2/11/2016 9/11/2016 16/11/2016 23/11/2016 30/11/2016 14/12/2016 28/12/2016 11/01/2017 25/12/2017 22/02/2017

10:50 11:15 9:35 9:24 7:44 8:38 7:30 8:20 7:50 8:16 8:47

26/10/2016 2/11/2016 9/11/2016 16/11/2016 23/11/2016 30/11/2016 14/12/2016 28/12/2016 11/01/2017 25/12/2017 22/02/2017

9:35 10:00 9:25 10:00 7:57 9:41 7:11 8:11 7:30 8:30 8:42

28/10/2016 4/11/2016 11/11/2016 18/11/2016 25/11/2016 2/12/2016 16/12/2016 30/12/2016 13/01/2017 27/01/2017 24/02/2017

13:20 10:35 16:48 9:30 9:05 8:00 8:22 6:47 7:55 8:43 9:00

2/11/2016 9/11/2016 16/11/2016 23/11/2016 30/11/2016 7/12/2016 21/12/2016 4/01/2017 18/01/2017 1/02/2017 1/03/2017

241

11:05 10:15 9:10 9:50 9:07 9:50 8:27 8:05 8:17 8:02 8:41

7/11/2016 14/11/2016 21/11/2016 28/11/2016 5/12/2016 12/12/2016 26/12/2016 9/01/2017 23/1/201 6/02/2017 6/03/2017

9:40 9:40 9:12 9:28 8:50 8:38 8:30 8:10 8:22 8:27 8:15

7/11/2016 14/11/2016 21/11/2016 28/11/2016 5/12/2016 12/12/2016 26/12/2016 9/01/2017 23/1/201 6/02/2017 6/03/2017

10:20 9:50 10:00 9:00 9:00 8:45 8:40 8:00 8:30 8:22 8:30

11/11/2016 18/11/2016 25/11/2016 2/12/2016 7/12/2016 16/12/2016 30/12/2016 13/01/2017 27/01/2017 10/02/2017 10/03/2017

19:12 9:10 8:35 7:50 9:00 8:56 7:10 7:50 7:58 8:14 9:04

242

Appendix 3: BioMare Cubes® nutritional information.

BREEDING AND GROWING HORSES A fully extruded feed with readily digested energy, high quality protein and essential vitamins and minerals that support optimum fertility, milk production and growth and development in a convenient and easy to feed cube. Provides a balance of nutrients vital for optimum fertility, conception and milk production in broodmares, and sound growth and development of foals, weanlings and yearlings under all seasonal conditions. Provides highly digestible and available energy, quality protein, essential amino acids and balanced vitamins and minerals, including Bioplex® organic trace minerals to ensure optimum uptake and utilisation. The digestible nature of this feed coupled with the high quality protein also makes it perfect for spelling horses that need to put on condition and rebuild muscles. Nutrients Supplied by BioMare Cubes®

Pryde's have formulated Pryde's BioMare Cubes® to provide a balanced nutrient intake based on the knowledge of the specific needs of horses and the nutrient content of common feeds. Formulations are updated regularly as new research findings become available.

Pryde's BioMare Cubes® provide the following nutrients: Major Nutrients: Macro Minerals: Digestible Energy 14.2 MJ/kg Calcium 11.5 g/kg Crude Protein 15.80% Phosphorus 8.7 g/kg Crude Fibre 3.80% Sodium 5.8 g/kg Crude Fat 3.70% Chloride 9 g/kg Salt 1.40% Magnesium 1.7 g/kg Lysine 7.4 g/kg Potassium 5.6 g/kg Lysine Digestibility 85% Methionine 2.2 g/kg Methionine Digestibility 85%

Vitamins: Trace Minerals: Vitamin A 7500 IU/kg Copper 54 mg/kg Vitamin E 195 IU/kg Zinc 153 mg/kg Vitamin D 1050 IU/kg Selenium 0.7 mg/kg Vitamin K 3.1 mg/kg Manganese 116 mg/kg Vitamin B1 14.2 mg/kg Iodine 1.2 mg/kg

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Vitamin B2 3.9 mg/kg Iron 140 mg/kg Niacin 41 mg/kg Cobalt 0.5 mg/kg Vitamin B5 6.2 mg/kg Vitamin B6 4.5mg/kg Folic Acid 3.6 mg/kg Biotin 0.2 mg/kg

Ingredients; Extruded Corn. Extruded Barley, Extruded Wheat, Extruded Soybean, Extruded Faba Beans, Extruded Lupins, Calcium Phosphate, Salt, Cold Pressed Canola Oil, Dried Molasses, Pryde's trace mineral and vitamin premix, including Bioplex® trace minerals for breeding and growing horses, Limestone, Lysine and Vitamin E.

Daily Feeding Rates for BioMare Cubes® (kg/day): Body Weight (kg) Class of Horse 300 400 500 600 700 Dry and Early Pregnant Mares 1.0 - 2.0 1.5 - 2.5 2.0 - 3.0 2.5 - 3.5 3.0 - 4.0 Late Pregnant Mares 1.5 - 2.5 2.0 - 3.0 2.5 - 3.5 3.0 - 4.0 3.5 - 4.5 Lactating Mares 2.5 - 4.0 3.0 - 4.5 3.5 - 5.0 4.0 - 5.5 4.5 - 6.0 Breeding Stallions 1.0 - 2.0 1.5 - 2.5 2.0 - 3.0 2.5 - 3.5 3.0 - 4.0 Spelling Horses 1.0 - 2.0 1.5 - 3.0 2.0 - 3.5 2.5 - 4.0 3.0 - 4.5 Expected Mature Body Weight (kg) 300 400 500 600 700 Weanlings 1.5 - 2.0 2.0 - 2.5 2.5 - 3.0 3.0 - 3.5 3.5 - 4.0 Yearlings 1.5 - 2.5 2.0 - 3.0 2.5 - 3.5 3.0 - 4.0 3.5 - 4.5

Feeding Directions For the best results with Pryde's BioMare Cubes®: The daily feeding rates should be used as a guide. The amount fed will depend on the quality and quantity of pasture, hay and chaff available, along with the horse's body condition, growth rate and stage of development, pregnancy, lactation. Feed in conjunction with good quality pasture, hay and/or chaff. Divide the diet into two or more smaller feeds if feeding more than 0.5 kg per 100 kg of body weight/day. Introduce into your horse's ration gradually over a period of 1 - 2 weeks. Clean, fresh water and a salt lick should be made available at all times.

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Appendix 4: Analysis of pasture samples. Dried pasture samples were sent to Dairy One Forage Testing Laboratory, USA for analysis. Prior to dispatch, the pasture samples were dried as follows (Dairy One Forage Lab). Representative samples were mixed thoroughly and then a sub-sample was placed in a pre- weighed paper bag (weight A) and re-weighed (weight B). The sample was then microwaved with a glass of water for 3 minutes. Samples were then removed, weighed, and then returned to the microwave and heated for an additional minute. This heating process was repeated until the weight of the sample no longer changed and all water was removed (weight C). The dry matter was then calculated as follows: % Dry Matter = (C - A)/ (B - A) x 100; For example: Paper bag weight = 10g (weight A) Paper bag + sample weight = 110g (weight B) Paper bag + dry sample weight = 60g (weight C), % Dry Matter = (60 - 10)/ (110 - 10) x 100 = 50/100 x 100 = 50%.

A representative pasture sample collected in zip lock bags ready for drying and analysis.

Supplementary feed Supplementary concentrate samples were collected with a feed scoop from three different batches, placed in clean zip lock bags (35x27cm) and sent for complete nutrient analysis to Equi-Analytical Laboratories, USA.

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Table A.4.1: Nutrient analysis of pasture, hay and feed.

Units Pasture Hay BioMare Cubes® DE* MJ/kg DM 14.2 NDF* % DM 58.6 67.9 - ADF* % DM 36.2 46.7 - Ash % DM 8.3 12 - Starch % DM 1.2 0.2 - CP* g/kg DM 127 139 158.0 Crude fat g/kg DM 26 12 37.0 Calcium g/kg DM 3.1 3.6 11.5 Phosphorous g/kg DM 3.4 5.0 8.7 Magnesium g/kg DM 1.0 2.2 1.7 Potassium g/kg DM 19 22 5.6 Sodium g/kg DM 1 6.1 5.8 Iron mg/kg DM 332 246 140.0 Zinc mg/kg DM 45 44 153.0 Copper mg/kg DM 20 19 54.0 Manganese mg/kg DM 93 41 116.0 Cobalt mg/kg DM - - 0.5 Selenium mg/kg DM - - 0.7 Vitamin K mg/kg DM - - - Vitamin A IU/kg DM - - 7500.0 Vitamin E IU/kg DM - - 195.0 Vitamin D IU/kg DM - - 1050.0 Vitamin B1 mg/kg DM - - 14.2 Vitamin B2 mg/kg DM - - 3.9 Folic Acid mg/kg DM - - 3.6

*Digestible energy (DE), neutral detergent fibre (NDF), acid detergent fibre (ADF) and crude protein (CP).

246

Appendix 5: VKDP FASTA file search.

>sp|P83005|OSTCN_HORSE Osteocalcin OS=Equus caballus OX=9796 GN=BGLAP PE=1 SV=1 YLDHWLGAPAPYPDPLEPRREVCELNPDCDELADHIGFQEAYRRFYGPV >tr|F7B1M1|F7B1M1_HORSE Osteocalcin OS=Equus caballus OX=9796 GN=LOC100146589 PE=3 SV=2 MAEASGVNVGSGCEEKGPEGLSLEPVPPGTTISRVKLLDTMVDTFLQKLIAAGSYQRFTD CYKRFYQLQPEMTQRIYDKFITQLQTSIREEISEIKAEGNLEAVLNALDTIVEEGKDHKE PAWRPSGIPEKDLQRAMVPYFLQQRDALQRRVQKQEAENRQLADAVLAGRRQVEELQLQG QARQQAWQAQPREYKRCWRLGEAAQLSPEQQHQHTMRPLPLLALLALAALCLAGWADAKP SRAESGRGAAFVSKQEGSEVVKRFRRYLDHWLGAPAPYPDPLEPRREVCELNPDCDELAD HIGFQEAYRRFYGTA >tr|F6R013|F6R013_HORSE Matrix Gla protein OS=Equus caballus OX=9796 GN=MGP PE=3 SV=1 MKSLLLLSILAALAMAALCYESHESLESYELNPFLNRRNANTFMTPQQRWRARAQERIRE STKPAHELNREACEDFKLCERYAMVYGYDAAYNRYFRQRRWEK >tr|F7BFJ1|F7BFJ1_HORSE Prothrombin OS=Equus caballus OX=9796 GN=F2 PE=3 SV=2 MAHVRGLWLPGCLALATLFGLVHSQHVFLAPPHALSLLQRVRRANSGFLEELREGDLERE CVEEQCSHEEAFEALESSSVSGSFPACESVRKPREKLVECLEGNCAEGLGMNYRGHVNFT RSGIECQLWRSRYPHKPEINCTTHPGADLQENFCRNPDGSSSGPWCYTTDPTVRREECSI PVCGHGVTAQLTPYSSSTKNLSPSLESCVPDRGQQYQGRLAVTTHGSPCLVWASSDAEAL SKDQDFNREVKLVENFCRNPDGDEEGVWCYVAGKPGFFEYCDLNYCEDPLDEEAEDQFGE DPDAPIEGRTTDEDFPLFFDVKTFGSGEADCGLRPLFEKKSVEDKTEKELLDSYIDGRIV EGWDAELGLAPWWVMIFRKSPQELLCGASLISDRWVLTAAHCLLYPPWDKNFTENDLLVR IGKHSRTRYERGVEKISMLEKIYIHPKYNWRDNLDRDIALLKLRRPIAFSDHVHPVCLPD KETTTRLFHAGYKGRVTGWGNLKETWTGHIGEVQPSVLQVVNLPIVEHSVCKASTRIRIT DNMFCAGFKPDEGRRGDACEGDSGGPFVMKNPFNNRWYQIGVVSWGEGCDRNGKYGFYTH VFRLKKWIQKVIGRSGG >tr|F6TDH5|F6TDH5_HORSE Coagulation factor X OS=Equus caballus OX=9796 GN=F10 PE=3 SV=2 MAGPLCLVLLSASLAGLLLPGGSVFLSRDRAHGLLHRVRRANSFLEELKKGNLERECREE SCSFEEAREVFEDVEQTTEFWNKYKDGDQCDSNPCLNEGKCKDGLGEYTCTCLEGFEGKN CELSMRQLCSLDNGDCDQFCSEERNSVVCSCASGYILGDNGKSCISTEPFPCGKHTQGRG KRAADQATQSHEDPTQTDILEQYSPGDLAPTKSPGDLLGSNKTETNAENQQNLVRIVGGK ECQEGECPWQALLINEENEGFCGGTILNEYYILTAAHCLHQTRRFKVRVGDRNTEEEEGN EMAHEVEMIIKHNKFIRETYDFDIAVVKLKTPITFRMNVAPACLPEKDWAESTLMTQKSG IVSGFGRTHEKGRPSATLKMLEVPYVDRNTCKLSSSFVITQNMFCAGYDSNPEDACQGDS GGPHVTRFKDTYFVTGIVSWGEGCARKGKYGVYTKVTSFLKWIDRSMKARGGAQAERAAP VPHPH >tr|F7DGR1|F7DGR1_HORSE Coagulation factor XII OS=Equus caballus OX=9796 GN=F12 PE=3 SV=2 MRALLLLGSLLVSLELALSVPGQWGTRKVIKECVLPAVLTVTGEPCYFPFQYHRQLHYKC THRGRPGPRPWCATTPSFEQDQRWAYCLEPKKVKDHCSKHSPCQKGGTCVNTPSSPHCIC PERFTGKHCQREKCFEPQLLQFFHENEIWYRLGPAGVAKCQCKGPDGHCKPLASQVCRTN PCLNGGRCLEAEGRRLCRCPAGYAGRFCDVDTEASCYHGRGLGYRGTAGTTISGARCRPW ASEATYRNVTAEQARNWGLGDHAFCRNPDNDTRPWCFVWSGDRLSWEYCHLAQCQASAPA APQIPRPTQVPYGHQNLPSPSISALQKPQPTTPTPGSHATPEPPSHLPGTGLRGCGQRLR KRLSSLSRIVGGLVALPGAHPYIAALYSRHDFCAGSLIAPCWVLTAAHCLQNLRAPEELT VVLGQDRFNQSCEQCQTLAVRAYRLHEGFSPTTFQHDLALVRLEERADGSCALLSPFVQP

247

VCLPSSAVRPAEPKAAFCEVAGWGHQFEGAEEFSSFLQEAQVPLIPPELCSTLDAHGAAF TPGMLCAGFLEGGTDACQGDSGGPLVCEDETAEGQLILRGIISWGSGCGDRYKPGVYTDV SNYLGWIREHTAS >tr|F7ABW7|F7ABW7_HORSE Coagulation factor VII OS=Equus caballus OX=9796 GN=F7 PE=3 SV=2 MLSQPRGLALLCLLLCLQGSLAAVFITQEEAHSILHRQRRANWFLEELKPGSLERECKEE QCSFEEAREIFKDTERTKQFWLSYTDGDQCASNPCQNGGSCEDQLQSYICFCLDGFEGRN CETNTDDQLICMNNNGDCEQYCSDHAGARRSCWCHEGYTLQANGVSCTPTVEYPCGKIPV LEKRNDTKPQGRIVGGKVCPKGQCPWQALLKMNGELLCGGTLLDTTWVVSAAHCFDRIRS WKNLTVVLGEHDLSEEDGDEQEQQVAQIIVPDKYVRLKTDHDLALLRLRRPVTFTDYVVP LCLPEKAFSERTLTLVRFSSVSGWGQLLHRGATALELMLINVPRLRTQDCLEQSHRMEGS PALTENMFCAGYVDGTQDACKGDSGGPHATKFQGTWYLTGVVSWGEGCAAVGHFGVYTRV SQYIEWLRRLMRSEPHSEGLFRAPFP >tr|F6RFT9|F6RFT9_HORSE Coagulation factor IX OS=Equus caballus OX=9796 GN=F9 PE=3 SV=1 MRCLNMIMAESLGLVTICLLGYLLSAECTVFLDRENATKILNRPKRYNSGKLEEFVRGNL ERECMEEKCSFEEAREVFENTEKTTEFWKQYVDGDQCESNPCLHGGVCKDDINSYECWCQ PGFEGKNCELYATCSIKNGRCKQFCKNSADNKVICSCTAGYRLAEDQKSCEPAVPFPCGK VSVSHASMKVTRAETIFSNMNYENSTEAETIWDNITEFDLNRVVGGENAKPGQFPWQVLL HGKIAAFCGGSIINEKWVVTAAHCIEPGVKITVVAGEHNTEEIDHTEQKRNVIRAIPHHS YNATLNKYNHDIALLELDKPLTLNSYVTPICVADKDYTNIFLKFGSGYVSGWGRVFSRGR SASILQHLKVPLVDRATCLRSTKFTIHNNMFCAGFHEGGKDSCQGDSGGPHVTEVEGTSF LTGIISWGEECAVKGKYGIYTKVSRYVNWIKEKTKLT >tr|A0A3Q2HXW1|A0A3Q2HXW1_HORSE Coagulation factor XII OS=Equus caballus OX=9796 GN=F12 PE=3 SV=1 MLSCRKRIFQGCVLFPTRPRGEVPGQWGTRKVIKECVLPAVLTVTGEPCYFPFQYHRQLH YKCTHRGRPGPRPWCATTPSFEQDQRWAYCLEPKKVKDHCSKHSPCQKGGTCVNTPSSPH CICPERFTGKHCQREKCFEPQLLQFFHENEIWYRLGPAGVAKCQCKGPDGHCKPLASQVC RTNPCLNGGRCLEAEGRRLCRCPAGYAGRFCDVDTEASCYHGRGLGYRGTAGTTISGARC RPWASEATYRNVTAEQARNWGLGDHAFCRNPDNDTRPWCFVWSGDRLSWEYCHLAQCQAS APAAPQIPRPTQVPYGHQNLPSPSISALQKPQPTTPTPGSWEGVGSSHATPEPPSHLPGT GLRGCGQRLRKRLSSLSRIVGGLVALPGAHPYIAALYSRHDFCAGSLIAPCWVLTAAHCL QNLRAPEELTVVLGQDRFNQSCEQCQTLAVRAYRLHEGFSPTTFQHDLALVRLEERADGS CALLSPFVQPVCLPSSAVRPAEPKAAFCEVAGWGHQFEGAEEFSSFLQEAQVPLIPPELC STLDAHGAAFTPGMLCAGFLEGGTDACQGDSGGPLVCEDETAEGQLILRGIISWGSGCGD RYKPGVYTDVSNYLGWIREHTAS >sp|Q28380|PROC_HORSE Vitamin K-dependent protein C (Fragment) OS=Equus caballus OX=9796 GN=PROC PE=2 SV=1 ENGEVDLDIQEVIMHPNYSKSSSDNDIALLRLARPATFSQTIVPICLPDSGLSERELTQA GQETVVTGWGYRSETKRNRTFVLNFIKVPVVPHSECVRTMHNLVSENMLCAGILGDTRDA CEGDSGGPMVASFRGTWFLVGLVSWGEGCGRLHNYGV >tr|F6ZSU8|F6ZSU8_HORSE Gla domain-containing protein OS=Equus caballus OX=9796 GN=PRRG2 PE=4 SV=2 MRGHPSLLLLYLGLTTCLDTSAPGEQDQEVFLDSPEAQSFLGGRRRIPRANHWDLELLTP GNLERECQEERCSWEEARECFEDNTLTERFWEDYIYNGKGGRGRVDVAGLAVGLTSGILL IVLAGLGAFWYLHWRRRRGQQPSPQEAELVSPLSSLGPPTPLPPPPPLPPGLPTYEQALA ASGVHDAPPPPYTRYRAGLLPRGGAQGAGTAGGGAWARGRSLEFGAYYPGRGWESGGGAL CHGLGAWPSGGGVVCVCVCVCVCVCVCVCVCVCVCVCVCVCVCVCVCVCVCVCVCVCV

248

>tr|A0A3Q2HAN3|A0A3Q2HAN3_HORSE Protein S OS=Equus caballus OX=9796 GN=PROS1 PE=4 SV=1 MQGQDKNVDDQNYPICSILSVLCSLPGSLAARFWLTSSSPTSVSHEPSKPDALRSESSKS SWPLSPTPSPRPPGLCKCLRPSPFPLGNFTFRRDGRSAGVWDLEGKLSPQCLPGLDTLPE PAPSSEKLPGDVPLITPPGWGRERAVPSAPGPTVPRLPPGSGWPRLAPGSGCALCAVRSL QRATPAACSSAARRAPPPSPPARVPAARPRPGPRRAPPPPPRSRVPAARLDRPRLFAMRV PRGRCAALLACLALALPVSEANFLSKQHASQVLVRKRRANSILEETKKGNLERECIEELC NKEEAREIFENIPETEYFYPKYLGCLGSFRAGLFTAARQSTNAYPDLRSCVNAIPDQCNP LPCKEDGYMSCKDGQATFTCVCKSGWQGEMCESDVNECKDPLNVNGGCSQICNNTPGSYY CSCKGGFVMLSNEKDCKDVDECTTQPSICGTAVCKNVPGDYECECAEGYSYNPSSKSCED VDECSENMCAQLCVNYPGGYYCYCDGKKGFKLAQDQKSCEAVPVCLPLNLDKNYELLYLA EQFVGVVLYLKFRLPDITRFSAEFDFRTYDSEGVILYAESLDHSAWFLIALRDGKIEIQF KNEHTTQITTGGKVINNGLWNMVSVEELEYSISVKIAKEAVMNINKPENLFKLTNGFLET KVYFAGLPRKVENALIRPINPRLDGCIRGWNLMNQGASGVKEIIQEKQNKHCLVSVEKGS YFPGSGVAQFSINYDNTSSAEGWHVNVSLRIRPSTGTGVMFALVSGNTVPFALSLVDSTS EKLQDILVSVESMVIYQIEALSLCSNQQSYLEFRVNRDSLQVSTPLRNNVIYSEDLPRQF ANLDKAMQGTMATYLGGLPDVPFSATPVNAFYSGCMDVNVNGAQLDLDEAVSKHNDIRAH SCPSVWRNTKRS >tr|F6SR87|F6SR87_HORSE Protein Z, vitamin K dependent plasma glycoprotein OS=Equus caballus OX=9796 GN=PROZ PE=3 SV=2 MASCVPLLLVLVPLAVPAAEPSGGRLLRGGAVSRCRSSWVSPCPLTGSAFFPCHAVFLSA SKANTVLARWKRAGSYLLEELFEGNLEKECYEEICVYEEAREVFENDATTGEFWTRYMGG SPCTSQPCRNNGSCQDSIRSYTCTCAPGYEGRDCAFAKNECHPLRTDGCQHFCHPGHESY RCSCAKGYKLGRDRKSCIPHEKCACGILKSESVARPPNSTQSLQVFPWQVKLTNSKGEDF CGGVIIQENFVLTTAKCSLLHKNITVKTNFPRTSRDPLTIAVQSVHVHMRYEEETGDNDV SLLELGLPIQCPDAGLPVCMPERDFAERALIPRTEGLLSGWTLNGSRLGNAPTQLLVTHM DSEECGQALDVTVTTRTYCERGTVAGGVRWAEGSMAAREHEGTWFLTGILRSAPTDEHGR AFLLTKVSRYSLWFRQIMKQLSPANQKD >tr|F6WIB0|F6WIB0_HORSE Vitamin K epoxide reductase complex subunit 1 OS=Equus caballus OX=9796 GN=VKORC1 PE=4 SV=1 MGASWRSPGWVRLALCLAGLMLSLYALHVKAARARDKDYRALCDVGTAISCSRVFSSRWG RGFGLVEHVLGRDSILNQSNSIFGCIFYTLQLLLGCLQGRWASTLLLLSSLVSLAGSLYL AWILFFVLYDFCIVCITTYAINVGLMILSFREVQGPQGKVKGH >tr|F7CXX9|F7CXX9_HORSE Proline rich and Gla domain 3 OS=Equus caballus OX=9796 GN=PRRG3 PE=4 SV=2 MAVFLEAKNAHSVLKRFPRANEFLEELRQGTIERECMEEICSYEEVKEVFEDKEKTMEFW KGYPNAVYSVRDPAQSSDAMYVVVPLLGVALLIVIALFIIWRCQLQKATRHHPSYAQNRY LASRAGHSLPRVMVYRGTVHSQGESSGHREAGSNPQVALGPSRGGRTTVRLESTLYLPEL ALSRLSSATPPPSYEEVTAPQESSSEEASVSYSDPPPKYEEIVAANPGSDK >tr|F7AD96|F7AD96_HORSE Proline rich and Gla domain 4 OS=Equus caballus OX=9796 GN=PRRG4 PE=4 SV=2 MFALVVLLSQLPPVVAAFPRHAGEDVFRSKEEANFFIRRHLLYNRFDLELFTPGDLEREC QEELCNYEEAREIFVDEDKTMTFWQEYSIKGLNTKSDGNREKIDVMGLLTGLIAAGVFLV IFGLLGYYLCITKCNRQRHPGSSATCRRRGRHTPSIVFRRPEEAVLCPSPPPEEDAGLPS YEQAMALTRKHNVSPPPPYPGPAKGFGVFKKSMSLPSH >tr|A0A3Q2I8M6|A0A3Q2I8M6_HORSE Proline rich and Gla domain 1 OS=Equus caballus OX=9796 GN=PRRG1 PE=4 SV=1 VFLTEEKANSVLKRYPRANGLFEEIRQGNIERECKEEVCTFEEAREAFENNEKTKEFWST

249

YTKAQQGESNRGSDWFQFYLTFPLIFGLFIILLVIFLIWRCFLRNKTRRQTVTEGHIPFP QHLNIITPPPPPDEVFDSSGLSPGFLEYVVGRSDSVSTRLSNCDPPPTYEEATGQVNLRR SETEPHLDPPPEYEDIINSNSASAIAMVPVVTTIK r|F6Y0G5|F6Y0G5_HORSE Periostin OS=Equus caballus OX=9796 GN=POSTN PE=4 SV=2 MIPFLPIFSLLLLFAVNPANANGHYDKILAHSRIRGRDQGPNVCALQQILGTKKKYFSTC RNWYQGAICGKKTTVLYECCPGYMRMEGMKGCPAVLPIDHVYGTLGIVGATTTQGYSDVS KLREEIEGKGSFTYFAPSNEAWDNLDPDIRRGLESNVNVELLNALHSHMVNKRMLTKDLK NGMIVPSMYNNLGLFINHYTNGVVTVNCARIIHGNQIATNGVVHVIDRVLTQIGTSIQDF IEAEDDLSSFRAAAITSDILEALGRDGHFTLFAPTNEAFEKLPRGVLERIMGDKVASEAL MKYHILNTLQCSEAIMGGAVFETLEGNTIEIGCDGDSITVNGIKMVNKKDIVTNNGVIHL IDQVLIPDSAKQVIELAGNQQTTFTDLVAQLGLASALRPDGEYTLLAPVNNAFSDDTLSM DQRLLKLILQNHILKVKVGLNELYNGQKLETIGGKQLRVFVYRTAVCIENSCMVRGSKQG RNGAIHIFREIIKPAEKSLHEKLKQDKRFSIFLSLLEAADLKELLTQPGDWTLFVPTNDA FKGMTNEEKEILIRDKNALQNIILYHLTPGVFIGKGFEPGVTNILKTTQGSKIYLKGVND TLLVNELKSKESDIMTTNGVIHVVDKLLYPADTPVGNDQLLEILNKLIKYIQIKFVRGSS FKEIPMTVYTTKIITKVVEPKIKVIEGSLQPIIKTEGPTITKVKIEGEPEFRLIKEGETV TQVIHGEPIIKKYTKIIDGVPVEITEKETREERIITGPEIKYTRISTGGGETEETLKKLL QEEVTKVTKFIEGGDGHLLEDEEIKRLLQGDTPVRKMQANKRVQGSRRRSREGRSQ >sp|O19011|TGFB1_HORSE Transforming growth factor beta-1 proprotein OS=Equus caballus OX=9796 GN=TGFB1 PE=2 SV=1 MPPSGLRLLPLLLPLLWLLVLTPGRPAAGLSTCKTIDMELVKRKRIEAIRGQILSKLRLA SPPSQGEVPPGPLPEAVLALYNSTRAQVAGESAETEPEPEADYYAKEVTRVLMVEKENEI YKTVETGSHSIYMFFNTSELRAAVPDPMLLSRAELRLLRLKLSVEQHVELYQKYSNNSWR YLSNRLLTPSDSPEWLSFDVTGVVRQWLSQGGAMEGFRLSAHCSCDSKDNTLRVGINGFS SSRRGDLATIDGMNRPFLLLMATPLERAQQLHSSRHRRALDTNYCFSSTEKNCCVRQLYI DFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQV LEPLPIVYYVGRKPKVEQLSNMIVRSCKCS

250

Appendix 6: Example of protein search summary report in ProteinPilot™ software showing identification and database search properties of assembled data from equine plasma of data dependent acquisition (DDA) experiments using TripleTOF5600 (SCIEX) instrument for the construction of a peptide spectral library. This is an automatically generated report.

251

Appendix 7: Example of mascot search summary report.

252

Appendix 8: List of 25 equine aligned gene entries (elute tissue fraction) from 44 protein entries submitted to the PANTHER classification tool.

No. Gene name; Gene symbol

1 Creatine kinase, M-type; CKM

2 Glyceraldehyde-3-phosphate dehydrogenase; GAPDH

3 Betaine--homocysteine S-methyltransferase; BHMT

4 Alpha-1,4 glucan phosphorylase; PYGM

5 Aldehyde dehydrogenase, mitochondrial; ALDH2

6 Carbonic anhydrase 3; CA3

7 Creatine kinase, mitochondrial 2; CKMT2

8 Regucalcin; RGN

9 ATP synthase subunit beta; ATP5F1B

10 tr, alpha 1, skeletal muscle; ACTA1

11 Tropomyosin alpha-4 chain; TPM4

12 Alcohol dehydrogenase E chain; unassigned

13 Calreticulin; CALR

14 Myosin-2; MYH2

15 Serum albumin; ALB

16 Actin, cytoplasmic 1; ACTB

17 Epoxide hydrolase 1; EPHX1

18 Myosin-1; MYH1

19 Apolipoprotein A1; APOA1

20 Tropomyosin 3; TPM3

21 Clusterin; CLU

22 Fibrinogen gamma chain; FGG

23 Myoglobin; MB

24 Serpin family C member 1; SERPINC1

25 Uncharacterized protein; CFB

253

Appendix 9: Swath (DIA) extracted synthetic asp-n peptides.

254

255

Appendix 10: List of equine plasma proteins identified from wash and elute fractions – CACHE.

Table A.7.3.1 Wash Proteins

No. Accession Name

1 gi|969812922 Chain A, Crystal Structure Of Equine Serum Albumin In Complex With Diclofenac And Naproxen Obtained In Displacement Experiment

2 gi|823630974 serotransferrin precursor [Equus caballus]

3 gi|706945 transferrin precursor [Equus caballus]

4 gi|664708399 serotransferrin-like [Equus przewalskii]

5 gi|664753781 alpha-2-macroglobulin-like [Equus przewalskii]

6 gi|958800570 serotransferrin isoform X2 [Equus asinus]

7 gi|664715914 apolipoprotein A-I [Equus przewalskii]

8 gi|953857243 complement C3-like [Equus caballus]

9 gi|194212541 complement C3-like [Equus caballus]

10 gi|958695472 haptoglobin-like [Equus asinus]

11 gi|958666372 fibrinogen beta chain [Equus asinus]

12 gi|664771918 complement C3 [Equus przewalskii]

13 gi|545209946 fibrinogen alpha chain [Equus caballus]

14 gi|958666378 fibrinogen gamma chain [Equus asinus]

15 gi|194225326 alpha-1-antiproteinase 2-like [Equus caballus]

16 gi|664771780 alpha-2-HS-glycoprotein [Equus przewalskii]

17 gi|197631767 alpha-1-antitrypsin [Equus caballus]

18 gi|664758590 complement C4-A [Equus przewalskii]

19 gi|953873926 apolipoprotein B-100 [Equus caballus]

20 gi|953848624 liver carboxylesterase-like [Equus caballus]

21 gi|958696132 complement factor B [Equus asinus]

22 gi|958737872 antithrombin-III [Equus asinus]

23 gi|664738192 plasminogen isoform X1 [Equus przewalskii]

256

24 gi|953864745 alpha-1B-glycoprotein [Equus caballus]

25 gi|953877247 ceruloplasmin isoform X1 [Equus caballus]

26 gi|664730291 hemopexin [Equus przewalskii]

27 gi|664763108 vitamin D-binding protein [Equus przewalskii]

28 gi|149716543 apolipoprotein A-IV [Equus caballus]

29 gi|664734892 afamin isoform X2 [Equus przewalskii]

30 gi|338722817 complement factor H isoform X1 [Equus caballus]

31 gi|3892519 transferrin, partial [Equus caballus]

32 gi|664740416 beta-2-glycoprotein 1 [Equus przewalskii]

33 gi|664705962 prothrombin [Equus przewalskii]

34 gi|953855943 alpha-2-macroglobulin isoform X2 [Equus caballus]

35 gi|953879542 histidine-rich glycoprotein [Equus caballus]

36 gi|85541966 Full=Clusterin; Contains: RecName: Full=Clusterin beta chain; Contains: RecName: Full=Clusterin alpha chain; Flags: Precursor

37 gi|99032240 Chain B, Atp Bound Gelsolin

38 gi|664771774 kininogen-1 isoform X2 [Equus przewalskii]

39 gi|958800575 serotransferrin [Equus asinus]

40 gi|664744007 plasma protease C1 inhibitor [Equus przewalskii]

41 gi|953868106 alpha-2-antiplasmin [Equus caballus]

42 gi|958794606 alpha-2-antiplasmin [Equus asinus]

43 gi|6176199 transferrin, partial [Equus caballus]

44 gi|953891026 complement factor H isoform X5 [Equus caballus]

45 gi|958669510 apolipoprotein E [Equus asinus]

46 gi|958790455 heparin cofactor 2 [Equus asinus]

47 gi|664749348 apolipoprotein A-II [Equus przewalskii]

48 gi|93279207 Chain B, Crystal Structure Of Carbonmonoxy Horse Hemoglobin Complexed With L35

49 gi|958724897 protein AMBP [Equus asinus]

50 gi|664752634 angiotensinogen [Equus przewalskii]

51 gi|93279206 Chain A, Crystal Structure Of Carbonmonoxy Horse Hemoglobin Complexed With L35

257

52 gi|664771706 apolipoprotein C-II [Equus przewalskii]

53 gi|953847626 complement factor I isoform X3 [Equus caballus]

54 gi|953851958 C4b-binding protein alpha chain [Equus caballus]

55 gi|953867178 membrane primary amine oxidase isoform X1 [Equus caballus]

56 gi|958711855 transthyretin [Equus asinus]

57 gi|958794798 pigment epithelium-derived factor [Equus asinus]

58 gi|664758132 hemoglobin subunit alpha [Equus przewalskii]

59 gi|958705700 fibronectin isoform X10 [Equus asinus]

60 gi|958730872 alpha-2-macroglobulin-like [Equus asinus]

61 gi|958747354 apolipoprotein C-III [Equus asinus]

62 gi|953870419 zinc-alpha-2-glycoprotein isoform X2 [Equus caballus]

63 gi|958704796 adiponectin [Equus asinus]

64 gi|958809428 complement component C9 isoform X2 [Equus asinus]

65 gi|664774476 serum paraoxonase/arylesterase 1 [Equus przewalskii]

66 gi|958675522 polymeric immunoglobulin receptor [Equus asinus]

67 gi|958705025 carboxypeptidase N subunit 2 [Equus asinus]

68 gi|958769434 vitronectin [Equus asinus]

69 gi|197631763 alpha-1-antitrypsin [Equus caballus]

70 gi|197631757 alpha-1-antitrypsin [Equus caballus]

71 gi|68067996 Full=Alpha-1-antiproteinase 2; AltName: Full=Alpha-1-antitrypsin 2; AltName: Full=Alpha-1- proteinase inhibitor 2; AltName: Full=SPI2; Flags: Precursor

72 gi|953876238 inter-alpha-trypsin inhibitor heavy chain H4 isoform X2 [Equus caballus]

73 gi|958793286 inter-alpha-trypsin inhibitor heavy chain H1 isoform X1 [Equus asinus]

74 gi|664703084 inter-alpha-trypsin inhibitor heavy chain H2 [Equus przewalskii]

75 gi|953876243 inter-alpha-trypsin inhibitor heavy chain H3 [Equus caballus]

76 gi|291478 lambda-immunoglobulin, partial [Equus caballus]

77 gi|291468 lambda-immunoglobulin [Equus caballus]

78 gi|42528291 immunoglobulin gamma 7 heavy chain, partial [Equus caballus]

79 gi|15026997 immunoglobulin gamma 1 heavy chain constant region, partial [Equus caballus]

258

80 gi|953894035 Ig gamma-1 chain C region [Equus caballus]

81 gi|42528293 immunoglobulin gamma 4 heavy chain, partial [Equus caballus]

82 gi|18996195 immunoglobulin gamma 5 heavy chain constant region, partial [Equus caballus]

83 gi|9858135 immunoglobulin G heavy chain, partial [Equus caballus]

84 gi|18996197 immunoglobulin gamma 6 heavy chain constant region, partial [Equus caballus]

85 gi|32442221 immunoglobulin gamma 3 heavy chain constant region, partial [Equus caballus]

86 gi|723943109 immmunoglobulin lambda light chain variable region, partial [Equus caballus]

87 gi|51831151 immunoglobulin mu heavy chain constant chain secreted form, partial [Equus caballus]

88 gi|300387668 immunoglobulin lambda light chain V-J region, partial [Equus caballus]

89 gi|723943082 immmunoglobulin lambda light chain variable region, partial [Equus caballus]

90 gi|723782931 immunoglobulin lambda light chain constant region, partial [Equus caballus]

91 gi|723782934 immunoglobulin lambda light chain constant region, partial [Equus caballus]

92 gi|300387704 immunoglobulin lambda light chain V-J region, partial [Equus caballus]

93 gi|488146 immunoglobulin kappa light chain [Equus caballus]

94 gi|300386770 immunoglobulin heavy chain V-D-J region, partial [Equus caballus]

95 gi|958718212 immunoglobulin J chain [Equus asinus]

259

Table A.7.3.2 Elute proteins

No. Accession Name

1 gi|969812922 Chain A, Crystal Structure Of Equine Serum Albumin In Complex With Diclofenac And Naproxen Obtained In Displacement Experiment

2 gi|194212541 complement C3-like [Equus caballus]

3 gi|194225326 alpha-1-antiproteinase 2-like [Equus caballus]

4 gi|545202733 alpha-1-antiproteinase 2-like [Equus caballus]

5 gi|6176199 transferrin, partial [Equus caballus]

6 gi|664708399 serotransferrin-like [Equus przewalskii]

7 gi|664715914 apolipoprotein A-I [Equus przewalskii]

8 gi|664729363 liver carboxylesterase-like [Equus przewalskii]

9 gi|664731106 fibrinogen alpha chain [Equus przewalskii]

10 gi|664734892 afamin isoform X2 [Equus przewalskii]

11 gi|664752634 angiotensinogen [Equus przewalskii]

12 gi|664753781 alpha-2-macroglobulin-like [Equus przewalskii]

13 gi|664758590 complement C4-A [Equus przewalskii]

14 gi|664763108 vitamin D-binding protein [Equus przewalskii]

15 gi|664771918 complement C3 [Equus przewalskii]

16 gi|664780060 complement factor H-like, partial [Equus przewalskii]

17 gi|706945 transferrin precursor [Equus caballus]

18 gi|953847626 complement factor I isoform X3 [Equus caballus]

19 gi|953855492 complement C1s subcomponent [Equus caballus]

20 gi|953855941 alpha-2-macroglobulin isoform X1 [Equus caballus]

21 gi|953857243 complement C3-like [Equus caballus]

22 gi|953864745 alpha-1B-glycoprotein [Equus caballus]

23 gi|953867178 membrane primary amine oxidase isoform X1 [Equus caballus]

24 gi|953870419 zinc-alpha-2-glycoprotein isoform X2 [Equus caballus]

25 gi|953877247 ceruloplasmin isoform X1 [Equus caballus]

260

26 gi|953879542 histidine-rich glycoprotein [Equus caballus]

27 gi|953885462 alpha-1-antiproteinase 2 isoform X1 [Equus caballus]

28 gi|958666372 fibrinogen beta chain [Equus asinus]

29 gi|958666378 fibrinogen gamma chain [Equus asinus]

30 gi|958691477 hemoglobin subunit beta [Equus asinus]

31 gi|958695472 haptoglobin-like [Equus asinus]

32 gi|958696132 complement factor B [Equus asinus]

33 gi|958704051 serpin A3-8-like [Equus asinus]

34 gi|958704648 clusterin [Equus asinus]

35 gi|958704766 alpha-2-HS-glycoprotein [Equus asinus]

36 gi|958705761 complement C3 [Equus asinus]

37 gi|958706284 complement factor H-like [Equus asinus]

38 gi|958711855 transthyretin [Equus asinus]

39 gi|958718289 alpha-fetoprotein [Equus asinus]

40 gi|958724897 protein AMBP [Equus asinus]

41 gi|958735085 hemopexin [Equus asinus]

42 gi|958737872 antithrombin-III [Equus asinus]

43 gi|958739433 serum paraoxonase/arylesterase 1 [Equus asinus]

44 gi|958756651 plasminogen isoform X2 [Equus asinus]

45 gi|958766862 prothrombin [Equus asinus]

46 gi|958769434 vitronectin [Equus asinus]

47 gi|958784600 apolipoprotein B-100 [Equus asinus]

48 gi|958790455 heparin cofactor 2 [Equus asinus]

49 gi|958791279 apolipoprotein C-II [Equus asinus]

50 gi|958794606 alpha-2-antiplasmin [Equus asinus]

51 gi|958794798 pigment epithelium-derived factor [Equus asinus]

52 gi|958797407 plasma protease C1 inhibitor [Equus asinus]

53 gi|958823918 apolipoprotein A-II [Equus asinus]

261

54 gi|99032240 Chain B, Atp Bound Gelsolin

55 gi|197631757 alpha-1-antitrypsin [Equus caballus]

56 gi|953876238 inter-alpha-trypsin inhibitor heavy chain H4 isoform X2 [Equus caballus]

57 gi|953876243 inter-alpha-trypsin inhibitor heavy chain H3 [Equus caballus]

58 gi|953876250 inter-alpha-trypsin inhibitor heavy chain H1 isoform X4 [Equus caballus]

59 gi|958697530 inter-alpha-trypsin inhibitor heavy chain H2 [Equus asinus]

60 gi|15020816 immunoglobulin gamma 1 heavy chain constant region, partial [Equus caballus]

61 gi|18996193 immunoglobulin gamma 3 heavy chain constant region, partial [Equus caballus]

62 gi|18996195 immunoglobulin gamma 5 heavy chain constant region, partial [Equus caballus]

63 gi|291474 lambda-immunoglobulin, partial [Equus caballus]

64 gi|32331167 immunoglobulin alpha constant heavy chain, partial [Equus caballus]

65 gi|32442221 immunoglobulin gamma 3 heavy chain constant region, partial [Equus caballus]

66 gi|346448 Ig lambda chain V region - horse (fragment)

67 gi|356494361 immunoglobulin lambda light chain constant region, partial [Equus caballus]

68 gi|42528291 immunoglobulin gamma 7 heavy chain, partial [Equus caballus]

69 gi|42528293 immunoglobulin gamma 4 heavy chain, partial [Equus caballus]

70 gi|488146 immunoglobulin kappa light chain [Equus caballus]

71 gi|51831151 immunoglobulin mu heavy chain constant chain secreted form, partial [Equus caballus]

72 gi|723943046 immmunoglobulin lambda light chain variable region, partial [Equus caballus]

262

Appendix 11: Elute and wash fraction Mascot VKDP searches.

Elute fraction Mascot VKDP database search results

263

Wash fraction Mascot VKDP database search results

m/z M(expt) M(calc) Peptide Score Expect Hits

437.7421 873.4696 873.4709 R. VTGWGNLK.E 29 0.0012 Prothrombin

590.7962 1179.5778 1179.5771 K.ELLDSYIDGR.I (61) 7.4e-07

590.7962 1179.5778 1179.5771 K.ELLDSYIDGR.I 61 7.3e-07

787.3750 1572.7348 1572.7348 R. TTDEDFPLFFDVK.T 104 3.9e-11

787.3750 1572.7348 1572.7348 R. TTDEDFPLFFDVK.T (80) 1.1e-08

787.3750 1572.7348 1572.7348 R. TTDEDFPLFFDVK.T (92) 6.2e-10

787.3750 1572.7348 1572.7348 R. TTDEDFPLFFDVK.T (85) 3.1e-09

264

Washes – HORSE_NCBI database

265