Application of Mass Spectrometry in Biology and Physiology

Application of Mass Spectrometry in

A dissertation submitted to the Graduate School of the University of Cincinnati in Partial Fulfillment of the Requirements for the Degree of

DOCTOR OF PHILOSOPHY (Ph.D.)

In the Department of Chemistry of McMicken College of Arts and Sciences by

Jiawei Gong

Bachelor of Science (B.S.), Chemistry Xiamen University, 2012

Dissertation Advisor: Joseph A. Caruso, Ph.D

Abstract

Mass spectrometry, as an analytical technique that sorts ions based on their mass to charge ratio, is playing significant roles in analysis not only in chemistry field, but also in other areas such as biology and physiology. In this dissertation, the application of mass spectrometry, including both atomic and molecular mass spectrometry, was investigated in those two areas mentioned above. Inductively coupled plasma mass spectrometry (ICPMS), a typical atomic analysis technique, is a powerful tool for elemental detection and speciation. Instrumental advances, such as the dynamic reaction cell and triple quad alignment, gave rise to monitoring sulfur and phosphorus that suffered a lot from polyatomic interferences and ionization issue in previous study. Additionally, ICPMS is capable of element specific detection, where the intensity of each element is directly proportional to the element present in the samples, allowing for element quantification via peak area integration. All these capabilities mentioned above opened a new window for the detection and quantification of DNA protein crosslinks (DPCs) as there is always sulfur in proteins and phosphorous in DNA. In this dissertation, an approach for purification and quantitative analysis of DPCs was established by increasing the sensitivity of sulfur and phosphorus signal via ICPMS. Further structural determination was completed by electron spray ion trap mass spectrometry (ESI-ITMS), a typical molecular analysis technique for DNA or proteins. This new approach achieved the quantitative analysis of DPCs with low i detection limit and high sensitivity and enabled the identification of the binding sites on the protein involved in the crosslinking reaction.

There are a variety of endogenous compounds affecting the cellular functions.

Purification and characterization of these compounds laid the foundation for the further research on physiology. Endogenous cardiotonic steroids (CTS) were considered important for human health as inhibitors of the Na+/K+-ATPase. In this dissertation, an approach was established to purify the CTS from pig skeletal muscles including batch affinity extraction (BAE), gel filtration separation and reverse phase chromatography. NADH coupled method was used to checked the activity of the each fraction after sequences of purification. The molecular weight of the active compounds was determined by ESI-ITMS. This new approach with the combination of serious separation methods saved time and labor even though

2D nuclear magnetic resonance (NMR) analysis is still needed for the structural determination.

iii Acknowledgement

I would like to acknowledge many people for helping me during my Ph.D. studies.

First of all, I would like to give special thanks to my research advisor, Dr. Joseph

Caruso for his tutorage and guidance. As a successful scientist and chemist himself, his is always encouraging me to think and work form the scientific perspective. In addition, his kindness and optimistic enthusiasm comfort me a lot, especially in the first year I came to Cincinnati. Unfortunately, he passed away at the end of 2015. However, his smile will stay in my memory forever. Hope him rest in peace in the other world.

I would like to express my deep appreciation to my committee members: Dr.

Landero, who trained me from beginning and helped me grow to an analytical chemist; Dr. Merino, who always inspire me by new ideas and explained biological issues in details; Dr. Zhang, who treats me as a friend; Dr. Guan, who is always helpful and encourage me a lot. Also, Dr. Heiny gave me a lot of support during the collaboration.

I would like to thank my colleagues in my research group, Cory Stiner, Skyler

Smith, Keaton Nahan, Ryan Saadawi, Megan Schmale, Traci Hanley, Amberlie

Clutterbuck, Anna Donnell, Nicole Hanks, for all the help they provided to me.

iv I am also grateful to the department of chemistry, University of Cincinnati, for the financial support in the form of teaching assistantship and to Agilent technologies for the instrument support.

v Table of Contents

Chapter 1 Introduction

1.1 Introduction to research 2 1.2 Application of mass spectrometry in a biochemistry model study: DNA protein crosslinks characterization using model molecules under UV radiation 3 1.2.1 Introduction 3 1.2.1.1 Formation of DPCs 4 1.2.1.1.1 Formaldehyde-induced DPCs 4 1.2.1.1.2 Cisplatin-induced DPCs 5 1.2.1.1.3 Metal-induced DPCs 6 1.2.1.1.4 Reactive oxygen species (ROS) -induced DPCs 7 1.2.1.2 Biological consequences of DPCs 8 1.2.1.3 Enzymatic repair of DPCs 8 1.2.1.4 Detection of DPCs 10 1.2.2. Research Overview 12 1.2.2.1 Overview of Chapter 2 12 1.2.2.2 Overview of Chapter 3 13 1.3 Application of mass spectrometry in physiology study: cardiotonic steroids project 14 1.3.1 Introduction 14 1.3.3.1 Na+/K+-ATPase 14 1.3.3.2 Compounds displaying ouabain-like activity: Cardiotonic Steroids (CTS) 15 1.3.3.3 Purification of CTS 18 1.3.2 Overview of Chapter 4 18

vi Chapter 2 Simultaneously Detection and Quantification of synthesized DNA

Protein Crosslinks by Triple Quad ICPMS (ICPQQQ)

2.1 Introduction 27 2.2 Experimental 31 2.2.1 Materials 31 2.2.2 Methods 31 2.2.2.1 DNA-Ribonuclease A Oxidative Cross-Linking 31 2.2.2.2 Optimization of the reaction in the octopole cell and other parameters 32 2.2.2.3 Quantification of sulfur and phosphor by using cap-RPHPLC-QQQ-ICPMS 33 2.2.2.4 Purification of the DPCs (DNA-Ribonuclease A cross-links) 33 2.2.2.5 Trypsin proteolysis of the DNA-Ribonuclease A cross-links 34 2.2.2.6 Pepetide identification after trypsin proteolysis by cap-RPLC-QQQ-ICPMS 35 2.3 Results and Discussion 35 2.3.1 Optimization of the instrument parameters 35 2.3.2 Quantification of S and P standards 37 2.3.3 Basic quantification and evaluation of laboratory synthesized DPCs 38 2.3.4 Cap HPLC-ICPMS detection of DPCs after trypsin proteolysis 39 2.4 Conclusions 40

Chapter 3 Structurally Characterization of DNA Protein Crosslinks (DPCs)

by nanoLC-ESI-MS

3.1 Introduction 49 3.2 Experimental 52

vii 3.2.1 Materials 52 3.2.2 Methods 53 3.2.2.1 2’-Deoxyguanosine-Lys derivative Oxidative Cross-Linking 53 3.2.2.2 Purification of the dG-AcLysOMe adduct by RP-HPLC 53 3.2.2.3 Structure determination of the dG-AcLysOMe adduct by ESI-ITMS 54 3.2.2.4 Thermal stability study of the cross-linking covalent bond 55 3.2.2.5 DNA-Ribonuclease A Oxidative Cross-Linking 55 3.2.2.6 Purification of the synthesized DPCs 56 3.2.2.7 Thermal Hydrolysis of DNA-Ribonuclease A cross-links and purification of dG-Ribonuclease 56 3.2.2.8 Trypsin proteolysis of the dG-Ribonuclease A 56 3.2.2.9 Peptide separation and identification by HPLC-Chip-ESI-ITMS 57 3.2.2.10 MASCOT protein data base search 58 3.2.2.11 Calculations 58 3.3 Results and Discussion 59 3.3.1 A model DPC: the dG-AcLysOMe adduct 59 3.3.2 Thermolysis of the dG-AcLysOMe adduct 60 3.3.3 Purification of laboratory synthesized DPCs by SEC 61 3.3.4 Expansion to identification of whole protein DPCs 61 3.3.5 Identification of the dG-containing peptide-by-peptide sequence matching 62 3.3.6 Cross-linking prediction 64 3.4 Conclusion 64

Chapter 4 Purification and Identification of Novel Endogenous Cardiotonic

Steroids (CTS) from pig skeleton muscle

4.1 Introduction 79

viii 4.2 Experimental 83 4.2.1 Materials 83 4.2.2 Methods 84 4.2.2.1 Homogenization of pig skeletal muscle 84 4.2.2.2 Batch affinity extraction 84 4.2.2.3 Post affinity gel filtration chromotography 85 4.2.2.4 Reverse Phase separation of OLC 86 4.2.2.5 Activity determination of OLC by NADH coupled essay 87 4.2.2.6 Mass spectrum analysis of active OLC fraciton 88 4.3 Results and Discussion 89 4.3.1 Post affinity gel filtration 89 4.3.2 Reverse Phase separation of OLC 90 4.3.3 Activity determination of OLC by NADH coupled essay 90 4.3.4 Mass spectrum analysis of active OLC fractions 91 4.4 Conclusion 92

Chapter 5 Future Directions

5.1 Future directions for DPCs project 110 5.2 Future directions of OLC project 112

ix List of Figures

Chapter 1:

Figure 1.1 Preferably targeted sited within DNA in the crosslinking reaction

Figure 1.2 Mechanism of formaldehyde-induced DPCs

Figure 1.3 Mechanism of cisplatin-induced DPCs

Figure 1.4 Chelation mechanism of Cr-induced DPCs

Figure 1.5 Structure of a) Marinobufagenin; b) ouabain; c) digoxin

Chapter 2:

Figure 2.1 Reaction Cell optimization for 47PO+ AND 48SO+

Figure 2.2 CapHPLC-QQQ-ICPMS chromatograms of organic standards

Figure 2.3 SEC-QQQ-ICPMS chromatograms of reactants and solution after

crosslinking reaction

Figure 2.4 CapHPLC-QQQ-ICPMS Chromatograms from tryptic digested samples

Chapter 3:

Figure 3.1 Reverse Phase chromatograms of AcLysOMe, 2-dG reations

Figure 3.2 MS & MS2 of the dG-AcLysOMe crosslinking compound

Figure 3.3 MS of the dG-AcLysOMe crosslinking compound after heating at 170

oC for 1 hour

Figure 3.4 Crosslinking reaction between 2-deoxguanosine (dG) and

N-acetyl-Lysine-Methyl ester (AcLysOMe)

Figure 3.5 Isoabsorbance plot after thermal hydrolysis of DPC x Figure 3.6 Mass spectrum for Identification of the dG-containing peptide by

peptide sequence matching

Figure 3.7 3D Structure of the Ribonuclease A

Chapter 4:

Figure 4.1 Structure of the Na+/K+-ATPase

Figure 4.2 NADH coupled method mechanism

Figure 4.3 Gel filtration chromatograms of molecular weight standards (black) and

OLC (red) sample after batch affinity extraction

Figure 4.4 Reverse phase chromatogram of Fa0

Figure 4.5 Reverse phase chromatogram of Fb0

Figure 4.6 Reverse phase chromatogram of Fc0

Figure 4.7 Reverse phase chromatogram of Fd0

Figure 4.8 Inhibition of Na+/K+-ATPase activity from ouabain

Figure 4.9 Dose response curves of Fa0 and all the fractions after reverse

separation from Fa0

Figure 4.10 Dose response curves of Fb0 and all the fractions after reverse

separation from Fb0

Figure 4.11 Dose response curves of Fc0 and all the fractions after reverse

separation from Fc0

Figure 4.12 Dose response curves of Fd0 and all the fractions after reverse

separation from Fd0 xi Figure 4.13 Mass spectra of active fraction Fb1

Figure 4.14 Mass spectra of active fraction Fc1

Figure 4.15 Mass spectra of active fraction Fc2

Figure 4.16 MS of active fractions, Fd1. and Fd2

Chapter 5:

Figure 5.1 Chelation mechanism to illustrate the Cr-induced crosslinking reaction

Figure 5.2 Free radical mechanism to illustrate the Cr-induced crosslinking

reaction

Figure 5.3 Oxidation mechanism to illustrate the Cr-induced crosslinking reaction

xii

Chapter 1

Introduction

1 1.1 Introduction to research

The goal of this dissertation was to investigate the application of mass spectrometry, including both atomic and molecular mass spectrometry, in the biochemistry and physiology fields. In the area of biochemistry, a method was established to detect and quantify DNA proteins crosslinks (DPCs) by monitoring sulfur and phosphorus signals simultaneously with a Triple Quad ICPMS

(QQQ-ICPMS). Furthermore, based on the purification of DPCs, structure characterization was achieved, as a result the amino acids and the nucleotides involved in the crosslinking bond were identified. This approach of detection, quantification and structure determination of DPCs opens a new window for further investigate the crosslinking mechanism and may contribute to the study of

DNA damage repair. In the physiology research field, an approach was established for the purification of novel cardiotonic steroids (CTS) by series of extraction and chromatographic methods. The active fractions were identified with electrospray ionization ion trap mass spectrometer (ESI-ITMS), providing the molecular weight of most intensive molecules. While the molecular structure of novel CTS compound still needed to be determined with high resolution mass spectrometer (HRMS) and nuclear magnetic nuclear magnetic resonance (NMR) spectrometer, this fundamental study laid milestones for the future study in physiology. This research advances the field of biochemistry and physiology by providing the analytical instrumental approaches, which are effective in solving problems in different areas.

2 1.2 Application of mass spectrometry in a biochemistry model

study: DNA protein crosslinks characterization using model

molecules under UV radiation

1.2.1 Introduction

Non-covalent DNA protein interactions, resulting from the binding of a protein to a molecule of DNA, are at the heart of normal cell activities since they always regulate the biological functions of DNA, usually the expression of a gene[1-4]. In eukaryotic cells, genomic DNA is surrounded with histones to package and order the DNA into structural unities, nucleosomes, in the nucleus[5]. Other proteins involved in the non-covalent binding to DNA motifs are called transcription factors, which activate or repress gene expression[6]. In a word, the non-covalent binding of proteins to DNA guarantee the biological processes in the cell, including the accurate propagation and expression of genetic information and even the regulation of cellular responses to DNA damage. In this case, the dynamic and reversible associations of DNA with proteins play a significant role in the cell survival. However, the covalent binding of DNA to proteins, in other words, the formation of DPCs, is distinctive due to their bulky size and helix-distorting nature, which may interrupt the normal process in the cell and lead to serious biological consequences[7-9]. In this part, the introduction of DPCs started from the formation induced with various agents undergoing different mechanisms and then went to the biological consequences resulting from DPCs. The popular approaches applied in the detection of DPCs were discussed as well. In addition, a brief

3 overview of chapter 2 and chapter 3 introduced the methods that I established in the detection, quantitative analysis and structure characterization of DPCs.

1.2.1.1 Formation of DPCs

Various endogenous factors such as reactive oxygen species (ROS)[10] from normal cellular metabolism and exogenous agents such as ultra-violet light[10], ionizing radiation[11], aldehydes[12], some chemotherapeutic drugs[13, 14], and metal complexes[15, 16] can induce the covalent trapping of proteins on DNA, resulting in

DPCs. In this crosslinking reactions, multiple sites within DNA have been investigates as preferably targets including the N7 of guanine[17], the exocylic amino groups of guanine[18], the C- 5 methyl group of thymine[19], cytosine[20], and adenine[21] (Figure 1.1).

O O

H H N N HN HN

N N H N N H2N N 2 N7 of guanine exocylic amino groups of guanine

NH H C 3 N NH N NH2

N N O N O H2N N O H H methyl group of thymine cytosine adenine

Figure 1.1. Preferably targeted sited within DNA in the crosslinking reaction

1.2.1.1.1 Formaldehyde-induced DPCs

4 DPCs can be initiated with general exposure to industrial chemical formaldehyde, resulting in tissue-specific tumor[22, 23]. DPCs induced by formaldehyde have been well studied. Formaldehyde is an active reagent that preferably reacts with nucleophilic amino and imino groups in the proteins, resulting in Schiff base intermediates, which then continue to crosslink with bases in the DNA, generating methylene DPCs (Figure 1.2)[24, 25]. This covalent trapping of proteins in DNA is reversible and efficient in the biological application of chromatin immunoprecipitation (ChIP)[26].

O H H O H OH + H H transfer -H2O N Protein H2N Protein H N H N Protein Protein H H2 H

NH2

N N

DNA

HN N Protein N H H+ transfer Protein HN N N H N N H N N 2 DNA N DNA N

Figure 1.2. Mechanism of formaldehyde-induced DPCs

1.2.1.1.2 Cisplatin-induced DPCs

Cisplatin is a chemotherapeutic drug that widely used in the targeted treatment of different types of cancer[27]. As it was illustrated in Figure 1.3, the chloride ligands in cisplatin are substituted by water in the process of hydrolysis. The aqua ligands resulting form hydrolysis are easily displaced by binding to N7-guanine positions

5 of DNA. These platinum complexes binding to DNA cause its strands to crosslink, which ultimately triggers cells to die in a programmed way[28]. However, more and more evidence shows that the aqua ligands resulting form hydrolysis can also be displaced by cysteine, arginine, and lysine side chains of proteins, resulting in large amounts of DPCs[29, 30].

2+ NH3

Pt NH3 Cl Cl hydrolysis OH2 Nu-Protein

O Pt(NH3)2 O Pt(NH3)2 O 2+ N Pt(NH3)2(OH2)2 Nu-Protein NH N N NH NH N N NH2 N N N NH2 N NH2 DNA DNA DNA

Figure 1.3. Mechanism of cisplatin-induced DPCs

1.2.1.1.3 Metal-induced DPCs

Environmental and occupational exposure to transition metals such as chromium can also drive DNA protein crosslinking reactions in cells and tissues.

Crosslinking by chelation has been proved the predominant mechanism in the formation of chromium-induced DPCs. Cr(VI) is transported into cells via a nonspecific anion carrier, where it can be reduced to Cr(III)[31]. The ligands on

Cr(III) are easily substituted by phosphate groups and guanine bases of DNA.

Also, nucleophillic amino acids including the amido side chains of proteins preferably attack the Cr(III) to generate DPCs in form of Cr(III) coordination complexes (Figure 1.4)[32, 33].

6 O

Cr OH O O- Cr (VI) OH2 Nu-Protein OH2

O Cr(III)L4 Cr(III)L4 O Cr(III)L4 O Nu-Protein N OH NH 2 N N NH NH

N N NH2 N N N NH2 N NH2 DNA DNA DNA

Figure 1.4. Chelation mechanism of Cr-induced DPCs

1.2.1.1.4 Reactive oxygen species (ROS) -induced DPCs

Reactive oxygen species (ROS) is a collective term that includes both oxygen

- radicals such as superoxide (O2 ), hydroxyl (OH), peroxyl (RO2) and hydroperoxyl

(HO2) radicals and certain non-radical oxidizing agents, such as hydrogen peroxide (H2O2), hyperchlorous acid (HOCl) and ozone that can be easily converted to radicals[34]. ROS are physiologically produced as a natural byproduct of the normal metabolism of oxygen during various cellular activities such as respiration, immune response and inflammation[35]. However, the ROS levels increase dramatically with exposure cells to ultra-violet light, ionizing irradiation or heating conditions, generating oxidative stress to cell, which may cause interruption of cell functions[36]. Exposure DNA to the oxidative stress resulting from ROS, cause the oxidation of bases on the DNA[37]. Guanine is preferably converted to 8OG due to its lowest redox potential among all the four bases[38].

Proteins and amino acids are susceptible to be attacked by ROS as well, generating protein hydroperoxides or other reactive protein species as well as additional free radicals[39]. These oxidative formats of DNA and proteins are very reactive and contribute to the covalent binding into DPCs[40-43].

1.2.1.2 Biological consequences of DPCs

In comparison to other DNA damage, DPC are distinctive due to the steric hindrance. DPCs are expected to hinder DNA metabolic processes such as replication, transcription and even further DNA repair since they are bulky, relatively stable and helix-distorting[8]. It has been proved by several groups that the formation of DPCs contributed to lots of genetic damage including sister chromatid exchange, mutagenicity, malignant transformation, and cell toxicity[44-47].

Evidence has shown that the formation of formaldehyde-induced DPCs is dose-dependent and is implicated to correlate with nasal tumors in animal experiments[48]. Additionally, research by different groups has proved that the occurrence of cellular mutagenesis increased as the exposure of cells to environment, which correlates to the increased incidence of respiratory cancers[49].

Futhermore, the oxidative stress caused by ROS during cellular metabolism can generate not only DPCs, but also DNA-DNA crosslinks and protein-protein crosslinks that are permanent cellular damage and difficult to repair[50].

1.2.1.3 Enzymatic repair of DPCs

Recent study provided increasing evidence of DPCs formation in mammalian cells[51, 52]. In addition, Balansky and his co-workers demonstrated that the levels of DPCs increased with age in the mice owning to the increases of

8-hydroxyl-2’-deoxyguanosine[53]. The processes of aging, and other cellular stress driven by metabolism, illness, exposure to ultra violet, pollutants, heavy

8 metals, etc. are suspected to lead to the accumulation of various DPCs. However, evidence has shown that most of the DPCs that induced by exogenous agents were clearly removed from the genome as time went on[54]. For example,

Dizdaroglu and his co-workers reported that the levels of DPCs induced by ferric nitrilotriacetate, crosslinked between thymine-tyrosine, in renal cells of Wistar rats peaked at 24 h (corresponding with the onset of mitosis), but returned to control level by the 19th day of ongoing retreatment[55]. This decrease of DPCs levels suggested active repair processes of these lesions.

There are different repair pathways owning to the size, proteins, crosslinking reaction chemistry involved in DPCs. Direct reversal pathways was effective when the breakage of the phosphodiester backbone was not involved in the crosslinking reaction. Direct reversal by chelation is possible in the case where the protein is bound under chelation mechanism with a metal while direct reversal by hydrolysis was published in the repair of DPCs induced by aldehyde[56]. Nucleotide excision repair (NER) pathway is another mechanism for DPCs repair. Recognition of these lesions resulted in the removal of a short single-stranded DNA segment in which the damage involved. The undamaged single-stranded DNA remained and it was used as a template for to synthesize a short complementary sequence. The formation of double-stranded DNA was carried out by DNA ligase to complete[57,

58]. Besides, Homologous recombination repair (HRR) pathway is another repair mechanism for DPCs. In this mechanism, nucleotide sequences are exchanged between two similar or identical molecules of DNA to fix theses lesions[59, 60].

9 Nevertheless, proteases were needed to de-bulked the proteins involved in the

DPCs before they can be processed the NER pathways or HRR repair pathways.

1.2.1.4 Detection of DPCs

The complex reaction between DNA and proteins, even at the level of model standards, possesses a challenge for product’s characterization, even at the most fundamental level, reaction yield and stoichiometry of products. In this regard, the current methods for detection and characterization include several indirect techniques, such as alkaline elution method[61], nitrocellulose filter-binding method[62], comet assay method[63] and SDS/K+ precipitation method[64, 65]. These methods all depend on the different elution or migration rates of DNA and DPCs on various materials and further distinguish them. These methods only provide the information on relative amounts of DPCs and yield neither quantitative information nor stoichiometric characterization of the adducts[66]. Additionally, I−[67] and fluorescein post-labeling methods[66] are very powerful for the direction and quantitative analysis of DPCs, even though they require the purification of chromosomal DNA. What is required is absolute quantification with the ability to identify the exact type of DPC formed. In chapter 2, a method was established to achieve these goals.

Many efforts have been done to characterize the structure of the DPCs as the development of mass spectrometry grows[68]. However, direct characterization of intact DPCs by mass spectrometry has still been challenging due to the different

10 ionization modes of DNA and protein and an appropriate matrix for MALDI

(matrix-assisted laser desorption/ionization mass spectrometry) analysis of DPCs is not yet available. Two general approaches have been widely applied for synthesizing DPCs that involve structurally defined, site-specific DNA strands and proteins. Enzymatic methodologies for DPC formation are site specific and efficient since these methods can trap DNA modifying proteins on their DNA substrate[69]. However, they are less effective in the systematic studies of DPC lesion structure on their biological outcomes. Synthetic methodologies for DPC formation are based on synthesis of oligonucleotide strands containing more protein-reactive DNA bases such as guanine[70-74]. This approach allows for a wider range of proteins to be crosslinking reacted with DNA and provides a greater flexibility for structural analysis of DPCs. In this case, peptide sequencing as analytical techniques are playing increasingly important roles for the structure determination of DPCs. With this method, DPCs are firstly proteolytically digested to smaller DNA-peptides, followed by separation and purification for further sequence analyses[75]. This method can provide some evidence of the cross-linking sites in the protein since the adducted fragment will have a higher molecular weight compared to the predicted fragment that result from proteolytic digestion. To further simplify mass spectral analysis, the DNA portion of the left crosslinking adduct can also be digested into nucleotides. A MS/MS analysis is then conducted on the resulting peptide-nucleotide adduct to reveal the peptide sequence involved in crosslinking reactions as well as the exact locations of the crosslinking amino acids[76]. This approach is effective in the structure

11 determination of DPCs, but requires two digestion processes including both protein and DNA, resulting many and complex steps of purification, which are time consuming and make the result doubtful. In chapter 3, a method similar with peptide sequencing approach was established for the structural characterization of laboratory synthesized DPCs. In this method, only one step of proteolytically digestion of proteins was required and thermal hydrolysis was conducted to get rid of DNA strand, which dramatically reduce the separation steps and save more time.

1.2.2. Research Overview

1.2.2.1 Overview of Chapter 2

Various endogenous and exogenous agents can drive reactions between nuclear proteins and DNA, generating damages that if remain unrepaired, permanent mutations or replication stops are formed leading to cytotoxicity. However, they have not fully studied, mainly because of the lack of good analytical procedures with sufficient detection capabilities and the necessity of synthetic standards to quantify it. Techniques, based on ultra-trace level detection of S and P, are good, but they are not without interferences. The state-of-the-art for interference removal is to use the newer Triple Quad ICPMS (QQQ-ICPMS) approach by passing 31P+ in the first quadrapole (Q1), passing to the dynamic reaction cell

47 + (Q2) operating in the reaction mode and adding O2, therefore generating PO in the second quadrapole (Q3) and leaving the usual NOH+ etc. interferences behind.

Monitoring 47PO+ results a signal absent from polyatomic interferences. The lower

12 detection limits and high sensitivity when compared with those from cells using the collision or energy discrimination modes are a major plus for the QQQ-ICPMS.

In conjunction with the high resolving power of high performance liquid chromatography (HPLC), the new QQQ-ICPMS technology was applied to study a synthetically made DNA-Protein (27 mer nucleotide-Ribonuclease A) cross-link model, by following both 47PO+ and 48SO+ in the intact complex and in its enzymatic digestion products. The involved peptide has a sequence containing three atoms of sulfur, NGQTNCYQSYSTMSITDCR, this peptide, associated with the synthetic DNA fragment was successfully identified by combining ICP-MS and

LC-MS results.

1.2.2.2 Overview of Chapter 3

To date, many different analytical methods have been used to investigate crosslinking reaction mechanism and to obtain the chemical structure of DPCs.

Direct MS analysis of DPCs is challenging because the ionization properties of

DNA and the protein. However, peptide sequencing and mass spectrometry as analytical techniques are playing increasingly important roles for the structure determination of DPCs. In our previous study, a novel approach was presented for purification, detection and quantification of DPCs by newly developed

QQQ-ICPMS/MS, which allows sub-ppb detection of S and P, the key heteroelements in DNA and proteins.

In this study, we enhanced our previously developed method to allow complete

13 characterization of DPCs. First, a small molecule model was utilized to identify the adduct structure that will likely occur in an intact DNA protein crosslink. We investigated the thermal stability of DPCs, both in an intact DPC and a small molecule adduct to determine feasibility of digestion/thermal hydrolysis of DNA without the crosslinking information being lost. Thermal hydrolysis was conducted to reduce the cross-linked DNA into a single nucleoside. The remaining protein-nucleoside adduct then is proteolytically digested, generating a peptide-nucleoside adduct. The absence of the phosphate moiety allows for facile structural characterization via electrospray ionization mass spectrometry

(ESI-MS). Additional calculations were done for peptide matching allowing us to determine the cross-link location in the protein, made possible via MS/MS analysis. Additionally, we showed that steric effects play an important role in DPC formation.

1.3 Application of mass spectrometry in physiology study:

cardiotonic steroids project

1.3.1 Introduction

1.3.3.1 Na+/K+-ATPase

Na+/K+-ATPase is an enzyme found in the plasma membrane of all animal cells. It was discovered in the 1950s by Jens Christian Skou, which is a significant step in the hundreds years of study on the cell as a basic unit of animal life[77]. His group first proved the existence of a protein-based structure, incorporated in the living cell membrane, which regulates the sodium and potassium ions concentration

14 against their concentration gradient, during the crab nerve experiment. The

Na+/K+-ATPase behaves like an ion solute pump, pumping three sodium ions out of cells while pumping two potassium ions into cells each time[78]. The

Na+/K+-ATPase binds to Adenosine Triphosphate (ATP) first, followed by binding three intracellular Na+ ions. The further hydrolysis of ATP, releasing ADP from the pump, generates the phosphorylated form of Na+/K+-ATPase at a highly conserved aspartate residue, which has a low affinity for Na+ ions but a high affinity for K+ ions. The conformation of Na+/K+-ATPase changes, releasing Na+ ions out. Then the Na+/K+-ATPase binds to two extracellular K+ ions with the dephosphorylation of the pump, reverting it to its previous conformational state, which has a high affinity for Na+ but a low affinity for K+. In this state, K+ bound to the pump are released[79]. This pumping activity of Na+/K+-ATPase is responsible to keep the Na+ and K+ electrochemical gradients through the cell membrane, leaving low Na+ concentration and high K+ concentration in the cell. These concentration gradients determine the cellular membrane potential[80, 81], which control a broad range of cellular functions, such as signal transducing[82] and cell volume adjusting[83].

1.3.3.2 Compounds displaying ouabain-like activity: Cardiotonic Steroids (CTS)

Cardiotonic steroids (CTS), which are also referred to as digitalis-like fators, are the groups of compound that behave like inhibitors of the Na+/K+-ATPase[84]. They are classified as two categories: endogenous cardenolides, including ouabain, digoxin and endogenous bufadienolides. Their structures were shown in Figure

15 1.5.

O O O O O a) b) c) OH O

H HO HO H OH HO H O O HO H H O OH O O OH

O H HO O OH O OH HO OH OH OH OH

Figure 1.5. Structure of a) Marinobufagenin; b) ouabain; c) digoxin

A. Endogenous Ouabain

Endogenous ouabain, which was proved to have the same molecular structure with ouabain extracted from plant, was discovered in 1991 during the experiment of investigation of natriuresis in salt loading[85]. Endogenous ouabain functions like endogenous hormone that inhibit the activity of Na+/K+-ATPase like digitalis[86].

Once ouabain binds to the Na+/K+-ATPase, the “pumping process” will be inhibited, leading to an increase of intracellular sodium. This increase in the concentration of intracellular sodium ions interrupts the activity of the sodium-calcium exchanger (NCX), which is another membrane protein pumping

Na+ to flow down its gradient across the plasma membrane in exchange for the countertransport of calcium ions (Ca2+)[87]. Therefore the decrease in the concentration gradient of sodium ions hampers the ability of the NCX to function, which in turn increases the concentration of intracellular calcium ions. This results in higher cardiac contractility and an increase in cardiac vagal tone, which can be used in the treatment of heart failure. In addition, this change in ionic gradients have effect the membrane voltage of the cell, which could be applied in the treatment of hypertension and cardiac arrhythmias with low does[88].

B. Endogenous digoxin

Digoxin is one of the most important medications in a basic health system around the world, extracted from foxglove plant more than 200 years ago[89]. Endogenous digoxin functions the same way as endogenous ouabain, resulting in an increase in the concentration of intracellular Ca+ ions. Increased concentration of intracellular Ca+ ions lengthens phase 4 and phase 0 of the cardiac action potential, leading to a decrease in heart rate[90]. Increased concentration of intracellular Ca+ ions also strengthens the force of heart contraction without increasing heart energy expenditure. Therefore, the endogenous digoxin is one if the most popular medications for the treatment of various heart conditions, namely atrial fibrillation, atrial flutter sometimes heart failure that cannot be controlled by other medication[91-93].

C. Endogeous bufadienolides

Bufadienolide was first extracted from toads its derivatives are known as bufadienolides[94]. Bufadienolides work as endogenous steroidal hormones through inhibition of the Na+/K+-ATPase, leading to an increase of intracellular Ca+ concentration[95]. This capability raises sodium excretion of the cell, produce vasoconstriction resulting in hypertension, and act as cardiac inotropes.

Bufadienolides have been implicated in instances of volume expansion-mediated hypertension, syndromes in which they are considered capable of causing a vascular leak, interfering with cellular proliferation, and inhibiting cellular

17 maturation. However, they can increase the risk of death with large doses[96].

1.3.3.3 Purification of CTS

Evidence has showed that CTS play very important roles in a lot of physiological processes and functions by inhibiting the ions transporting activity of the

Na+/K+-ATPase[97]. CTS can be extracted from various plants and animals. At least 10 CTS have been partially purified from mammals[98-102]. However, only three of them have been structurally analyzed by mass spectrometer and nuclear magnetic resonance (NMR) spectrometer. At least six HPLC separations are needed to purify one compound owning to the complex matrix 40 years ago. The exploration of antibody affinity column highly reduced the HPLC separation steps by specially binding to CTS[103]. However, this method can only be used to purify known compounds. Batch affinity extraction (BAE) was first introduced to purify

CTS in human plasma by using the CTS binding sites on the Na+/K+-ATPase itself to capture its endogenous ligands[104]. This method highly reduces the HPLC separation steps and provides the possibility of purifying the endogenous CTS in large scale. In my study, BAE is also used as the first basic separation method for the purification of endogenous CTS from pig skeleton muscles.

1.3.2 Overview of Chapter 4

CTS play a significant role in inhibiting the activity of Na+/K+-ATPase. In this chapter, an approach was established to purify the CTS from pig skeletal muscles.

BAE was first carried out to extract endogenous ouabain like compound from the

18 homogenized skeletal muscle solution. Serious of chromatographic methods were applied for further purification, including gel filtration chromatography and reverse phase separation. NADH coupled method was used to checked the activity of the each fraction. The molecular weight of the active compounds was determined by

ESI-ITMS. This approach laid the foundation for the further physiological study on endogenous ouabain like compound.

19 Reference:

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Chapter 2

Simultaneously Detection and Quantification of Synthesized DNA Protein Crosslinks by

Triple Quad ICPMS (ICPQQQ)

26 2.1 Introduction

It is well known that genomic DNA can be transiently associated with various structural, regulatory, repair and transcription-involved proteins. The dynamic and reversible association between DNA and proteins guarantee the accurate expression and propagation of genetic information[1]. However, various endogenous and exogenous agents, including free radicals from normal cellular metabolism[2], UV light[3, 4], ionizing radiation[5], chemotherapeutic agents, such as cisplatin and aldehydes[6], metal complexes, such as nickel[7], chromium[8] and arsenic[9] can induce covalent association of proteins with DNA, resulting in

DNA-protein cross-links (DPCs), which may have serious affects in cellular processes[10].

Compared with other DNA lesions, DPCs are distinctive in that they are very bulky, making them likely to impair various DNA processes including replication, transcription and repair via steric hindrance[1]. In this case, detection, quantification and characterizing the structure of DPCs are of high significance.

An absolute detection and purification of DPCs is the foundation for this study.

Identifying the nucleotides and amino acids involved in cross-linking is valuable in understanding the crosslinking mechanisms, which is valuable to avoid or even repair this type of lesion[10].

Many different analytical methods have been used to detect and quantify the

DPCs. The alkaline elution method relies on the different elution abilities from a

27 filter under alkaline conditions between DNA and DPCs. Lysed Cells are filtered on to a polyvinylchloride filter. DNA retained on the filter is eluted at pH 12.1 while the elution rate of DPCs is decreased due to the adsorption of crosslinked proteins to the filter[11]. The nitrocellulose filter-binding method is based on the different abilities of DNA and DPCs to bind to a nitrocellulose filter. The nitrocellulose filter retains proteins and DPCs while allows the free DNA passing through[12]. The comet assay method is based on the different migration rate between DNA and DPCs. Pretreatment of lysed cells with proteinase K clearly distinguishes DNA and DPCs[13]. The SDS/K+ precipitation method is based on different binding abilities to sodium-dodecyl sulphate (SDS) of DNA and DPCs since SDS binding tightly to proteins and forming insoluble precipitates with K+.

Cells are lysed with SDS, and SDS-bound proteins and DPCs, but not free

DNA[14]. These indirect DPCs detection methods have provided valuable information on relative amounts of DPCs. However, as well as there being many technical issues. First, the adsorption of DNA on the filters and precipitation of

DNA by SDS/K+ depend on not only the crosslinked proteins but also on the length of DNA which varies significantly depending on DNA-damaging agents and their doses. Second, there is no linear relationship between the amounts of DNA and crosslinked proteins. Thus to determine the amount of crosslinked proteins from that of DNA inevitably requires some assumptions, which have not been rigorously examined and verified by compelling evidence[15]. Recently reported techniques, 125I post-labeling[16] method and fluorescence-labeling method[15] have been applied in the detection of DPCs. These methods used either 125I or

28 fluorescein isothiocyanate to label crosslinked proteins to detect DPCs. These methods were proved powerful for detection and quantitative analysis of DPCs, however, both of them required the purification steps to get rid of DNA.

Inductively Coupled Plasma Mass Spectrometry (ICPMS) is a powerful tool for trace element detection[17]. Sulfur is an important intra-molecular elemental-tag for the analysis of cysteine and/or methionine containing proteins by ICPMS, while phosphorus is a target for DNA. This opens the possibility of using the matrix-independent and compound-independent detection and quantification capabilities of the ICPMS to analyze DPCs through the detection of sulfur and phosphorus by ICPMS. However, the detection of metalloids and non-metals that are poorly ionized in the plasma source due to their high ionization potential remains a challenge, as well as important for DPCs are sulfur and phosphorus that suffer from isobaric and polyatomic interferences[18]. Scientists have been working on improving the detection of S and P by ICPMS for many years. High resolution sector field ICPMS was used to ensure the detection limit in 1991. This instrument can be applied to separate the isobaric interferences that affect S and

P, based on a mass difference of 0.0178 to 0.0416 Da; but these instruments are very costly and less robust on its operation when compared with a quadrupole based instrument[19]. Dynamic Reaction Cell was first introduced in 2002 and a reaction with oxygen has been explored with promising result. The O-atom affinity between analytes and all interferences was investigated. O-atom transfer from O2 to ionized S and P was calculated as thermodynamically allowed while for NO+

29 + + NOH and O2 they were forbidden under thermal conditions since they were calculated as endothermic[19]. This provided the theoretical milestone of this method to remove interferences with m/z of 31 and 32. However, two issues stand in the way of this approach: the matrix-dependent reactivity of the analytes and the spectral interferences from 47Ti+, 48Ca+ and 48Ti+ that cannot be distinguished from the oxidation products of P and S at m/z 47PO+ and 48SO+.

Most recently the implementation of a commercial Triple Quad ICPMS

(QQQICPMS) opens the possibility of eliminating most of the limitations of analyzing P and S via reaction with oxygen[20]. By filtrating out all m/z ions except

31 or 32, the reaction takes place in a much cleaner environment than in a single quadrupole instrument, and because m/z= 47 and 48 are filtrated out in the first quadrupole, no interferences are present from Ti or Ca reach the detector, but just the reaction products of P or S oxidation. Furthermore, by coupling ICPMS detection with high performance liquid chromatography, HPLC, virtually simultaneous separation and detection can be achieved. Additionally, the use of capillary HPLC allows the use of organic based mobile phases as eluents, necessary for the use of reverse phase columns. With this combination,

CapHPLC-QQQ-ICPMS, the DPCs, its precursors and its byproducts can be separated with high efficiency and detected in a very specific manner simultaneously.

In our study, quantitative determination of sulfur and phosphorus was done with

30 different organic standards and achieved a sub nanogram level detection limit.

Also, with the low-level S and P detection, the HPLC separation allows a high quality purification and basic quantification of the DNA protein cross-link.

2.2 Experimental

2.2.1 Materials

All experiments were done in triplicate or greater. All chemicals were purchased from Fisher Scientific (Pittsburgh, PA, USA) unless otherwise noted. The

27-nucleotide DNA, 5’-GGGGCCCCGTCGTTTTACAACGTCGTG-3’, and its complement 5’-CACGACGTTGTAAAACGACGGGGCCCC-3’ were purchased from Eurofins Genomics Operon (Huntsville, AL, USA). Ribonuclease A (RNase),

Rose Bengal, L-glutathione, cysteine and Adenosine Triphosphate (ATP) were obtained from Sigma Aldrich (St. Louis, MO, USA). The sequence-grade modified trypsin was purchased from Promega (Madison, WI，USA).

2.2.2 Methods

2.2.2.1 DNA-Ribonuclease A Oxidative Cross-Linking

For the cross-linking reaction, the Ribonuclease A and single stranded

27mer-nucletide DNA were mixed in a 0.5 ml centrifuge tube without lid and then rose bengal was added as the photo oxidant [500µL; 0.35 mM ribonuclease A, 0.5 mM DNA and 1 mg/mL rose Bengal, 25mM sodium phosphate, pH=8]. The mixture was irradiated for 1 hour with a LED lamp located 2 cm above the solution and providing 3 mW of radiative flux at 375nm.

2.2.2.2 Optimization of the reaction in the octopole cell and other parameters

An Agilent 8800 Triple Quad ICP-MS (QQQ-ICPMS) was utilized (Agilent

Technologies, Santa Clara, CA), equipped with an Agilent micro flow nebulizer kit.

The ICP-QQQ included a sampler cone made of platinum, a platinum skimmer cone, a shield torch, two quadrupole mass analyzers separated by an octopole cell (referred to as Q2) with pressurized oxygen gas (purity of 99.999%), and an electron multiplier detector. The spectral interferences are eliminated when pressurized gas comes into the octopole cell and is reacted with some of the ions selected at the m/z setting of the first quadrupole, Q1. After the reaction, the second quadrupole, (referred to as Q3) allows those ions at its specific m/z to pass through. This combination offers exceptional performance with higher sensitivity and lower backgrounds, especially important for non-metal detection, such as with sulfur and phosphor. To optimize the reaction in the octopole (Q2), the O2 flow rate was systematically changed from 0 to 50% of 1 ml while keeping the analyte (AMP and cysteine) concentration constant at 200 ppb. Both signal of

31P+ and 47PO+, 32S+ and 48SO+ were followed and the signal to noise ratio (S/N) was manually calculated with a blank solution at each point of the ramping procedure. In addition, a tuning solution of Y+ (m/z = 89) at 20 ppb was injected from syringe pump to the QQQ-ICPMS and the shifted Y+ signal at m/z = 105

(105YO+) was monitored. Lenses and other parameters were adjusted to get the highest intensity. Table 2.1. shows the final instrument parameters.

32 2.2.2.3 Quantification of sulfur and phosphor by using cap-RPHPLC-QQQ-ICPMS

For the quantification of sulfur and phosphorus signal, L-glutathione and cysteine were used as organic sulfur standards [50ppb, 100ppb, 200ppb, 500ppb,

1000ppb, 2500ppb] while ATP were chosen as organic phosphorus standard

[0ppb, 50ppb, 200ppb, 500ppb, 1000ppb]. HPLC was carried out on an Agilent

1200 series system (Agilent Technologies, Santa Clara, CA, USA) equipped with a vacuum degasser, two binary pumps, a well plate auto-sampler and a thermostatically controlled column compartment. All lines within the capillary system were PEEK covered fused silica tubing with 50 µm internal diameter. The

QQQ-ICPMS was used as detector with the tuned parameters in Table 2.1. An

Agilent Zorbax SB C18 [300A, 5µm, 150x0.5mm] revered phase column was used for separation. To get the signal intensity of the standards, an isocratic separation was run with 60% of solvent A (100% water with 0.1% formic acid) and

40% of solvent B (20% water, 80% acetonitrile with 0.08% formic acid) for 7 minutes at a flow rate of 10 µL/min. The injection volume was 2 µL for each standard.

2.2.2.4 Purification of the DPCs (DNA-Ribonuclease A cross-links)

To purify the DNA-Ribonuclease A cross-links from the reagents, size exclusion chromatography (SEC) coupled with QQQ-ICPMS as the detectors was used for the separation. A TSK gel 3000SW column from Tosoh Bioscience LLC. (King of

Prussia, PA, USA) was used. Two reagents, the 27mer-nucleotide DNA and the

RNase, and the solution after the cross-linking reaction were injected to the

33 instrument separately. The injected volume was 30µL for each injection. The mobile phase consisted of 99.5% DDI and 0.5% methanol with 50mM ammonium acetate. The LC separation was performed for 30 minutes with the flow rate at 1 mL/min. The fraction of the DNA-Ribonuclease A corsslinks was collected to get rid of the unreacted reagents and by product. Freeze drying was followed to concentrate the fraction to 100 µL.

2.2.2.5 Trypsin proteolysis of the DNA-Ribonuclease A cross-links

After the purification of DPCs from the unreacted reagents, a trypsin proteolysis digestion was carried out to slice RNase involved in the DPCs into small peptides.

In this digestion, water was used as blank and a solution of pure RNase with concentration of 1 mg/mL was used to observe the S-containing peptides and its retention time. The digestion was conducted in a pH buffered solution (mixing

15µL of digestion buffer [50mM ammonium bicarbonate], 1.5µL of reduction buffer

[100 mM Dichloro-Diphenyl-Tricgloroethane (DDT)], and 10 µL of the

DNA-Ribonuclease A cross-link solution in a 0.5 mL tube). The mixture was adjusted to 30µL with ultrapure water and the sample was incubated at 95oC for

15 minutes. Then 3 µL of Alkylation Buffer (100 mM iodoacetamide) were added to the tube and incubated in the dark at room temperature for 20 min. 2µL of sequence grade modified trypsin (1mM) were added to the reaction tube and incubated at 37oC overnight. To stop the digestion, 1 µL of formic acid was added to decrease the pH. Ultrafiltration was then carried out in a 0.5 mL MWCO spin filter, with a nominal 10,000 Da cut off membrane. The sample was loaded onto

34 the filter and was centrifuged for 10 min at 10,000 g. The filtered solution was analyzed by CapHPLC-QQQ-ICPMS.

2.2.2.6 Pepetide identification after trypsin proteolysis by cap-RPLC-QQQ-ICPMS

The blank, proteolized Ribonuclease A and digested DNA-Ribonuclease A cosslink samples were injected at 6 µL per injection from an Agilent 1200 series capillary system to a triple Quad ICPMS. The column and mobile phase used in this separation were same with those in the sulfur and phosphorus quantification step. All the instrumental parameters were kept the same except for changing from isocratic runs to gradient elution. The gradient conditions, also at a flow rate of 10 µL/min, were 4 min for 2% solvent B, followed by a linear ramp to 50% B in

60min. Then the gradient was ramped to 80% B in 3 min and held for 5 min before linearly decreasing to 2% B. 15 minutes of column equilibration was needed in preparation for the next run.

2.3 Results and Discussion

2.3.1 Optimization of the instrument parameters

To optimize the instrument sensitivity and detection limits. O2 flow rate was systematically changed from 0 to 50 % of 1 ml while keeping the analytes concentration constant (ATP or Cys) at 200 ppb with respect to P or S (Figure.

2.1). We followed both m/z=31 and 47 for P while m/z=32 and 48 for S. A blank solution was used to compare the instrumental response to the analyzed m/z vs. the standard solution at different O2 flows. The signal to noise ratio was manually

35 calculated with a blank solution at each point of the capLC ramping procedure. It

31 + was found that at the standard solution, as O2 increases, the signal for P decreased until reaching a minimum. The non-zero minimal value likely reflects detection of polyatomic interferences. A simultaneous increase at m/z=47 was attributed to the increase in the 47PO+ formation. The maximum S/N value was

−1 observed at 30 % of 1 ml min of O2 (the manner this flow is described by the instrument). The blank solution showed a small decrease at m/z=31, while m/z=47 went from no-counts to less than 1000 cps, which can be attributed to P impurities in the formic acid or water.

For sulfur, the plot looks different than the one for P, with m/z=32 being in the tens of millions cps, and virtually identical for the blank and for the 200 ppb S standard all along the O2 ramping. This means that the level of interference for m/z=32 is much more intense than that for m/z=31 under current conditions. Only at m/z=48 a difference was observable between the blank and the 200 ppb S standard. Two orders of magnitude in the difference were found vs. to the more than three for P.

This observation can be attributed to the immense signal from the nitrogen-, oxygen- and hydrogen-based interferences, which can exceed the peak filtering capacity of a quadrupole-based ICP-MS, along with the higher level of contamination in the reagents and water used for the blanks. This substantial difference with background removal for P and S directly affects the limits of detection (LOD) for these elements under the MS/MS mode.

The lenses were tuned with a solution containing 20 ppb of Y at a flow rate of 10

μL/min by using a syringe pump connected to the capillary nebulizer with a 50 μm internal diameter capillary to maximize the signal of 89Y+→105YO+. The argon carrier gas flow was set to 0.9 L/min, which is lower than normal, but necessary for capillary LC systems. With these tune parameters, the instrument shows a signal with a 105YO+ target above 130,000 cps, while for 89Y+, it was below 2000 cps, yielding a 98 % conversion to 105YO+.

2.3.2 Quantification of S and P standards

Capillary HPLC was coupled to QQQ-ICPMS for separation and detection.

Acetonitrile was used as the mobile phase, since the microflow produces only a small organic flow to the ICPMS, minimizing the carbon residue and plasma shutdown that would occur with normal analytical HPLC flow.

Cysteine were used as standards for sulfur with concentrations ranging from

50-2500 ppb and ATP was used as the standard for phosphorus with a concentration from 0 to 1000 ppb for quantitative purpose. From Figure 2a, it can be seen that the phosphorous standards, ATP was consistent with > 5000 cps/ppb and the calculated limit of detection (LOD) was ≈ 0.1ppb. The limit of detection was calculated as three times the standard deviation of the blank intensity at the base line of the chromatograms, divided by the slope of the calibration curve based on the height of the chromatographic signals from the standards. Consider

37 from Figure 2b, for the sulfur standard, cysteine, about 3200 cps/ppb, lower than the phosphorus standards, and the LOD was calculated as 5.5 ppb. This 50 times larger of sulfur LOD than that of phosphorus resulted from of the extremely low and stable the phosphorus background. Additionally, all the calibration curves show a good linearity, as the R values were greater than 0.999 in both cases, which establish confidence for quantification. Thus this method provides unrivaled performance with higher sensitivity and lower backgrounds for sulfur and phosphorus detection and can achieve the separation and detection with small volume samples as necessary.

2.3.3 Basic quantification and evaluation of laboratory synthesized DPCs

The separation of synthesized DPCs from the reactants is becoming an increasingly important problem due to the low yield of the cross-linking reactions.

However, the high sensitivity and low limit of detection available with the sulfur and phosphorus detection with the ICP-MS/MS is promising for purification and quantification proposes. Size exclusion chromatography (SEC) coupled with QQQ

ICP-MS/MS was the technique used for purification of the synthesized DPC. Also, desalting and changing to a volatile solvent was achieved by fraction collection.

To determine the retention time of the reactants and products, the two main reactants (oligonucleotide and RNase) and the solution from the cross-linking reaction were injected into the instrument separately. From the chromatogramin

Figure 2.3, it may be seen that the protein itself provided a 48SO+ signal at a retention time of 17 to 20 min, while DNA gives a signal of 47PO+ at ca. 17 min.

38 DPC gives signals for both sulfur and phosphorus at a single retention time earlier than both of the reactants due to its higher hydrodynamic radius. By collecting this fraction, crosslinking product purification can be achieved, and by comparing with the peak area applied in the following equation, the mole ratio of DNA to protein in forming the DPCs is determined to be ca. 0.8, Eq. 1.

Eq. 1. Molar ratio calculation performed with the DNA and protein parts involved in DPCs, after sensitivity adjustment.

2.3.4 Cap HPLC-ICPMS detection of DPCs after trypsin proteolysis

The ICP-MS/MS coupled with capillary RPLC provides the capability for separation and detection of DPCs post-trypsin proteolysis. Trypsin cleaves peptide chains primarily at the carboxyl side of lysine or arginine, except when either is followed by proline, which makes it attractive for further mass spectrometric analysis. Once the DPC has been cleaved, capillary LC with a 0.5 mm i.d. C18 column was used to separate the generated peptides, one of which was attached to the oligonucleotide residue. To simplify the problem of identifying the peptide involved, the ICP-MS/MS chromatographic signal of the pure protein digest was compared with the DPC digest. As can be seen from Figure 4a, the inorganic materials elute in the void volume between 0 and 5 min. The S signal for the DPC was low, as a very small amount was purified and injected to the system, but the co-elution of an S containing peptide and the P containing oligonucleotide

39 can be observed at 48.5 min. This signal can be correlated to the large S signal at

51.5 min from the digested protein, by taking into account that the addition of the polar nucleotide residue will decrease the retention time. Given the poorer detection limit of S compared with P, it is not surprising that the intact oligonucleotide, containing 27 P atoms, shows a much larger signal than the sulfur containing peptide. The peptide involved in the DPC is amenable to two ways of identification. The retention time for the protein-derived peptide (51.5 min from Figure 4a) was identified by LC-MS analysis, in a traditional bottom up proteomic approach. And secondly, the elution order of the S containing peptides produced from tryptic digestion of the protein was estimated by taking account the hydrophobicity and confirmed by semi-quantitatively comparing the S content of each peptide according to the predicted elution order. These, notwithstanding, the aim of this study are to report a new approach to improve the characterization and purification steps at the synthesis for a DPC model. When the proposed sequence

NGQTNCYQSYSTMSITDCR matches with the elution order and contains three atoms of S, from cysteine and methionine, assured by the S ICP-MS/MS signal, its identity is only suggested and not confirmed.

2.4 Conclusions

The improved capabilities for P and S detection of a Triple Quad ICP-MS were successfully applied to the initial structural studies of a DPC model. From the purification of the reaction products with stoichiometry estimation, to the final assignment of the protein fragment associated with the DNA moiety, the ICP-MS

40 signal was a very useful tool, when combined with SEC and CapRP-HPLC. The possibility of following the S related signal of tryptic digested proteins open a new window of opportunities for protein studies, and facilitates the quantification DNA protein conjugates. However, there is a limitation of this method when the peptides involved in the formation of DPCs don’t contain S signal amino aid

(cysteine and methionine).

Table 2.1. Parameters for QQQ-ICPMS Tune parameters for QQQ-ICPMS Scan type parameters Scan type MS/MS Plasma Parameters RF Power 1600W RF matching 1. 60V Saml Depth 8.0 mm Carrier Gas 0.9 L/min S/C Temp 2o C Dilution Gas 0.7 L/min Lenses Parameters (V) Extract 1 0 Extract 2 -200 Omega Bias -110 Omega Lens 9.6 Q1 Entrance -14 Q1 Exit 1 Defect 4.5 Plate Bias -60 Cell Focus -2 Cell Entrance -60 Cell Exit -70 Q1 Parameters Q1 Bias 0 Q1 Postfilter Bias -24 Q1 Prefilter Bias -14 Cell Parameters

Use Gas O2 Gas Flow 30% Meters Water RF/W 1.27 L/min Refected Power 14W

41 a )

b )

Figure 2.1. Reaction Cell optimization for 47PO+ AND 48SO+. a) Doted lines represent the response to m/z = 31 of the blank (--□--) and 200 ppb P standard as ATP (--■--); while continuous lines represent the signal for m/z= 47 of the blank (-△-) and 200 P standard as ATP (-▲-). b) Doted lines represent the response to m/z =32 of the blank (--□--) and 200 ppb S standard as cysteine (--■--);while continuous lines represent the signal for m/z= 48 of the blank (-△-) and 200 S standard as cysteine (-▲-). The abscissa axis corresponds to the oxygen flow in mL min-1, while the ordinate axis represents the signal response in a logarithmic scale. c) Signal to noise ratio was calculated and represented by (-■-)

Standard Cysteine ATP Linear fitting expression y=942.22x+115257 y=5335.3x-759171

R2 0.9994 0.9991

Sensitivity 942.2CPS/ppb 5335CPS/ppb

Limit of Detection 5.5 ppb (11pg) 0.1 ppb (0.2pg)

Limit of Quantification 18 ppb 0.3 ppb

Figure 2.2. CapHPLC-QQQ-ICPMS chromatograms of organic standards in different concentration a) cysteine; b) ATP; c) Linear fitting of the area and calculated LOD and LOQ

Figure 2.3. SEC-QQQ-ICPMS chromatograms for a) Ribonuclease A, 48SO+ signal in black; b) 27mer-nucleotide, 47PO+ signal in blue; c) DNA Ribonuclase A crosslinks after crosslinking reaction merged 48SO+ in black and 47PO+ signal in blue.

Figure 2.4. a) CapHPLC-QQQ-ICPMS Chromatograms from tryptic digested samples a SO+ RNase A digest; b) SO+ signal of DPC digest, in black, the reagent blank and in red, the digested DPC; c PO+ signal of DPC digest, in black, the reagent blank and in red, the digested DPC. The indicated signal at 48.5 min in b correspond to the peptide fragment associated to the oligonucleotide as it co-elutes with the P signal from the oligonucleotide moiety

45 Reference:

1. Ide, H., et al., Repair and biochemical effects of DNA–protein crosslinks. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 2011. 711(1–2): p. 113-122. 2. Barker, S., M. Weinfeld, and D. Murray, DNA-protein crosslinks: their induction, repair, and biological consequences. Mutation research, 2005. 589(2): p. 111-135. 3. Alexander, P. and H. Moroson, Cross-linking of Deoxyribonucleic Acid to Protein following Ultra-Violet Irradiation of Different Cells. Nature, 1962. 194(4831): p. 882-883. 4. Smith, K.C., Dose dependent decrease in extractability of DNA from bacteria following irradiation with ultraviolet light or with visible light plus dye. Biochemical and Biophysical Research Communications, 1962. 8(3): p. 157-163. 5. Frankenberg-Schwager, M., Induction, repair and biological relevance of radiation-induced DNA lesions in eukaryotic cells. Radiation and Environmental Biophysics. 29(4): p. 273-292. 6. Costa, M., et al., DNA-PROTEIN CROSS-LINKS PRODUCED BY VARIOUS CHEMICALS IN CULTURED HUMAN LYMPHOMA CELLS. Journal of Toxicology and Environmental Health, 1997. 50(5): p. 433-449. 7. Chakrabarti, S.K., C. Bai, and K.S. Subramanian, DNA–Protein Crosslinks Induced by Nickel Compounds in Isolated Rat Lymphocytes: Role of Reactive Oxygen Species and Specific Amino Acids. Toxicology and Applied Pharmacology, 2001. 170(3): p. 153-165. 8. Kuykendall, J.R., et al., Waterborne and dietary hexavalent chromium exposure causes DNA-protein crosslink (DPX) formation in erythrocytes of largemouth bass (Micropterus salmoides). Aquatic Toxicology, 2006. 78(1): p. 27-31. 9. Gebel, T., et al., Arsenic(III), but not antimony(III), induces DNA-protein crosslinks. Anticancer research, 1998. 18(6A): p. 4253-4257. 10. Marnett, L.J. and J.P. Plastaras, Endogenous DNA damage and mutation. Trends in Genetics, 2001. 17(4): p. 214-221. 11. Kohn, K.W. and R.A.G. Ewig, DNA-protein crosslinking by trans-platinum(II)diamminedichloride in mammalian cells, a new method of analysis. Biochimica et Biophysica Acta (BBA)-Nucleic Acids and Protein Synthesis, 1979. 562(1): p. 32-40. 12. Chiu, S.-M., et al., Differential Processing of Ultraviolet or Ionizing Radiation-induced DNA—protein Cross-links in Chinese Hamster Cells. International Journal of Radiation Biology and Related Studies in Physics, Chemistry and Medicine, 1984. 46(6): p. 681-690. 13. Merk, O., K. Reiser, and G. Speit, Analysis of chromate-induced DNA-protein crosslinks with the comet assay. Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 2000. 471(1–2): p. 71-80. 14. Costa, M., et al., Interlaboratory validation of a new assay for DNA-protein crosslinks. Mutation Research/Genetic Toxicology, 1996. 369(1–2): p. 13-21. 15. Shoulkamy, M.I., et al., Detection of DNA–protein crosslinks (DPCs) by novel direct fluorescence labeling methods: distinct stabilities of aldehyde and radiation-induced DPCs. Nucleic Acids Research, 2012. 40(18): p. e143-e143. 16. Zhuang, Z. and M. Costa, Development of an 125I-postlabeling assay as a simple, rapid, and sensitive index of DNA-protein cross-links. Environmental Health Perspectives, 1994. 102(Suppl 3): p. 301-304.

46 17. Jenner, G.A., et al., ICP-MS — A powerful tool for high-precision trace-element analysis in Earth sciences: Evidence from analysis of selected U.S.G.S. reference samples. Chemical Geology, 1990. 83(1–2): p. 133-148. 18. Gießmann, U. and U. Greb, High resolution ICP-MS — a new concept for elemental mass spectrometry. Fresenius' Journal of Analytical Chemistry. 350(4): p. 186-193. 19. Bandura, D.R., V.I. Baranov, and S.D. Tanner, Detection of Ultratrace Phosphorus and Sulfur by Quadrupole ICPMS with Dynamic Reaction Cell. Analytical Chemistry, 2002. 74(7): p. 1497-1502. 20. Fernández, S.D., et al., Triple Quad ICPMS (ICPQQQ) as a New Tool for Absolute Quantitative Proteomics and Phosphoproteomics. Analytical Chemistry, 2012. 84(14): p. 5851-5857.

Chapter 3

Structurally Characterization of DNA Protein

Crosslinks (DPCs) by nanoLC-ESI-MS

48 3.1 Introduction

Oxidative DNA damage has been implicated in aging, diseases and environmental toxicity[1, 2]. Oxidative damage to DNA can be produced by reactive oxygen species (ROS), including superoxide radical anion, singlet oxygen, hydrogen peroxide and hydroxyl radical, which are present in the cell as a consequence of endogenous reactions, or from exogenous sources, including ionizing radiation and UV irradiation[3, 4]. If reactions occur on the nucleobase, the preferential site is guanine owing to its lowest redox potential (1.3 V vs NHE) among the four DNA bases[5]. The formats of products driven by different ROS species were complex. For example, 2’-deoxyguanosine (dG) preferably converts to 7,8-dihydro-8-oxo-2’-deoxyguanosine (8OdG) which is more reactive to be attacked and oxidized by different ROS species owning to its even lower redox potential (0.7 V vs NHE) compared with dG. More complex products are formed with C4-C5 attack[6, 7]. A number of in vitro studies have shown that 8OdG within a double helix is highly prone to subsequent oxidative damage, forming what are termed hyper-oxidation products (i.e. four electron products) including spiroiminodihydantoin (Sp), guanidinohydantoin (Gh), Imidazolone (Iz) and its hydrolysis product, oxazolone[8-13]. For instance, cross-linking in the presence of copper-generated reactive oxygen produces adducts with Gh[14], while singlet oxygen induced oxidative conditions generates Sp-based adduct[15, 16]. If water is substituted by the side-chain of a nucleophilic amino acid like lysine, a nucleophilic addition will occur resulting in a base–amino acid (nucleophilic) crosslinking reaction, or even DPCs in large scale.[15]

As mentioned above, an important event that stems from the secondary oxidation of 8OdG is the formation of DPCs. DPCs are extremely bulky lesions that are likely to impair various DNA processes including replication, transcription and repair[17, 18]. It has been estimated that the endogenous level of DPCs in human white blood cell range between 0.5 to 4.5 in 107 bases[19] with elevated levels linked to oxidative conditions such as exposure to heavy metals such as nickel and chromium[20-24], which are known carcinogens, and are also shown to be the major lesion associated with arsenite exposure[25]. Importantly, DNA-protein cross-links were observed to increase as a function of age in mouse organs and this correlates strongly with the occurrence of

7,8-dihydro-8-oxo-2’-deoxyguanosine (8OdG), a known marker of oxidative stress[26]. Most of the previous studies are conducted on small molecule model systems involving the nucleoside dG and a nucleophilic amino acid such as lysine and tyrosine, since these models are relatively easy to control and can be further analyzed[27-32].

Many efforts have been done to characterize the structure of the DPCs as the growing development of mass spectrometry[33, 34]. However, direct characterization of intact DPCs by mass spectrometry has still been challenging due to the different ionization modes of DNA and protein. DNA is preferably ionized in negative mode, while proteins in the positive mode. Additionally, an appropriate matrix for MALDI (matrix-assisted laser desorption/ionization mass

50 spectrometry) analysis of DPCs is not yet available. More recently, peptide sequencing as analytical techniques are playing increasingly important roles for the structure determination of DPCs[35]. With this method, DPCs are proteolytically digested to smaller DNA-peptides, followed by separation and purification for further sequence analyses. This method can provide some evidence of the cross-linking site in the protein since the adducted fragment will have a higher molecular weight compared to the predicted fragment that result from proteolytic digestion. To further simplify mass spectral analysis, the DNA portion of the left crosslinking adduct is also digested into a nucleotides[36, 37]. A MS/MS analysis is then conducted on the resulting peptide-nucleotide adduct to reveal the peptide sequence involved in cross-linking as well as the exact location of the adduct. By using this method, scientists have completed the determination of the structure of some DPCs with different mechanisms driven by various agents, such as

CH2BrCH2Br, methylglyoxal, cisplatin and 1,2,3,4-Diepoxybutane. However, this method requires two hydrolysis processes including both protein and DNA, resulting too many steps of purification, which are time consuming and make the result doubtful[38-41].

In our previous study, a novel approach was presented for the sub-ppb detection and quantification of DPCs by a newly developed ICPMS, the Triple Quad ICPMS

(QQQ-ICPMS).[42] This method is an effective technique for purifying DPCs. DPCs were readily isolated and purified via fraction collection of species containing both a sulfur and phosphorus signal from a HPLC-QQQ-ICPMS system, which is

51 indicative of a molecule containing both protein and DNA, and is likely a

DNA-protein cross-link. We aim here to enhance our developed method to allow complete characterization of DNA-protein cross-links. First, a small molecule model was utilized to identify the adduct structure that would likely occur in an intact DNA-protein cross-link. We investigated the thermal stability of DPCs, both in an intact DPC and a small molecule adduct to determine feasibility of digestion/thermal hydrolysis of DNA without cross-link information being lost.

Then, thermal hydrolysis is conducted to break the cross-linked DNA into a single nucleoside. The remaining protein-nucleoside adduct is proteolytically digested generating a peptide-nucleoside adduct, which in the absence of the phosphate moiety allows for facile structural characterization via nano HPLC coupled with electrospray ionization mass spectrometric (nanoHPLC-ESI-MS) analysis.

Additional calculation was done for peptide matching allowing us to determine the location of the cross-link in the protein. Importantly, using peptide MS/MS analysis, we were able to locate the specific amino acid position involved. Additionally, we show that steric effect places an important role in DNA-protein cross-link formation.

3.2 Experimental

3.2.1 Materials

All experiments were done in triplicate or greater. All chemicals were purchased from Fisher Scientific (Pittsburgh, PA, USA) unless otherwise noted. The

27-nucleotide DNA, 5’-GGGGCCCCGTCGTTTTACAACGTCGTG-3’, and its

52 complement 5’- CACGACGTTGTAAAACGACGGGGCCCC-3’ were purchased from Eurofins Genomics Operon (Huntsville, AL, USA). Ribonuclease A, Rose

Bengal, 2’-Deoxyguanosine, and N-acetyl-Lysine-Methyl ester were obtained from Sigma Aldrich (St. Louis, MO, USA). The sequence-grade modified trypsin was purchased from Promega (Madison, WI,USA).

3.2.2 Methods

3.2.2.1 2’-Deoxyguanosine-Lys derivative Oxidative Cross-Linking

For the cross-linking reaction, the 2’-Deoxyguanosine (dG) and

N-acetyl-Lysine-Methyl ester (AcLysOMe) were mixed in a 1.5 ml centrifuge tube without lid and then rose bengal was added as the photo oxidant [500 µL; 10 mM dG, 20 mM AcLysOMe and 1 mg/mL rose Bengal, 25mM sodium phosphate, pH=8].

The mixture was irradiated with a LED lamp located 2 cm above the solution and providing 3 mW of radiative flux at 375nm. Four reactions were carried out in same condition at the same time and last for 1 hour, 2 hours and 4 hours separately.

3.2.2.2 Purification of the dG-AcLysOMe adduct by RP-HPLC

Purification of the cross-linking adduct from the reaction of 2’-deoxyguanosine and N-acetyl-lysine-methyl ester was carried out by high performance liquid chromatography (HPLC) on an Agilent 1100 series system (Agilent Technologies,

Santa Clara, CA, USA), equipped with a vacuum degasser system, two binary

HPLC pumps, a well plate autosampler, a temperature column compartment, and

53 a diode array detector. All lines within this HPLC system were PEEK covered fused silica tubing with 100µm internal diameter. The UV absorbance was recorded at 240nm. A Beckman Coulter Ultrasphere C18 [5 µm, 4.6 mm x 15 cm] revered phase (RP) column was used. The column was pre-equilibrated with water containing 0.1% formic acid for 20 minutes before the samples were injected. To identify peaks on the chromatogram after the cross-linking reaction, the mixture containing all components of the three reactions and the controls, which are the two reactants, were injected separately into the system with an injection volume of 2 µL, and the chromatograms overlapped. The other parameters for this RP-HPLC separation are given in Table. 3.1. The dG-AcLysOMe adducts was eluted between 5.0 minute and 5.6 minute and the 0.6-minute fraction was collected and concentrated to 100 µL by freeze-drying for the following step.

3.2.2.3 Structure determination of the dG-AcLysOMe adduct by ESI-ITMS

The identity verification of the dG-acyLysme adduct was performed on a 6340 series

MSD Ion Trap XCT Ultra system with direct infusion via a syringe pump at a flow rate of 18 µL/h (Bruker Corporation, Billerica, MA) with the micro fluidic nanospray chip cube interface, which is automatically loaded and positioned into the MS nanospray chamber. The system was operated in ultra-scan positive ion mode with the maximum accumulation time of 300 msec. The ESI-ITMS was tuned under positive ion mode with the ESI Tuning Mix for Ion Trap (Agilent

Technologies, Santa Clara, CA) containing compounds at the following m/z: 118,

54 322, 622, 922, 1,522, and 2,122. All MSn experiments were performed with an isolation width of 1.2 mass units and the fragmentation amplitude was 0.6

(arbitrary units). Six parent ions were selected and were isolated and fragmented, producing MS2 and MS3 spectra by collision induced dissociation with He gas. Full scan mass spectra were acquired over the m/z range 50–800 in the positive ion mode. For MS/MS experiments, experimental conditions consisted of: m/z range:

50–500; isolation width, 2 m/z units; fragmentation energy, 30–200%; fragmentation time, 40 ms.

3.2.2.4 Thermal stability study of the cross-linking covalent bond

The thermo stability study of the cross-linking bond was done with a voltage-controlled heater using an oil bath. A 50 µL volume of the samples from the RP-HPLC peak containing the dG-AcLysOMe adduct were heated to 170 0C for

1 hour and then reconstituted in 50 µL of water. The product after heating was also analyzed by the ESI-ITMS under the conditions given above.

3.2.2.5 DNA-Ribonuclease A Oxidative Cross-Linking

For the cross-linking reaction, the Ribonuclease A and single stranded

27mer-nucletide DNA were mixed in a 0.5 ml centrifuge tube without lid and then rose bengal was added as the photo oxidant [500 µL; 0.35 mM ribonuclease A,

0.5 mM DNA and 1 mg/mL rose Bengal, 25 mM sodium phosphate, pH=8]. The mixture was irradiated for 1 hour with a LED lamp located 2 cm above the solution and providing 3 mW of radiative flux at 375nm.

3.2.2.6 Purification of the synthesized DPCs

Prussia, PA, USA) was used. Two reagents, the 27mer-nucleotide DNA and the

RNase, and the solution after the cross-linking reaction were injected to the instrument separately. Other parameters for SEC and QQQ-ICPMS are shown in

Table 3.1. The chromatographic signal containing both sulfur and phosphorus identified as the DPC, was collected and concentrated to 100 µL by freeze drying for the following step.

3.2.2.7 Thermal Hydrolysis of DNA-Ribonuclease A cross-links and purification of dG-Ribonuclease

The solution containing the purified DNA-protein cross-link was heated to 170 oC for 1 hour as earlier described and was re-dissolved in 100 µL of water. Size

Exclusion Chromatography (SEC) in same condition is used to separate the components into molecular weight fractions following thermal hydrolysis.

3.2.2.8 Trypsin proteolysis of the dG-Ribonuclease A

After the purification of dG-RNase A from the components post-thermal hydrolysis, trypsin was added to the dG-RNAse A adduct to digest the RNAse A portion of the adduct into its peptide components. Water was used as blank and 1 mg/mL

56 Bovine Serum Albumin (BSA) was used as standard to evaluate the extent of proteolysis. Digestion was conducted in a pH buffered solution (mixing 15µL of 50 mM ammonium bicarbonate, 1.5 µL of 100 mM DDT, and 10 µL of sample for digestion in a 0.5 mL tube). The mixture was adjusted to 27 µL with ultrapure water and the sample was incubated at 95oC for 15 minutes. A 3 µL volume of 100 mM iodoacetamide was then added into the mixture and incubated in the dark at room temperature for 20 min, followed by addition of 2 µL of sequence grade modified trypsin to a final trypsin concentration of 0.1 mg/mL. The mixture is subsequently incubated overnight at 37oC. A 1 µL volume of 88% formic acid was added to the mixture to stop the reaction. The mixture is then subjected to ultrafiltration by loading into a 0.5 mL MWCO spin filter with a 10,000 Da (nominal) cut-off membrane and centrifuged for 10 minutes at 10,000 g. The filtrate containing the peptide fragments was injected into the nanoLC-Chip-ESI-ITMS system.

3.2.2.9 Peptide separation and identification by HPLC-Chip-ESI-ITMS

LC-MS/MS analysis of the peptide mixture was performed on an Agilent 6340 series HPLC- Chip-IonTrap XCT system (Agilent Technologies, Santa Clara, CA), equipped with a Chip Cube interface with an electrospray ionization source, a well plate sampler, a nano-flow binary pump, and a capillary-flow binary pump. The

Chip Cube can be operated in two modes, enrichment and analysis modes. A

Large Capacity Chip II (Agilent Technologies, Santa Clara, CA) contains an enrichment column and an analysis column, both made of stable-bond C18 silica

57 with 300 pore size packed with 5 µm diameter particles. In the enrichment mode, the sample was loaded on to the enrichment column with the isocratic flow (3% B pumped by the capillary-flow pump, and then, while in analysis mode, the sample was then separated on the analytical column with the gradient elution flow pumped by the nano-flow pump. The gradient for nano-flow pump is shown in

Table 3.1, along with other parameters for HPLC-Chip-ITMS. A 2 µL volume of the peptide mixture and the digested standard BSA solution were injected into the system separately.

3.2.2.10 MASCOT protein data base search

MS and MS/MS spectra obtained from HPLC-Chip-ESI-ITMS were extracted and deconvoluted before they were submitted for MASCOT protein database search

(Matrix Science–London, UK). They were searched against the species

Viridplantae under the database NCBInr. “Carbamidomethyl (C)” was selected as variable modification. The MASCOT score of significance threshold was 40. The

MS/MS tolerance was set as ±0.6 Da, the peptide tolerance as ± 1.2 Da, and peptide charges as 1+ to 3+. All the hits reported in this study have ion scores above the significance thresholds.

3.2.2.11 Calculations

Tryptic digest of RNAse A will result to 45 peptide fragments as predicted by the

UCSF MS Digest website: http://prospector.ucsf.edu/prospector/cgibin/msform.cgi?form=msdigest

58 The peptides are listed in Column B in Table 3..2. and their mono protonated mass to charge ratios are listed in Column C. Lysine containing peptides are predicted to form adducts with guanine in DNA. Addition of one dG molecule will result in Δm/z of 281.07. Because there are multiple lysine residues in the RNAse

A sequence, each of these residues can potentially form adducts with dG resulting to peptide fragment cross-linking with multiple equivalents of dG. For example, if the molecular weight of one peptide is M, it is possible to observe [M-H-ndG]+,

[M-2H-ndG]++, and [M-3H-ndG]+++ (n could be 1 or 2 or 3). In addition, each of the M-dG, M-2dG, M-3dG can also be ionized with two or three protons (the mass of one proton is 1.007825). All the results from the calculation were shown in

Table 3.2. and were used to identify the site of cross-linking.

3.3 Results and Discussion

3.3.1 A model DPC: the dG-AcLysOMe adduct

It was previously shown that under singlet oxygen oxidative conditions,

2-deoxguanosine was oxidized forming 8OdG and subsequently reacted with a nucleophile (i.e. water) to form the hyper-oxidation products such as Sp and Gh.

In this work, we investigated adduct formation between the amino acid

N-acetyl-Lysine-Methyl ester (AcLysOMe), containing a nucleophilic side chain with dG to produce the dG-AcLysOMe adduct. The mixture after the crosslinking reation is injected and separated via HPLC. To identify the retention times of each reactant and product, solutions containing the individual reactants,

2-deoxguanosine and AcLysOMe and the cross-linking reaction mixture were

59 injected separately. Reactions were allowed to proceed for 1 hour, 2 hours and 4 hours before HPLC analysis. As shown in Figure 3.1, the retention times of un-reacted AcLysOME and dG was determined to be 3.5 and 7 minutes, respectively.

Overlaying the chromatograms from the three reactions, 1, 2 and 4 hours, a peak unique to the cross-linking reaction at 5.2 minutes is increasing in intensity with irradiation time. This peak possessed a retention time that is halfway between that of unreacted AcLysOME and dG suggesting a combined hydrophobicity of the two components, and is likely the adduct between AcLysOME and dG. This adduct was purified via fraction collection, for further analysis.

3.3.2 Thermolysis of the dG-AcLysOMe adduct

The dG-AcLysOMe cross-link peak was collected and analyzed by flow Injection

ESI-ITMS via syringe pump . Mass spectral analysis reveals a species with a m/z value of 484.1, which is consistent with the of [Sp-5-AcLysOMeH]+. Figure 3.2a shows the resulting mass spectra and the proposed structure of dG-AcLysOMe adduct. MS2 fragmentation generated a m/z value of 386.1, which corresponded to the loss of ribose and a proton from the molecule, suggesting a nucleoside component of the adduct. The mass spectra and the proposed structure was shown in Figure 3.2b. Mass spectral analysis supported the formation of Sp-

AcLysOMe as the predominant product during singlet oxygen-induced oxidation. In order to investigate the thermal stability of the crosslinking covalent bond, the purified adduct was heated at 170oC for one hour, then re-suspended in 50 µL doubly distilled water and injected into the ESI-MS with a flow injection. As it was

60 shown in Figure 3.3, the resulting mass spectra a 484 m/Z corresponded to the intact adduct, and 368.1 m/Z corresponded to the adduct minus ribose. A m/z of

203 corresponds to AcLysOMe, and indicated a fragmentation at the covalent bond between AcLysOMe and dSp. The reaction of crosslinking formation and thermal hydrolysis is described in Figure 3.4.

3.3.3 Purification of laboratory synthesized DPCs by SEC

The separation of laboratory synthesized DPCs from the other reaction components is an increasingly important issue due to the low yield of the cross-linking reactions. However, the method established in chapter 2 with the high sensitivity and low limits of detection via QQQ-ICPMS for sulfur and phosphorus detection, is a promising approach when using Size Exclusion

Chromatography (SEC) for separation and purification of the synthesized DPC.

The product peak is isolated via fraction collection for subsequent analyses with the protocol described in chapter 2.

3.3.4 Expansion to identification of whole protein DPCs

Having shown that the covalent bond between AcLysOMe and dG is thermally stable, the purified DNA-RNase cross-linking adduct was heated and hydrolyzed under the same conditions to determine if similar stability would be observed. The solution after thermal hydrolysis was injected into the system and separated by

SEC-HPLC with UV detection. In Figure 3.5, an iso-absorbance plot of the chromatogram obtained is shown. Due to the affect of its larger size, the earliest

61 eluting peak with the maxim absorbance at 260 nm is the intact DNA-RNAse A adduct. Additionally, two other peaks are observed with maximum absorbance around 260 nm. By comparing with SEC standards, the peak with a retention time of 24.2 min is the DNA strand while the peaks eluting after 30 minutes are the free nucleotides post thermal hydrolysis. Most importantly, there was a peak with two higher UV absorbances, 280nm, matching protein specific absorbance and

240nm, matches the cross-linking bond specific absorbance. We concluded that the peak mentioned above, which exhibited the unique absorbance for protein at

280nm and for oxidized dG at 240nm is the RNAse A-dG residue following thermal hydrolysis. This fraction was isolated via fraction collection and concentrated to 100 µL by freeze drying for further studies.

3.3.5 Identification of the dG-containing peptide-by-peptide sequence matching

The isolated dG-RNAse A adduct from the preceding section was subjected to trypsin digestion and analyzed using nanoLC-Chip-ESI-ITMS. The resulting mass spectrum was used to search the MASCOT database for peptide identification.

The search results are shown in Table 3.2. For the trypsin proteolysis of the

RNase A, the maximum missed cleavage was set as 2 and the minimum peptide length was set as 5. Variable carbamidomethyl modification of Cysteine was accepted. The detection range was set from m/z from 250 to 3000. Tryptic digest of RNAse A will result to 45 peptide fragments as predicted by the PeptideCutter search tool (http://web.expasy.org/peptide_cutter/). Lysine containing peptides

62 are predicted to form an adduct with guanine in DNA. Addition of one dG molecule will result in Δm/z of 281.1. Because there were multiple lysine residues in the

RNAse A sequence, each of these residues could potentially form adducts with dG resulting to peptide fragment cross-linking with multiple equivalents of dG. For example, if the molecular weight of one peptide is M, it is possible to observe

[M-H-ndG]+, [M-H-ndG]++, and [M-H-ndG]+++. All the possibilities for the m/z value were calculated and shown in Table 3.2. The extracted ion chromatogram (EIC) for each of these possibilities was checked and both MS and MS2 were also extracted to determine if any distinct peptide feature is observed.

Two values from Table 3.2, 810.36 m/z and 640.78 m/z, fit the requirements for a cross-linked peptide. Each of their EICs showed an apparent peak and MS and

MS2 indicated distinctive characteristics and peptide. The 810.36 m/z matched the m/z value of [M-3H-1dG]+++, where M corresponded to the peptide,

ETGSSKYPNCAYKTTQANK. Since there are three lysine residues in this peptide, dG can be cross-linked to any or all of these residues. Additionally, dG could form adducts with tyrosine residues in the sequence giving an m/z that is equal to that of dSp-Lysine adduct. To account for all the possible masses, a theoretical fragmentation table for each cross-linking possibility was constructed.

The thirty-one most intense peaks during MS2 were compared with the predicted values in the fragmentation table, with the highest coverage of ten of the thirty-one peaks matching the theoretical fragmentation table for the adduct,

ETGSSK(dG)YPNCAYKTTQANK. Two cross-links had low coverage, that is, 7 in

63 one of the cross-links and 2 in the other of the 31 most intensive peaks, show a match on the fragmentation table for the MS2 from the corresponding EICs.

Although 640.78 m/z fit the MS characteristics of KETAAK, further investigation shows that 0 of the 31 peaks in the MS2 spectrum matched with the theoretical fragmentation table, meaning this fit was coincidental.

3.3.6 Cross-linking prediction

Therefore, it can be concluded that the cross-linking reaction first took place on lysine91 in the peptide, ETGSSKYPNCAYKTTQANK, on the protein ribonuclease

A. The structure of ribonuclease A was shown in Figure 3.7 and the peptide sequence was labeled in red chain with lysine91 shown in balls. From this structure scheme it can be seen that this lysine is exposed, making it sterically possible to associate with the DNA. Secondly, this peptide appears as a loop, which is preferable to trap the DNA.

3.4 Conclusion

In this work, we demonstrate thermal stability of DNA-protein adducts, both in the nucleoside-amino acid model, and more importantly, in the DNA-protein model.

These findings suggest that these lesions are stable and are not easily reversed.

There are two important implications. The first one is biological in that,

DNA-protein cross-link formation will have significant consequences when generated inside the cell if it remains unrepaired. It is likely to block important cellular processes such as replication (producing the highly detrimental double

64 strand breaks that can lead to genetic instability and cancer) and can also hamper the transcription process and impede production of important proteins. The second implication of these findings is that because of the stability of the adduct, a facile method of DPC characterization (i.e. the cross-linking site, the amino acid and base involved and more importantly identifying the proteins that participate in cross-linking) can be developed. In this study, we have done the first step in intact cross-link characterization by successfully hydrolyzing the adduct between DNA and protein, removing only the un-cross-linked nucleosides. This was followed by tryptic digestion, which was initially thought to induce cross-link hydrolysis and would hinder characterization of intact DPCs by mass spectrometry. We are able to identify the cross-linking site following proteolysis of cross-linked RNAse A. In our synthesized DPCs model, ETGSSKYPNCAYKTTQANK was the peptide involved in the crosslinking reaction and the first lysine is the crosslink site with guanine. This is a significant advance from the small molecule models and will be useful for total characterization of DPCs, protein identification and in biomarker discovery, where the cross-link identity has potential use as fingerprint for disease.

Table 3.1. Operating conditions for ICPMS/MS, SEC and HPLC-CHIP-ESI-ITMS

RF power (W) 1550 Carrier gas flow rate (L min-1) 0.9 Plasma gas flow rate (L min-1) 14 ICPMS Oxygen gas flow rate (L min-1) 0.1 paramete Ions monitered 47 PO+ 48 SO+ rs RF matching (V) 1.7 Smpl Depth (mm) 8.0 Smpl Period (s) 0.6 Column A, 25mM ammonium acetate in acetonitrile/water (3/97, v/v) mixture RP Mobile phases B, 25mM ammonium acetate in Chromato acetonitrile/water (95/5,v/v) mixture graphic Flow rate (mL min-1) 1 paramete 0-7 min, 0% B; 7-22 min,0- 100% B; rs Gradient 22-24 min, 100% B; 24-26 min, 100-0% B Injection volume (uL) 4 Large Capacity Chip II Zorbax Column 300SB-C18 (150mm x 75um, 5um) 3 for capillary flow pump; 0.3 for nano Flow rate (uL min-1) flow pump HPLC-Chi A, 0.1% formic acid in water; B, 0.1 % p-ESI-IT Mobile phases formic acid in acetonitrile/water (90/10, MS v/v) mixture paramete Gradient rs Nano spray needle voltage (V) 1850 Injection volume (uL) 2 Drying temperature (°C) 300 -1 Drying gas (L min N2) 3.0 mass range 100-2200 m/z

66 Table 3.2 Results of Crosslink Calculations

Carb m/z amid [M+2H] [M+3H+ [M+H+d [M+2H+ [M+3H+ [M+H+2 [M+2H+ [M+3H+ [M+H+3 [M+2H+ [M+3H+ No Peptide K (mono) omet ++ ++ G]+ dG]++ dG]+++ dG]+ 2dG]++ 2dG]++ dG]+ 3dG]++ 3dG]+++ hyl 1 ETGSSKYPNCAYKTTQANK 2090.98 0 3 1045.99 697.67 2372.05 1186.53 791.36 2653.12 1327.06 885.05 2934.19 1467.60 978.74

2 ETGSSKYPNCAYKTTQANK 2148.00 1 3 1074.51 716.67 2429.07 1215.04 810.36 2710.14 1355.58 904.05 2991.21 1496.11 997.74

3 CKPVNTFVHESLADVQAVCSQK 2403.18 0 2 1202.09 801.73 2684.25 1342.63 895.42 2965.32 1483.16 989.11

4 CKPVNTFVHESLADVQAVCSQK 2460.20 1 2 1230.60 820.74 2741.27 1371.14 914.43 3022.34 1511.67 1008.12

5 CKPVNTFVHESLADVQAVCSQK 2517.22 2 2 1259.12 839.75 2798.29 1399.65 933.44 3079.36 1540.19 1027.13

6 KETAAAKFER 1150.62 0 2 575.81 384.21 1431.69 716.35 477.90 1712.76 856.88 571.59

7 ETGSSKYPNCAYK 1447.65 1 2 724.33 483.22 1728.72 864.87 576.91 2009.79 1005.40 670.60

8 YPNCAYKTTQANK 1501.71 1 2 751.36 501.24 1782.78 891.89 594.93 2063.85 1032.43 688.62

9 KETAAAK 718.41 0 2 359.71 240.14 999.48 500.24 333.83 1280.55 640.78 427.52

10 DRCKPVNTFVHESLADVQAVCSQK 2731.33 1 2 1366.17 911.11 3012.40 1506.70 1004.80 3293.47 1647.24 1098.49

11 DRCKPVNTFVHESLADVQAVCSQK 2788.35 2 2 1394.68 930.12 3069.42 1535.21 1023.81 3350.49 1675.75 1117.50

12 CKPVNTFVHESLADVQAVCSQKNVACK 2918.43 0 2 1459.72 973.48 3199.50 1600.25 1067.17 3480.57 1740.79 1160.86

13 CKPVNTFVHESLADVQAVCSQKNVACK 2975.45 1 2 1488.23 992.49 3256.52 1628.77 1086.18 3537.59 1769.30 1179.87

14 NVACK 534.27 0 1 267.64 178.76 815.34 408.17 272.45

15 NVACK 591.29 1 1 296.15 197.77 872.36 436.68 291.46

16 ETAAAK 590.31 0 1 295.66 197.44 871.38 436.20 291.13

17 ETGSSK 608.29 0 1 304.65 203.43 889.36 445.18 297.12

18 TTQANK 662.35 0 1 331.68 221.45 943.42 472.21 315.14

19 SRNLTK 718.42 0 1 359.71 240.15 999.49 500.25 333.84

20 NLTKDR 746.42 0 1 373.71 249.48 1027.49 514.25 343.17

21 YPNCAYK 858.38 0 1 429.69 286.80 1139.45 570.23 380.49

22 YPNCAYK 915.40 1 1 458.21 305.81 1196.47 598.74 399.50

67 23 SRNLTKDR 989.55 0 1 495.28 330.52 1270.62 635.81 424.21

24 ETAAAKFER 1022.53 0 1 511.77 341.51 1303.60 652.30 435.20

26 QHMDSSTSAASSSNYCNQMMK 2307.91 0 1 1154.46 769.98 2588.98 1294.99 863.67

27 QHMDSSTSAASSSNYCNQMMK 2364.93 1 1 1182.97 788.98 2646.00 1323.50 882.67

28 QHMDSSTSAASSSNYCNQMMKSR 2551.04 0 1 1276.03 851.02 2832.11 1416.56 944.71

29 QHMDSSTSAASSSNYCNQMMKSR 2608.06 1 1 1304.54 870.03 2889.13 1445.07 963.72

30 NVACKNGQTNCYQSYSTMSITDCR 2687.13 0 1 1344.07 896.38 2968.20 1484.60 990.07

31 FERQHMDSSTSAASSSNYCNQMMK 2740.12 0 1 1370.56 914.05 3021.19 1511.10 1007.74

32 FERQHMDSSTSAASSSNYCNQMMK 2797.14 1 1 1399.08 933.05 3078.21 1539.61 1026.74

33 NVACKNGQTNCYQSYSTMSITDCR 2744.15 1 1 1372.58 915.39 3025.22 1513.12 1009.08

34 NVACKNGQTNCYQSYSTMSITDCR 2858.20 3 1 1429.60 953.40 3139.27 1570.14 1047.09

35 NVACKNGQTNCYQSYSTMSITDCR 2801.17 2 1 1401.09 934.40 3082.24 1541.63 1028.09

36 NGQTNCYQSYSTMSITDCRETGSSK 2818.17 1 1 1409.59 940.06 3099.24 1550.12 1033.75

37 NGQTNCYQSYSTMSITDCRETGSSK 2761.15 0 1 1381.08 921.06 3042.22 1521.61 1014.75

38 NGQTNCYQSYSTMSITDCRETGSSK 2875.19 2 1 1438.10 959.07 3156.26 1578.64 1052.76

39 TTQANKHIIVACEGNPYVPVHFDASV 2810.39 0 1 1405.70 937.47 3091.46 1546.24 1031.16

40 TTQANKHIIVACEGNPYVPVHFDASV 2867.41 1 1 1434.21 956.48 3148.48 1574.75 1050.17

41 FERQHMDSSTSAASSSNYCNQMMKSR 2983.26 0 1 1492.13 995.09 3264.33 1632.67 1088.78

42 NGQTNCYQSYSTMSITDCR 2171.88 0 0 1086.44 724.63

43 NGQTNCYQSYSTMSITDCR 2228.90 1 0 1114.95 743.64

44 NGQTNCYQSYSTMSITDCR 2285.92 2 0 1143.46 762.65

45 HIIVACEGNPYVPVHFDASV 2224.09 1 0 1112.55 742.03

Figure 3.1. a) Reverse Phase chromatogram of solutions after 1 hour, 2hour and 4 hour reaction times. b) Reverse Phase Chromatogram of two reactants (AcLysOMe, 2-dG) and and purified dG-AcLysOMe

O O HN C a O

H N O N O NH HO N O N NH2

Sp-5-lys Chemical Formula: C19H29N7O8 Exact Mass:483.21

Figure 3.2. a) MS of the dG-AcLysOMe crosslinking compound. b) MS2 of the dG-AcLysOMe crosslinking compound

Figure 3.3. MS of the dG-AcLysOMe crosslinking compound after heating at 170 0C for 1 hour

O O HN C O

HO H N O N O O O + OH NH N NH HN HO N NH2 N NH2 HO O N

HO Chemical Formula: C14H21N7O5 Exact Mass:367.16

H2O oxidant heat

O O O H O O N NH HN C HN C O O HO O N NH2 O N H N O H N N N O O O NH HO O NH HO 2 HO N + N HN C Lysine O O 8O-dG O N NH2 N NH2 H2N HO HO Chemical Formula: C9H18N2O3 Exact Mass:202.13 Sp-5-lys Gh-5-lys Chemical Formula: C19H29N7O8 Chemical Formula: C18H31N7O7 Exact Mass:483.21 Exact Mass:457.23

Figure 3.4. Crosslinking reaction between 2-deoxguanosine (dG) and N-acetyl-Lysine-Methyl ester (AcLysOMe)

Figure 3.5. Isoabsorbance plot after thermal hydrolysis of DPC showing that the heating process partially broke the glucosidic bond, leaving a DNA strand and the Rnase-dG adduct, At the same time, some of the DPC remained stable, which eluted earlier. In addition, some of the nucleoside was removed from the DNA strand, which eluted after 30 minute.

Figure 3.6. a) Mass spectrum for Identification of the dG-containing peptide by peptide sequence matching. b) b and y ion description by table with matches to the MS/MS spectrum of Figure 3.6 a)

Figure 3.7. 3D Structure of the Ribonuclease A

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76 33. Rhee, Ho S. and B.F. Pugh, Comprehensive Genome-wide Protein-DNA Interactions Detected at Single-Nucleotide Resolution. Cell. 147(6): p. 1408-1419. 34. Geyer, H., R. Geyer, and V. Pingoud, A novel strategy for the identification of protein–DNA contacts by photocrosslinking and mass spectrometry. Nucleic Acids Research, 2004. 32(16): p. e132-e132. 35. Doneanu, C.E., et al., Mass Spectrometry of UV-Cross-Linked Protein−Nucleic Acid Complexes: Identification of Amino Acid Residues in the Single-Stranded DNA-Binding Domain of Human Replication Protein A. Analytical Chemistry, 2004. 76(19): p. 5667-5676. 36. Wang, Y. and Y. Wang, Structure Elucidation of DNA Interstrand Cross-Link by a Combination of Nuclease P1 Digestion with Mass Spectrometry. Analytical Chemistry, 2003. 75(22): p. 6306-6313. 37. Catalano, M.J., et al., Chemical Structure and Properties of Interstrand Cross-Links Formed by Reaction of Guanine Residues with Abasic Sites in Duplex DNA. Journal of the American Chemical Society, 2015. 137(11): p. 3933-3945. 38. Loeber, R., et al., Cross-Linking of the Human DNA Repair Protein O6-Alkylguanine DNA Alkyltransferase to DNA in the Presence of 1,2,3,4-Diepoxybutane. Chemical Research in Toxicology, 2006. 19(5): p. 645-654. 39. Wickramaratne, S. and N.Y. Tretyakova, Structure Elucidation of DNA–Protein Crosslinks by Using Reductive Desulfurization and Liquid Chromatography–Tandem Mass Spectrometry. ChemBioChem, 2014. 15(3): p. 353-355. 40. Petrova, K.V., et al., Characterization of the Deoxyguanosine–Lysine Cross-Link of Methylglyoxal. Chemical Research in Toxicology, 2014. 27(6): p. 1019-1029. 41. Li, H., et al., Mass Spectrometry Evidence for Cisplatin As a Protein Cross-Linking Reagent. Analytical Chemistry, 2011. 83(13): p. 5369-5376. 42. Gong, J., et al., Developing ICP-MS/MS for the detection and determination of synthetic DNA-protein crosslink models via phosphorus and sulfur detection. Analytical and bioanalytical chemistry, 2015. 407(9): p. 2433-2437.

Chapter 4

Purification and Identification of Novel Endogenous Cardiotonic Steroids (CTS) from pig skeleton muscle

78 4.1 Introduction

Cardiotonic steroids (CTS), which are also referred to as digitalis-like fators, are one of the most useful groups of drugs in therapeutics as they behave like inhibitors of the Na+/K+-ATPase[1, 2]. Na+/K+-ATPase is an enzyme found in the plasma membrane of all animal cells. It was discovered in the 1950s by Jens

Christian Skou, which is a significant step in the hundreds years of study on the cell as a basic unit of animal life. His group first proved the existence of a protein-based structure, incorporated in the living cell membrane, which regulates the sodium and potassium ions concentration against their concentration gradient, during the crab nerve experiment[3]. The Na+/K+-ATPase behaves like an ion solute pump, pumping three sodium ions out of cells while pumping two potassium ions into cells each time, hydrolyzing Adenosine Triphosphate (ATP) to

Adenosine Diphosphate (ADP) to provide energy[4].

The sodium pump is directly responsible to control multiple essential cellular functions[5-8]. The generation of the resting membrane potential results from the action of the Na+/K+-ATPase, removing one positive charge carrier from the intracellular space[9]. Another important function of the Na+/K+-ATPase is to provide Na+ gradient that is the driving force for several secondary active transporters membrane transport proteins, importing glucose, amino acids, and other nutrients into the cell[10]. The Na+/K+-ATPase contributes to control the cell volume as well owning to its ability to help maintain a cell’s osmolarity by adjusting the concentration of ions species in the cell[11]. In addition, more and more

79 research proved that Na+/K+-ATPase could also relay extracellular ouabain-binding signal into the cell through regulation of protein tyrosine phosphorylation, mediating as signal transducer[12]. Furthermore, it has been shown recently that the Na+/K+-ATPase plays an important role in controlling the intrinsic activity mode of cerebellar Purkinje neurons[7].

The structure of Na+/K+-ATPase has been fully investigated[13, 14]. The minimum functional entity of the Na+/K+-ATPase consists of two α and β polypeptides in equimolar ratios[15]. Each of these subunits has different isoenzymes[16]. There are ten transmembrane segments with about 112 kD and 1012 amino acids on the α catalytic subunit. They schematically present in an “unfolded” disposition with N- terminal and C terminal ends into the cytoplasmic side[17]. However, there is only one single-transmembrane segment with about 55 kD and 300 amino acids on the

β subunit[18]. Additionally, there is a third γ subunit, associating with α,β-subunit complex on the Na+/K+-ATPase. While it is not an integral part the Na+/K+-ATPase, it behaves as the modulator of the enzyme transport activities[19]. There are some special binding sites of CTS on the extracellular segment of α subunit, including

TM1-TM2, TM5-TM6, TM7-TM8 loops and several amino acids from the transmembrane regions M4, M6 and M10[20]. Once the CTS were bound, the pumping activity of Na+/K+-ATPase will be inhibited.

Figure 4.1. Structure of the Na+/K+-ATPase

Evidence has showed that CTS play very important roles in a lot of physiological processes and functions by inhibiting the ions transporting activity of the

Na+/K+-ATPase[1]. CTS can be extracted from various plants and animals. At least

10 CTS have been partially purified from mammals[21-25]. However, only three of them have been structurally analyzed by mass spectrometer and NMR spectrometer. At least six HPLC separations are needed to purify one compound owning to the complex matrix 40 years ago. The exploration of antibody affinity column highly reduced the HPLC separation steps by specially binding to CTS[26].

However, this method can only be used to purify known compounds. Batch affinity extraction (BAE) was first introduced to purify CTS in human plasma by using the

CTS binding sites on the Na+/K+-ATPase itself to capture its endogenous ligands[27]. This method highly reduces the HPLC separation steps and provides the possibility of purifying the endogenous CTS in large scale. In my study, BAE is also used as the first basic separation method for the purification of endogenous

81 CTS from pig skeleton muscles.

NADH coupled method, or NADH turnover method, is proved effective in checking the activity of Na+/K+-ATPase inhibitors[28]. In the perspective of physiology,

Na+/K+-ATPase stimulates the hydrolysis of ATP, converting it to ADP[29]. However, pyruvate kinase[30], an enzyme involved in glycolysis, can at the same time catalyze the transfer of a phosphate group from phosphoenolpyruvate (PEP) to

ADP, yielding one molecule of pyruvate and one molecule of ATP[31]. Pyruvate is easily converted to lactate under the catalysis of lactate dehydrogenase (LDH), with the conversion of NADH to NAD+[32]. As NADH has maximum absorption around 340 nm while the absorption at the same wavelength for NAD+ is 0[33], the decline of absorption around 340 nm represents the conversion efficiency of APT to ADP. CTS are a group of compounds, which can specifically bind to

Na+/K+-ATPase, inhibiting the conversion of ATP to ADP and finally interrupting the consuming of NADH. In a word, the inhibition activity of the CTS, or OLC on the Na+/K+-ATPase can be determined by calculating the rate of decline in absorption of NADH at 340 nm.

Figure 4.2. NADH coupled method mechanism.

In this chapter, a method to purify endogenous CTS from pig skeleton muscles was established via batch affinity extraction, gel filtration chromatography and reverse phase separation. The activity of the purified compound was tested with

NADH coupled method. Further molecular analysis was carried out by electrospray ionization ion trap mass spectrometry (ESI-ITMS).

4.2 Experimental

4.2.1 Materials

All experiments were done in triplicate or greater. All chemicals were purchased from Fisher Scientific (Pittsburgh, PA, USA) unless otherwise noted. Ouabain standard were obtained from Sigma Aldrich (St. Louis, MO, USA). Molecular markers for gel filtration chromatography were purchased from Bio-Rad

Laboratories (Hercules, CA).

83 4.2.2 Methods

4.2.2.1 Homogenization of pig skeletal muscle

8 g of frozen pig skeletal muscle were mixed with 560 mL of the chilled 0.1% trifluoroacetic acid (TFA) / 50% methanol in a big beaker chilled in an ice bath.

The pig skeletal muscle was homogenized with the Tissue Tearor (TT), with the running setting of #4 for 120 sec and then of #5 for 30 sec. Forming needed to be avoided carefully. Once the skeletal muscle was homogenized, it was placed on the VWR® Standard Orbital Shaker (VWR Corporate, Radnor, PA) to shake for 20 min, keeping it still in the ice bath. After shaking, the solution was divided into 10 x

60 mL Beckman ultracentrifuge tubes equally and centrifuged in a at 11400 g,

4 °C for 20 min. in a Beckman Coulter Avanti J-E High-Performance Centrifuge

(Beckman Coulter, Hebron, KY). The supernatant was filtrated with P5 filter paper by flow meter and then dried with LabTech EV311 Rotary Evaporator (LabTech

International, East Sussex, TN) to remove methanol. The water bath was set at

40 °C and keep the rpm at 50. The dry sample was reconstituted in 50 mL of double deionized water and shaked on the shaker for 30 min at 40 rpm. Then the solution was centrifuged at 11400 g, 4°C for 20 min. The supernatant was spinned on the centrifuge at 29400 g, 4°C for 20 min again. The supernatant was lyophilized until dry for the following step. I named it as ouabain-like compound

(OLC)

4.2.2.2 Batch affinity extraction

The binding buffer (BB) was prepared in a volumetric flask (1 L: 280 mM Sucrose,

84 10 mM 2-hydroxyethyl, 5 mL MgCl, 5mM Na2HPO4, 1 mM NaVO3, 0.02%

Saponin), titrated to pH = 7.4 and incubated at 37.4 °C. Dried sample and purified lamb kidney Na+/K+-ATPase (NKA) were reconstituted in the BB in a polycarbonated capped centrifuge tube (15 mL, 100 nM OLC, 6.25 µM NKA). The extraction was carried out at 37.4 °C for 3 h. Then 45 mL of ice cold Binding Buffer was added into the tube to stop the reaction. The solution was centrifuged at

38,000 rpm, 4 °C for 60 min. The supernatant was discarded and the pellet was resolublized into 60 mL of BB and homogenized with TT at 10 -20 K after washing with nanopure water for three times. The solution was centrifuged and washed again in the same condition mentioned above, however, at this time it was resolublized into 60 mL of release buffer (1L: 280 mM sucrose, 10 mM

2-hydroxyethyl, 5 mM Ethylene Diamine Tetraacetic Acid (EDTA), 0.02% Saponin, pH = 7.4). The solution was homogenized with TT at 10-20 K and incubated at

37 °C for 16 h to unbind the OLC and then centrifuged in the same condition. The pellet was discarded and the supernatant was concentrated to 5 mL by freeze-drying for the following step.

4.2.2.3 Post affinity gel filtration chromotography

To basic purify the OLC from large proteins and small molecules from last step, gel filtration was carried out on an Agilent 1100 series system (Agilent

Technologies, Santa Clara, CA, USA) equipped with a vacuum degasser, one quaternary pump, a manual injector with 500 µL loading loop, a thermostatically controlled column compartment and a UV detector. All lines within the capillary

85 system were PEEK covered fused silica tubing with 100 µm internal. An Superdex

Peptide 10/300 GL gel filtration column (13µm, 300x10mm) was used for separation. The monitored UV spectrum was set at 220 nm. The mobile phase consisted of 99.5% DDI and 0.5% methanol with 50mM ammonium acetate. The gel filtration separation was performed for 45 minutes with the flow rate at 0.5 mL/min. To estimate the molecular weight time of OLC, 50 µL of the molecular weight markers for gel filtration chromatography [0.5 mL: 10 mg/mL thyroglobulin

(bovine), 10 mg/mL γ-globulin (bovine), 10 mg/mL ovalbumin (chicken), 5 mg/ml myoglobin (horse), 1mg/mL Vitamin B12] were injected into the HPLC system with the UV absorbance set at 280 nm. Then the OLC solution was injected into the system with the maximum injection volume of 500 µL. Fractions from 16 to 23 min

(Fa0), 28 to 34 min (Fb0), 34 to 38 min (Fc0) and 38 min to 40 min (Fd0) were collected in different vials. The mobile phase consisted of 99.5% DDI and 0.5% methanol with 50mM ammonium acetate. The gel filtration separation was performed for 45 minutes with the flow rate at 1 mL/min. Freeze-drying was followed to concentrate the fraction to 100 µL for the following step.

4.2.2.4 Reverse Phase separation of OLC

The reverse phase separation of the OLC for each fractions collected above was carried out by HPLC on an Agilent 1100 series system equipped with a vacuum degasser system, two binary HPLC pumps, a well plate autosampler, a temperature column compartment, and a diode array detector. All lines within this

HPLC system were PEEK covered fused silica tubing with 100 µm internal

86 diameter. The UV absorbance was recorded from 190 to 600 nm. The response time (peak width) was set < 0.01 min (0.1s) and the slit was 4nm. The gradient conditions, at a flow rate of 1 mL/min, were 3 min for 97% solvent A (100% water with 0.1% formic acid) and 3% solvent B (100% acetonitrile with 0.1% formic acid), followed by a linear ramp to 55% B in 52min. Then the gradient was ramped to

100% B in 5 min and held for 5 min before linearly decreasing to 3% B in 5 min. 20 minutes of column equilibration was needed in preparation before each run. For

Fa0 from the gel filtration, fractions from 1 - 1.5 min (Fa1), 1.5 -2 min (Fa2), 2 -2.2 min (Fa3), 2.2 - 2.4 min (Fa4), 24 - 34 min (Fa5), 34 -50 min (Fa6), 50 -70 min (Fa7) were collected; for Fb0, fractions from 0.8 -2.1 min (Fb1), 2.1 -2.6 min (Fb2), 24 -34 min (Fb3), 34 -70 min (Fb4) were collected; for Fc0, fractions from 0.8 -2.0 min (Fc1)

2 -2.2 min (Fc2), 2.2 -2.5 min (Fc3), 2. 5 -5 min (Fc4), 5 -14 min (Fc5), 24 -34 min

(Fc6), 34 -70 min (Fc7) were collected; for Fd0, fractions from 1.3 -3.5 min (Fd1) and 24 -34 min (Fd2) were collected. All the fractions were concentrated to 100

µL.

4.2.2.5 Activity determination of OLC by NADH coupled essay

The activities of OLC fractions after gel filtration and reverse phase separation were determined by the NADH coupled essay. 15 μL of NKA samples was incubated with 40 μL of OLC fractions with different concentration (1:1 to 1:104) or

40 μL Ouabain (1mM to 10-4mM) at 37.4 °C . After 15 minutes incubation in 96 multi-well plates, 36 μL of ATPase activity buffer [10 mL: 110 mM NaCl, 20 mM

KCl, 5 mM MgCl2, 30 mM 2-hydroxyethyl, 2 mM Ethylene Glycol Tetraacetic Acid

87 (EGTA), 0.5 mM sodium azide, 1 mM phosphoenolpyruvate, 0.2 mM 1, Beta

Nicotinamide adenine dinucleotide hydrate, 30 U.mL-1 lactate dehydrogenase, 10

U.mL-1 pyruvate kinase, 0.01% Saponin , pH = 7.4] was added to each well. After

5 min incubation, absorption of samples was measured simultaneously by

PerkinElmer Envision 2100 series Multimode Plate Reader (PerkinElmer,

Waltham, MA). The NKA phosphorylation reaction was started by adding 15 µL

ATP (30 mM) to each well. The rate of decline in absorption at 340 nm, caused by

NADH oxidation, is an index of rate of ATPase hybridization.

4.2.2.6 Mass spectrum analysis of active OLC fraciton

Mass spectrum analysis of active OLC fracitons was performed on a 6340 series

MSD Ion Trap XCT Ultra system with direct infusion via a syringe pump at a flow rate of 25 µL/h (Bruker Corporation, Billerica, MA) with the micro fluidic nanospray chip cube interface, which is automatically loaded and positioned into the MS nanospray chamber. The system was operated in ultra-scan positive ion mode with the maximum accumulation time of 300 msec. The ESI-ITMS was tuned under positive ion mode with the ESI Tuning Mix for Ion Trap (Agilent

Technologies, Santa Clara, CA) containing compounds at the following m/z: 118,

322, 622, 922, 1,522, and 2,122. All MSn experiments were performed with an isolation width of 1.2 mass units and the fragmentation amplitude was 0.6

88 mode. For MS/MS experiments, experimental conditions consisted of: m/z range:

50–500; isolation width, 2 m/z units; fragmentation energy, 30–200%; fragmentation time, 40 ms. Acetonitrile and nanopure water were used to rinse the system between each injections.

4.3 Results and Discussion

4.3.1 Post affinity gel filtration

The molecular weight markers for gel filtration chromatography were used to estimate the molecular weight time of OLC. The molecular weight of each marker is shown in Table 4.1.

Table 4.1. Gel filtration marker

Marker Molecular weight (kDa)

Thyroglobulin (bovine) 600

γ-globulin (bovine) 158

Ovalbumin (chicken) 44

Myoglobin (horse) 17

Vitamin B12 1.3

Size-exclusion chromatography (SEC) is a chromatographic method in which molecules in solution are separated by their size, and in some cases molecular weight. Within the column separation range, the higher the molecular weight of a compound, the earlier it will be eluted from the column. By compared with the retention time of OLC after BAE with the molecular weight markers on Figure 4.1, it can be concluded that the OLC fraction eluted at the similar retention time with

89 Vitamin B12, which meant that the molecular range of the OLC fraction is below

1.3 kDa. In addition, the chromatogram of OLC showed that several peaks overlapped, indicating that further purification is still need for OLC samples.

4.3.2 Reverse Phase separation of OLC

Four fractions collected from gel filtration separation were injected into the HPLC system and separated by reverse phase chromatography after concentrated.

Figure 4.2 showed the four chromatograms and a series of peaks with strong absorption at 220 nm. All of these peaks, and the left over fractions after gel filtration were collected to check which one behaved like ouabain by NADH coupled essay.

4.3.3 Activity determination of OLC by NADH coupled essay

The turnover activity of NKA is determined by calculating the rate of decline in absorption of NADH at 340 nm for various concentrations of ouabain and OLC samples. The dose–response relationship was obtained by drawing the slope of the absorption decay of NADH according to logarithm of the ouabain or OLC samples concentration. These curves described the inhibition of ouabain or OLC samples in effect on NKA caused by differing levels of doses after a certain exposure time. The half maximal effective concentration (EC50) was obtained from the does-response curve. Apparent affinity constant K0.5 were determined based on ATPase activity of individual samples in presence of various concentrations of ouabain/OLCs by nonlinear regression with a Hill equation in the following form

90 with a variable Hill coefficient b:

Figure 4.3 showed the does-response curve of each fraction compared with ouabain as standard. No inhibition was observed in all of the Fa fractions. Among the Fb fractions, Fb0 and Fb1 showed activity and the Log EC50 was calculated equal to -2.2 and -1.9. Fc1 and Fc2 also behaved like ouabain standard with the

Log EC50 equal to -1.2. All of the Fd fractions showed activity and Log EC50 for Fd1 is -0.5, for Fd2 is -2.4, for Fd3 is -2.8. These results was consistent with our prediction that Fa0, Fb0, Fc0 and Fd0 showed more abilities of NKA inhibition since they are the fractions before reverse phase chromatography, a combination of all the other fractions.

4.3.4 Mass spectrum analysis of active OLC fractions

All of the active OLC fractions after reverse phase separation, Fb1, Fc1, Fc2, Fd1 and Fd2 were analyzed by ESI-ITMS with flow injection in positive mode. As it shown in Figure 4.13a, for active fraction Fb1, the most intensive signal is the ion with mass to charge ratio (m/z) of 477.4 and its fragmentation information (MS2) is shown in Figure 4.13b. The ion after collision with m/z of 238.1 has the exactly same m/z with the second intensive signal on Fb1a. For active fraction Fc1, the most intensive signal is the ion with mass to charge ratio (m/z) of 260.6 and its fragmentation information (MS2) is shown in Figure 4.14b. In the MS2, there is a signal giving m/z of 651.1, which indicated that the ion with m/z of 260.6 was a triple charged ion. Its molecular weight was supposed to be 778.8. Same thing

91 happened to the second intensive signal, 282.8, and the third intensive signal

393.5, as shown in Figure 4.14c and Figure 4.15e, and their molecular weight were calculated as 846.0 and 1177.5. Also, the fragmentation of the ion with m/z of 810.5 was same with the second intensive signal, which implicated that those two ions were charged from same molecular with different ionization. The MS of active fraction Fc2 was shown in Figure 4.15a. The fragmentation of the second intensive signal, 372.0, gave a signal of 355.2, which meant that those two signal were all from the same molecular. For active fraction Fd1 and Fd2, since there are too much soluble salts in the solution, the background is too noise to draw any convinced conclusion (Figure 4.16).

4.4 Conclusion

CTS play a significant role in inhibiting the activity of Na+/K+-ATPase. Based upon the SEC chromatogram the purification protocol has been successfully applied, started with 8 g of skeletal muscle, as no HMM molecules are present after the

BAE. Based on UV-VIS data and activity check via NADH Couple method, five fractions, including Fb1, Fc1, Fc2, Fd1 and Fd2, after reverse phase separation, contain CTS that can inhibit the activity of Na+/K+ pump. Their molecular weight was measured with ESI-ITMS. This approach laid the foundation for the further physiological study on endogenous ouabain like compound. Nevertheless, more research is needed to investigate the molecular structure of each active fraction.

Figure 4.3. Gel filtration chromatograms of molecular weight standards (black) and OLC (red) sample after batch affinity extraction

Figure 4.4. Reverse phase chromatogram of Fa0

Figure 4.5. a) Reverse phase chromatogram of Fb0; b) Magnified chromatogram of Figure 4.5 a) from 0 to 4 min

Figure 4.6. a) Reverse phase chromatogram of Fc0; b) Magnified chromatogram of Figure 4.6 a) from 0 to 5 min

Figure 4.7. a) Reverse phase chromatogram of Fd0; b) Magnified chromatogram of Figure 4.7 a) from 0 to 5 min

Figure 4.8. a) NADH absorption decline over time; b) Dose response curve of ouabain; c) Trendline of dose response curve of ouabain; d) Ouabain affinity presented by EC50

Figure 4.9. Dose response curves of Fa0 and all the fractions after reverse separation from Fa0

Figure 4.10. Dose response curves of Fb0 and all the fractions after reverse separation from Fb0

100

Figure 4.11. Dose response curves of Fc0 and all the fractions after reverse separation from Fc0

101

Figure 4.12. Dose response curves of Fd0 and all the fractions after reverse separation from Fd0

102 Intens. 477.4 +MS, 9.5min #531 x108 477.1 a 238.3

1.5

1.0

292.8 0.5

531.0 715.0

315.0 791.1 129.6 499.1 553.0 737.1 158.7 975.1 0.0 100 200 300 400 500 600 700 800 900 1000 m/z

Figure 4.13. a) MS of active fraction Fb1 ; b) MS2 of 477.1

103 Intens. +MS, 3.7min #148 x106 260.6 a b 5

4 282.8

393.3

984.3 782.5 976.3 874.6 2 722.7 658.6 226.1 983.4 1 548.6

1437.01502.0 1666.1 1744.7 0 200 400 600 800 1000 1200 1400 1600 m/z

c d

2 Figure 4.14 a) MS of Fc1 b) MS of ions with m/z of 2 260.9 in active fraction Fc1 c) MS of ions with m/z 2 of 183.0 in active fraction Fc1 d) MS of ions with 2 m/z of 810.5 in active fraction Fc1 e) MS of ions with m/z of 393.5 in active fraction Fc1

104 Intens. +MS, 4.9min #218 x107 355.4 1.25 a

1.00

0.75

393.3

0.50

238.6

445.3

0.25

467.3 260.8297.2 147.7 922.1 520.1 600.7 767.8 0.00 200 400 600 800 1000 1200 1400 1600 m/z

Intens. +MS2(372.0), 4.6min #205 x107 355.2 b 2.0

1.5

1.0

0.5

0.0 200 400 600 800 1000 1200 1400 1600 m/z

2 Figure 4.15 a) MS of Fc2 b) MS of ions with m/z of 372.0 in active fraction Fc2

105 a

Figure 4.16. MS of active fractions a) Fd1. and b) Fd2.

106 Reference:

1. Bagrov, A.Y., J.I. Shapiro, and O.V. Fedorova, Endogenous Cardiotonic Steroids: Physiology, Pharmacology, and Novel Therapeutic Targets. Pharmacological Reviews, 2009. 61(1): p. 9-38. 2. Lingrel, J.B., The Physiological Significance of the Cardiotonic Steroid/Ouabain-Binding Site of the Na,K-ATPase. Annual Review of Physiology, 2010. 72(1): p. 395-412. 3. Skou, J.C., The influence of some cations on an adenosine triphosphatase from peripheral nerves. Biochimica et Biophysica Acta, 1957. 23: p. 394-401. 4. Lingrel, J.B. and T. Kuntzweiler, Na+,K(+)-ATPase. Journal of Biological Chemistry, 1994. 269(31): p. 19659-19662. 5. Yuan, Z., et al., Na/K-ATPase Tethers Phospholipase C and IP3 Receptor into a Calcium-regulatory Complex. Molecular Biology of the Cell, 2005. 16(9): p. 4034-4045. 6. Tian, J., et al., Binding of Src to Na(+)/K(+)-ATPase Forms a Functional Signaling Complex. Molecular Biology of the Cell, 2006. 17(1): p. 317-326. 7. Forrest, M.D., et al., The Sodium-Potassium Pump Controls the Intrinsic Firing of the Cerebellar Purkinje Neuron. PLoS ONE, 2012. 7(12): p. e51169. 8. Forrest, M.D., The sodium-potassium pump is an information processing element in brain computation. Frontiers in Physiology, 2014. 5: p. 472. 9. Tanaka, Y. and S. Ando, Synaptic aging as revealed by changes in membrane potential and decreased activity of Na+,K+-ATPase. Brain Research, 1990. 506(1): p. 46-52. 10. Levy, B., et al., Relation between muscle Na+K+ ATPase activity and raised lactate concentrations in septic shock: a prospective study. The Lancet. 365(9462): p. 871-875. 11. Jensen, M.K., S.S. Madsen, and K. Kristiansen, Osmoregulation and salinity effects on the expression and activity of Na+,K+-ATPase in the gills of European sea bass, Dicentrarchus labrax (L.). Journal of Experimental Zoology, 1998. 282(3): p. 290-300. 12. Xie, Z. and A. Askari, Na+/K+-ATPase as a signal transducer. European Journal of Biochemistry, 2002. 269(10): p. 2434-2439. 13. Morth, J.P., et al., A structural overview of the plasma membrane Na+,K+-ATPase and H+-ATPase ion pumps. Nat Rev Mol Cell Biol, 2011. 12(1): p. 60-70. 14. Rice, W.J., et al., Structure of Na+,K+-ATPase at 11-Å Resolution: Comparison withCa2+-ATPase in E1 and E2 States. Biophysical Journal, 2001. 80(5): p. 2187-2197. 15. Lingrel, J.B., Na,K-ATPase: Isoform structure, function, and expression. Journal of Bioenergetics and Biomembranes. 24(3): p. 263-270. 16. Gerbi, A., et al., Alteration of Na,K-ATPase isoenzymes in diabetic cardiomyopathy: effect of dietary supplementation with fish oil (n-3 fatty acids) in rats. Diabetologia. 40(5): p. 496-505. 17. Jørgensen, P.L. and J.P. Andersen, Structural basis for E1–E2 conformational transitions in Na, K-pump and Ca-pump proteins. The Journal of Membrane Biology. 103(2): p. 95-120. 18. von Heijne, G. and Y. Gavel, Topogenic signals in integral membrane proteins. European Journal of Biochemistry, 1988. 174(4): p. 671-678. 19. Arystarkhova, E., et al., The γ Subunit Modulates Na+ and K+Affinity of the Renal Na,K-ATPase. Journal of Biological Chemistry, 1999. 274(47): p. 33183-33185.

107 20. Mobasheri, A., et al., Na+, K+-ATPase Isozyme Diversity; Comparative Biochemistry and Physiological Implications of Novel Functional Interactions. Bioscience Reports, 2000. 20(2): p. 51-91. 21. Li, S.-q., et al., Bovine adrenals and hypothalamus are a major source of proscillaridin A- and ouabain-immunoreactivities. Life Sciences, 1998. 62(11): p. 1023-1033. 22. Schneider, R., et al., Bovine Adrenals Contain, in Addition to Ouabain, a Second Inhibitor of the Sodium Pump. Journal of Biological Chemistry, 1998. 273(2): p. 784-792. 23. Lichtstein, D., et al., Identification of digitalis-like compounds in human cataractous lenses. European Journal of Biochemistry, 1993. 216(1): p. 261-268. 24. D’Urso, G., et al., Production of Ouabain-Like Factor in Normal and Ischemic Rat Heart. Journal of Cardiovascular Pharmacology, 2004. 43(5). 25. Manunta, P. and M. Ferrandi, Cardiac Glycosides and Cardiomyopathy. Hypertension, 2006. 47(3): p. 343-344. 26. Hunter, M.M., et al., High-affinity monoclonal antibodies to the cardiac glycoside, digoxin. The Journal of Immunology, 1982. 129(3): p. 1165-1172. 27. Hamlyn, J.M., et al., Identification and characterization of a ouabain-like compound from human plasma. Proceedings of the National Academy of Sciences of the United States of America, 1991. 88(14): p. 6259-6263. 28. De La Cruz, E.M., H.L. Sweeney, and E.M. Ostap, ADP Inhibition of Myosin V ATPase Activity. Biophysical Journal, 2000. 79(3): p. 1524-1529. 29. Reinhard, L., et al., Na+,K+-ATPase as a docking station: protein–protein complexes of the Na+,K+-ATPase. Cellular and Molecular Life Sciences, 2012. 70(2): p. 205-222. 30. Ibsen, K.H., Interrelationships and Functions of the Pyruvate Kinase Isozymes and Their Variant Forms: A Review. Cancer Research, 1977. 37(2): p. 341-353. 31. The Enzymes. Edited by Paul D. Boyer, ed. E.G. Krebs, P.D. Boyer, and D.S. Sigman. 1970, New York: Academic Press. 32. Rajendrakumar, C.S.V., B.V.B. Reddy, and A.R. Reddy, Proline-Protein Interactions: Protection of Structural and Functional Integrity of M4 Lactate Dehydrogenase. Biochemical and Biophysical Research Communications, 1994. 201(2): p. 957-963. 33. Hatefi, Y., [3] Preparation and properties of NADH: Ubiquinone oxidoreductase (complex I), EC 1.6.5.3, in Methods in Enzymology, F. Sidney and P. Lester, Editors. 1978, Academic Press. p. 11-14.

108

Chapter 5

Future Directions

109 5.1 Future directions for DPCs project

The method that I established in chapter 2 could be applied in the purification of

DPCs via detection of sulfur and phosphorus simultaneously. However, the method will be out of effect if there is no sulfur-containing amino acids in the proteins involving in the crosslinking reaction. To solve this problem,

N-hydroxysuccinimido biotin (NHS-Biotin) could be used to react with the primary amino groups (-NH2) in the proteins to form stable amide bonds. In this case,

DPCs is easy to be detected by monitoring both sulfur and phosphorus at the same time since there is always sulfur in the NHS-Biotin.

In addition, environmental and occupational exposure to transition metals such as chromium can also drive DNA protein crosslinking reactions in cells and tissues[1].

There are three mechanisms published to illustrate this Cr-induced crosslinking reaction: chelation mechanism, free radical mechanism and oxidation mechanism.

As it was illustrated in Figure 5.1, Cr(VI) is transported into cells via a nonspecific anion carrier, where it can be reduced to Cr(III). The ligands on Cr(III) are easily substituted by phosphate groups and guanine bases of DNA. Also, nucleophillic amino acids including the amido side chains of proteins preferably attack the Cr(III) to generate DPCs in form of Cr(III) coordination complexes[2]. On the other hand, in the redox cycle of Cr(VI)/Cr(III), ROX are produced, which can contribute to the formation of DPCs by the free radical mechanism shown in Figure 5.2. In this case, tyrosine in the protein is reactive to the free radical on the thymine in the

DNA, forming DPCs[2]. Moreover, it has also been proved that the redox cycling of

110 Cr(VI)/(III) can produce an “oxidative stress” environment, in which case a large proportion of Cr(VI)-induced DPCs could be formed by oxidation mechanism showing in Figure 5.3[3]. Cr(III) is involved in the crosslinking reaction in the chelation mechanism by linking proteins and DNA together while it is the oxidative species that actually induce the crosslinking reaction between proteins and DNA in the other two mechanisms. In other words, Cr(III) behaves as a crossing linker in the chelation mechanism but an oxidative agents driver in the free radical mechanism and oxidation mechanism. In this case, QQQ-ICPMS could be applied in distinguishing DPCs generated by three mechanisms. There will be signal of sulfur, phosphorus and chromium in the DPCs forming under the chelating mechanism. However, there will be only sulfur and phosphorus signal with no chromium signal in the DPCs forming under the other two mechanisms.

This application of QQQ -ICPMS is valuable in investigating the distribution of the

DPCs generated by chromium with different mechanisms. Furthermore, method established in chapter 3 could be applied in determination of the crosslinking amino acids and nucleotides as well.

Cr OH O O- Cr (VI) OH2 Nu-Protein OH2

O Cr(III)L4 Cr(III)L4 O Cr(III)L4 O Nu-Protein N OH NH 2 N N NH NH

N N NH2 N N N NH2 N NH2 DNA DNA DNA Figure 5.1 Chelation mechanism

111 Protein

O O O Protein O Protein CH 2 CH2 HN HN OH HN HN

O N O N -H O N HO O N HO DNA DNA DNA DNA

Figure 5.2 Free radical mechanism

R-CH2NH2 R-CH2NH2 Cr (VI)

R-CH NH Redox metabolism 2 2 Cr(V) Cr(III) R-CH2NH2 Cr (V) Cr (III) O H2O2 H2O2 O R H H O +OH-+ NH H2O 2 R H 3 - +OH + NH3 R-CH NH R-CH NH R-CH NH Cr (III) R-CH2NH2 2 2 Cr (V) Cr (VI) OH Cr (IV) OH OH OH R-CH NH R-CH2NH 2 Cr (VI) Cr (IV) OH OH Figure 5.3 Oxidation mechanism

5.2 Future directions of OLC project

In Chapter 4, a method was established to purify the cardiotonic steroids from pig skeletal muscles via batch affinity extraction, gel filtration chromatography and reverse phase separation. The molecular weight of the active compounds was determined by ESI-ITMS. However, there are still some limitations for this method.

At first, the molecular weight of the active fractions is not distinctive. Some mass spectra of these compounds are complex. Therefore, further separation is required. Since all the active fractions are eluted at early retention time, which indicated that they are vey polar, a hypercarb porous graphitic carbon column is needed to extend the separation capability. Additionally, all methods is completed 112 in small scale (starting with 8 g of pig skeletal muscle). In order to get enough purified active compounds to determine the structure, separation in large scale with the application of preparative column is needed. At least 1 μg of the dry active compound needs to be collected for 2D NMR analysis. Additionally, high resolution mass spectrometer could also be applied to obtain the exact mass of the active compounds.

113 Reference:

1. Taioli, E., et al., Increased DNA-protein crosslinks in lymphocytes of residents living in chromium-contaminated areas. Biological Trace Element Research. 50(3): p. 175-180. 2. Macfie, A., E. Hagan, and A. Zhitkovich, Mechanism of DNA−Protein Cross-Linking by Chromium. Chemical Research in Toxicology, 2010. 23(2): p. 341-347. 3. Mattagajasingh, S.N., B.R. Misra, and H.P. Misra, Carcinogenic chromium(VI)-induced protein oxidation and lipid peroxidation: implications in DNA–protein crosslinking. Journal of Applied Toxicology, 2008. 28(8): p. 987-997.

114