Selenium Mediated Arsenic Toxicity Modifies Cytotoxicity, Reactive Oxygen

Selenium mediated arsenic toxicity modifies cytotoxicity, reactive oxygen

species and phosphorylated proteins

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

in the Department of Chemistry

of the College of Arts and Sciences

Karnakar Reddy Chitta

Bachelor of Pharmacy, Nagarjuna University 2004

M.S., Chemistry, Western Illinois University 2007

M.S., Chemistry, University of Cincinnati 2011

Committee Chair: Joseph. A. Caruso, Ph.D.

Abstract of dissertation

The effect of selenium on modulating arsenic cytotoxicity is well known in mammals, but not well understood. Cell cytotoxicity and reactive oxygen (ROS) changes were performed in combinations of As(III) and selenomethionine (SeMet) toxic mixes on, HEK 293, human embryonic kidney cells. Cell growth is readily restored from 20% to 60% when switching from

30 M As(III) as toxin to a mix of 30 M As(III) and 100 M SeMet. As(III) alone triggers ROS formation, primarily hydrogen peroxide, in a concentration dependent manner as observed through changes in the fluorescence from 2’,7’-dichlorofluorescin diacetate. Importantly,

SeMet induces lower ROS levels at the same concentrations used to modulate As(III) cytotoxicity (IC50). Elevated ROS is important to As(III) cytotoxicity and minimizing it is essential to the SeMet modulating function. Changes in cell signaling, through analysis of signaling changes via differential protein phosphorylation to uncover molecular level changes occurring in HEK 293 human kidney cells as SeMet modulates the As(III) cytotoxicity. To discover changes in the phosphoproteome, cells were incubated under three conditions: 30 M As(III), 100 M

SeMet, and 30 M As(III)+ 100 M SeMet. After total protein isolation the three samples were separated into fractions using size exclusion chromatography by detecting 31P+. Each sample was analyzed for the phosphorylated peptides by enzymatic digestion, selective enrichment of phosphorylated peptides via TiO2, followed by nanoLC-ESIMS. Phosphorylated proteins unique to the As(III)/SeMet mixture were then identified. The molecular level changes to the cells show uniquely that the As(III)/SeMet mixture details proteins involved in ROS detoxification, cell cycle arrest, and protein/DNA damage. This study shows that SeMet not only lowers the total

amount of ROS in a cell but also confers upon HEK 293 cells the ability to detoxify. Thus, SeMet is not only a potent antioxidant in this system, but induces molecular changes that confer survival.

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Acknowledgements

I am dedicating this dissertation to my parents Koti Reddy Chitta and Venkata Lakshmi

Chitta. I would like to thank them a lot for allowing me to pursue my goals and supporting me throughout these years. Their unconditional love always makes me feel indebted for them. I would like to acknowledge my brother Venugopal Reddy Chitta for sharing my happiness and sadness. This journey of mine is not possible without the families - love, support and encouragement.

I want to express my deep heartfelt gratitude to Professor Joseph A Caruso; I along with many people call him by “DOC”. It was for me more than pleasure to work with him and would be honored to graduate as his student. The joy and enthusiasm I had working with him is great.

He is unconditionally excellent as advisor, great person by nature and his motivation is contagious. When I was going through extremely hardships in the middle of my Ph.D, he gave me not only hope but chance to work with him. The freedom he provides in the project along with his valuable guidance is always unforgetful. I can probably write another thesis, how I am indebted to doc for opportunities he has provided me, so I would stop now. I would specially thank Judy Caruso for kindness, hospitality and treating me as a part of family.

I would specially thank Dr.Edward J.Merino for his invaluable suggestions, collaborator and being a co-advisor in my work. I am grateful to Professor Thomas H.Ridgway for serving in my committee more than 5 years and guiding me all of the time.

I would like to thank my dearest friend Phani Chand Kodali for all his help and support for this long time. I would like to extend my special greeting to Dr.Julio Alberto Landero for teaching me some mass spectrometry in my initial days and being a helpful guide.

I would like to acknowledge the past and present members of the Caruso group for their support and friendship. Many thanks to, Dr. Karolin Kroening, Dr. Qilin Chan, Dr.Irena,

Dr.Uma Tiwari, Dr.Necate Kaval, Dr.Eme Amba, Dr. Yaofang Zhang, Dr. Renee Easter, Dr.

Brittany Catron, Dr. Cheolho Yoon, Dr. Daniel Persson, Dr. Pablo Pacheco, Traci Hanley, Ryan

Saadawi, Anna Daigle, Christopher Medley, Nicole Hanks, Keaton Nahan, Kaitlyn Taylor,

Morwina Solivia, Tiffany Bell, Josh Rohman.

Table of Contents

Abstract of dissertation Acknowledgements Chapter 1: Introduction………………………………………………………………………………….1 1.1 Arsenic, Arsenic toxicity, Selenium, Arsenic and Selenium antagonism....2 1.1.1 Arsenic………………………………………………………………………………………………2 1.1.2 Arsenic toxicity………………………………………………………………………………….3 1.1.3 Selenium……………………………………………………………………………………………4 1.1.4 Arsenic and selenium antagonism……………………………………………………..5 1.2 Phosphorylation……………………………………………………………………………………….5 1.3 Metallomics………………………………………………………………………………………………8 1.3.1 Introduction to Metallomics………………………..…………………………………….8 1.3.2 Metallomics approach……………………………………………………………………….8 1.3.3 Molecular Mass Spectrometry for metallomics and metal-binding proteins……………………………………………………………………………………………………10 1.4 Inductively Coupled Plasma Mass Spectrometry (ICPMS)………………………11 1.4.1 Introduction to ICPMS……………………………………………………………………..11 1.4.2 Advantages and limitations of ICPMS…………………………………..………….14 1.5 High Performance Liquid Chromatography (HPLC)……………..………………….15 1.5.1 HPLC-ICPMS…………………………………………………………………………………….17 1.6 References………………………………………………………………………………………………18

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Chapter 2: Selenium mediated arsenic toxicity modifies cytotoxicity, reactive oxygen species and phosphorylated proteins……………………………………………..22 2.1 Abstract…………………………...... ……………………………………………………………….23 2.2 Introduction………………………………..…………………………………………………………25 2.3 Experimental………………………………………………………………………………………….29 2.3.1 Reagents…………………………………………………………………………………………29 2.3.2 Cell culture, cell cytotoxicity and cell lysate…………………………………….30 2.3.3 ROS detection…………………………………………………………………………………31 2.3.4 Size Exclusion Chromatography (SEC)……………………………………………..31 2.3.5 Inductively Coupled Plasma Mass Spectrometry (ICPMS)……..………..32 2.3.6 ESI-MS/MS, NanoLC-Chip and Tryptic digestion……………………….………32 2.4 Results…………………………………………………………………………………………….………34 2.4.1 Effect of SeMet on arsenic cytotoxicity……………………………………..…….34 2.4.2 Effect of SeMet on the As(III) generated ROS…………………………………..35 2.4.3 Using SEC-ICPMS as part of the identification scheme……………………..37 2.4.4 Identification of -casein using phosphochip®………..……………………...41 2.4.5 Identification of phosphorylated proteins using the phosphochip®…41 2.5 Conclusion………………………………..…………………………………………………………...50 2.6 Acknowledgements…………………………………………………………………………….….51 2.7 References...... 52

Chapter 3: Identification of selenium-containing proteins in HEK 293 kidney cells using multiple chromatographies, LC-ICPMS and nano-LC-ESIMS……………….61 3.1 Abstract………………………………………………………………………………………………….62

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3.2 Introduction…………………………………………………………………………………………..63 3.3 Experimental………………………………………………………………………………………….65 3.3.1 Reagents…………………………………………………………………………………………65 3.3.2 Cell culture and Cell lysate………………………………………………………………66 3.3.3 Size Exclusion Chromatography (SEC)……………………………………………..66 3.3.4 Inductively Coupled Plasma Spectrometry (ICPMS)…………………………67 3.3.5 Capillary Reversed Phase Liquid Chromatography (capRPLC)……..…..67 3.3.6 Tryptic digestion, ESI-MS and Nano-LC-Chip……………………………………68 3.4 Results and Discussion…………………………………………………………………………..69 3.4.1 capRPLC………………………………………………………………………………………….72 3.4.2 Identification of Se-containing proteins………………………………..………..74 3.5 Conclusion……………………………………………………………………………………………..78 3.6 Acknowledgements………………………………..……………………………………………..79 3.7 References……………………………………………………………………………………………..80

Chapter 4: Separation of Peptides by HPLC using a Surface Confined Ionic Liquid Stationary Phase………………………………………………………………………………………….84

4.1 Abstract………………………………………………………………………………………………….86 4.2 Introduction……………………………………………………………………………………………89 4.3 Experimental……………………………………………………………………………..…………..89 4.3.1 Materials…………………………………………………………………………………………89 4.3.2 Methods………………………………………………………………………………………….90 4.3.2.1 Stationary Phase Synthesis……………………..……………………………………90

4.3.2.2 HPLC analysis…………………………………………………………….……………….90 4.3.2.3 Capillary Electrophoresis Analysis……………………………………………….91 4.4 Results and Discussion………………………………………………………………………….91 4.4.1 Post-void volume eluting peptides……………………………………..………….92

4.4.1.1 Effect of organic modifier on enkephalin retention………..…………..92 4.4.1.2 Effect of TFA……………………………………………………………………………….95 4.4.2 Pre-void volume eluting peptides…………………………………………………..97 4.5 Conclusions…………………………………………………………………………………………100 4.6 Acknowledgements…………………………………………………………………………….101 4.7 References……………………………………………..…………………………………………..102 Chapter 5: Preliminary work with Human Keratinocytes, Conclusions and Future work………………………………………………………………………………………………………….104 5.1 Preliminary work with Human Keratinocytes (KC)…………..…………………105 5.2 Conclusion and future work...... 107

Appendix………………………………………….……………………………………………………..109

Chapter 1

Introduction

1.0 Introduction

1.1 Arsenic, Arsenic toxicity, Selenium, Arsenic and Selenium antagonism

1.1.1 Arsenic

Arsenic is the 33rd element of periodic table with only one natural isotope at mass 75 amu.

It is a toxic metalloid, thus displaying both metal and nonmetal characteristic behavior.

However in the context of toxicology is referred to as a heavy metal. Arsenic is a naturally occurring toxic element present in air, water and thus making it into the food chain [1].

Weathering of rocks converts arsenic sulfides present in rocks to arsenic trioxide, which enters the environment as the dust or dissolution by rain, further entering into water or ground water.

Thus arsenic is a serious threat to the humankind worldwide [2].

Arsenic is present in environment as organic and inorganic forms. The most common oxidation numbers of arsenic are +5, +3 and -3, in which the element is able to form both inorganic and organic compounds, both in the environment and within the human body [3].

Inorganic forms of arsenic are more toxic relative to the organic forms. Of the inorganic forms, under oxidizing conditions, in aerobic environments, arsenate (iAsV) is the stable species, while under reducing conditions arsenite (iAsIII) is the predominant arsenic species. Trivalent arsenite

(iAsIII) is more toxic than pentavalent arsenate (iAsV). Organoarsenicals generally involve methylation and occur as methylarsonic acid (MMA), dimethylarsinic acid (DMA), trimethylarsine oxide (TMAO) and tetramethylarsonium ion (TETRA) and are moderately toxic.

These compounds represent precursors of more complex organic and relatively less toxic forms, including arsenobetaine (AsB), arsenocholine (AsC) and arsenosugars (AsS). The metabolism of inorganic arsenic is shown in figure 1. In the environment dimethylarsinic acid (DMA) is the 2

major form of organic arsenic and had been used in general herbicide or pesticide for many years. It is the major metabolite of inorganic arsenic in the urine of many mammals including humans [4].

Figure 1: Metabolism of inorganic arsenic in to different forms

1.1.2 Arsenic Toxicity

Arsenic is a known toxic element with carcinogen effects. In addition, arsenic exposure has been related to several diseases including cardiovascular, dermal, hepatic, renal and type II diabetes mellitus, gastrointestinal disturbances, neurological disorders and reproductive health effects. Several mechanisms are considered responsible for metabolized arsenic induced carcinogenesis. Arsenic is known to elevate reactive oxygen species (ROS) and nitrogen species

[5, 6]. Arsenic directly induces ROS leading to the formation of mutagenic 8-oxo-7,8-dihydro-2'- deoxyguanosine in vitro and in-vivo [7, 8]. Damaged DNA can give rise to active oncogenes and 3

sensitize cells to other environmental toxins [9-11]. Many of the metabolized arsenic forms are capable of methylating DNA [12]. Both hyper or hypo methylation of DNA has been observed

[13]. Finally, arsenic has been shown to specifically interact with selenium-containing thioredoxin, causing specific cancer inhibition.

Further, arsenic causes depletion of cell antioxidant defenses leading to the imbalance or disruption of cell antioxidant/prooxidant equilibrium. For its ability to bind with the sulfydryl group, arsenic can also inhibit the activity of numerous enzymes, especially by involvment in glucose uptake, glutathione production and fatty acid oxidation. Although the arsenic toxic and carcinogenic effects had been well documented, the exact mechanism of arsenic induced carcinogenesis or an effective means to limit these adverse effects is not known [14].

1.1.3 Selenium

Selenium is an essential micronutrient for plants and animals. Identification of how essential selenium is to human nutrition and its role in the prevention of some types of cancer, resulted in the proliferation of food supplements fortified with this element in recent years

[15]. Selenium incorporation in to the proteins through genetically encoded amino acid, selenocysteine forms selenoproteins. However, if the incorporation of selenium into proteins is via selenomethionine (SeMet), then the term is selenium-containing proteins. Selenoproteins predominantly function as antioxidants and participate in thyroid hormone production, DNA synthesis and fertilization [16-18]. Selenium acts as an active center in the formation of a selenol group (-SeH) on a selenocysteinyl (SeCys) residue in a selenoprotein sequence [19].

Selenium exerts its biological functions through the presence of selenoproteins. However, few low-molecular weight selenium compounds, such as methyl selenic acid, methylselenocysteine and SeMet have been found to contain antitumorigenic effects in animal studies and in vitro models [20-22]. Numerous selenoproteins have not yet been found and their specific biological functions are not clearly elucidated. However TrxR1, TrxR2 and GPx4 are essential for life, as demonstrated in knock out mouse models.

1.1.4 Arsenic and Selenium antagonism

Gailer et al. stated that “Among the most startling observations in mammalian toxicology is that a lethal dose of selenium can be overcome by an otherwise lethal dose of arsenic” [23]. In the study of Gailer et al., the arsenic selenium antagonism was due to the formation of

- selenium-glutathione-arsenic compound ([(GS)2AsSe] ), which was confirmed by use of X-ray fluorescence spectrometry. Furthermore in a 2007 review, the authors show that selenium can modify arsenic toxicity by forming As-Se bonds, in addition to Hg-Se bonds [24]. In a recent publication Leslie discusses transport of the arsenic-glutathione complexes [25]. Because of all these works, our group has been trying to understand what effects selenium species have on arsenic-induced toxicity at the phosphoproteomic level.

1.2 Phosphorylation

Protein phosphorylation is one of the most abundant and important post-translational modifications (PMTs) and is also responsible for numerous essential biological processes [26-

28]. Some of the important functions for protein phosphorylation is the effect on signal transduction, cell growth, proliferation, intercellular communications and apoptosis [29].

Reversible protein phosphorylation occurs on specific protein sites of serine (Ser), threonine

(Thr) and tyrosine (Tyr) with the relative abundance of 1800:200:1 controlled by enzymes of kinase and phosphatase families, although the mole fraction of phosphorylated amino acids is only 1-2% of the entire proteome [30]. Phosphorlyation is often sub-stoichiometric; hence at given time points a given protein/peptide sites may or may not be phosphorylated, thus requiring highly sensitive methods for isolation, detection and quantification of low abundant phosphorylation sites.

The traditional approach for analyzing phosphoproteins involved incorporation of radioactive 32P in to the phosphoproteins [31]. Incorporation of 32P is achieved by incubation of cells, tissues with [32P] ATP or [32P] phosphate. The radioactive proteins are further subjected to fractionation procedures (e.g. high-performance liquid chromatography [HPLC] or two- dimensional gel electrophoresis). Proteins thus identified can be subjected to complete hydrolysis and the phosphoamino acid content determined. The site(s) of phosphorylation can be determined by proteolytic digestion of the radiolabeled protein, separation and detection of phosphorylated peptides (e.g. by two-dimensional peptide mapping), followed by peptide sequencing by Edman degradation [32]. These techniques can be tedious, require significant quantities of the phosphorylated protein and produce potentially high levels of radioactivity.

Though full characterization of phosphoproteins remains an analytical challenge, although over the past decade and half, mass spectrometry (MS) has emerged as an alternative

to the traditional methods of phosphorylation analysis [33, 34]. Recent developments in selective and efficient strategies for the enrichment of phosphopeptides/phophoproteins along with mass spectrometry have made it feasible to study protein phosphorylation on a much larger scale.

There are many phosphoproteomic strategies available. Most common involves enrichment using phosphorylation-specific antibodies at both protein and peptide levels.

However, the poor specificity of anitphosphoserine and antiphosphothreonine antibodies has limited their use [35, 36], though specific antiphosphotyrosine antibodies remain in use [37,

38]. For many years, immobilized metal ion affinity chromatography (IMAC) had been the popular method for phosphopeptide enrichment prior to MS analysis [39, 40]. Phosphoproteins are bound to the stationary phase by electrostatic interactions of the phosphate moiety with positively charged metal-ions (Fe3+, Ga3+, Al3+ or Zr4+), which are immobilized on the column material via iminodiacetic acid, nitriloacetic acid or Tris-(carboxymethyl)-ethylendiamine linkers

[41]. Non-phosphorylated species can be washed away and the phosphoproteins may be eluted by salt and/or pH-gradients. IMAC had a limited selectivity when used for complex samples, and further requiring O-metylesterification step for improving selectivity [42].

Recent introduction of Al(OH)3 [43], ZrO2 [44], HfO2 [45], Nb2O5 [46], SnO2 [47] and TiO2

[48] based stationary phases served as an alternative to the IMAC resins. Only organic phosphates bind specifically to the column under acidic conditions, allowing removal of all non- phosphorylated peptides. Elution of phosphopeptides is done at an alkaline pH. Though numerous metal oxides were used for phosphopeptide enrichment, TiO2 chromatography

enrichment initiated significant progress in the development of new and highly sensitive strategies for phosphoproteomic studies [49, 50].

In summary, the current phosphorylation-enrichment methods, along with TiO2 is highly efficient in isolation of phosphoproteins/phosphopeptides from small amounts of starting material containing numerous proteins. Their combination with the efficient MS technology has made it possible to study numerous biological questions heretofore not feasible.

1.3 Metallomics

1.3.1 Introduction to Metallomics

The term “Metallome” was coined by Williams RPJ as term analogous to the proteome in the context of metal distribution in the cells. The meaning of the term “Metallome” was then further proposed to extend to the metal and metalloid species present in a cell or tissue type.

With this concern, a scientific area defined by “metallomics” was firstly proposed by Haraguchi in 2004 to elucidate the importance of revealing the mystery of metals in biological system, but the term was finalized in 2010 [51]. Metallomics is a study of the metallome, interactions, and functional connections of metal ions and other metal species with genes, proteins, metabolites, and other biomolecules in biological systems (defined by IUPAC). Metallomics refers to the entirety of research activities aimed at the understanding of the molecular mechanisms of metal-dependent life processes [52]. It is a comprehensive investigation that not only focuses on concentration or speciation of metals or metalloids, but also emphasize the statistical, structural or functional significance and contribution of metals/metalloids and their species to biological systems. Still the metallomics concept is relatively new area to explore, further to 8

show the growth of this area a new peer-reviewed journal called “Metallomics” has been started in 2009 by Royal Society of Chemistry (RSC).

1.3.2 Metallomics Approach

Several analytical approaches are used for metallomics. The predominant and widely used approach is shown in figure 2. In general metals associated with biological molecules are the most studied, such as metalloproteins. The presence of trace levels of metalloproteins or metal-biomolecule complexes requires combination of techniques, a high-resolution separation by HPLC or electrophoresis coupled with sensitive detection of Inductively Coupled Plasma

Mass Spectrometry (ICPMS) or other MS. A common strategy involves screening of metalloproteins by HPLC-ICPMS and then identification of proteins by a molecular mass spectrometry. The purpose of the separation technique is to provide sufficient resolution to avoid the co-elution of two species of the same element, when ICPMS detection is used or that of any easily ionized concomitant species capable of suppressing the analyte’s ionization, when molecular MS is used.

X-ray absorption spectroscopy (XAS), which can provide useful data on the metal- coordination environment and oxidation state, the requirement of having a relatively high level of metal in the sample along with the purity required, limits its applications. Over the years

ICPMS had emerged as powerful tool for the elemental analysis due to its sensitivity and wide range of elemental detection capabilities.

HPLC-ESI MALDI MS MS/MS

Immobilized Metal affinity or Metal Oxide Affinity Chromatography

Experimental Approach for X-ray absorption Metallomics Spectrometry

1 or 2-D gel Liquid Chromatography or electrophoresis Capillary Electrophoresis

MALDI MS, HPLC- Laser ablation ICP MS ESI MS/MS ESI MS/MS ICP MS

Figure 2: Analytical approached employed in the metallomics

1.3.3 Molecular Mass Spectrometry for metallomics and metal-binding proteins

Mass Spectrometry (MS) has became a primary analytical tool for proteomic analysis, since the introduction of two soft ionization techniques – electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI). The main advantage of the soft ionization techniques is due to little or no fragmentation that the thermally labile biomolecules such as proteins under go when carried from sample to the mass analyzer. MS based proteomics are commonly used for large-scale identification, quantification of proteins, characterization of post-translational modifications (PTMs) and investigation of protein-protein interactions. MS- based proteomics can be classified in to two categories, bottom-up proteomics and top-down proteomics [53].

The bottom-up approach is the most popular method for tackling high-complexity samples for large scale analysis [54]. In the bottom-up approach, proteins are proteolytically digested into peptides prior to mass analysis, then peptide masses and sequences are used to identify corresponding proteins. Most bottom-up approaches require tandem data acquisition in which peptides are subjected to collision-activated dissociation and protein identification is done by experimental MSn data compared to the database search. Of all the bottom-up approaches, shotgun proteomics has gained wide acceptance [55]. This approach involves batch digestion of an unseparated or separated protein mixture, further separation of the resulting peptides by multidimensional liquid chromatography and analyzing peptides using mass spectrometric for identification of proteins.

Top-down proteomics involves the ionization of intact proteins or larger protein fragments followed by tandem mass spectrometric analysis [56]. In general, protein molecular ions generated by electrospray ionization (ESI) are introduced in to a quadrupole ion trap (QIT) or Fourier transform ion cyclotron resonance (FTICR) for mass analysis. The fragmentation used for top-down proteomics is electron capture dissociation (ECD) and electron transfer dissociation (ETD).

1.4 Inductively Coupled Plasma Mass Spectrometry (ICPMS)

1.4.1 Introduction to ICPMS

Inductively coupled plasma mass spectrometry (ICPMS) is undoubtedly the fastest- growing trace element technique available today. ICPMS not only offers extremely low

detection limits in the sub parts per trillion (ppt) range, but also enables quantification at the high parts per million (ppm) levels. The first ICPMS, an ICP source coupled to a quadrupole- based mass analyzer, was developed in 1980 by Houk. Since its commercialization in 1983, approximately 6000 systems have been installed worldwide, carrying out many varied and diverse applications. ICP-MS has been a versatile tool for any type of samples, gaseous, liquid and solid. In addition to total concentration measurements, it is also a useful online detection technique for common separation techniques, GC, HPLC and CE. There are number of different

ICP-MS designs available today, which share many similar components, such as nebulizer, spray chamber, plasma torch and detector, but differ significantly in the design interface, ion focusing system, mass analyzer and vacuum chamber. The introduction of collision/reaction cells in 2001 increased the capability of ICPMS via interference removal for high matrix samples. A typical design of an Agilent Technologies7700 ICP-MS is shown in figure 3.

Figure 3: A Schematic diagram of Agilent 7700

A brief description of how a sample is processed and analyzed is explained. The sample usually in the liquid form is pumped at 1mL/min using a peristaltic pump into a nebulizer, where it is converted in to a fine aerosol with argon gas at about 1L/min. The fine droplets of the aerosol are separated from the larger droplets by means of spray chamber. The fine aerosol emerges from the spray chamber and is transported to the plasma torch. The plasma in formed by the interaction of an intense magnetic field generated by radiofrequency (RF) passing through the copper coil on a tangential flow of argon gas at about 15 L/min flowing through a concentric quartz tube (torch). The effect of ionizing the argon gas, when seeded with a source of electrons from a high-voltage spark, forms a very high temperature plasma discharge of ~

10000 K at the open end of the torch. Fine aerosol is converted in to ions upon entering in to plasma and directed into the mass spectrometer via the interface region, which is usually maintained at vacuum of ~1-2 Torr with a mechanical roughing pump. The interface consists of two metallic cones called sampler cone and a skimmer cone, both have a small orifice to allow the ions to pass through into the ion optics. Once the ions have been successfully extracted from the interface region, they are directed into the main vacuum chamber by a series of electrostatic lens called ion optics. The vacuum in this region is maintained at about 10-3 Torr with a turbo pump. If a collision/ reaction cell is installed between ion optics and mass analyzer, collision/reaction gas may be introduced in to the cell to eliminate the polyatomic interference ion by Kinetic Energy Discrimination (KED) or collisional deactivation or chemical reaction depending on the operating mode chosen based on analytical requirement.

The ion beam coming from collision/reaction cell containing analytes and matrix ions passes to the mass spectrometer, which is operated at 10-6 Torr by a turbo pump. There are 13

many different mass separation devices - quadrupole, magnetic sector, time of flight, etc. All serve the same purpose of allowing the analyte ions of a particular mass-to-charge ratio through to the detector and filter out the non analyte and matrix ions. The ions emerging from the mass separation device are converted in to an electrical signal using an ion detector. The discrete dynode detector is the most commonly used, which consists of a series of dynodes.

Ions impinge on the first dynode and are converted in to electrons. Electrons generated from one dynode are attracted to the next dynode, this step is repeated resulting in electron multiplication, and finally a beam of electrons emerge from the final dynode. This electrical signal is processed and converted into analyte concentration.

1.4.2 Advantages and limitation of ICPMS

Trace and ultratrace measurement of greater than 70 elements with a wide range from parts per trillion (ppt) to parts per million (ppm), spectral simplicity, speed of multi-elemental analysis, flexibility to optimize for specific applications, directly coupling to different chromatographies, fast semi-quantitative analysis and isotope ratio measurement in nuclear, geological, environmental and nutrition studies.

ICP is a “hard ion” source breaking down most of the molecules to atoms or ions, giving

ICPMS a robust elemental detection, even in complex matrices. However the molecular structure is destroyed in harsh ICP conditions rendering ICPMS not capable of structural characterization; this is considered the primary limitation. For obtaining structural information, molecular mass spectrometry as a complimentary technique for ICPMS is more often used.

Though numerous applications were developed based on HPLC-ICPMS, sudden varying organic concentrations in Reversed Phase Chromatography (RP-HPLC) could lead to turning off plasma and further huge accumulation of carbon in the skimmer and sampler cones. Nevertheless,

ICPMS advantages are far more outweighed than its few limitations, leading to its applications in numerous areas.

1.5 High Performance Liquid Chromatography (HPLC)

Liquid chromatography (LC) is a physical separation technique conducted using liquid phases. Sample is separated in to its constituent components or analytes by distribution between the mobile phase (flowing liquid) and a stationary phase (sorbent packed inside a column). The rebirth of liquid chromatography in the present state-of-the-art, the High

Performance Liquid Chromatography (HPLC) started in the early sixties. HPLC has developed in to a widely used technique, which had evolved in to an indispensable tool in modern analytical laboratories. It is a versatile analytical technology widely used for the analysis of pharmaceuticals, biomolecules, polymers and many organic and ionic compounds.

Numerous chromatographies are employed using HPLC, for example Normal-phase,

Reversed-phase, size-exclusion, ion-exchange, affinity, chiral, hydrophilic interaction, hydrophobic interaction, supercritical fluid and electrochromatography. The most prominent and widely used chromatographies of all are normal phase, reversed-phase, size exclusion and ion exchange chromatography.

Normal phase chromatography (NPC) or adsorption chromatography is the traditional separation mode based on adsorption/desorption of the analyte on to a polar stationary phase, typically silica or alumina [57]. NPC is used for the separation of non-polar compounds and isomers. Reversed phase chromatography (RPC) is the by far the most popular HPLC mode use in today. A hydrophobic stationary phase, usually an octadecyl or octyl functional group chemically bonded on to the silica, is used in conjunction with a polar mobile phase. The separation is based on analyte partition coefficients between a polar mobile phase and a hydrophobic (nonpolar) stationary phase. The mechanism of separation is primarily attributed to solvophobic or hydrophobic interaction. Ion-pairing agents are extensively used in RP-HPLC depending upon the charge of the peptides or proteins or polar analytes to be separated. The homologous series of volatile perfluorinated acids - trifluoroacetic acid, pentafluoropropionic acid, heptafluorobutyric acid, and formic acid, proved to be excellent ion-pairing agents for RP-

HPLC. These ion-pairing agents affect the polar analyte retention behavior by interaction of the ions with oppositely charged functional groups causing net reduction in the hydrophilicity of polar analytes; further the alkyl groups of the ion-pairing agent impart hydrophobic character to the ion pair resulting in the increased hydrophobic interaction with the reversed-phase.

Size-exclusion chromatography is a separation mode based on the analyte’s molecular size. The large molecules are excluded from the pores and migrate quickly, while small molecules can penetrate the pores and migrate slowly down the column. It is often called gel permeation chromatography (GPC) when used for the determination of molecular weights of organic polymers and gel filtration chromatography (GFC) when used in the separation of water- soluble biological materials. In ion-exchange chromatography separation is based on the 16

exchange of ionic analytes with the counter ions of the ionic group attached on a solid support.

Typically phases are cationic exchange and anionic exchange, where concerned ionic groups bonded to polymeric or silica material. Mobile phases consist of buffers, often with increasing ionic strength, for the elution of analytes.

1.5.1 HPLC-ICPMS

A few years after the introduction of ICPMS, HPLC coupling to ICPMS was done and soon became popular for elemental speciation studies [58, 59]. HPLC, GC and CE have been the most common online separation techniques coupled to ICP-MS, HPLC-ICPMS is the most popular hyphenated technique for elemental speciation due to numerous advantages such as very simple straight forward interface between HPLC and ICPMS and different chromatography’s

(normal phase, reversed phase, ion exchange etc) that could be employed.

Interfacing of HPLC to ICPMS is by simply connecting HPLC exit line to the ICPMS nebulizer. Though the interfacing is relatively easy, the performance of the ICPMS depends on the nebulizer and spray chamber. Great care should be taken when using high concentrations of organics and salts, since HPLC can easily handle these, while in the ICPMS buildup of carbon on the cones and further non-volatile salts causes loss of sensitivity along with matrix effects.

Recent uses of capillary or nano flow HPLC-ICPMS suggest an increasingly attractive option since it would overcome some of problems associates with traditional approach.

References:

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Chapter 2

Selenium mediated arsenic toxicity modifies cytotoxicity, reactive oxygen

species and phosphorylated proteins

2.1 Abstract

amount of ROS in a cell but also confers upon HEK 293 cells the ability to detoxify. Thus, SeMet is not only a potent antioxidant in this system, but induces molecular changes that confer survival.

2.2 Introduction:

Arsenic is a toxic naturally occurring metalloid that is widely distributed in nature [1]. In several countries the occurrences at high levels in drinking water remains a major public health concern affecting tens of millions of people worldwide, not only from the drinking water, but through the arsenic laden croplands such as rice [2, 3]. Inorganic arsenic is more toxic than the organic forms; among inorganic forms arsenite As(III) is more toxic than arsenate (As(V)) [4, 5].

Once inside the cell As(III) and As(V) are metabolized into forms that exert profound changes to a cell [6]. Several mechanisms are considered responsible for metabolized As(III) induced carcinogenesis. As(III) is known to elevate reactive oxygen species (ROS) and nitrogen species

[7, 8]. As(III) directly induces ROS leading to the formation of mutagenic 8-oxo-7,8-dihydro-2'- deoxyguanosine in vitro and in-vivo [9, 10]. Damaged DNA can give rise to active oncogenes and sensitize cells to other environmental toxins [11-13]. Many of the metabolized As(III) forms are capable of methylating DNA [14]. Both hyper or hypo methylation of DNA has been observed

[15]. Finally, As(III) has been shown to specifically interact with selenium-containing thioredoxin, causing specific cancer inhibition [16]. The exact mechanism of As(III) induced carcinogenesis or an effective means to limit these adverse effects is not known [17].

Selenium is an essential micronutrient for animals and plants over a narrow concentration window. Selenium incorporation into proteins, through the genetically encoded amino acid, selenocysteine, is termed a selenoprotein, however if the incorporation of selenium into the protein is through selenomethionine, the proper terminology is selenium-containing protein, since the incorporation of selenomethionine is not owing to genetic coding. Selenium

related biomolecules have numerous health benefits including antioxidant and anti- inflammatory functions (notably as GPx), antiviral properties and exhibits a putative role in cancer chemoprevention and cell reproduction [18, 19]. It is, however, known that As(III) cytotoxicity is limited by selenium in its various forms or species. Gailer et al. state that “Among the most startling observations in mammalian toxicology is that a lethal dose of selenium can be overcome by an otherwise lethal dose of arsenic” [20]. This study poses a glutathione- arsenic-selenium compound found by X-ray methods. In the 2007 Gailer review paper, a thorough discussion of As-Se and Hg-Se bonds is presented [21]. In a recent publication Leslie discusses transport of the arsenic-glutathione complex [22].

Selenium, supplemented as selenite, selenomethionine (SeMet), selenocysteine (SeCys) and Se‐methylselenocysteine (MeSeCys) forms can minimize As(III) cytotoxicity [22-25].

Selenium, when incorporated into selenoproteins is a strong antioxidant as it is more easily oxidized than equivalent thiol groups. Along with its antioxidant function in selenoproteins, selenium has been investigated regarding its role in mitigating the cytotoxicity of arsenic in vivo and in vitro [26, 27]. The changes in protein expression induced by selenomethionine supplementation in cells can be tracked and compared between As(III) toxified cells and

As(III)/SeMet supplemented cells. It is also important to track these differential cell signaling pathways in order to have an enhanced understanding of possible molecular mechanisms underlying SeMet modulation of As(III) cytotoxicity. Proteomic changes that SeMet induces in cells when modulating As(III) cytotoxicity may provide insight into ways of reducing the As(III) toxicity. Therefore, a key question to ask is whether SeMet acts alone as a strong antioxidant to detoxify As(III) or does it induce specific changes to the proteome that further mediate As(III) 26

cytotoxicity? Answering the question may provide a means of evaluating how cells cope with stressors like As(III) via any changes that might take place in the cell signaling cascades.

The effect of SeMet on cell signaling, as visualized through protein phosphorylation, is assessed in this study. Protein phosphorylation is one of the most abundant post-translational modifications and is also responsible for numerous essential biological processes [28-30].

Protein phosphorylation and dephosphorylation in vertebrate cells occur on protein side chains of serine (Ser), threonine (Thr) and tyrosine (Tyr) with the relative abundance of 1800:200:1.

Despite being ubiquitous, phosphorylation remains challenging to assess. The mole fraction of phosphorylated amino acids is only 1-2% of the entire proteome [31]. In general phosphorylation is sub-stoichiometric; hence, phosphorylated proteins are in lower abundance than non-phosphorylated proteins. Traditional strategies for assessing protein phosphorylation need refinement. The oldest method is identification of phosphorylated proteins by incorporation of radioactive 32P into cellular proteins, requiring further methods for detection

[32-34]. Apart from using radioactive material, this method is challenging and time consuming with other shortcomings. More recently, phosphorylation-specific antibodies and site-specific mutagenesis have been used for identification of phosphorylated proteins through the identification of phosphorylated peptide amino acids [35, 36], but this is also time consuming and restricted to a specific peptide or amino acid. Furthermore, no site specificity can be determined and quantification is difficult. We have developed methods to rapidly screen phosphorylation status in a cell. The methods involves direct detection of proteins containing phosphorylated amino acids by liquid chromatography coupled to inductively coupled plasma mass spectrometry (LC-ICPMS) in tandem with nano-liquid chromatography electrospray mass 27

spectrometry (nanoLC-ESIMS) [27, 37-39]. This experimental setup offers several advantages for these global phosphorylation studies. First, ICPMS is selective for 31P+, the only natural isotope of phosphorus, at sub-ppb detection levels and is less time consuming in identification of phosphorylated peptides or proteins [27, 37, 40-42]. When coupled with liquid chromatography, ICPMS can establish the elution times of phosphorylated peptides or proteins as a function of hydrodynamic radii (Size Exclusion Chromatography) or by hydrophobicity

(Reversed Phase Chromatography). Inherent in the ICPMS technique is its ability to quantify the number of phosphorylated amino acids, peptides or proteins by normalizing to sulfur abundance. In short, by using the mass spectrometric methods (ICPMS and ESIMS) in tandem it is possible to identify multiple peptides/proteins and site locations from a single experiment using 31P+ as the elemental target, rather than radioactive 32P.

Polyatomic interferences are a known problem in 31P+ ICPMS analysis, so the use of the collision/reaction cell or a higher resolution mass spectrometer to remove them is essential to have reliable results. Further, the specificity of the 31P+ signal provided by ICPMS, results in simpler chromatograms of tryptic digested protein fractions than those obtained using UV or

ESIMS alone because selective 31P+ detection is an excellent screening tool. ICPMS is thus, ideal for a fast screening of complex samples when searching specifically for phosphorylated peptides or phosphorylated proteins. By fraction collection multiple times at various chromatographic points, sample concentrating prior to nanoLC-ESIMS is achieved, as necessary.

Further characterization of these fractions by nanoLC-ESIMS/MS can be readily accomplished at reduced complexity owing to the ICPMS screening.

In this study phosphorylation differences are compared across control cells (cells with no As(III) or SeMet dosing), As(III) toxified and As(III)/SeMet toxified human kidney cells

(HEK293), as a means of further exploring the mechanism of As(III)/SeMet antagonism.

Interestingly, the cells toxified with As(III)/SeMet showed enhanced protein phosphorylation signaling highly favoring cell health with respect to the cells inoculated with As(III) alone. This is supported by a corresponding cytotoxicity reduction and a reduction in ROS species, particularly H2O2.

2.3 Experimental:

2.3.1 Reagents:

All reagents are of analytical grade unless otherwise stated. All the aqueous based solutions are prepared in 18 MΩ cm‐1 doubly deionized water (Sybron Barnstead, Boston, MA,

USA). HEK 293 human kidney cell lines, Dulbecco’s Modified Eagle’s Medium (DMEM), Fetal

Bovine Serum (FBS), M‐Per protein extraction buffer, Halt phosphatase inhibitor cocktail, formic acid (FA), acetic acid (HOAc), sodium arsenite (As(III)), urea and ammonium bicarbonate were purchased from Fisher Scientific (Fairlawn, NJ, USA). L‐selenomethionine (SeMet), Dithiothreitol

(DTT), iodoacetamide (IAA), protein standards β-casein, bovine serum albumin (BSA) and 2’7’- dichlorofluorescin diacetate (DCFH-DA) were purchased from Sigma-Aldrich Chemical Company

(St. Louis, MO, USA). The HPLC grade solvents, water and acetonitrile (ACN), were of high purity and purchased from Burdick and Jackson (Muskegon, MI, USA). Sequence grade modified trypsin and acetic acid buffer were obtained from Promega (Madison, WI, USA). Trypsin‐EDTA:

0.05% trypsin 0.53 mM EDTA × 4Na, was purchased from Gibco Invitrogen Corporation

(Carlsbad, CA, USA). 3-(4,5-Dimethyl-2-thazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) was purchased from MP Biomedicals (Solon, OH, USA).

2.3.2 Cell culture, cell cytotoxicity and cell lysate:

Under humid conditions, human kidney cells (HEK293 cells) were cultured in 75 cm2

o tissue culture plates using DMEM medium containing 10% FBS at 37 C with 5% CO2. Fresh solutions of sodium arsenite (As(III)) and selenomethionine (SeMet) were made using DMEM medium prior to dosing. After cells reached 80% confluence, they were grown for 24hrs with no dosing, 30 µM of As(III) alone, 100 µM of SeMet alone, 30 µM As(III) and 100 µM SeMet together.

Cell cytotoxicity concentrations were chosen based on obtained results and previous work [37]. For the determination of the cytotoxicity of each chemical, HEK293 human kidney cells were seeded in 24-well plates at a density of 1x104 mL-1 and incubated for 24 hours before adding As(III), and SeMet. Cells were treated with As(III) and SeMet in the concentration ranges of 10-100 μM and 10-200 μM, respectively, for 24 hours to indicate the cytotoxic affect of each chemical. A working solution of MTT reagent was dissolved in 1 mL phosphate saline buffer

(PBS; Gibco Invitrogen Corporation, Carlsbad, CA, USA) at a concentration of 5 mg mL-1 and diluted to a volume of 25 mL with media. After removing the media in each well at the end of exposure time, 0.5 mL of MTT-media solution was placed in each well and incubated for 3 hours. After removing the media containing MTT, 0.2 mL DMSO was added to each well. Then the culture plate was placed on a micro-plate reader and the background corrected absorbance measured at 550 nm.

After cell incubation for 24 hrs with the selected concentrations of As(III) and SeMet,

Trypsin-EDTA was used to isolate the cells, followed by washing with PBS to remove the traces of leftover media. M‐Per protein extraction buffer, including Halt phosphatase inhibitor cocktail, was used for lyses and extraction. Cells were centrifuged at 10,000g for 10 minutes and supernatants were used for further analysis.

2.3.3 ROS Detection:

HEK293 human kidney cells were seeded in 24-well plates at a density of 1x104 mL-1 and incubated for 24 hours. The media was then removed and washed with 1X PBS followed by addition of HBSS media and incubated with 100 μM of DCFH-DA for 30min. Cells were treated with 10-100 μM of As(III), 10-200 of μM SeMet and 100 μM of tert-butyl hydroperoxide (TBHP) and incubated for 4 hrs. Accumulation of 2’7’-dichlorofluorescin (DCF) is measured by an increase in fluorescence at 520 nm, when the sample is excited at 485 nm. The DCF measurements were taken immediately after the 4 hr. incubation to assure a strong correlation of DCF signal with the amounts of ROS.

2.3.4 Size Exclusion Chromatography (SEC):

An Agilent 1100 series HPLC system equipped with a binary pump, vacuum membrane degasser, thermostated auto sampler, column oven, and diode array detector with a semi- micro flow UV-Vis cell was used for SEC chromatographic analysis. The entire system was controlled using Chemstation software (all from Agilent Technologies, Santa Clara, CA, USA).

SEC was performed using TSK Gel 3000SW 7.5 x 300 mm (Tosoh Bioscience LLC, PA, USA). The size exclusion column was calibrated using a UV detector (wavelength, 280 nm) by using a gel filtration standard mixture (MW of thyroglobulin, 670 kDa; MW of γ-globulin, 158 kDa; MW of 31

ovalbumin, 44 kDa; MW of myoglobin, 17 kDa; MW of vitamin B12, 1.3 kDa) purchased from

Bio-Rad Laboratories (Life Science Research, CA, USA) R = 0.997. Mobile phases were; A: 50 mM NH4OAc, B: MeOH, Isocratic: 5% B at Flow rate of 0.5 ml/min. The fractions isolated, based on the SEC-ICPMS 31P signal, were collected and taken to dryness by lyophilization.

2.3.5 Inductively Coupled Plasma Mass Spectrometry (ICPMS):

An Agilent Technologies 7700 inductively coupled plasma mass spectrometer, equipped with a Micromist nebulizer, a Peltier-cooled spray chamber (2oC), and an octopole collision/reaction cell with helium gas pressurization (purity of 99.99%) was used. The entire system was controlled using Mass Hunter software (all from Agilent Technologies, Santa Clara,

CA, USA). The ICP-MS conditions were as follows: forward power, 1500 W; plasma gas flow rate

15 L min-1, carrier gas flow, 0.91 L min-1; make-up gas 0.12 L min-1; collision gas, He, 4.0 ml min-

1; quadrupole bias, -16.0 V; octopole bias -18.0 V for a +2 volt energy discrimination setting. The isotope monitored was phosphorus 31P+.

2.3.6 ESI-MS/MS, NanoLC-Chip and Tryptic digestion:

Electrospray experiments were performed using an Agilent 6300 Series nanoLC Chip/Ion

Trap XCT system (Agilent Technologies, Santa Clara, CA). An Agilent 1200 LC, equipped with both capillary and nano pumps, was used for loading and flushing the on-chip nano column. For phosphorylated peptide analysis, the Agilent LC phosphochip® (Agilent Technologies, Santa

Clara, CA, USA) was used, which has a three sectioned trapping column consisting of the first

100 nL C18 trapping column (Zorbax Extend 5 μm), a 40 nL TiO2 column (10 μm, GL Sciences), and a second 100 nL Zorbax Extend C18 trapping column. Also the Phosphochip® has a Zorbax

300Extend C18 (150 mm × 75 μm, 5 μm) analytical column. All the samples were loaded and 32

peptide trapping was performed at a flow rate of 4 μL min-1 using 0.6% HOAc (acetic acid) and

2% FA (formic acid) in water. Solvents used for the nanoflow experiment were 0.6%

HOAc/0.5% FA (solvent A) and 0.6% HOAc/0.5% FA/80% ACN (solvent B). Samples were loaded on the enrichment column at a flow rate of 3 µL min-1 with a 97:3 ratio of solvent A (0.1% FA

(v/v) in water) and B (90% ACN, 0.1% FA (v/v) in water). After the enrichment column was loaded, the on-chip microfluidics switched to the analytical column at a flow rate of 0.3 µL min-

1. The following gradient conditions were used in the analysis: 0–2 min, 2% B; 2–20 min, 30% B;

20–60 min, 50% B; 60–70 min, 60% B; 70–90 min, 90% B. Full scan mass spectra were acquired over the m/z range 100–2200 in the positive ion mode. For MS/MS experiments, experimental conditions consisted of: m/z range: 100–2200, isolation window: 2 m/z units, 2 precursors, maximum accumulation time 150 ms and active exclusion after 3 spectra from 0.5 minutes, fragmentation energy: 30% – 200%, fragmentation.

A tryptic digestion was performed as follows: the protein pellets obtained from SEC were re-suspended in 25 µL of 50 mM ammonium bicarbonate, 2 µL of 100 mM DTT were added as reducing buffer and the mixture was heated at 95 °C for 5 minutes; this step unfolds the proteins and reduces the disulfide bonds. After cooling the sample, an alkylation was carried out to protect the thiol groups of the cysteine residues by adding 3 µL of 100 mM iodoacetamide. The mixture was incubated in the dark for 20 minutes at room temperature.

After the alkylation, 1 µL of modified sequence grade trypsin solution was added and incubated at 37 oC for 2 hours. Then, 2 µM of additional trypsin was added to complete the reaction, followed by incubation at 35 oC for 8 hours. 1 µL of formic acid was added to stop the reaction,

and the solution was ultra-filtered through 5 kDa filters to eliminate the undigested proteins and the trypsin.

2.4 RESULTS:

2.4.1 Effect of SeMet on arsenic cytotoxicity:

MTT assays were performed to determine the cytotoxicity. As(III) cytotoxicity was assessed at various concentrations. The IC50 for HEK293 cells is 28 +/- 3 M. Cell viability at 30

μM As(III) was found to be 41 +/- 2 %. This concentration was used for this study and is shown in Figure 1A. We then determined the cytotoxicity of SeMet, both with and without As(III).

SeMet alone proved non-cytotoxic up to 200 μM, as shown in grey (Figure 1B). When the As(III) concentration was kept constant at 30 μM and various concentrations of SeMet were used, cells became viable in a concentration dependent manner (Figure 1B, black). When low concentrations of SeMet were present, cell viability was 16.8 +/- 3.0%. Enhanced SeMet- dependent viability reached a plateau at 100 M to 62.7 +/- 3.8 percent viability. Thus, SeMet enhanced viability by a factor of 3.7. To further understand the molecular level changes, detection of ROS as a function of the different toxins, or combinations thereof, could provide additional molecular level insights for the SeMet role in reducing As(III) toxicity. An initial study of ROS H2O2 follows.

Figure 1: A) A plot of cell viability with no As(III) (-) and with As(III) 30 µM (+). As can be seen in the plot with no As(III) (-) almost 100% cell viability, while with 30 µM arsenic (+) only 40% cell viability. B) A plot of cell viability with various concentrations of SeMet ranging from 10 µM to 200 µM. The no As(III) (darker trace) represents cells grown with only SeMet and shows almost 100% cell viability, indicating even the higher doses of SeMet are not toxic. As(III) (lighter trace) represents all cells grown with 30 µM As(III) together with different concentrations of SeMet. From the plot at 100 µM SeMet, cells show increased viability from 20% to 60%.

2.4.2 Effect of SeMet on the As(III) generated ROS:

The As(III) induction of cellular reactive oxygen species, ROS, and the anti-oxidant function of SeMet were assessed. The magnitude of ROS scavenging by SeMet was quantified using 2’,7’-dichlorofluorescin diacetate (DCFH-DA). DCFH-DA crosses cell membranes and is enzymatically hydrolyzed by intracellular esterases to impermeable non-fluorescent 2’,7’- dichlorofluorescin (DCFH). In the presence of ROS, in particular toward hydrogen peroxide

(H2O2), DCFH is rapidly oxidized to highly fluorescent 2’,7’-dichlorofluorescein (DCF). The DCF emission increase or decrease is proportional to the increase or decrease of ROS. There is some

controversy regarding the proportionality of DCF emission [43] with the amounts of ROS; however, our measurements were taken immediately after the 4 hr. incubation. Extending the measurement period beyond this can compromise the correlation. Figure 2 shows the relative

DCF fluorescence (%Max) vs. different concentrations of As(III). As can be seen in the figure, increasing arsenic concentrations resulted in increasing ROS levels (via increasing fluorescence) up to 95 +/- 6% at 30 μM As(III). One important observation is that elevated ROS, induced by

As(III) correlates with the IC50 value, but at lower concentrations than those of cell viability. The

IC50 of As(III) is 28 +/- 3 μM, but the midpoint of maximal ROS level is approximately 2 M.

Upon the addition of 100 μM SeMet, the ROS levels decreased by 25 +/- 2 at the 30 μM As(III) toxic point. Clearly, the ROS levels differ for cells treated with 30 μM As(III) alone or together with 30 μM As(III) and 100 μM SeMet, providing evidence for molecular level changes, and supportive of the cytotoxicity changes we observed (Figure 1) plus further confirming the role of SeMet as an antioxidant. These concentrations of SeMet and As(III) were used in the ESIMS experiments in evaluating differential cell signaling via phosphorylation cascades. Additional studies are planned along different ROS, arsenic and selenium species.

Figure 2: Relative DCF fluorescence changes induced by As(III). A) Concentration-dependent DCF changes. As(III) alone (light trace) leads to elevated ROS. Addition of SeMet (dark trace) lowers the DCF fluorescence in a concentration dependent manner. A 25% decrease in ROS is seen for cells grown with 30 µM As(III) alone compared with those grown with As(III)+100 µM SeMet. B) Kinetics of DCF fluorescent changes. At 10 M As(III) the elevation of DCF fluorescence is both rapid and sustained. Error bars represent experiments in triplicate.

2.4.3 Using SEC-ICPMS as part of the identification scheme:

This study identifies differential protein phosphorylation changes occurring in HEK293 cells treated with sodium arsenite As(III) alone and in combination with selenomethionine

(SeMet). The protein phosphorylation changes occurring in cells treated with As(III)/SeMet could provide insights into the SeMet antagonist role in mitigating As(III) toxicity by tracking some cell signaling pathways. A better understanding at the molecular level of this antagonism, through protein phosphorylation differences may lead to significant ways of reducing As(III) toxicity when As(III), or its toxic source cannot be removed or ameliorated. The strategy

employed in the identification of phosphorylated proteins in this work can be seen in Figure 3.

While SEC-ICPMS can readily provide quantification for Se, As and P, we use it in this study only to inform us of which peaks contain any one of these elements or all of the three of them and at what approximate concentration levels. In this way the enrichment level necessary prior to the nanoLC-ESIMS will be known and addressed. For example, if in the chromatogram, peaks of

20,000 counts per second are seen (fractions), then for the ESIMS mass spectrometer used in this study, a good signal/background will be seen in the MS or MS/MS spectra produced from

ESIMS. However, if lower counts than this are observed further fraction concentration is necessary prior to the ESIMS portion of the scheme.

Size Exclusion Chromatography (SEC) done using Conventional LC + ICPMS (detecting for 31P+)

Fraction collection and Pre-concentrate the fractions to the necessary level for ESIMS (depending on intensity seen on the ICPMS)

Tryptic digestion performed on the fractions obtained from SEC and filtered through 5kDa (MCO) filter

Nano LC ESI-MS with Phosphochip followed by Spectrum Mill and Mascot data base searches

Figure 3: Protein rich supernatant was obtained through lysis of HEK293 cells followed by extraction and centrifugation. Only a part of the sample supernatant was subjected to the SEC with in line monitoring of isotope 31P using ICPMS. With the plasma off, rest of the supernatant is subjected to the SEC and fractions are collected. Fractions isolated were pre-concentrated to

the necessary level for further ESIMS experiments. A tryptic digestion on the pre-concentrated fractions were performed and subjected to nanoLC ESI-MS/MS with phosphochip.

Figure 4A shows the chromatograms (UV detection at 280 nm) of SEC separated fractions for cells grown with no As(III), SeMet or both together (Cell Control), 30 µM of arsenite alone (Cells + 30 µM As(III)), 100 µM of SeMet alone (Cells + 100 µM SeMet), 30 µM arsenite and 100 µM SeMet together (Cells + 30 µM As(III)+ 100 µM SeMet). Differences are observed in the UV profiles, but the differences obtained through the ICPMS 31P+ signals shown in Figure 4B are considerable and also demonstrate that an element specific detection usually provides simpler to understand results relative to universal detection, as is the case with UV. Through this SEC-ICPMS coupling, monitoring the 31P+ signal resulted in easy identification of phosphorus containing species and resulted in the separation of four fractions as a function of molecular weight (hydrodynamic radii). For fraction collection, the SEC fractions were collected multiple times as necessary to achieve an appropriate concentration prior to the ESIMS experiments

(after initially establishing the fraction retention times via ICPMS, the plasma is turned off and post-UV fraction collection is done). After fraction collection, the samples were lyophilized and reconstituted with 200 μL of 50 mM NH4HCO3. Tryptic digestions were done on the four fractions before analyzing them by ESI-MS/MS.

670 KDa 158 KDa 44 KDa 17 KDa 180

) Cells + 30 µm As(III) + 100 160 µm SeMet

Cells Control mAU 140 1 2 3 4 Cells + 30 µm As(III) 120 Cells + 100 µm SeMet 100 1.35 KDa Salts 80 60 40

20 Absorbance at 280 nm ( at 280 nm Absorbance 0 0 5 10 15 20 25 30 Time (min)

6 1.5 x10 Cells + 30 µm As(III) + 100 µm SeMet Cells Control Cells + 30 µm As(III)

+ Cells + 100 µm SeMet P

31 1 1 2 3 4

0.5 Abundance CPS CPS Abundance

8.0 16.0 24.0 Time (min)

Figure 4: A) SEC chromatograms showing the UV signals monitored at 280nm. B) SEC-ICPMS chromatograms showing counts of 31P isotope (y-axis) vs. Time (x-axis). The color traces, sample description, column, flow rate, solvent conditions are similar to Figure 4A. It is apparent that the ICPMS chromatograms are simpler and more useful for screening 31P.

2.4.4 Identification of -casein using phosphochip®

In addition to the above sample treatments, prior to SEC protein fraction analysis by

ESIMS, the TiO2 nanochip performance was studied for its role in enriching and eluting the phosphorylated peptides. A brief discussion of the phosphochip function is as follows. It can be operated in a flow-through mode and an elution mode. In flow-through mode the non- phosphorylated peptides are separated based on the gradient, while the phosphorylated peptides are retained on the TiO2. In the elution mode, the buffer elutes the phosphorylated peptides and separates them on the C-18 reversed phase analytical column using a gradient.

The data obtained from the flow-through and elution mode chromatograms were searched using both Spectrum Mill and Mascot search engines for identification of proteins and the full complement are shown in the Supplement. For the evaluation of the phosphochip, a 2l of standard containing BSA and -casein were injected to the nanoLC-Chip/ESIMS system at a concentration of 100 pmol L-1 under optimized conditions. The total ion chromatogram, extracted ion chromatogram, MS and MS/MS of -casein also are shown in the appendix. The results for -casein obtained from both search engines gave high scores confirming the functionality and reliability of the phosphochip to isolate and enrich phosphorylated peptides.

2.4.5 Identification of phosphorylated proteins using the phosphochip®

The cell lysate from the cells grown with 100 M SeMet + 30 M As(III) was used as the sample for phosphorylated protein identification and site location. The reason for using the particular concentration of SeMet (100 M) is because of the enhanced viability imparted to

the As(III) inoculated cells (30 M), and this increased viability may result from changes in cell signaling via appropriate protein phosphorylation. Thus, identifying phosphorylated peptides/proteins, clearly relates to cell survival and cell signaling events. The cells grown with no inoculation (cell control), only with 30 µm As(III) or only with 100 µm SeMet serve as comparison experiments to the cells inoculated with the combination As(III)/SeMet mix.

Protein scores are used to identify proteins. Tryptic digestion generates a number of peptides that are analyzed by nanoLC-ESIMS/MS. The mass spectrometric data show peaks representing each peptide (MS) and certain of these peptide fragment masses (MS/MS) taken over a much narrower mass range (the isolation window was 2 m/z taken from the full spectrum MS). These spectra then were searched against peptide sequence databases containing known protein amino acid sequences. The protein score generated is a statistical score based on how well the experimental data match the peptide sequence from the database. An acceptable peptide score (p<0.05) for Spectrum Mill is 14 and for Mascot is usually 30-40 or higher, but is derived from each individual search. For example, a Mascot score of 40 means that there is a 1 in 10+40 chance of the report being random.

The functions found for phosphorylated proteins present in the cells grown with the

As(III)/SeMet mix, differed from those grown with only As(III) or SeMet. The proteins found in cells grown alone with As(III) or SeMet are listed in the appendix tables from S7 to S14. For example, alpha-enolase is present with predominantly high scores in almost all fractions of

HEK293 cells toxified with As(III) only or SeMet only. It takes part in inhibiting cell growth, hypoxia tolerance, allergic responses, and it is used as a diagnostic marker for many tumors [44,

45]. Though the alpha enolase is readily found in these cells under the conditions indicated, 42

dosing with the As(III)/SeMet mix showed this protein, but with a poor score. Several apoptotic proteins that are triggered by the presence of As(III) alone are missing when inoculated with only SeMet. Interestingly, the anti-apoptotic proteins, triggered in the cells with As(III) alone, appeared in the cells grown with the mix of As(III)/SeMet.

Though numerous apoptosis related proteins were found in the cells grown with only

As(III) toxification, only those of high scores are discussed as those with lower scores have lower statistical meaning. Ubiquitin-like modifier-activating enzyme has a role in cell death [46], eukaryotic translation initiation factor 5A-1 functions as a regulator of p53/TP53 and p53/TP53- dependent apoptosis [47] and further regulates TNF-alpha-mediated apoptosis [48], is expressed in umbilical vein endothelial cells and several cancer cell lines [49]. Tryptophanyl- tRNA synthetase inhibits the stress-activated responses [50] and is not found in cells grown with SeMet or together with As(III)/SeMet; however, it is seen in cells grown with As(III). This may suggest decreased stress levels in cells with the addition of SeMet. Spectrin alpha chain has a role in breakdown of the structures such as organelles, proteins, or other macromolecular structures during apoptosis [51] and appears in cells grown with As(III) only.

The protein 14-3-3 protein zeta/delta was identified with high scores in cells grown with only As(III) inoculation and As(III)/SeMet inoculation. Its main role involves anti-apoptosis, found in numerous signaling events and it can enhance p53 transcriptional activity [52, 53]. This protein is an adapter protein implicated in the regulation of a large spectrum of both general and specialized signaling pathways. It generally binds to the numerous other proteins through recognition of a phosphoserine or phosphothreonine, thereby resulting in the modulation of the binding protein activity [54, 55]. 43

The total number of proteins identified with cells grown with 30 µM As(III) + 100 µM

SeMet is 154 and upon subtracting those from the ones appearing in the cell control, the 30 µM

As(III) and the 100 µM SeMet, the significant proteins, found only in cells inoculated with the

As(III)/SeMet mix number 107 for all 4 SEC fractions. Further, separation of 107 proteins according to the number of proteins in the 4 SEC fractions was 24, 37, 24 and 22, respectively.

Figure 5 shows a pie chart containing total number of proteins 107, identified with cells grown with 30 µM As(III) + 100 µM SeMet classified base on their role in apoptosis, metabolism and signaling.

Figure 5: A pie diagram showing total number of proteins 107, identified with cells grown with 30 µM As(III) + 100 µM SeMet classified base on their role in apoptosis, metabolism and signaling.

Table 1 shows the most significant of the 107 proteins from the As(III)/SeMet mix, primarily cell proliferation and anti-apoptosis proteins, but not in control, As(III) alone or SeMet

alone inoculated cells. A full table of all 107 proteins is shown in the appendix. These data show that the molecular level phosphorylation events track the cytotoxic studies, the ROS studies as well, and support the selenium literature dealing with selenium species as antioxidant favoring cell health. For the As(III)/SeMet mix the proteins chosen to present directly in the manuscript are those that have positive or negative effects on the cell. These proteins, their scores, modifications and a summary of their functions are shown in Table 1 and discussed below. a. Fraction 1 from SEC-ICPMS followed by nanoLC-ESIMS and database searching

Deoxyribonucleoside 5'-monophosphate N-glycosidase catalyzes the cleavage of the N- glycosidic bond of deoxyribonucleoside 5'-monophosphates to yield deoxyribose 5-phosphate and a purine or pyrimidine base. Deoxyribonucleoside 5'-monophosphates containing purine bases are preferred to those containing pyrimidine bases. Its important biological functions include: nucleotide metabolism through cleavage of the N-glycosidic bond, cell proliferation, deoxyribonucleoside monophosphate catabolic process and positive regulation of cell growth

[56, 57]. Figure 6 shows the mass spectrum followed by the MS/MS spectrum, which is then taken for database searching. The score provided by Spectrum Mill is 16.7 (database search engines define their own score). Identified as one of the most responsive targets to c-Myc

(regulative gene coding for transcription factors), it can induce independent cell growth and tumor formation [58]. Significant up regulation is noticed in selective cancers, such as breast cancer, prostate cancer, lymphoma, while in untransformed cells, its expression is kept at basal level [59]. In this study, presence of arsenic may have caused the up regulation of this protein.

Figure 6: A figure showing the mass spectra of Deoxyribonucleoside 5'-monophosphate N- glycosidase peptide (1050.0) as indicated with the red line upper trace, followed by MS/MS spectra. The peptides found in the MS/MS spectrum are taken for database searching to search for these peptide combinations which best suggest protein identification.

Proteasome subunit alpha type-4 is a multicatalytic proteinase complex, which is characterized by its ability to cleave peptides with Arg, Phe, Tyr, Leu, and Glu adjacent to the leaving group at neutral or slightly basic pH. The proteasome has an ATP-dependent proteolytic activity. Its important biological functions include DNA damage response, signal transduction by p53 class mediator resulting in cell cycle arrest and regulation of apoptosis [60, 61].

DNA (cytosine-5)-methyltransferase is required for genome wide de novo methylation and is essential for the establishment of DNA methylation patterns during development.

Numerous evidences indicate that many CpG islands can undergo abnormal de novo methylation in cancer cases. Initially shown as a silencing mechanism for tumor suppressor genes, but later found to be much more wide spread [62].

Baculoviral IAP repeat-containing protein 6 (BIRs), protects cells from undergoing apoptosis [63]. Caspases once activated initiate an irreversible cascade of events resulting in rapid cell death [64]. The BIRs are involved in inhibition of caspases cascade, thus preventing cells from undergoing apoptosis [63]. b. Fraction 2 from SEC-ICPMS followed by nanoLC-ESIMS and database searching

The first protein of fraction 2 with high Spectrum Mill score (19.8) is 60 kDa heat shock protein (Hsp60). It is located in mitochondria and is implicated in mitochondrial protein import and macromolecular assembly. This protein is highly up regulated, usually attributed to its role in mitochondria and plays an important role in cytoprotection [65, 66]. Though lower levels are up regulated with As(III), the presence of SeMet may facilitate additional up regulation, thus promoting cell survival. It may facilitate the correct folding of imported proteins and promote the refolding and proper assembly of unfolded polypeptides generated under stress conditions in the mitochondrial matrix. Important biological functions include de novo protein folding, B cell cytokine production, B cell proliferation, T cell activation, chaperone-mediated protein complex assembly. The 70 kDa heat shock protein (Hsp70), stabilizes pre-existent proteins against aggregation and mediates the folding of newly translated polypeptides in the cytosol as 47

well as within organelles [67, 68]. These chaperones participate in all these processes through their ability to recognize non-native conformations of other proteins. They bind extended peptide segments with a net hydrophobic character exposed by polypeptides during translation and membrane translocation, or following stress-induced damage. In general, this protein production is triggered in response to unfolded proteins. The up regulation of these heat shock proteins could be from the stress generated by the As(III) .

The 78 kDa glucose-regulated protein may play a role in facilitating the assembly of multimeric protein complexes inside the endoplasmic reticulum (ER). Important biological functions include ER-associated protein catabolic process, anti-apoptosis, platelet activation, platelet degranulation and regulation of protein folding in ER [69, 70].

Thioredoxin participates in various redox reactions through the reversible oxidation of its active center dithiol to a disulfide and catalyzes dithiol-disulfide exchange reactions [71-73].

It plays a role in the reversible S-nitrosylation of cysteine residues in target proteins, and thereby contributes to the response to intracellular nitric oxide, nitrosylates the active site Cys of CASP3 in response to nitric oxide (NO), and thereby inhibits caspase-3 activity. Important biological processes include cell proliferation, cell-cell signaling, positive regulation of DNA binding, regulation of protein import into nucleus, translocation, signal transduction, transcription (DNA-dependent).

Superoxide dismutase destroys free radicals which are normally produced within the cells, thus reducing toxicity associated with free radicals in biological systems. It’s important biological processes include DNA fragmentation involved in apoptotic nuclear change, 48

activation of MAPK activity, double-strand break repair, glutathione metabolic process, muscle cell homeostasis, removal of superoxide radicals, response to drugs and spermatogenesis [74].

Programmed cell death protein may function in the process of apoptosis and its induction. c. Fraction 3 from SEC-ICPMS followed by nanoLC-ESIMS and database searching

Protein arginine N-methyltransferase 2, methylates the guanidino nitrogens of arginyl residues in proteins such as STAT3, FBL, histone H4. It may inhibit NF-kappa-B transcription and promote apoptosis [75-77]. A key biological process is growth regulation, induction of apoptosis, developmental cell growth, negative regulation of G1/S transition of mitotic cell cycle and NF-kappaB transcription factor activity, positive and negative regulation of transcription (DNA-dependent), regulation of androgen receptor signaling pathways.

Tyrosine-protein kinase ABL1, non-receptor tyrosine-protein kinase plays a role in many key processes linked to cell growth and survival such as cytoskeleton remodeling in response to extracellular stimuli, cell motility and adhesion, receptor endocytosis, autophagy, DNA damage response and apoptosis [78-80]. Important biological processes include affecting apoptosis,

DNA damage induced protein phosphorylation, DNA damage response, signal transduction resulting in induction of apoptosis, cell adhesion, mismatch repair, negative regulation of protein serine/threonine kinase activity, peptidyl-tyrosine phosphorylation and regulation of transcription involved in S phase of the mitotic cell cycle. d. Fraction 4 from SEC-ICPMS followed by nanoLC-ESIMS and database searching

E3-UFM1-protein ligase mediates ufmylation of target proteins such as

DDRGK1/C20orf116. Important biological roles involve tumor suppressor by inhibiting cell invasion, blocking NF-kappa-B signaling and increasing stability of CDK5RAP3 [81, 82].

The majority of these proteins involving cell well-being or demise are clearly antioxidant, signaling and growth proteins. Proteins regulating apoptosis and anti-apoptosis are crucial in the death or survival of cells.

2.5 Conclusion:

SeMet when inoculated along with As(III) results in reduced cytotoxicity in human embryonic cells, HEK293. It was noted that 20% cell survival with As(III) inoculation alone was increased to 60% survival when SeMet was added to the inorganic As(III) toxin. Similarly it was found that with the As(III) in comparison with the As(III) + SeMet, the amount of ROS was reduced by ~ 25% with the latter mix as determined by DCF fluorescence.

The coupling of ICPMS with SEC for protein phosphorylation screening via 31P+ detection with the use of He as collision gas, followed by nanoLC-ESIMS resulted in identifying phosphorylated proteins. Further, the use of the phosphochip containing TiO2 for phosphorylated peptide enrichment resulted in lower level phosphorylated protein identification. These studies reveal marked differences in protein phosphorylation among HEK control cells, As(III) toxified cells and As(III)/SeMet toxified cells. In the As(III)/SeMet toxic mix, many more proteins related to anti-oxidation, anti-tumor activity and cell growth are seen vs. the As(III) toxified or general control cells. Though the exact mechanism of action of selenomethionine in mitigating the toxicity of As(III) is not yet known, this study helps to 50

further understand the mechanism through the cytotoxicity reduction and production/ up- regulation of key signaling proteins due to the presence of SeMet. The next steps will be to progress to more specific arsenic target cells; namely primary keratinocytes and include other

Se, As and ROS species.

2.6 Acknowledgements:

The authors would like to thank Agilent Technologies for their continued support with chromatography and mass spectrometry instrument loans. We are also grateful to CEM for microwave digestion equipment.

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Fraction 1 Score Peptide Sequence Entry name Variable Sites Uniprot KB Briefly - Important found, NR none Variable Sites functions recorded 16.67 (R)FQVWDYEEGEV Deoxyribonucl NR S : 12, 28, 169 Nucleotide metabolism, EALLDR(Y) eoside 5'- cell proliferation, monophospha deoxyribonucleoside te N- monophosphate catabolic glycosidase process, positive regulation of cell growth 10.07 (R)YLLQYQEPIPCE Proteasome NR S : 13, 75 Activated upon DNA QLVTALCDIK(Q) subunit alpha damage response, signal type-4 transduction by p53 class mediator resulting in cell cycle arrest, regulation of apoptosis 7.26 (R)LQAFFtsDTGLE DNA(cytosine- S : 552 T : 551 NR Essential for the YEAPKLYPAIPAAR( 5)- establishment of DNA R) methyltransfe methylation patterns rase during development. 6.50 (K)LsQLKsQQVAA Nucleosome- S : 788, 792 S : 216, 572, 763, May regulate AAHEANKLFK(E) remodeling 817, 1231, 1300, transcription through factor subunit 1827, 1833, 2098, direct binding to DNA or BPTF 2465 T : 909, transcription factors. 1064 Y : 839 Phosphorylation enhances DNA-binding.

6.19 (K)RVEIMAQCEEW Baculoviral S : 4781 S : 473, 480 Protects cells from IADIQQYsSDK(R) IAP repeat- undergoing apoptosis containing protein 6 Fraction 2 19.79 (R)IQEIIEQLDVTTS 60 kDa heat NR S : 70 Implicated in EYEK(E) shock protein, mitochondrial protein mitochondrial import and macromolecular assembly. 15.54 (K)ATAGDTHLGGE Heat shock 70 NR NR In cooperation with other DFDNR(L) kDa protein chaperones, Hsp70s stabilize preexistent proteins against 58

aggregation and mediate the folding of newly translated polypeptides in the cytosol as well as within organelles. 10.8 (K)TAFQEALDAAG Thioredoxin NR T : 100 Cell proliferation, cell-cell DK(L) signaling, positive regulation of DNA binding, regulation of protein import into nucleus, translocation, signal transduction, transcription (DNA- dependent) 8.51 (K)GDGPVQGIINFE Superoxide NR S : 99 Destroys radicals which QK(E) dismutase are normally produced within the cells and which are toxic to biological systems. 8.31 (R)DVVCEDEDAFC Protein S : 211 S : 195, 265, 501, Key regulator of LsLENIATLQKLLR( regulator of 513, 571, 592, cytokinesis. Q) cytokinesis 615 T: 470, 481, 616 7.91 (R)SVGDGETVEFD DNA-binding NR S : 2, 34, 38, 79, Negative regulation of VVEGEK(G) protein A 134, 201, 203, apoptotic process. 287, 369, 370 T : 287 7.69 (R)HPEIKVTAK(Y) Zinc finger NR S : 1076, 1079, May be involved in protein 462 1082, 2024 Y : transcription regulation. 2083 6.45 (K)HGDPGDAAQQ Programmed NR S : 119 May function in the EAK(H) cell death process of apoptosis. protein Fraction 3 7.21 (R)HMSVALSWAVt Protein T : 414 NR induction of apoptosis, SRQDPTSQK(V) arginine N- developmental cell methyltransfe growth, negative rase 2 regulation of G1/S transition of mitotic cell cycle and NF-kappaB transcription factor activity, positive and

negative regulation of transcription (DNA- dependent), regulation of androgen receptor signaling pathway 6.43 (R)GPPEGGLNEDE Tyrosine- NR Partial List - S : Non-receptor tyrosine- RLLPKDK(K) protein kinase 50, 446, 569… protein kinase that plays a ABL1 620, 659, 683, role in many key 718, … 917, 919, processes linked to cell ... 977 T : 392 ... growth and survival such 613, 735, 781… as DNA damage response 852 Y : 70, 185, and apoptosis. 226 ... 393, 469 Fraction 4 7.72 (K)QNESRTTECKQ Nipped-B-like S : 666 T : 667 S : 139, 150, 162, Phosphorylated upon DNA NEstIVEPK(Q) protein 274, 280, 284, damage. 306, 318, 350, 353, 553, 849, 850, 912, 1089, 1090, 1096, 1150, 1152, 1154, 1160, 1459, 2509, 2511, 2513, 2658, 2672 T : 558, 646, 746, 914, 1458, 2667 Y : 1159 7.52 (K)LSEETKVALTKL E3UFM1- S : 629 S : 458 May act as a tumor HNsLNEK(S) protein ligase suppressor by inhibiting 1 cell invasion.

Table 1: Shows the scores, peptides identified, names of the proteins, modifications found in

the study, Uniprot reported modifications and a brief summary of the important functions.

Chapter 3

Identification of selenium-containing proteins in HEK 293 kidney cells using

multiple chromatographies, LC-ICPMS and nano-LC-ESIMS

3.1 Abstract:

Our previous studies using HeLa and HEK 293 cells demonstrated that selenomethionine, SeMet, exerts more of an antagonistic effect on arsenic than other selenium species. These studies attributed the antagonistic effect of SeMet to decreased levels of reactive oxygen species, ROS, changes in protein phosphorylation and possible incorporation of

SeMet into proteins. The present study employs a metallomics approach to identify the selenium-containing proteins in HEK 293 cells raised with SeMet. The proteins were screened and separated using two dimensional high performance liquid chromatography (HPLC)- inductively coupled plasma mass spectrometry (ICPMS), size exclusion chromatography (SEC) and reversed-phase chromatography (RPC). The Se-containing proteins were identified by peptide mapping using nano-HPLC-Chip-electrospray ionization mass spectrometry (ESIMS).

3.2 Introduction:

Selenium (Se) is an essential micronutrient for humans and animals [1] and is considered to be associated with the function of numerous major cellular metabolic pathways [2-4]. Se is required for the activity of a number of selenium-dependent enzymes, such as glutathione peroxidases, thioredoxin reductases, iodothyronine deiodinases and selenophosphate synthetases [5, 6]. The proteins containing selenium are termed as selenium-containing proteins. However, if the selenium incorporation is through selenocysteine, SeCys, these coded proteins are termed as selenoproteins. Though selenium-containing proteins are considered physiologically not as important as selenoproteins, they serve in synthesis of selenoproteins, thus having an indirect effect. It is thought that SeMet is incorporated into proteins through the sulfur metabolic pathway [7, 8]. In other words, methionines are replaced by SeMet during the corresponding peptide synthesis. Selenium exerts its biological functions largely through its presence in selenoproteins; however, some low molecular weight selenium compounds, such as methyl selenic acid (MeSeA), methyl-selenocysteine (MeSeCys) and selenomethionine

(SeMet) also have profound cellular effects. Recent studies have shown that Se-containing small molecules modulate tumor growth in animal studies and in vitro models [4, 9-11]. Lack of dietary selenium, has been attributed to the increased risk of cancer, infections [12, 13], cardiomyopathy [14], male infertility [15], Parkinson’s disease [16], Alzheimer’s disease [17], depleted immune response [18] and thyroid function [19]. Further, selenium deficiencies lead to decreased levels of selenoproteins, such as GPx, thus generating increased levels of ROS leading to numerous detrimental effects [2, 20, 21].

Selenium species can mitigate the toxicity of ingested and inhaled arsenic and mercury

[22-24]. Our earlier studies using HeLa and HEK 293 cells demonstrated that SeMet exerts a greater antagonistic effect on arsenic than other selenium species [24, 25]. Examination showed that SeMet decreased levels of reactive oxygen species (ROS) through its anti-oxidant function. Importantly, key changes in signaling cascades were noted via our phosphoproteomic analysis. It has been recently proposed that ROS is a key cellular signal transducer for cell growth and angiogenesis [26, 27]. Thus, SeMet not only quenches the ROS induced from arsenic and mercury but also lowers the initiation capacity of these key cancer-signaling cascades. We hypothesize that this occurs through possible incorporation of SeMet into proteins. The first step in exploring this hypothesis is to demonstrate enhanced non-specific incorporation of

SeMet into cellular proteins. To demonstrate the non-specific incorporation of SeMet into HEK

293 cell proteins, this study was undertaken.

Currently the most predominant method for identification of selenium-containing proteins is chromatographic separation followed by mass spectrometry [28, 29]. Coupling High

Performance Liquid Chromatography (HPLC) with Inductively Coupled Mass Spectrometry

(ICPMS) or Electrospray Ionization Mass Spectrometry (ESIMS) provides a means of identifying selenoproteins and selenium-containing proteins, and this coupling technique was reviewed by

Sanz-Medel [30, 31] and Szpunar [32]. Lobinski et al. did extensive work on identifying selenoproteins and selenium-containing proteins in selenized yeast [28, 33].

The aim of this study is to identify selenium-containing proteins in HEK cells cultured in the presence of SeMet. First, size exclusion chromatography (SEC) coupled to ICPMS was used

to confirm the presence of selenium in various molecular weight regions, and then followed by fraction collection based on molecular weight ranges. To assess whether the selenium found through SEC-ICPMS is covalently bound to proteins or not, each fraction was further analyzed using capillary reversed-phase liquid chromatography (capRPLC) coupled to ICPMS. Any free selenium that is not covalently bound to the proteins would be found in this procedure, since denaturation of SEC protein fractions is done prior to capRPLC. To overcome the small capRPLC amounts, the developed method was transferred to normal bore reversed-phase liquid chromatography (RPLC, with peptide MW standards used for to assure the MW assignments for the fractions were correct). With the ICPMS offline, the fractions were isolated. The isolated fractions were tryptically digested and protein identification from their corresponding peptides was achieved through nanoLC-ESIMS2 followed by database searching.

3.3 Experimental:

3.3.1 Reagents:

All the aqueous solutions were prepared in 18 MΩ cm‐1 doubly deionized water (Sybron

Barnstead, Boston, MA, USA). HEK 293 human kidney cell lines, Dulbecco’s Modified Eagle’s

Medium (DMEM), Fetal Bovine Serum (FBS), M‐Per protein extraction buffer, formic acid (FA), urea, ammonium acetate and ammonium bicarbonate were purchased from Fisher Scientific

(Fairlawn, NJ, USA). Trypsin‐EDTA: 0.05% trypsin 0.53 mM EDTA × 4 Na, was purchased from

Gibco Invitrogen Corporation (Carlsbad, CA, USA). L‐selenomethionine (SeMet), Dithiothreitol

(DTT), iodoacetamide (IAA) and bovine serum albumin (BSA) were purchased from Sigma-

Aldrich Chemical Company (St. Louis, MO, USA). The HPLC grade solvents, water and 65

acetonitrile (ACN), were of high purity and purchased from Burdick and Jackson (Muskegon, MI,

USA). Sequence grade modified trypsin and acetic acid buffer were obtained from Promega

(Madison, WI, USA).

3.3.2 Cell culture and Cell lysate:

Under humid conditions, human kidney cells (HEK293 cells) were cultured in 75 cm2

o tissue culture plates using DMEM medium containing 10% FBS at 37 C with 5% CO2. Fresh solutions of selenomethionine were made using DMEM medium prior to dosing. After cells reached 80% confluence, they were raised for 24hrs with no SeMet dosing or with 100 µM of

SeMet.

After incubation for 24 hrs with 100 µM SeMet, Trypsin-EDTA was used to isolate the cells, followed by washing with PBS to remove the traces of leftover media. M‐Per protein extraction buffer was used for lyses and extraction. Cells were centrifuged at 10,000g for 10 minutes and supernatants were used for further analysis.

3.3.3 Size Exclusion Chromatography (SEC):

Bio-Rad Laboratories (Life Science Research, CA, USA), and R = 0.997 for the calibration. Mobile phases were: A: 50 mM NH4OAc, B: MeOH, Isocratic: 5% B at Flow rate of 0.5 ml/min. The fractions isolated based on the SEC-ICPMS signal were collected and pre-concentrated to dryness by lyophilization.

3.3.4 Inductively Coupled Plasma Mass Spectrometry (ICPMS):

An Agilent Technologies 7700 inductively coupled plasma mass spectrometer, equipped with a Micromist nebulizer, a Peltier-cooled spray chamber (2 oC), and an octopole collision/reaction cell with hydrogen gas pressurization (purity of 99.99%) was used. The entire system was controlled using Mass Hunter software (all from Agilent Technologies, Santa Clara,

CA, USA). The ICPMS conditions were as follows: forward power, 1500 W; plasma gas flow rate

-1 -1 -1 -1 15 L min , carrier gas flow, 0.91 L min ; make-up gas 0.12 L min ; collision gas, H2, 4.0 ml min ; quadrupole bias, -16.0 V; octopole bias -18.0 V for a +2 volt energy discrimination voltage. The isotope monitored was selenium 78Se.

3.3.5 Capillary Reversed Phase Liquid Chromatography (capRPLC):

An Agilent 1200 HPLC system equipped with a binary pump, vacuum membrane degasser, thermostated auto sampler, column oven, and diode array detector with a semi- micro flow UV-Vis cell was used for chromatographic analysis. Reversed phase chromatography was performed using C-4 phase, Jupiter 150 x 0.50 mm, 5-μ-particle size (Phenomenex, CA,

USA). The solvents used were 0.1% FA in H2O (Solvent A) and 0.1% FA in 90% ACN (Solvent B). A flow rate of 10 μl/min was used. The following gradient was used in the analysis: 3 min 3% B; 3-

60 min 65% B, 60-70 min 90% B, 70-75 min 90% B, 75-80 min 2% B and 80-90 min 3% B.

3.3.6 Tryptic digestion, ESI-MS and Nano-LC-Chip:

Tryptic digestions were performed as follows: the protein pellets obtained from the SEC fractions collected were re-suspended in 50 µL of 50 mM ammonium bicarbonate, 4 µL of 100 mM DTT were added as reducing buffer and the mixture was heated at 95 °C for 5 minutes; this step unfolds the proteins and reduces the disulfide bonds. After cooling the sample, an alkylation was carried out to protect the thiol groups of the cysteine residues by adding 6 µL of

100 mM iodoacetamide. The mixture was incubated in the dark for 20 minutes at room temperature. After the alkylation, 2 µL of modified sequence grade trypsin solution was added and incubated at 36 oC for 2 hours. Then 2 µM of additional trypsin was added to complete the reaction, followed by incubation at 36 oC for 8 hours. 2 µL of formic acid was added to stop the reaction, and the solution was ultra-filtered through 5 kDa filters to eliminate the undigested proteins and the trypsin.

Electrospray experiments were performed using an Agilent 6300 Series nanoLC Chip/Ion

Trap XCT system (Agilent Technologies, Santa Clara, CA). An Agilent 1200 LC, equipped with both capillary and nano pumps, was used for loading and flushing the chip nano column. This is equipped with both capillary and nano pump and used for mass identification. The chip used for the analysis consists of a Zorbax 300SB C18 enrichment column (4 mm x 75 µm, 5 µm) and a

Zorbax 300SB C18 analytical column (150 mm x 75 µm, 5 µm). Two microliters of sample were loaded via the capillary pump onto the on-chip enrichment column. Samples were loaded on to the enrichment column at a flow rate of 3 µL min-1 with a 97:3 ratio of solvent A (0.1% FA (v/v) in water) and B (90% ACN (acetonitrile), 0.1% FA (v/v) in water). After the enrichment column was loaded, the on-chip microfluidics switched to the analytical column at a flow rate of 0.3 µL 68

min-1. The following gradient conditions were used in the analysis: 0–5 min, 10% B; 5–85 min,

35%B; 85–90 min, 75% B; 90–95 min, 75% B; 95–98 min, 3% B; 98–105 min, 3% B. For MS/MS experiments, experimental conditions consisted of: m/z range: 100–2200, isolation width: 2 m/z units, 2 precursors, maximum accumulation time 150 ms and active exclusion after 3 spectra from 0.5 minutes, fragmentation energy: 30% – 200% fragmentation.

The MS/MS data obtained from the experiments were exported to the online MASCOT program (Matrix Science Inc.) and Spectrum Mill database search engines, and submitted with the following parameters: Taxonomy (Mammals), Enzyme (Trypsin), Missed Cleavages (Two),

Fixed modifications (Carbamidomethyl), Peptide tolerance (2 Da), MS/MS tolerance (0.8 Da),

Peptide charge (+1,+2,+3) and Instrument (ESI-TRAP). MASCOT then searched against Uniprot database and the reported hits were validated doing a blast analysis of the reported peptides.

3.4 Results and Discussion:

This study identifies the selenium containing proteins in HEK cells grown with and without100 µM SeMet (control vs. analytical sample). The strategy employed in the identification of selenium-containing proteins is shown in Figure 1. Size Exclusion

Chromatography was used for separation of proteins based on their molecular weight and the removal of low MW free selenium with ICPMS detection monitoring 78Se. Insights into which fraction includes selenium containing proteins were therefore obtained. Figure 2a shows the chromatograms (UV detection at 280 nm) of SEC fractions for cells grown with 100 µM SeMet and the control. Although slight differences are observed in the UV profiles, the differences obtained via the ICPMS 78Se signal is considerable, as shown in Figure 2b and it may be noted that element specific detection usually provides simpler to understand results, relative to 69

universal detection, such as UV. Thus, the incorporation of SeMet into the higher molecular weight regions is extensive. Four fractions, as a function of molecular weight (hydrodynamic radii) were separated. For fraction collection, the ICPMS plasma is turned off and SEC fractions are collected multiple times to achieve an appropriate concentration prior to the capRPLC experiments. After fraction collection, the samples were lyophilized and denatured prior to the injection into capRPLC.

Size Exclusion Chromatography (SEC) done using Conventional LC + ICPMS (detecting for 78 Se)

Separation of concentrated fractions using CapRPLC Coupled to the ICPMS (Detecting for 78Se), CapRPLC method trasfered to normal RPLC and fraction isolated and concentrated

Tryptic digestion performed on the fractions obtained from RPLC and filtered through 5kDa (MCO) filter

Nano-LC-ESI-MS followed by Spectrum Mill and Mascot data base searches

Figure 1: Protein rich supernatant was obtained through lysis of HEK293 cells followed by extraction and centrifugation. Only a part of the sample supernatant was analyzed using SEC with in line monitoring of isotope 78Se using ICPMS. With the plasma off, remaining supernatant is subjected to the SEC and fractions were collected. Fractions isolated were concentrated to the necessary level for further analysis using capRPLC-ICPMS. After monitoring the isotope 78Se on line using ICPMS, the capRPLC method was transferred to normal RPLC and fractions isolated. Isolated fractions were further concentrated. Tryptic digestions on the concentrated fractions were performed and subjected to nanoLC-ESIMS analysis.

3000

) 670 KDa 158 KDa 44 KDa17 KDa __

Control mAU 2a __ 2000 SeMet aaaa0a

1000 Absorbance at 280 nm nm ( 280 at Absorbance

0 0 5 10 15 20 25 30 35 Time(Minutes)

25000 4 1 2 3 __ Control 20000 2b __

Se Se SeMet 78

15000 CPS

10000

Abundacne 5000

0 5 10 15 20 25 30 35 Time(Minutes)

Figure 2: a) SEC chromatograms showing the UV signals monitored at 280nm. b) SEC-ICPMS chromatograms; counts of 78Se isotope (y-axis) vs. Time (x-axis). The color traces, sample description, column, flow rate, solvent conditions are similar to Figure 2a. It is apparent that the ICPMS chromatograms are simpler and generally more useful than with UV detection for screening selenium containing proteins.

3.4.1 capRPLC

SeMet incorporation into proteins is nonspecific and the incorporation of selenium in the selenium-containing proteins can be through covalent and non-covalent bonding. Though SEC separation based on hydrodynamic volume (an indication of molecular weight) provides purification of proteins from the free inorganic selenium, a confirmation of covalently bound selenium in these proteins is established by denaturing the protein fractions.

Denaturing the fractions liberates free selenium not covalently associated. Further, these denatured protein fractions are separated using capRPLC and 78Se signal is monitored by

ICPMS.

Figures 3a and 3b represent the 78Se signal monitored over time using ICPMS for first and second SEC fractions, which contain various substances including proteins whose molecular weights are greater than 44 kDa. As can be seen from the figure there is no loss of selenium upon injection, suggesting all the selenium present is covalently bound. Figures 3c and 3d represent the 78Se chromatography for the third and fourth SEC fractions, whose molecular weights are less than 44 kDa. As can be seen from the figures there is high amount of selenium loss upon injection. This suggests that selenium is not covalently associated with proteins of <

44 kDa.

Thus, coupling capRPLC with ICPMS provides greater insights about the covalent or non- covalent bonding of selenium in SEC separated fractions. As can be seen from Figures 3a-d, fractions 1 and 2 (isolated by SEC) are important for further analysis, since the association of selenium in these protein fractions is through covalent bonding. It is not practical to collect fractions on capRPLC to the necessary levels to further perform ESIMS protein analysis. To 72

overcome this, the gradient solvent conditions employed in the capRPLC were transferred to the normal bore RPLC to separate a much larger protein amount. These reversed phased fractions were further concentrated to necessary levels and tryptic digestion performed prior to nanoLC-ESIMS analysis.

160000

Se 140000 3a __ 78 78Se (Control) 120000 __ 100000 78Se (SeMet) 80000 60000

40000 Abundance Abundance CPS 20000 0 0 20 40 60 80 100 Time (Minutes)

160000

140000 3b Se

78 120000 __ 78Se (Control) 100000 __ 80000 78Se (SeMet) 60000 40000 Abundance Abundance CPS 20000 0 0 20 40 60 80 100 Time(Minutes)

500000 3c

Se 400000 78

300000

200000

Abundance Abundance CPS 100000

0 0 20 40 60 80 100 Time(Minutes)

140000 3d

Se 120000 78 78 100000 80000 60000 40000

Abundance Abundance CPS 20000 0 0 20 40 60 80 100 Time(Minutes)

Figure 3: All figures 3a-3d represent counts of 78Se isotope (y-axis) vs. Time (x-axis) and color traces, sample description, column, flow rate and solvent conditions are similar. a&b) First fraction and second fraction of SEC analyzed using capRPLC. c&d) Third fraction and fourth fraction of SEC analyzed using capRPLC, the arrow points the free selenium eluted upon injection.

3.4.2 Identification of Se-containing proteins:

Based on the 78Se signal provided by capRPLC-ICPMS, 12 fractions were collected from the scaled up normal bore experiment, digested with trypsin and analyzed by nano-LC-Chip- 74

ESIMS. The resulting MS/MS spectra were database searched for peptide identification.

Numerous proteins were found in the fractions isolated containing Se, though it would be presumptuous to declare these are Se-containing proteins. A generally accepted hypothesis is that SeMet is incorporated into proteins through non-specific pathways, thus SeMet and Met can be incorporated into the same type of proteins, further indicating the presence of both peptides, a S-peptide, the peptide containing Met, and a Se peptide, the peptide with the same sequence as the S-peptide except that the Met is replaced by SeMet. Calculations were made to predict the m/z of the Se-peptide based on the m/z of S-peptide using the formula shown below [34] .

80 32 m/z (S-peptide) + n × ( Se - S)/z = m/z (Se-peptide)

In the above formula, m/z (S-peptide) would be obtained from standard peptide mapping, n

= 1,2,3…..N, where N is the number of Met in the S-peptide. The difference in atomic mass between 80Se and 32S is 48 and z is the charge of the S-peptide precursor ion (it is assumed that the charge is the same for the Se-peptide). Since a Se-peptide may contain more than one Met and SeMet may replace more of them, it is possible that multiple Se-peptides may be predicted from an S-peptide with multiple Met. It is expected that an S-peptide and its corresponding Se- peptide elute closely together (unless a carbamidomethylation is present on S-peptide and absent in Se-peptide) from the reversed-phase column during nano-LC-ESI-MS/MS analysis. The extracted ion chromatograms (EIC) of the m/z S-peptide and the predicted m/z Se-peptide were extracted from the total ion chromatogram (TIC). If an S-peptide and its corresponding Se- peptide are both present, a retention time match will be observed as shown in Figures 4a and

4b, since they are structurally identical except the difference between S atoms (Met) and Se 75

atoms (SeMet). A use of the formula is illustrated below.

Intensity

5 VETGVLKPGMVVTFAPVNVTTEVK VETGVLKPGSeMVVTFAPVNVTTEVK x10 5 838.6 x10 6 5 854.8

5 880.2 4 1050.0 4

3 3 958.5

2 789.9 2 937.7 808.4 990.4 1 1

0 0 800 m/z 1000 800 m/z 1000

Figure 4: a) Extracted Ion chromatogram (EIC) of an S-peptide (VETGVLKPGMVVTFAPVNVTTEVK) belongs to Elongation factor 1-alpha and has m/z of 838 b) EIC of Se-peptide (VETGVLKPGSeMVVTFAPV-NVTTEVK) belongs to Elongation factor 1-alpha and has m/z of 854; the retention time overlap is to be noted c) MS/MS spectra of S-peptide (VETGVLKPGMVVTFAPVNVTTEVK) belong to Elongation factor 1-alpha and has m/z of 838 d) MS/MS spectra of Se-peptide (VETGVLKPGSeMVVTFAPVNVTTEVK) belongs to Elongation factor 1-alpha and has m/z of 854.

All the selenium-containing proteins identified in 12 fractions isolated using normal bore

LC are presented in Table 1. For a better understating on how the selenium-containing proteins were isolated and identified, a discussion follows for one protein. The first peptide in the table is taken for the explanation. This peptide sequence confirms the presence of protein Elongation factor 1-alpha. The S-peptide (VETGVLKPGMVVTFAPVNVTTEVK) belongs to Elongation factor 1- alpha and has m/z of 838. The total ion chromatogram, extracted ion chromatogram for S- peptide and Se-peptide, MS/MS spectra of S-peptide and Se-peptide are shown in Figures 4a-d.

As it is expected that the S-peptide would elute at the same retention time compared to the Se- peptide (at least very close, since the change in net hydrophobicity would be small), the retention time matches should serve for identification. The Se-peptide

(VETGVLKPGSeMVVTFAPVNVTTEVK) has an m/z of 854 and its EIC matches with EIC of S- peptide at the same retention time confirming the both peptides. The S-peptide mass is

2515.38 and its charge is +3, so the observed m/z is 838.46. The Se peptide mass is 2515.38 plus the difference between the two atomic weights of selenium and sulfur is 48. So, the net mass is

2515.38+48 = 2563.38, divided by its charge +3. Final observed m/z is 2563.38/3 = 854.46. A similar strategy was employed in identifying all the Se-containing proteins reported in Table 1

based on their S-peptide. Table 1 also shows the differences between the S and Se peptide masses for all proteins reported as obtained from the MS/MS spectra.

Though numerous proteins were found to contain the SeMet, only a few of those showing significance are discussed here. Further information can be found by following the accession numbers. WD repeat and HMG-box DNA-binding protein 1 has a role in DNA binding and acts as a replication initiation factor that brings together the mini-chromosome maintenance proteins (MCM2-7) helicase and the DNA polymerase alpha/primase complex in order to initiate DNA replication [35]. Suppressor of tumorigenicity 14 protein has a similar role to serine protease and is proposed to play a role in breast cancer invasion and metastasis [36].

Collagen alpha-2(IV) chain is the major structural component of glomerular basement membranes (GBM). Canstatin, a cleavage product corresponding to the collagen alpha 2(IV)

NC1 domain, possesses both anti-angiogenic and anti-tumor cell activity [37].

3.5 Conclusion:

A metallomics approach was used for identification of Se-containing proteins in HEK 293 cells cultured in the presence of SeMet. The use of SEC-ICPMS, resulted in preliminary peak screening and molecular weight fraction separation for selenium containing proteins by monitoring the 78Se isotope. The fractions were isolated based on the MW with the ICPMS offline. Further use of cap-RPLC on SEC fractions clearly established the differences in fractions containing Se association by means of covalent or non-covalent bonding. Se incorporation in higher molecular weight fractions are through covalent bonding, while lower molecular weight fractions are through non-covalent bonding. The selected RPLC Se-containing fractions were

analyzed by peptide mapping with HPLC-Chip-ESIMS and MASCOT database searches.

Numerous selenium-containing proteins were identified based on the approach, thus providing evidence of the SeMet incorporation into the HEK proteins is non-specific.

3.6 Acknowledgements

3.7 References

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17. Yoo MH, Gu X, Xu XM, Kim JY, Carlson BA, Patterson AD, Cai H, Gladyshev VN, Hatfield DL: Delineating the role of glutathione peroxidase 4 in protecting cells against lipid hydroperoxide damage and in Alzheimer's disease. Antioxid Redox Signal 2010, 12(7):819-827. 18. Pagmantidis V, Meplan C, van Schothorst EM, Keijer J, Hesketh JE: Supplementation of healthy volunteers with nutritionally relevant amounts of selenium increases the expression of lymphocyte protein biosynthesis genes. Am J Clin Nutr 2008, 87(1):181- 189. 19. Kohrle J, Jakob F, Contempre B, Dumont JE: Selenium, the thyroid, and the endocrine system. Endocr Rev 2005, 26(7):944-984. 20. Prabhu KS, Zamamiri-Davis F, Stewart JB, Thompson JT, Sordillo LM, Reddy CC: Selenium deficiency increases the expression of inducible nitric oxide synthase in RAW 264.7 macrophages: role of nuclear factor-kappaB in up-regulation. Biochem J 2002, 366(Pt 1):203-209. 21. Seyedrezazadeh E, Ostadrahimi A, Mahboob S, Assadi Y, Ghaemmagami J, Pourmogaddam M: Effect of vitamin E and selenium supplementation on oxidative stress status in pulmonary tuberculosis patients. Respirology 2008, 13(2):294-298. 22. Gailer J, George GN, Pickering IJ, Prince RC, Younis HS, Winzerling JJ: Biliary excretion of [(GS)(2)AsSe](-) after intravenous injection of rabbits with arsenite and selenate. Chem Res Toxicol 2002, 15(11):1466-1471. 23. Korbas M, Percy AJ, Gailer J, George GN: A possible molecular link between the toxicological effects of arsenic, selenium and methylmercury: methylmercury(II) seleno bis(S-glutathionyl) arsenic(III). J Biol Inorg Chem 2008, 13(3):461-470. 24. Alp O, Zhang Y, Merino EJ, Caruso JA: Selenium effects on arsenic cytotoxicity and protein phosphorylation in human kidney cells using chip-based nanoLC-MS/MS. Metallomics 2011, 3(5):482-490. 25. Alp O, Merino EJ, Caruso JA: Arsenic-induced protein phosphorylation changes in HeLa cells. Anal Bioanal Chem 2010, 398(5):2099-2107. 26. Finkel T: Intracellular redox regulation by the family of small GTPases. Antioxid Redox Signal 2006, 8(9-10):1857-1863. 27. Ushio-Fukai M, Alexander RW: Reactive oxygen species as mediators of angiogenesis signaling: role of NAD(P)H oxidase. Mol Cell Biochem 2004, 264(1-2):85-97. 28. Encinar JR, Ouerdane L, Buchmann W, Tortajada J, Lobinski R, Szpunar J: Identification of water-soluble selenium-containing proteins in selenized yeast by size-exclusion- reversed-phase HPLC/ICPMS followed by MALDI-TOF and electrospray Q-TOF mass spectrometry. Anal Chem 2003, 75(15):3765-3774. 29. Becker JS, Jakubowski N: The synergy of elemental and biomolecular mass spectrometry: new analytical strategies in life sciences. Chem Soc Rev 2009, 38(7):1969- 1983. 30. Sanz-Medel A, Montes-Bayon M, Luisa Fernandez Sanchez M: Trace element speciation by ICP-MS in large biomolecules and its potential for proteomics. Anal Bioanal Chem 2003, 377(2):236-247.

31. Bettmer J, Montes Bayon M, Encinar JR, Fernandez Sanchez ML, Fernandez de la Campa Mdel R, Sanz Medel A: The emerging role of ICP-MS in proteomic analysis. J Proteomics 2009, 72(6):989-1005. 32. Szpunar J: Advances in analytical methodology for bioinorganic speciation analysis: metallomics, metalloproteomics and heteroatom-tagged proteomics and metabolomics. Analyst 2005, 130(4):442-465. 33. Tastet L, Schaumloffel D, Bouyssiere B, Lobinski R: Capillary HPLC-ICP MS mapping of selenocompounds in spots obtained from the 2-D gel electrophoresis of the water- soluble protein fraction of selenized yeast. Anal Bioanal Chem 2006, 385(5):948-953. 34. Chan Q, Caruso JA: A metallomics approach discovers selenium-containing proteins in selenium-enriched soybean. Anal Bioanal Chem 2012, 403(5):1311-1321.

35. Im JS, Ki SH, Farina A, Jung DS, Hurwitz J, Lee JK: Assembly of the Cdc45-Mcm2-7-GINS complex in human cells requires the Ctf4/And-1, RecQL4, and Mcm10 proteins. Proc Natl Acad Sci U S A 2009, 106(37):15628-15632. 36. Ge W, Hu H, Ding K, Sun L, Zheng S: Protein interaction analysis of ST14 domains and their point and deletion mutants. J Biol Chem 2006, 281(11):7406-7412. 37. Kamphaus GD, Colorado PC, Panka DJ, Hopfer H, Ramchandran R, Torre A, Maeshima Y, Mier JW, Sukhatme VP, Kalluri R: Canstatin, a novel matrix-derived inhibitor of angiogenesis and tumor growth. J Biol Chem 2000, 275(2):1209-1215.

Matched Matched Parent Parent Parent Accession Score Sequence Protein Name charge Mass of S- Mass of Se Number Peptide Peptide

3 16.8 (R)VETGVLKPGMVVTFAPVNVTTEVK(S) 2515.3 2563.3 P68104 Elongation factor 1-alpha 3 16.51 (K)EKIRLDEAGVTDEVLDSAMQAFLLEIIVK(H) 3245.7 3293.7 O60507 Protein-tyrosine sulfotransferase 3 15.88 (K)FRLDASDKPLKVGLGMYSK(E) 2125.1 2173.1 Q9UFH2 Dynein heavy chain 3 15.83 (R)NTEELDMAGVQSLVPR(L) 1758.8 1806.8 Q68DX3 FERM and PDZ domain-containing protein 3 15.62 (K)LHLMFSLMDKVPNGIEPMLK(D) 2313.2 2361.2 Q93034 Cullin-5 3 14.16 (K)LEFAQDAHGQPDVSAFDFTSMMRAESSAR(V) 3201.4 3249.4 Q8TDZ2 NEDD9-interacting protein 3 14.1 (K)GGDIMLWNFGIKDKPTFIKGIGAGGSITGLK(F) 3191.7 3239.7 Q92466 DNA damage-binding protein 3 13.49 (K)EENLICSECGDEFTLQSQLAVHMEEHR(Q) 3261.4 3309.4 Q9UL36 Zinc finger protein 3 13.36 (K)EYSSEHQAADMAEIDARLKALQEYMNR(L) 3169.4 3217.4 Q9Y6R9 Coiled-coil domain-containing protein 3 13.27 (K)ELMGCQCCEEKPSIMVSNLHKEYDDK(K) 3200.3 3248.3 Q8WWZ7 ATP-binding cassette sub-family A member 3 13.15 (R)VHEKPFVAFGSGMESLVEATVGERVRIPAK(Y) 3240.7 3288.7 P35968 Vascular endothelial growth factor receptor 3 13.13 (K)GYLGAKGIQGMPGIPGLSGIPGLPGRPGHIKGVK(G) 3279.8 3327.8 P08572 Collagen alpha-2(IV) chain 3 13.05 (R)RQASSMPTGTTMGSPASPPGPNSKTGR(V) 2658.2 2706.2 Q9Y2E6 Protein deltex-4 3 12.96 (R)GVELMRFTTPGFPDSPYPAHARCQWALR(G) 3260.5 3308.5 Q9Y5Y6 Suppressor of tumorigenicity 14 protein 3 12.91 (R)VANIARTNATNNMNLSRSSSDNNTNTLGR(N) 3106.5 3154.5 Q9ULT8 E3 ubiquitin-protein ligase HECTD1 3 12.9 (K)LMSCPLAGLISKDAINLKAEALLPTQEPLK(A) 3234.7 3282.7 Q9NR48 Probable histone-lysine N-methyltransferase 3 12.73 (R)QGRVISVIAVSIGFLASVTGAMITSAAVAGIYR(V) 3278.8 3326.8 Q5T4T1 Transmembrane protein 3 12.63 (R)HGLPIPGSTPTPMVGSGRLGAPVGRSGGGASAR(S) 3054.6 3102.6 Q96S07 Proline-rich protein 3 12.45 (K)EMEEFVQSSGENGVVVFSLGSMVSNTSEER(A) 3264.4 3312.4 P06133 UDP-glucuronosyltransferase 3 12.44 (K)RCLTLMDRGFIFNLINDYISGFSPK(D) 2977.5 3025.5 Q5JSL3 Dedicator of cytokinesis protein 3 12.43 (R)YGGNLSLQSAMSVRFNSNGTQLLALRR(R) 2953.5 3001.5 Q96JK2 DDB1- and CUL4-associated factor 3 12.42 (K)SYLAWIGFSAEGTPCYVDSEGIVRMLNR(G) 3191.5 3239.5 O75717 WD repeat and HMG-box DNA-binding protein

3 12.4 (K)NKVNFIPTGSAFCPVKLLGPLLPASDLMLK(N) 3240.7 3288.7 Q86VQ1 Glucocorticoid-induced transcript 1 protein 3 12.23 (K)KAFATCSSHVAVVGLFYGAGIFTYMRPK(S) 3078.5 3126.5 Q8NG77 Olfactory receptor 2T12 OS=Homo sapiens 3 11.05 (R)GIVARLVQKCLPPEIIMEYGEEVLEEIK(N) 3255.7 3303.7 P51956 Serine/threonine-protein kinase 3 12.22 (R)SRYGESYLDQILNGMDEELGSLEELEK(K) 3117.4 3165.4 P16157 Ankyrin-1 OS=Homo sapiens 3 12.1 (K)KLPFIYGNLSQGMVSEPLEDVDPYYYKK(K) 3293.6 3341.6 Q01118 Sodium channel protein type 7 subunit alpha 3 11.98 (R)WSSYQNQTDNSVSNDPLLMTQYFK(K) 2866.2 2914.2 Q6PJP8 DNA cross-link repair 1A protein NACHT, LRR and PYD domains-containing 3 11.95 (R)YTRLLLVKEHSNPMQVQQQLLDTGR(G) 2967.5 3015.5 P59046 protein 3 11.71 (R)LGIPMSVLMGANIASEVADEKFCETTIGCK(D) 3241.5 3289.5 P21695 Glycerol-3-phosphate dehydrogenase [NAD+] 2 11.59 (R)MNNGDVDLTSDR(Y) 1336.5 1384.5 Q12860 Contactin-1 3 11.44 (K)AGMNIARLNFSHGSHEYHAESIANVR(E) 2881.3 2929.3 P30613 Pyruvate kinase isozymes R/L 3 11.38 (R)LTFPDREALAEHADLKSMVELIK(R) 2626.3 2674.3 Q8NE63 Homeodomain-interacting protein kinase 3 11.29 (K)TKQFAPIHAEAPEFMEMSVEQEILVTGIK(V) 3273.6 3321.6 P06576 ATP synthase subunit beta, mitochondrial 3 11.23 (K)QKDSLLQAPMHIDRNILMLILPLILLNK(C) 3253.8 3301.8 Q5PT55 Sodium/bile acid cotransporter

Table 1 - parent ion charge, score, peptides identified, accession number and protein name*

* The observed MS/MS spectra show the m/z of the peptides to be 1/3 of the peptide masses shown above because of the +3 charge, except for Contactin-1, which has a charge of +2.

Chapter 4

Separation of Peptides by HPLC using a Surface Confined Ionic Liquid Stationary Phase

4.1 Abstract:

A butylimidazolium bromide surface confined ionic liquid stationary phase (SCIL) was synthesized in-house. The synthesized phase was investigated for the separation of 5 peptides

(Gly-Tyr, Val-Tyr-Val, leucine enkephalin, methionine enkephalin and angiotensin-II). The peptides were successfully separated in less than five minutes. The effect of trifluoroacetic acid

(TFA) on the separation of peptides was evaluated with results confirming that TFA was not acting as ion pairing agent in separation of peptides on this phase.

4.2 Introduction:

Peptides have significant roles in biochemical function, physiological processes, molecular biology, clinical research and in the identification of proteins in proteomics [1,2].

These important applications have led to the increased momentum for techniques that effectively separate them. The technique of choice for the separation of peptides and proteins is high performance liquid chromatography (HPLC) [3,4].

The challenges associated with peptide separations are due to nature of peptides themselves such as their diverse chemical nature (e.g., hydrophobic and hydrophilic sites, acidic and basic groups, aromatic rings) and variability in size, shape and charge. Typically, they are present as complex mixtures. Thus, the separation of peptides often requires the application of several strategies and chromatographies in their separation.

In HPLC, different chromatographic modes such as reverse phase [5,6,7,8], ion exchange

[9,10], size exclusion [11,12], hydrophilic interaction [13,14] and affinity chromatography

[15,16] are predominantly used for successful separation of peptides. Depending upon the complexity of peptide mixture, a series of chromatographic techniques are sometimes employed for their effective separations.

The method of choice for over two decades in separation of peptides is reversed-phase high performance liquid chromatography (RP-HPLC), where stationary phases made of hydrocarbon ligands (e.g., C-4, C-8, C-18, phenyl phases). Recently, multimodal macrocyclic antibiotic phases and porphyrin based phases have demonstrated utility for peptide separations [2,17]. In RP-HPLC different gradient elution schemes with polar and non-polar solvents are used for the separation of peptides and the primary mode of interaction of 86

peptides with these phases is purely hydrophobic; hence, more hydrophobic peptides are more retained on the hydrophobic phases. In RP-HPLC of peptides, ion-pairing agents are commonly used as mobile phase additives.

Ion pairing agents are selected depending upon the charge of the peptides of interest to be separated. The homologous series of volatile perfluorinated acids-trifluoroacetic acid (TFA), pentafluoropropionic acid (PFPA), heptafluorobutyric acid (HFBA) - proved to be excellent ion- pairing agents for RP-HPLC of peptides [18,19,20,21]. These ion pairing agents affect the peptide retention behavior during RP-HPLC by interaction of the ions with oppositely charged functional groups on the peptides (e.g., basic side-chains Lys, Arg, His; free terminal α-amino group interacting with ion pairing agent anion), causing a net reduction in the hydrophilicity of peptides; the alkyl chains of the ion pairing ions impart hydrophobic character to the ion pair resulting in increased hydrophobic interaction with the reverse phase sorbent.

Of all the perfluorinated acids, TFA is the most extensively used mobile phase additive due to its UV transparency, excellent solubilizing properties, volatility, readily availability, and high purity [22]. Generally, the concentration of acidic agents employed is 0.05% - 0.1% (v/v) with increasing concentration causing longer retention of peptides. However, concentrations of these ion-pairing agents must be limited because of the vulnerability of silica based bonded phases to acid hydrolysis.

Molten salts, also known as ionic liquids, are defined as liquids which are composed solely of ions [23,24]. Ionic liquids are typically composed of large asymmetrical organic cations coupled to inorganic anions [25,26]. Room temperature ionic liquids (RTILs) are molten salts with melting points at or below approximately 100 oC. RTILs have a diverse range of 87

applications [23,25] (e.g., solvents in organic synthesis [27,28,29], matrices in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry [30,31], additives or coatings in capillary electrophoresis [32,33,34] and stationary phases in gas chromatography

[35,36,37,38,39,40]). However, they do have limited applicability in high performance liquid chromatography (HPLC) as mobile phase additives [41]. Because of the relatively high viscosities of RTILs, their concentrations in mobile phases must be kept relatively low (1-10mM)

[42]. Immobilization of RTIL motifs onto a silica substrate does not technically constitute an ionic liquid; nevertheless, it has been shown that these phases may retain some ionic liquid-like properties (e.g., partitioning behavior) [43] and affords some unique selectivities as stationary phases in HPLC lead to further applications of RTILs in separation science [44].

Currently, a number of research groups are utilizing similar methods to covalently attach various RTIL cations, primarily alkylimidazolium, with a variety of associated anions (e.g., chloride, bromide, iodide, tetrafluoroborate) to silica sorbents, forming surface-confined ionic liquid (SCIL) stationary phases for HPLC [43,45,46,47,48,49,50,51]. Changing the identity of cation and anion combination on the surface of silica during the synthesis leads to the development of numerous new SCIL phases and the multiple retention modalities (i.e., hydrophobic, electrostatic, hydrogen bonding) in which these new bonded phases can participate have shown utility in the separation of different classes of compounds

[43,45,48,51]. The reports published by various groups have primarily focused on the characterization of surface-confined ionic liquid (SCIL) phases in terms of physical characteristics [25,47,48] and their ability to separate aromatic carboxylic acids [25], ephedrines [45], inorganic ions [47,48], nucleotides [43] and geometric isomers [51]. 88

The retention mechanism on these SCILs relies on complex intermolecular interactions which are dependent upon the kind of analytes used and solvent system employed. Indeed, it was shown that despite the presence of a positively charged imidazolium ring, these phases had a phenyl type reverse-phase chromatographic mechanism for neutral aromatics. However, published results on use of SCILs [43] for the separation of nucleotides have shown that these phases behave like strong anion exchange stationary phases; further making these phases an interesting point of study. As both modes have proven useful in the separation of peptides

[6,10], our work here is focused on assessing the utility of this SCIL phase in the separation of peptides.

4.3 Experimental:

4.3.1 Materials:

All reagents used in the synthesis of the stationary phase; hydrogen hexachloroplatinic(IV) acid hydrate, 8-bromo-1-octene, trichlorosilane, chlorotrimethylsilane, trichlorooctylsilane, 1-butyl imidazole, 2,6-lutidine, anhydrous toluene; were purchased from the Sigma–Aldrich Chemical Company (St. Louis, MO, USA). The silica sorbent used is spherical

5µm, 300 Å pore Symmetry® silica provided by the Waters Corporation (Milford, MA, USA).

The peptides used in the study glycine-tyrosine (Gly-Tyr), valine-tyrosine-valine (Val-Tyr-

Val), leucine enkephalin, methionine enkephalin and angiotensin-II were obtained from Sigma.

Mobile phase components (HPLC-grade water and acetonitrile) were supplied by Sigma.

Trifluoroacetic acid (TFA), sodium hydroxide and nitromethane was obtained from Fisher

Chemical Company (Fair Lawn, NJ, USA). Formic acid (FA) was obtained from Sigma. 89

4.3.2 Methods:

4.3.2.1 Stationary phase synthesis:

Stationary phase synthesis has been reported previously [46]. Briefly, the butyl imidazolium bromide-modified silica phase was prepared by hydrosilylation of the alkenylbromide followed by immobilization of the trichlorosilane ligand on to the surface of the silica substrate. The phase was then endcapped with chlorotrimethylsilane and the butyl- imidazole was subsequently attached. An octylsilane-modified stationary phase [C8] was also prepared on the same lot of silica. In the case of the SCIL phase, elemental analysis (Galbraith

2 Laboratories Inc., Knoxville, TN, USA) revealed that loading of the linker was ~3.68 µmol/m , the

2 loading of the endcapping agent was ~0.66 µmol/m and the loading of the butylimidazolium

2 2 cation was ~1.79 µmol/m . The loading for the octyl phase was found to be 3.4 µmol/m . The butylimidazolium modified silica was packed into a stainless steel HPLC column

(150mm×4.6mm ID; Waters Corporation, Milford, MA, USA).

4.3.2.2 HPLC analysis:

All HPLC studies were carried at room temperature, using a Shimadzu (Columbia, MD,

USA) LC-10AT solvent pump and a Shimadzu (Columbia, MD, USA) SPD-10A UV detector set at

214nm. Chromatographic retention data was recorded with Shimadzu Class-VP server. Sample introduction was accomplished with a Shimadzu SIL-10AF automatic sample injector fitted with a 20-µL loop. All experiments were performed in triplicate with flow rates of 1mL/min with mobile phases composed of mixtures of acetonitrile and water (v/v). TFA or FA is added to both the water and acetonitrile in equal concentrations. All the peptides were prepared in concentration range of 0.004-0.5 gL-1 in HPLC-grade water, and were refrigerated when not in 90

use. Because of the difficulty in identifying a solute which does not interact with this multimodal stationary phase, the void volume of the column was determined by measuring the weight difference of the column when filled with either dichloromethane or hexane [46]. The void volume of the column was 1.82 mL.

4.3.2.3 Capillary Electrophoresis Analysis:

All CE studies were carried, using a P/ACE MDQ Capillary Electrophoresis System

(Beckman Coulter, Fullerton, CA) controlled by 32 Karat Software (v. 8.0). Detection was carried out using UV/Vis at 214nm. Fused-silica capillaries (75 µm i.d.) purchased from

Polymicro Technologies Inc. (Phoenix, AZ) was used. The capillary was conditioned prior to initial use using NaOH (1 M) for 10 minutes at 10 psi, and followed by rinsing with deionized (DI) water for 10 minutes at 10 psi. All experiments were performed in triplicate by using background electrolytes composed of 10% acetonitrile and water (v/v) with varying TFA percentages (0.001% - 0.06% v/v). Nitromethane (1% v/v) was used as the electroosmotic flow marker. All injections were performed hydrodynamically (0.5 psi for 3 seconds). The capillary was rinsed with DI water for 10 minutes at a pressure of 10 psi between each run.

4.4 Results and Discussion:

A schematic for the phase that was used for the study is shown in Figure 1. Previous work done in our group has reported cation loading ranging from 0.7 µmol/m2 to 1.0 µmol/m2

(% linkerModified= 20% to 30%) on a 100 Å pore silica [46,51]. Interestingly, cation loading on the

2 300 Å pore silica used in this work is 1.8 µmol/m (% linkerModified = 49%) which is significantly higher than reported previously. This may be attributed to steric effects at the mouth of the 91

smaller pores and is consistent with increased loading of bulky pendant groups on larger pore silica reported previously [52].

Si O

- Si Br

O OH + N N

Figure 1: SCIL stationary phase

Successful separation of five peptides under a time scale of less than five minutes was obtained using 0.005%TFA with 10%ACN under isocratic conditions as can be seen in Figure 2a.

From the chromatogram, it can be seen that, surprisingly, three peptides out of the five eluted before the void volume (1.82 mL) while the other two eluted after the void volume. To elucidate the mechanism responsible, it is convenient to first examine the retention behavior of the solutes eluting after the void volume.

4.4.1 Post-void volume eluting peptides:

4.4.1.1 Effect of organic modifier on enkephalin retention:

The retention of enkephalins is plotted as elution volume vs %ACN in 0.001% TFA shown in Figure 3. As can be seen in the figure, increasing %ACN corresponds to a decrease in the elution volume which is consistent with general reverse phased behavior of peptides on hydrophobic phases (e.g., C-4, C-8, C-18) [53,54,55]. However, further increase in acetonitrile concentration causes further decreases in elution volumes; at acetonitrile concentrations above

40%, the enkephalins elute before the void volume. This behavior is in stark contrast to the

behavior of peptides on conventional alkyl phases where a minimum retention is observed at some intermediate organic modifier concentration [2,53,56,57]. This behavior is also in contrast when compared with the behavior of basic peptides on a weak anion exchange phases [58] where increasing acetonitrile concentrations causes the increase of elution times. Similar behavior of peptides i.e., decreased elution volumes with increased acetonitrile concentrations were observed even at higher concentrations of TFA.

Figure 2: Chromatogram obtained of peptide mixture using 10% ACN a) with 0.005% TFA on SCIL phase (elution order is angiotensin-II, Val- Tyr-Val, Gly-Tyr, leucine enkephalin, and 94

methionine enkephalin), b) with 0.005% FA on SCIL phase (elution order same as in a), and c) with 0.005%TFA on C8 phase (elution order is Gly-Tyr, Val-Tyr-Val, methionine enkephalin, and leucine enkephaline). Angiotensin-II did not elute.

10 9 Leu-Enkephalin 8 Met-Enkephalin

5 Elution Volume 4 3 2 1 0 0% 20% 40% 60% 80% 100% %ACN

Figure 3: A plot of the elution volume obtained on SCIL phase for the two enkephalins vs %ACN in 0.001%TFA.

4.4.1.2 Effect of TFA:

In conventional reversed phase chromatography of peptides using TFA as an ion pairing agent, increased concentration of TFA leads to increased retention [59]. A plot of log k for the enkephalins vs %TFA is shown in Figure 4. As can be seen in the figure, increasing TFA concentration produces decreased retention for these two peptides. Interestingly, these results suggest that TFA is not acting as ion pairing agent for these solutes on this SCIL phase under reverse phase conditions. To further understand the role of TFA in separation of these peptides, the TFA was replaced with formic acid. Again, a successful separation of five peptides under a time scale of less than five minutes was observed in 0.005% formic acid with 10%ACN under

isocratic conditions shown in Figure 2b. As can be seen in figure, virtually identical separation achieved using formic acid as a substitute for TFA is supporting evidence that TFA is not acting as ion pairing agent in this separation. Further, the similarities of the chromatograms using FA and TFA suggest that the mobile phase anion does not play a significant role in the separation.

0.8

0.5 Leu-enkephalin

0.2 Met-enkephalin

-0.1 log k log -0.4

-0.7

-1 0.000 0.003 0.006 0.009 0.012

%TFA

Figure 4: A plot of the log k for the two enkephalins vs %TFA in 10% ACN.

To further understanding the role of stationary phase in the separation of these peptides, an alkyl phase (C-8 phase, 300 Å, 5µm) was subjected to the best separation conditions (0.005%

TFA, 10% ACN) found on the SCIL phase. Separation of peptides on alkyl phase is shown in

Figure 2c. While all of the peptides eluted after the void volume on the C8 phase, they demonstrated a much wider range of elution volumes from the C8 phase than from the SCIL phase. The most significant differences in the elution order between the two phases are that while the angiotensin is the first to elute on the SCIL phase, it failed to elute after 200 minutes

from the C8 phase under these mobile phase conditions. In addition, the elution order of the two enkephalins from the C8 phase was reversed relative to their elution order from the SCIL phase. Hence, the retention properties of the two phases are very different.

4.4.2 Pre-void volume eluting peptides:

To elucidate the mechanism responsible for the solutes eluting before the void volume, it is helpful to examine the peptide structures. The first peptide eluting is angiotensin, which is the largest peptide followed by the tri- and dipeptide. While this elution order is consistent with size exclusion chromatography (SEC), these analytes are too small for a size exclusion mechanism. Further examination of the peptide structures reveals the presence of three amines in angiotensin-II whereas the di- and tripeptides contain only one. The concentration of

TFA that is used for the study (0.001% - 0.1% v/v) is sufficient to protonate the three amine- containing angiotensin-II residues (Asp, Arg, His).

The stationary phase used here has an imidazolium ring that is positively charged and could promote Donnan exclusion as in ion exclusion chromatography [60,61] where similar charged species to the phase are repelled while oppositely charged and neutral species are retained. This kind of chromatography is predominately used in the separation of ions [61].

The higher charge on angiotensin-II (+3) charged relative to other peptides, causes an increased exclusion of this peptide from the pores of the stationary phase. A plot of elution volume of angiotensin-II vs %TFA is shown in Figure 5. As can be seen from the plot, increasing TFA concentration increases the elution volume which may be attributed to masking of positive charges of the peptide and stationary phase by TFA anions.

The next two eluting peptides are Val-Tyr-Val (tripeptide) and Gly-Tyr (dipeptide). Plots of the elution volumes of the dipeptide and tripeptide vs %TFA are also shown in Figure 5. As can be seen in Figure 5, these two peptides’ behavior with changing TFA concentrations is similar, further indicating the same mechanism that is involved for elution of both peptides before the void volume. At very low TFA concentrations, the elution volumes are high; at intermediate TFA concentrations, the elution volume decreases but at higher concentrations the elution volume increases again.

2.5 Val-Tyr-Val Gly-Tyr 2 Angiotensin

1.5

Elution Elution Volume

0.5

0 0.000 0.025 0.050 0.075 0.100 0.125

% TFA

Figure 5: A plot of the elution volume obtained on SCIL phase of angiotensin, Val-Tyr-Val, and Gly-Tyr vs %TFA in 10% ACN

At low concentrations of TFA, the elution behavior of the di- and tripeptide may be attributed to neutralization of the carboxylate resulting from addition of the acidic TFA modifier. A plot of the electrophoretic mobility of the tripeptide in 10% ACN vs %TFA is shown in Figure 6. In the absence of TFA, the peptide exists as the neutral zwitterion. Addition of small amounts of TFA begins to neutralize the carboxylate and the peptide becomes more

positively charged. As can be seen in the figure, the most dramatic change in electrophoretic mobility occurs in the same region of TFA concentrations as in the chromatographic

2.5

04)

- (E

1.5

µ 1

0.5

0 0.000 0.020 0.040 0.060 0.080

%TFA

Figure 6: A plot of electrophoretic mobility of Val-Tyr-Val vs % TFA in 10% ACN

experiments in which the elution volume is decreasing (Figure 5). As the TFA concentration is further increased in the electrophoresis experiment, the electrophoretic mobility starts to decrease which may be attributed to peptide:TFA anion association. In the chromatographic case, the elution volume also begins to increase at higher concentrations of TFA; the increased elution volume of the two peptides may be attributed to the masking of the positive charges of the peptide and stationary phase by TFA anions.

Plots of elution volumes of tripeptide and dipeptide vs %FA is shown in Figure 7. As can be seen in the figure, substitution of FA for TFA shows the same impact on elution volumes at low concentrations. However, in contrast to the TFA case, further increases in FA concentration

do not lead to increased elution volume. This is likely because FA is a much weaker acid than

TFA; hence, the low concentrations of its conjugate base are ineffective at counteracting the

Donnan exclusion. Nevertheless, better separation of the two peptides is obtained in the presence of FA than in the presence of TFA across a range of acid concentrations.

2.5 Val-Tyr-Val Gly-Tyr

1.5

Elution Volume

0.5

0 0.000 0.025 0.050 0.075 0.100 0.125

%FA

Figure 7: A plot of the elution volume obtained on SCIL phase of Val- Tyr-Val and Gly-Tyr vs %FA in 10% ACN

4.5 Conclusions:

The present study assesses the role of a SCIL stationary phase in the separation of peptides. A successful separation of peptides using this phase is seen under isocratic conditions. Despite the presence of covalently attached cations in the stationary phase, reversed-phase behavior is observed. TFA does not seem to be playing the role of ion- pairing agent on this phase for these solutes; hence, FA may be substituted for TFA in these systems.

The results obtained on the SCIL phase demonstrate that while the SCIL phase exhibits some

100

reversed-phase character, electrostatic interactions dominate at high organic and/or low pH modifier concentrations.

4.6 Acknowledgements

The authors gratefully acknowledge support from the Waters Corporation and the

National Institutes of Health (R01 GM 067991–02A2).

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4.7 References

1. Shi Y, Xiang R, Horváth C, Wilkins JA (2004) J Chromatogr A 1053:27-36. 2. Zhang B, Soukup R, Armstrong DW (2004) J Chromatogr A 1053:89-99. 3. Kwok SK,Wilson GS, Rabel SR, Stobaugh JF, Williams TD, Veldes DGV, Schöneich C (1993) Anal Chem 65:67R-84R. 4. Larive CK, Lunte SM, Zhong M, Perkins D, Wilson GS, Gokulrangan G, Williams T, Afroz F, Schoneich C, Derrick TS, Middaugh CR, B-Knipp S (1999) Anal Chem 71:389R-423R. 5. Hansen B, SØrensen HH, Welinder BS (1986) J Chromatogr 361:357-367. 6. Boyes BE, Walker DG (1995) J Chromatogr A 691:337-347. 7. Purcell AW, Zhao GL, Aguilar MI, Hearn MTW (1999) J Chromatogr A 852:43-57. 8. Chan KC, Blonder J, Ye X, Veenstra TD, Issaq HJ (2009) J Chromatogr A 1216:1825-1837. 9. St.Pierre S, Rioux F, Simic MG, Dizdaroglu M (1982) J Chromatogr A 245:158-162. 10. Mumford C, Streater M, Brandt-Nielsen A, Pathirana ND, Badger SE, Levison PR (1997) J Chromatogr A 760:151-158. 11. Irvine GB (1987) J Chromatogr 404:215-222. 12. Barth HG, Boyes BE, Jackson C (1998) Anal Chem 70(12):251-278. 13. Alpert AJ (1990) J Chromatogr 499:177–196. 14. Javendra P (2008) J Sep Sci 31:1421-1437. 15. Xiao-Chuan L (2006) Chin J Chromatogr 24(1):73-80. 16. Raftery MJ (2008) Anal Chem 80(9):3334-3341. 17. Charvátová J, Kašička V, Barth T, Deyl Z, Mikšík I, Král V (2003) J Chromatogr A 1009:73- 80. 18. Horvath C, Melander W, Molnar I, Molnar P (1977) Anal Chem 49(14):2295-2305. 19. Guo D, Mant CT, Hodges RS (1987) J Chromatogr A 386:205-222. 20. Shibue M, Mant CT, Hodges RS (2005) J Chromatogr A 1080:58-67. 21. Mant CT, Hodges RS (2006) J Chromatogr A 1125:211-219. 22. McCroskey MC, Pearson JD (1996) J Chromatogr A 746:277-281. 23. Forsyth SA, Pringle JM, MacFarlane DR (2004) Aust J Chem 57:113-119. 24. Bader GA, Baker SN, Pandey S, Bright FV (2005) Analyst 130:800-808. 25. Wang Q, Baker GA, Baker SN, Colόn LA (2006) Analyst 131:1000-1005. 26. Anderson JL, Armstrong DW, Wei GT (2006) Anal Chem 78(9):2893-2902. 27. Adams CJ, Earle MJ, Roberts G, Seddon KR (1998) Chem Commun 19:2097–2098. 28. Welton T (1999) Chem Rev 99:2071–2083. 29. Ranu BC, Saha A, Saha D (2009) Green Chem 11:733-737. 30. Armstrong DW, Zhang LK, He L, Ross ML (2001) Anal Chem 73:3679–3686. 31. Carda-Broch S, Berthod A, Armstrong DW (2003) Rapid Commun Mass Spec 17:553–560. 32. Yanes EG, Gratz SR, Baldwin MJ, Robison SE, Stalcup AM (2001) Anal Chem 73:3838– 3844. 33. Vaher M, Koel M, Kaljurand M (2002) J Chromatogr A 979:27–32.

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34. Cabovska B, Kreishman GP, Wassell DF, Stalcup AM (2003) J Chromatogr A 1007:179– 187. 35. Poole CF, Furton KG, Kersten BR (1986) J Chromatogr 24:400–409. 36. Arancibia EL, Castells RC, Nardillo AM (1987) J Chromatogr 398:21–29. 37. Furton KG, Poole CF (1987) J Chromatogr 399:47–67. 38. Anderson JL, Ding J, Welton T, Armstrong DW (2002) J Am Chem Soc 124:14247–14254. 39. Armstrong DW, He L, Liu YS (1999) Anal Chem 71:3873–3876. 40. Anderson JL, Armstrong DW (2003) Anal Chem 75:4851–4858. 41. He L, Zhang W, Zhao L, Liu X, Jiang S (2003) J Chromatogr A 1007:39-45. 42. Polyakova Y, Row KH (2006) Acta Chromatogr 17:210-221. 43. Van Meter DS, Sun Y, Parker KM, Stalcup AM (2008) Anal Bioanal Chem 390:897–905. 44. Valkenberg MH, deCastro C, Hölderich WF (2002) Green Chem 4:88–93. 45. Liu SJ, Zhou F, Xiao XA, Zhao L, Liu X, Jiang SX (2004) Chin Chem Let 15(9):1060–1062. 46. Sun Y, Cabovska B, Evans CE, Ridgway TH, Stalcup AM (2005) Anal Bioanal Chem 382:728–734. 47. Qiu H, Jiang S, Liu X (2006) J Chromatogr A 1103:256–270. 48. Qiu H, Jiang S, Liu X, Zhao L (2006) J Chromatogr A 1116:46–50. 49. Qiu H, Jiang Q, Wei Z, Wang X, Liu X, Jiang S (2007) J Chromatogr A 1163:63–69. 50. Van Meter DS, Stuart OD, Carle AB, Stalcup AM (2008) J Chromatogr A 1191:67–71. 51. Van Meter DS, Oliver NJ, Carle AB, Sabine D, Ridgway TH, Stalcup AM (2009) Anal Bioanal Chem 393:283-294. 52. Stalcup AM, Chang SC, Pitha J, Armstrong DW (1990) J Chromatogr 513:181-194. 53. Grego B, Hearn MTW (1981) J Chromatogr 218:497-507. 54. Grego B, Hearn MTW (1981) J Chromatogr 14(10):589-592. 55. Purcell AW, Zhao GL, Anguilar MI, Hearn MTW (1999) J Chromatogr 852:43-57 56. Blanquet RS, Bui KH, Armstrong DW (1986) J Liq Chromatogr 9:1933-49. 57. Simpson RJ, Mortiz RL (1989) J Chromatogr 474:418-423. 58. Alpert AJ (2008) Anal Chem 80:62-76. 59. Chen Y, Mehok AR, Mant CT, Hodges RS (2004) J Chromatogr A 1043:9-18. 60. Wheaton RM, Bauman WC (1953) Ind Eng Chem 45(1):228-233. 61. Haddad PR, Novič M (2006) J Chromatogr A 1118:19-28.

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Chapter 5

Preliminary work with Human Keratinocytes, Conclusions and Future work

104

5.1 Preliminary work with Human Keratinocytes (KC)

Further studies were done to evaluate the effects of As-induced cytotoxicity and SeMet detoxification by classical biochemistry using KC primary cultures. These primary cells more representative arsenic cytotoxicity targets than are the HEK 293 cells. Keratinocytes were treated with zero (control) or 5 M As for 24 h and the cellular morphological changes visualized by microscopy (Figure 1A). The control group showed cells with characteristic morphology presenting single nuclei representing cells in either the G1 or S phase of the cell cycle. The group treated with 5 M As, however, showed clear signs of cellular distress with more than 20% of cells displaying round shape, indicative of loss of substrate attachment and decreased viability. Additionally, we found that ~50% of cells showed two nuclei (Figure 1B).

The high percentage of cells presenting two nuclei indicates cell cycle arrest in the G2 phase after DNA synthesis. This G2 arrest suggests that after DNA synthesis many errors were encountered. These findings are consistent with As-induced mutagenesis.

The guardian of the genome, protein p53, is a known inducer of G2 arrest. We, therefore, examined the expression of p53 by Western blotting 48h after As-incubation and found a strong accumulation of p53 (Figure 1C) further supporting our observations.

Furthermore, in alignment with the increase in p53, we also observed elevated expression of its downstream target p21, which sustains G2 arrest. We also verified increased expression of critical factors in cellular antioxidant responses in KCs treated with As. The antioxidant transcription factor Nrf-2 and the antioxidant enzymes glutathione S transferase (GST) and catalase (CAT) were also observed to be elevated by Western blot analysis. Indeed, KC responds

105

to As-treatment by increasing the levels of anti-oxidants and displaying elevated percentage of cells in G2 arrest. Therefore, suggesting that mutated DNA has been synthesized and p53 is preventing mitosis until DNA repair is complete. Loss of p53 function through oncogenesis will lead to a higher mutation rate and likely a larger incidence of cancer.

Figure 1: Cytotoxic effects of As. (A) KCs were treated with increasing doses of 1, 5 and 10 μ t for 24h. On the left side of the panel are representative images of control and cells treated with As. The red arrows point to cells displaying signs of severe stress suggestive of apoptosis. On the right side of the panel cells were treated with As in the presence of SeMet. The images show cells with less signs of stress. (B) KCs were treated with increasing doses of As for 24h and cell viability determined by MTT 106

assay. The lethal dose of As that causes 50% of cell death (LD50) was calculated as 4.77 μmol/ml using the GraphPad Prism software. (C) Expression of p53, its downstream target p21; Nrf-2 and its downstream target glutathione S transferase (a phase II antioxidant enzyme) and catalase the major hydrogen peroxide detoxifying enzyme, in cells exposed to 1 μmol/ml As.

5.2 Conclusions and future work

This dissertation demonstrates the use of metallomic approaches for better understanding of the SeMet antagonism on arsenic. The coupling of element selective ICPMS with HPLC that can be operated in various modes of separation; along with the LC-MS/MS based proteomics, were used to further inform antagonistic studies.

SeMet when inoculated along with As(III) resulted in reduced cytotoxicity in human embryonic cells, HEK293. Similarly it was found that with the As(III) in comparison with the

As(III) + SeMet, the amount of ROS was reduced with the latter mix as determined by DCF fluorescence. These studies reveal marked differences in protein phosphorylation among HEK control cells, As(III) toxified cells and As(III)/SeMet toxified cells. In the As(III)/SeMet toxic mix, many more proteins related to anti-oxidation, anti-tumor activity and cell growth are seen vs. the As(III) toxified or general control cells. This study helps to further understand the mechanism through the cytotoxicity reduction and production/ up-regulation of key signaling proteins due to the presence of SeMet.

In the present study the arsenic and SeMet antagonism are attributed due to the decreased levels of ROS, phosphorylation changes and incorporation of selenium in to the proteins due to the SeMet. Furthermore mechanism of actions of selenomethionine in

107

mitigating the toxicity of As(III) could be possible owing form the fact of unknown mechanisms of arsenic toxicity.

The next steps will be to progress to more specific arsenic target cells; namely primary keratinocytes and include other Se, As and ROS species along with Identifying the expression levels of various proteins in cells treated with As and SeMet together.

108

APPENDIX

109

Figure 1a: A figure showing the total ion chromatogram (-casein peptide) representing intensity (y-axis) vs time (x-axis). Loading and peptide trapping was performed at a flow rate of

4 μL/min using 0.6% acetic acid (HOAc ) and 2% formic acid (FA) in water. Solvents used for the nanoflow experiment were 0.6% HOAc/0.5% FA (solvent A) and 0.6% HOAc/0.5% FA/80% acetonitrile (ACN) (solvent B). Samples were loaded on-to the enrichment column at a flow rate of 3 µL min-1 with a 97:3 ratio of solvent A (0.1% FA (v/v) in water) and B (90% ACN, 0.1%

FA (v/v) in water). After the enrichment column was loaded, the on-chip microfluidics switched to the analytical column at a flow rate of 0.3 µL min-1. Gradient: 0–2 min, 2% B; 5–50 min,

40%B; 50–60 min, 70% B; 60–70 min, 90% B; 70–90 min, 0% B.

FQsEEQQQTEDELQDK

110

Figure 1b: A figure of an extracted ion chromatogram for the -casein peptide containing intensity (y-axis) vs time (x-axis).

Figure 1c: A figure showing the mass spectra of -casein peptide (m/z-1093.7) at the red diamond.

Figure 1d: A figure showing the MS/MS of the m/z-1093.7.

111

Matched Data Base Parent Accession Score Sequence parent Entry name charge number mass Spectrum (R)DMPIQAFLLYQEPVL 2 19.74 2186.16 P02666 Beta-casein Mill GPVR(G) (R)DMPIQAFLLYQEPVL Mascot 2 90.84 186.86 P02666 Beta-casein GPVR(G)

Table 1: Shows the identification of Beta-casein using Spectrum Mill data base

112

Variable UniprotKB No Charge Score Peptide Total Sequence Modifications Matched Accession Entry name Sites Reported Intensity Intensity parent mass number found in Variable the study Sites 1 2 16.67 86.8 4980063 (R)FQVWD 2097.976 O43598 Deoxyribonucleoside 5'- NR S : 12, 28, YEEGEVEA monophosphate N- 169 LLDR(Y) glycosidase

2 3 11.01 81.8 1319656 (K)DQFPEV 2773.324 P61586 Transforming protein NR S : 34, 37, 3 YVPTVFEN RhoA 156 YVADIEVD GK(Q) 3 3 10.18 79.9 8300444 (K)TLRVLN C:Carbamidomethylation 2879.585 A4D1F6 Leucine-rich repeat and T : 293, NR LEYNQLttF t:Phosphorylated T death domain- 294 PKALCFLP containing protein K(L) 4 3 10.07 88.7 6941975 (R)YLLQYQ C:Carbamidomethylation 2694.352 P25789 Proteasome subunit NR S : 13, 75 EPIPCEQL alpha type-4 VTALCDIK( Q) 5 3 9.9 68.4 4812443 (R)ssNYEIIT s:Phosphorylated S 2531.249 O95551 Tyrosyl-DNA S : 195, NR GHEEGYFT phosphodiesterase 2 196 AIMLKK(S) 6 3 9.1 78.5 3489902 (R)EQPAPN t:Phosphorylated 2613.364 Q9P2P6 StAR-related lipid S : 3219 NR HRGsLPVtT T
s:Phosphorylated S transfer protein 9 T : 3223 IFSGPKHSR (S) 7 3 8.81 64.9 5251199 (K)ALNTtDs t:Phosphorylated 2902.403 P49116 Nuclear receptor T : 323 S: S : 19, 46, SSSPSLADG T
s:Phosphorylated S subfamily 2 group C 325 55, 68, 219 IDTSGGGSI member 2 T : 224 HVISR(D) 8 3 7.87 64.1 8633731 (R)sPIPIRV s:Phosphorylated S 2807.625 Q9H329 Band 4.1-like protein S : 650 NR ETAQPAVE 4B KPEIKPPR VR(K) 9 3 7.82 82.4 5297382 (K)MCLYF C:Carbamidomethylation 2699.42 P15954 Cytochrome c oxidase S : 47 NR GsAFATPFL s:Phosphorylated S subunit 7C, VVRHQLL mitochondrial KT(-)

113

10 3 7.67 88 3363724 (R)IAAQDL 2786.468 Q96P70 Importin-9 NR NR LLAVATDF QNESAAAL AAAATR(H) 11 3 7.4 60.4 6291673 (K)ASKEPV C:Carbamidomethylation 2735.389 Q86XD8 AN1-type zinc finger S : 568 NR GCVNNIsFL t:Phosphorylated and ubiquitin domain- T : 577 ASLAGStSR T
s:Phosphorylated S containing protein NR(L) 12 3 7.26 83.9 5962927 (R)LQAFFts t:Phosphorylated 2869.477 Q9UBC3 DNA (cytosine-5)- S : 552 T NR DTGLEYEA T
s:Phosphorylated S methyltransferase : 551 PKLYPAIP AAR(R) 13 3 7.25 100 2641592 (R)IDKAPSF s:Phosphorylated S 2232.13 Q8NHP7 Exonuclease 3'-5' S : 370 NR TsQDFHGD domain-containing VNLLK(E) protein 1 14 3 7.11 91.2 1798411 (K)RISDSEV C:Carbamidomethylation 3087.334 Q9UQ26 Regulating synaptic S : 973 S : 366, 400, sDYDCDDG s:Phosphorylated S membrane exocytosis 427, 429, IGVVSDYR protein 2 1030, 1038 HDGR(D) T : 973 15 3 7.01 85.8 5178341 (R)LLGAYL t:Phosphorylated T 2962.564 P36894 Bone morphogenetic T : 42 S : 215, 216, FIISRVQGQ protein receptor type- 218, 220 NLDSMLH 1A GtGMK(S) 16 3 6.84 62 3161782 (R)GSYTEK s:Phosphorylated S 3193.675 Q6P2M8 Calcium/calmodulin- S : 114 NR DAsHLVGQ dependent protein VLGAVSYL kinase type 1B HSLGIVHR( D) 17 3 6.75 93.3 1712349 (R)NLSFDS 2554.22 Q9NW13 RNA-binding protein NR S : 122, 202 EEEELGEL 28 LQQFGELK (Y) 18 3 6.63 62.1 6217155 (K)RLVLTH 2155.261 Q9H777 Zinc phosphodiesterase NR NR FSQRYKPV ELAC protein 1 ALAR(E) 19 3 6.62 90.1 4703006 (R)TDERLN y:Phosphorylated Y 2286.09 O15537 Retinoschisin Y : 165 NR WIyYKDQT GNNR(V)

114

20 3 6.5 100 2165855 (K)LsQLKs s:Phosphorylated S 2282.262 Q12830 Nucleosome-remodeling S : 788, S : 216, 572, QQVAAAA factor subunit BPTF 792 763, 817, HEANKLFK 1231, 1300, (E) 1827, 1833, 2098, 2465 T : 909, 1064 Y : 839 21 3 6.41 81.4 1761384 (K)QTEFIIt C:Carbamidomethylation 3006.55 P49815 Tuberin S : 41, 49 S : 939, 981, AEILRELs t:Phosphorylated 1097, 1132, MECGLNN T
s:Phosphorylated S 1155, 1334, RIR(M) 1337, 1338, 1341, 1364, 1387, 1388, 1411, 1418, 1420, 1449, 1452, 1462, 1798 T : 927

22 3 6.27 91 2842742 (K)LPSSVF s:Phosphorylated S 2816.447 Q96GA3 Protein LTV1 homolog S : 116 S : 182, 188, AsEFEEDV 244, 331 GLLNKAAP VSGPR(L)

23 3 6.19 87.6 5197425 (K)RVEIMA C:Carbamidomethylation 2699.244 Q9NR09 Baculoviral IAP repeat- S : 4781 S : 473, 480 QCEEWIAD s:Phosphorylated S containing protein 6 IQQYsSDK( R)

24 3 6.06 64.5 2327496 (R)GGDVAS C:Carbamidomethylation 2847.222 P03952 Plasma kallikrein T : 52 NR MYTPNAQ t:Phosphorylated T YCQMRCtF HPR(C)

Table 2: Proteins identified in fraction 1 of cells grown together with As(III) and SeMet, fraction isolated based on Size Exclusion

Chromatography (SEC).

115

Variable No Charge Score Peptide Total Sequence Modifications Matched Accession Protein name Sites UniprotKB Intensity Intensity parent mass number found in Reported the study Variable Sites 1 2 19.79 97.1 6288674 (R)IQEIIEQL 2038.02 P10809 60 kDa heat shock NR S : 70 DVTTSEYEK protein, (E) mitochondrial 2 2 15.54 88.7 6790895 (K)ATAGDT 1675.73 P34931 Heat shock 70 kDa NR NR HLGGEDFD protein NR(L)

3 2 14.5 100 1E+07 (R)VEIIAND 1228.62 P11021 78 kDa glucose- NR T : 166, Y : 466, QGNR(I) regulated protein S : 571 4 2 11.62 79.7 1.5E+07 (K)DCGATW C:Carbamidomethylatio 1586.73 P60174 Triosephosphate NR S : 21, 80 VVLGHSER( n isomerase R) 5 2 11.5 100 9763194 (K)DSYVGD 1198.52 P62736 Actin, aortic smooth NR S : 325 EAQSK(R) muscle

6 3 11.38 85.3 1.2E+07 (R)CTEPEDQ C:Carbamidomethylatio 2486.22 Q9H3G5 Probable serine Y : 328, NR LyyVKFLSLP n y:Phosphorylated Y carboxypeptidase 329 EVR(Q) CPVL 7 2 10.8 80.1 9369689 (K)TAFQEAL 1336.63 P10599 Thioredoxin NR T : 100 DAAGDK(L) 8 2 9.98 81.4 2552994 (K)GHYTEG 1958.98 Q13885 Tubulin beta-2A NR S : 78, 95 AELVDSVLD chain VVR(K) 9 3 9.84 95.4 3433608 (K)AEASSGD 2089.89 Q02790 Peptidyl-prolyl cis- NR T : 143, S : 451, HPTDTEMK trans isomerase 453 EEQK(S) FKBP4 10 3 9.84 74.2 2.2E+07 (R)ASDTLSA y:Phosphorylated Y 1879.99 Q9H254 Spectrin beta chain Y : 2178 T : 2111, 2112 S: EVRTRVGyV 2121 R(Q) 11 3 8.58 64.7 9424084 MSHLQsLLL t:Phosphorylated 2704.5 Q8NHR9 Profilin-4 S : 6, T : NR DtLLGTKHV T
s:Phosphorylated 11 DSAALIK(I) S

116

12 2 8.51 83 1.8E+07 (K)GDGPVQ 1501.76 P00441 Superoxide NR S : 99 GIINFEQK(E dismutase ) 13 3 8.31 90 1.4E+07 (R)DVVCED C:Carbamidomethylatio 2951.44 O43663 Protein regulator of S : 211 S : 195, 265, 501, EDAFCLsLE n s:Phosphorylated S cytokinesis 513, 571, 592, NIATLQKLL 615 T: 470, 481, R(Q) 616 14 3 8 100 2270809 (K)AGAAPY 2351.22 Q01518 Adenylyl cyclase- NR S : 290, 295, VQAFDSLLA associated protein 301, 308, 310 T : GPVAEYLK( 164, 307 I) 15 2 7.91 84.1 6794591 (R)SVGDGET 1795.82 P16989 DNA-binding NR S : 2, 34, 38, 79, VEFDVVEGE protein A 134, 201, 203, K(G) 287, 369, 370 T : 287 16 3 7.8 75.7 8236621 (K)MAKSFTS 2421.3 O00203 AP-3 complex NR S : 276, 609, 661 EDDLVKLQI subunit beta-1 LNLGAK(L) 17 2 7.69 91.8 3069351 (R)HPEIKVT 1022.59 Q96JM2 Zinc finger protein NR S : 1076, 1079, AK(Y) 462 1082, 2024 Y : 2083

18 2 7.52 83.3 6119478 (R)DADDAV 1309.59 Q08170 Serine/arginine-rich NR S : 113, 222, 255, YELNGK(D) splicing factor 4 300, 302, 304, 316, 322, 448, 458, 460, 462 Y : 53 T : 440 19 3 7.45 72.5 2476994 (R)DLLPSGS C:Carbamidomethylatio 3183.48 Q5JRA6 Melanoma S : 1900 S : 727, 1727, RDEPPPASQ n s:Phosphorylated S inhibitory activity 1739, 1740, 1892, STSQDCsQA protein 3 1906 T : 382 LKQSP(-) 20 3 7.4 68.2 2319204 (R)SLDAQSV y:Phosphorylated Y 2817.38 Q9H488 GDP-fucose protein Y : 307 NR yVATDSESY O- VPELQQLFK fucosyltransferase 1 (G) 21 2 7.1 83.8 5427945 (R)DPENFPF 1475.75 P51149 Ras-related protein NR S : 72 Y : 183 VVLGNK(I) Rab-7a 22 2 7.05 69.1 1.3E+09 (K)SSNVLLD t:Phosphorylated T 1428.76 O43187 Interleukin-1 T : 348 T : 368 QNLtPK(L) receptor-associated kinase-like 2

117

23 2 7 67.3 6831116 (K)EKtIsVSS t:Phosphorylated 1717.96 Q8N3X1 Formin-binding S : 285 T S : 18, 116, 172, SKSGPVIAK( T
s:Phosphorylated protein 4 : 283 432, 435, 442, R) S 499, 508, 963, 964, 965 T : 516, 517 Y : 113 24 3 6.92 85 4142276 (K)FTEWAY C:Carbamidomethylatio 2878.27 Q8NER1 Transient receptor S : 386 T S : 117, 502, 775, GPVHSSLYD n t:Phosphorylated potential cation : 390 801, 821 T : 145, LsCIDtCEK( T
s:Phosphorylated channel subfamily V 371, 705 N) S member 25 3 6.75 77.6 6825687 (K)STPSAPW t:Phosphorylated 2324.07 Q8WXI7 Mucin-16 NR ITEMMNsVS T
s:Phosphorylated EDtIK(E) S 26 3 6.73 84.7 7615616 (K)SEFPIRT C:Carbamidomethylatio 2057.15 Q14008 Cytoskeleton- T : 1762, S : 816 LKtLLHtLC n t:Phosphorylated T associated protein 1766 K(L) 27 2 6.73 93.6 9457260 (K)KVEEAEP 1661.82 Q13185 Chromobox protein NR S : 93, 95, 97, 99, EEFVVEK(V) homolog 176 28 2 6.72 75.2 1.1E+07 (R)yATALYs y:Phosphorylated 1515.78 P48047 ATP synthase S : 47 Y : NR AASKQNK(L Y
s:Phosphorylated subunit O, 41 ) S mitochondrial 29 3 6.67 67.4 3E+07 (K)LNTPSSL C:Carbamidomethylatio 2817.45 Q6N022 Teneurin-4 NR NR AVCADGEL n YVADLGNIR IR(F) 30 3 6.6 88 7855747 (MGKAENyE y:Phosphorylated Y 2584.2 P20648 Potassium- Y : 7 S : 954 LYSVELGPG transporting PGGDMAAK ATPase alpha chain (M) 31 2 6.5 100 4018747 (K)EKsEFVD t:Phosphorylated 1439.7 Q9Y6V0 Protein piccolo S : 1428 S : 2895 DITtR(R) T
s:Phosphorylated T : 1436 S 32 2 6.45 97.4 2492273 (K)HGDPGD 1323.59 O14737 Programmed cell NR S : 119 AAQQEAK(H death protein ) 33 2 6.23 73.8 3691102 (K)TENNDHI 1197.58 P61956 Small ubiquitin- NR NR NLK(V) related modifier 34 3 6.22 73 1.2E+07 (R)CQGsGDD C:Carbamidomethylatio 2108.96 P04150 Glucocorticoid S : 370 T S : 45, 134, 203, NLTSLGtLN n t:Phosphorylated receptor : 380 211, 226, 234, FPGR(T) T
s:Phosphorylated 267 T : 8 S

118

35 3 6.22 74.4 2327828 (K)LFFHTEY C:Carbamidomethylatio 2924.37 P17025 Zinc finger protein T : 199 Y NR EKtNPGMKP n t:Phosphorylated 182 : 208 YGyKECGK( T
y:Phosphorylated G) Y 36 3 6.21 73 1.8E+07 (K)TTAPFKI t:Phosphorylated 2357.35 Q8IYT8 Serine/threonine- S : 617 T NR PKtQASsNLL T
s:Phosphorylated protein kinase : 613 ALVTR(H) S ULK2 37 3 6.13 93.7 9979191 (K)AVGsLDP s:Phosphorylated S 2062.09 O14522 Receptor-type S : 529 NR SADLSSQRG tyrosine-protein KVFK(L) phosphatase 38 3 6.09 89.9 3329637 (R)DMQPLSP y:Phosphorylated 2458.2 P28749 Retinoblastoma-like S : 649 Y S : 640, 650, 749, ISVHERysSP Y
s:Phosphorylated protein : 648 762, 964, 975, TAGSAK(R) S 988, 1009 T : 332, 369, 385, 997

Table 3: Proteins identified in fraction 2 of cells grown together with As(III) and SeMet, fraction isolated based on Size Exclusion

Chromatography (SEC).

119

No Variable No Charge Score Peptide Total Sequence Modifications Matched Accession Entry name Sites UniprotKB Reported Intensity Intensity parent mass number found in Variable Sites the study 1 2 11.29 95.1 8.79E+06 (K)DVNFEF 1384.642 P62081 40S ribosomal NR NR PEFQL(-) protein S7 2 3 10.25 67.7 1.29E+08 (R)QSQPPSI t:Phosphorylated 1803.935 P82987 ADAMTS-like S : 1189 NR sFNKtINSR( T
s:Phosphoryl protein 3 T : 1193 I) ated S 3 3 8.21 67.6 1.84E+08 (R)yANLGN y:Phosphorylated Y 2237.276 O76090 Bestrophin-1 Y : 131 NR VLILRSVST AVYKR(F) 4 3 7.85 68.5 4.58E+08 (K)WQVSR s:Phosphorylated S 2158.1 Q9BWD1 Acetyl-CoA S : 185 NR EDQDKVA acetyltransferase, VLsQNR(T) cytosolic 5 3 7.21 84.6 5.67E+07 (R)HMSVAL t:Phosphorylated T 2229.108 P55345 Protein arginine N- T : 414 NR SWAVtSRQ methyltransferase 2 DPTSQK(V) 6 3 7.15 75.2 1.38E+08 (K)RsSSMA C:Carbamidomethy 2144.97 Q9ULV0 Myosin-Vb S : 1642 S : 1446 Y : 694 DGDNSyCL lation Y : 1652 EAIIR(Q) y:Phosphorylated Y
s:Phosphoryl ated S 7 3 7.09 64.8 2.06E+07 (R)WVDPNS C:Carbamidomethy 2236.168 P42330 Aldo-keto reductase NR NR PVLLEDPV lation family 1 member C3 LCALAK(K) 8 3 6.96 66.4 4.46E+07 (R)LTCEEE C:Carbamidomethy 1957.874 P51532 Transcription NR S : 609, 610, 613, 655, EEKMFGR lation activator BRG1 657, 660, 662, 695, GSR(H) 699, 721, 1382, 1452, 1570, 1575, 1586, 1627, 1631, 1640, 1642, 1644 T: 11, 609

120

9 3 6.82 60.8 1.64E+07 (K)SsPGtQD t:Phosphorylated 2175.042 Q5SW79 Centrosomal S : 313 T T : 377, 501, 628, 937, LLGIQTGM T
s:Phosphoryl protein of 170 kDa : 316 1533 S : 168, 354, MAPENK(V ated S 356, 359, 446, 502, ) 580, 630, 633, 636, 667, 838, 879, 930, 958, 1079, 1112, 1160, 1165, 1198, 1251, 1260, 1270, 1521, 1522, 1529, 1533 10 3 6.72 85.9 9.33E+07 (R)CGPRAN C:Carbamidomethy 2318.051 Q96N53 Putative NR NR GEEASSCA lation uncharacterized WVSRAPR( protein encoded by A) NCRNA00167 11 3 6.64 67.5 1.27E+08 (R)STESVK s:Phosphorylated S 2242.181 Q7Z6B7 SLIT-ROBO Rho S : 421 S : 906, 917, 999, STVSETYLs GTPase-activating 1005, 1008, 1010, KPSIAK(R) protein 1 1029, 1032 T : 1001, 1004 12 3 6.63 78.8 9.63E+06 (R)GGVSEG 892.419 Q9HCK5 Protein argonaute-4 NR S : 798 QMK(Q) 13 3 6.62 71.6 6.97E+06 (K)ETITKM t:Phosphorylated T 2149.205 Q5XG92 Carboxylesterase T : 383, NR LWStRtLLN 4A 385 ITK(E) 14 3 6.59 65.9 1.83E+08 (K)AKPMC C:Carbamidomethy 2299.11 Q9NPC7 Myoneurin T : 306, S S : 289 NtCGKVFS lation : 316 EASsLRR(H t:Phosphorylated ) T
s:Phosphoryl ated S 15 3 6.58 67.9 1.67E+08 (R)DIHsGDF s:Phosphorylated S 2162.967 P78332 RNA-binding S : 122 S : 360, 362, 891, RDREGPPM protein 6 1022, 1025 T : 923 Y DYR(G) : 914 16 2 6.58 87.4 2.59E+06 (R)IGDQEF 1984.99 P46108 Adapter molecule NR S : 41, 74 T : 42 Y : DSLPALLE crk 136, 221, 239, 251 FYK(I) 17 3 6.55 62.3 1.20E+08 (K)LRNRSS t:Phosphorylated T 2156.121 Q08828 Adenylate cyclase T : 554, NR FStNVVYTT type 1 563 PGtR(V)

121

18 3 6.43 60.2 1.29E+08 (R)GPPEGG 1964.008 P00519 Tyrosine-protein NR S : 50, 446, 569, 618, LNEDERLL kinase ABL1 619, 620, 659, 683, PKDK(K) 718, 805, 809, 855, 917, 919, 936, 949, 977 T : 392, 394, 613, 735, 781, 814, 844, 852 Y : 70, 185, 226, 253, 257, 264, 393, 469 19 3 6.24 73.2 1.69E+08 (R)AQNCNI C:Carbamidomethy 2229.202 Q9Y4D1 Disheveled- S : 701 NR LLsRLKLS lation associated activator NDEIK(R) s:Phosphorylated S of morphogenesis 1 20 3 6.2 73.7 1.56E+07 (K)VLySQG y:Phosphorylated 2400.271 A6NKT7 RanBP2-like and S : 1075 NR VKLFRFDA Y
s:Phosphoryl GRIP domain- Y : 1061 EVsQWK(E) ated S containing protein 3 21 3 6.05 72.5 1.24E+08 (K)ELHVVY C:Carbamidomethy 1882.923 P20936 Ras GTPase- S : 742 Y : 615 ALsHVCGQ lation activating protein 1 DR(T) s:Phosphorylated S 22 3 6.05 77.2 7.75E+07 (K)WDSEsN s:Phosphorylated S 1881.857 P30414 NK-tumor S : 889 S: 347, 379, 406, 408, SERDVTKN recognition protein 410, 463, 572, 887, SK(N) 889, 891, 1146 T : 343

23 3 6.04 70.4 1.84E+07 (K)HGLSYE s:Phosphorylated S 2485.418 Q9Y5Q0 Fatty acid S : 430 NR VKPFLTAL desaturase 3 VDIVRsLK( K) 24 3 6.03 61.2 2.74E+08 (R)SSGLSSR 2325.08 Q9NT68 Teneurin-2 NR S : 523 Y : 1150 ENSALTLT DSDNENK(S )

Table 4: Proteins identified in fraction 3 of cells grown together with As(III) and SeMet, fraction isolated based on Size Exclusion

Chromatography (SEC).

122

Variable

Accession Sites found UniprotKB Peptide Total Matched No Charge Score Sequence Modifications number Entry name in the Reported Intensity Intensity parent mass study Variable Sites (R)GVKSSPIQ Tctex1 domain- 1 3 8.61 63.2 1.07E+07 TPNQTPQQA 2459.336 Q8IZS6 containing NR NR

PVTPRK(E) protein 3 (K)tFLQREHQ C:Carbamidomethylati 2 3 8.47 69.2 1.76E+07 PFSLQCEAVY 2381.171 P08648 Integrin alpha-5 T : 944 NR on t:Phosphorylated T K(A) Potassium/sodium (R)QFGALLQ hyperpolarization 3 3 8.42 63.4 2.68E+06 PGVNKFSLR 2453.312 Q9UL51 -activated cyclic NR S : 146, 779

MFGSQK(A) nucleotide-gated channel 2 Serine/threonine- C:Carbamidomethylati (K)stLKIPAD protein on t:Phosphorylated S : 634 T : 4 3 8.22 63.6 2.47E+07 KHLLQQLEM 2380.284 Q6NUP7 phosphatase 4 NR T
s:Phosphorylate 635 CVR(K) regulatory d S subunit 4 Kynurenine/alpha (K)FLYTVPN -aminoadipate 5 3 8.04 66 1.34E+07 GNNPTGNSL 2309.152 Q8N5Z0 NR NR aminotransferase, TSERK(K) mitochondrial (R)GPSLLQTP Putative TBC1 6 3 8.01 62.6 4.21E+06 PRVPGQQAL 2302.263 Q9UFV1 domain family NR NR

SRGDK(G) member 29 (R)FGLQGSA Pantothenate 7 3 7.73 69.8 8.87E+06 VASsFGNMM s:Phosphorylated S 2232.09 Q8TE04 S: 496 NR kinase 1 SKEKR(D)

123

S : 139, 150, 162, 274, 280, 284, 306, 318, 350, 353, 553, 849, 850, 912, 1089, C:Carbamidomethylati 1090, 1096, (K)QNESRTT on t:Phosphorylated Nipped-B-like S : 666 T : 1150, 1152, 8 3 7.72 62.9 4.18E+07 ECKQNEstIV 2378.125 Q6KC79 T
s:Phosphorylate protein 667 1154, 1160, EPK(Q) d S 1459, 2509, 2511, 2513, 2658, 2672 T : 558, 646, 746, 914, 1458, 2667 Y : 1159 (K)LSEETKV E3 UFM1-protein 9 3 7.52 74.4 3.00E+06 ALTKLHNsLN s:Phosphorylated S 2154.177 O94874 S : 629 S : 458 ligase 1 EK(S) (R)VFEGPYK 10 3 7.26 63.4 9.31E+06 EYHEEAQKW 2311.078 Q9C0C4 Semaphorin-4C NR S : 832

DR(Y) C:Carbamidomethylati (K)yLGCKQD on y:Phosphorylated Zinc finger Y : 478 S : 11 3 7.05 69.6 4.59E+06 NsSSPKPSSVF 2157.039 Q7Z4V0 NR Y
s:Phosphorylate protein 438 486 R(N) d S S : 297, 374, 471, Histone-lysine N- 596, 775, 882, (R)RIVFTITtG methyltransferase 826, 834, 899, 12 3 6.94 61.3 1.49E+07 AGSAKQSPSS t:Phosphorylated T 2036.113 Q8TEK3 T : 1051 , H3 lysine-79 902, 1001, 1009, K(H) specific 1104, 1246, 1349 T : 480, 900 (K)ELHVVYA Ras GTPase- C:Carbamidomethylati 13 3 6.9 60.9 3.27E+06 LsHVCGQDR( 1882.923 P20936 activating S : 742 Y : 615 on s:Phosphorylated S T) protein1 S : 245, 301, 304, (R)SENGSGsT t:Phosphorylated Smoothelin S : 701 T: 341, 357, 503, 14 3 6.82 62 4.38E+06 MMQtKTFSSS T
s:Phosphorylate 2384.07 P53814 OS=Homo 706 514, 576, 729 T : SSSKK(M) d S sapiens 351, 360 (R)SVLDLVN Ubiquitin 15 3 6.61 65.3 3.93E+07 YFLSPEKLTA 2308.218 Q9P2H5 carboxyl-terminal NR S : 613

ENR(Y) hydrolase 35

124

(K)MEGTGK TNFAIP3- S : 99, 403 T : 16 3 6.51 81.1 1.86E+06 KAVAGQQQA s:Phosphorylated S 2047.06 Q15025 interacting S : 284 438 sVTAGK(V) protein 1 Potassium (R)DTNMIPGs voltage-gated 17 3 6.38 62.1 5.02E+06 PGSTELEGGF s:Phosphorylated S 2464.189 Q12809 channel S : 871 S : 320 SRQRK(R) subfamily H member 2 (R)NPAVLSA ASFDGRISVY Protein transport S : 527, 532, 799, 18 3 6.38 61 1.44E+07 t:Phosphorylated T 2841.42 O94979 T : 338 SIMGGStDGL protein Sec31A 1163 T : 1161 R(Q) (R)LMHKLSV IST1 homolog 19 3 6.28 62.3 5.48E+06 EAPPKILVER 1960.141 P53990 OS=Homo NR Y : 43

(Y) sapiens (R)TREEWTS Probable G- 20 3 6.19 73.7 5.09E+06 AQVPRGGEs s:Phosphorylated S 2046 Q6PRD1 protein coupled S : 1616 NR QK(D) receptor 179 (R)DPLMIFVL S : 338, 4264 T : 21 3 6.11 60.4 4.64E+06 NEKVDEFSG 2321.221 Q9NZJ4 Sacsin NR 4263 Y : 4333 VLR(V)

Table 5: Proteins identified in fraction 4 of cells grown together with As(III) and SeMet, fraction isolated based on Size Exclusion

Chromatography (SEC).

125

Peptide Total Matched Accession No Charge Score Sequence Modifications Entry name Intensity Intensity parent mass number

(K)LAYVAAGDLAPI Ubiquitin-like modifier-activating 1 3 18.76 90.2 6.61E+07 NAFIGGLAAQEVM 2603.39 P22314 enzyme 1 K(A) (R)LGFSEVELVQM Creatine kinase B-type OS=Homo 2 2 18.39 97 1.47E+07 1848.978 P12277 VVDGVK(L) sapiens (R)NDFQLIGIQDGY Eukaryotic translation initiation factor 3 3 16.45 88.6 7.81E+06 2580.294 P63241 LSLLQDSGEVR(E) 5A-1 (R)YVEPIEDVPCGN 4 3 15.21 84.1 4.67E+07 IVGLVGVDQFLVK( C:Carbamidomethylation 2759.432 P13639 Elongation factor 2 OS=Homo sapiens T) (K)DGPLNMILDDG 5 3 13.47 88 2.80E+07 2252.123 P23526 Adenosylhomocysteinase GDLTNLIHTK(Y) (K)QIVWNGPVGVF 6 2 13.42 80.7 2.63E+07 2105.06 P00558 Phosphoglycerate kinase 1 EWEAFAR(G) (R)DLFEDELVPLFE Heterogeneous nuclear 7 2 11.83 93.5 6.01E+06 1593.805 O60506 K(A) ribonucleoprotein Q (R)CLQILAAGLFLP 8 3 11.15 93.3 8.43E+06 GSVGITDPCESGNF C:Carbamidomethylation 2892.438 Q12905 Interleukin enhancer-binding factor 2 R(V) (K)ISFPAIQAAPSFS Tryptophanyl-tRNA synthetase, 9 3 10.83 86.3 1.08E+07 2325.203 P23381 NSFPQIFR(D) cytoplasmic (K)ALINADELASDV 10 2 10.32 83.5 2.39E+06 2127.093 Q13813 Spectrin alpha chain AGAEALLDR(H) (R)AVSTGVQAGIP 6-phosphogluconate dehydrogenase, 11 3 9.77 65.9 8.47E+06 MPCFTTALSFYDG C:Carbamidomethylation 2809.332 P52209 decarboxylating YR(H) (K)ASLVKNGAEFT 12 3 9.55 79.8 8.77E+06 2148.166 Q14562 ATP-dependent RNA helicase DHX8 DSLISNLLR(L) (K)LIIVSNPVDILTY 13 2 9.2 90.3 4.77E+06 1944.121 Q9BYZ2 L-lactate dehydrogenase A-like 6B VAWK(L) (R)DQEGQDVLLFID ATP synthase subunit beta, 14 2 9 100 2.02E+06 1921.965 P06576 NIFR(F) mitochondrial (K)VMIPQDEYPEIN 15 3 8.83 94.6 4.11E+06 2400.263 Q15637 Splicing factor 1 FVGLLIGPR(G) (R)YSLLPFWYTLLY 16 3 8.8 92.4 7.73E+06 2071.08 Q14697 Neutral alpha-glucosidase AB QAHR(E) 126

(R)NDFQLIGIQDGY Eukaryotic translation initiation factor 17 3 8.64 89.4 5.42E+06 2580.294 P63241 LSLLQDSGEVR(E) 5A-1 (K)FTASAGIQVVGD 18 2 8.5 100 3.61E+06 2033.055 P06733 Alpha-enolase DLTVTNPK(R) (R)DQEGQDVLLFID ATP synthase subunit beta, 19 2 8.5 100 2.10E+06 1921.965 P06576 NIFR(F) mitochondrial (K)LISVFyAIVMPLL 20 3 7.97 66.3 7.27E+06 y:Phosphorylated Y 2421.398 O95006 Olfactory receptor 2F2 NPVIySLR(N) (R)ASTAAAPLAAW 21 3 7.91 84.6 4.55E+06 VKANIQYSHVLER(I 2567.373 Q8NCM8 Cytoplasmic dynein 2 heavy chain 1

) (K)RTEDDPEVGGP C:Carbamidomethylation 22 3 7.85 62.8 1.09E+07 CyTLYLEYLGGLVP 3167.597 Q9NRJ4 Tubby-related protein 4 y:Phosphorylated Y ILK(G) (R)sGHVKAFAHITG Trifunctional purine biosynthetic 23 3 7.74 92.2 1.30E+07 s:Phosphorylated S 2174.183 P22102 GGLLENIPR(V) protein adenosine-3 (R)GITQALNLVDsL Zinc finger BED domain-containing 24 3 7.54 66.7 7.02E+06 SLKLETDTLLSAML s:Phosphorylated S 2887.606 P86452 protein 6 K(S) (K)DLVSSLTSGLLT 25 2 7.5 100 7.23E+06 1646.896 P53396 ATP-citrate synthase IGDR(F) (R)AFADAMEVIPST 26 3 7.41 85.2 6.66E+06 LAENAGLNPISTVT 3030.545 P50991 T-complex protein 1 subunit delta

ELR(N) (R)ALESLRQDWQA 27 3 7.3 67.4 9.19E+06 2487.249 Q8NF91 Nesprin-1 OS=Homo sapiens YQHRLSETR(T) (K)HHFGINKPEKII Phosphatidylinositide phosphatase 28 3 7.16 64.9 1.83E+07 s:Phosphorylated S 2159.124 Q9Y2H2 PsPDDsK(F) SAC2 (K)ItEELNLRSLETy t:Phosphorylated Vacuolar protein sorting-associated 29 3 7.12 82.9 6.52E+06 2422.246 Q15906 ERLEADK(K) T
y:Phosphorylated Y protein 72 homolog (R)ASSSGNQESSGQ 30 3 7.12 75.6 1.41E+07 SCIILLFDVIKSAIR( C:Carbamidomethylation 2867.457 Q9BXW9 Fanconi anemia group D2 protein Y) (R)RWGDSVLLVDL 39S ribosomal protein L4, 31 3 6.85 80 6.42E+06 THEEMPQSIVEATS 2968.483 Q9BYD3 mitochondrial R(L) (K)EEMLFEALVTR 32 3 6.69 73.5 1.23E+07 t:Phosphorylated T 2180.163 B2RTY4 Myosin-IXa KTVtVGEK(L) (K)AAFDDAIAELDT 33 2 6.5 100 3.43E+06 2087.965 P62258 14-3-3 protein epsilon LSEESYK(D) (R)AWNDVDALFtT t:Phosphorylated 34 3 6.5 100 4.70E+06 2371.208 Q9H9C1 VPS33B-interacting protein KNWLGyTKK(R) T
y:Phosphorylated Y 127

(K)HKPQAPVIVKTE Glutamate receptor, ionotropic kainate 35 3 6.5 100 5.30E+06 2703.403 Q13002 EVINMHTFNDR(R) 2 (K)LSSCIAAIAALSA C:Carbamidomethylation 36 3 6.41 81.6 1.98E+07 2223.147 Q9HCE3 Zinc finger protein 532 KKAAsDSCK(E) s:Phosphorylated S C:Carbamidomethylation (K)ESKSCtIGMVLR Protein phosphatase Slingshot homolog 37 3 6.32 82.2 3.83E+06 t:Phosphorylated 2111.062 Q8WYL5 LWsDTK(I) 1 T
s:Phosphorylated S (R)CLLAyFLDKGAD C:Carbamidomethylation 6-phosphofructo-2-kinase/fructose-2,6- 38 3 6.32 73.4 3.53E+06 ELPYLRCPLHTIFK( 3153.626 O60825 y:Phosphorylated Y biphosphatase 2 L) (R)YNEDLELEDAIH 39 3 6.3 94.3 4.75E+06 2201.134 P25787 Proteasome subunit alpha type-2 TAILTLK(E) (K)LyPGESIGQTSDI 40 3 6.16 77.4 6.91E+06 SSPELMGVGSLLK y:Phosphorylated Y 2806.454 O95714 E3 ubiquitin-protein ligase HERC2 K(Y) (K)QFLEALLRNSAA Zinc finger and BTB domain-containing 41 3 6.12 92.4 7.59E+06 s:Phosphorylated S 2725.344 Q8NAP8 PSKDDADHHFsR(S) protein 8B (R)AVSTGVQAGIP 6-phosphogluconate dehydrogenase, 42 3 6.1 73 7.24E+06 MPCFTTALSFYDG C:Carbamidomethylation 2809.332 P52209 decarboxylating YR(H)

Table 6: Proteins identified in fraction 1 of cells grown with only As(III), fraction isolated based on Size Exclusion Chromatography

(SEC).

128

Peptide Total Matched Accession No Charge Score Sequence Modifications Entry name Intensity Intensity parent mass number 1 2 19.31 92.7 2.11E+07 (K)VVIGMDVAASEFFR(S) 1540.78 P06733 Alpha-enolase

(R)ETYEVLLSFIQAALGD U5 small nuclear ribonucleoprotein 200 2 2 18.85 96.8 6.33E+06 2150.11 O75643 QPR(D) kDa helicase (K)NMITGTSQADCAVLIV 3 3 16.93 98.2 1.22E+07 C:Carbamidomethylation 2909.44 P68104 Elongation factor 1-alpha 1 AAGVGEFEAGISK(N) (K)ITGMLLEIDNSELLHM 4 3 11.23 87.3 2.16E+08 2740.39 P11940 Polyadenylate-binding protein 1 LESPESLR(S) (K)QYDADLEQILIQWITT 5 3 10.61 77.5 5.42E+06 C:Carbamidomethylation 2394.18 P37802 Transgelin-2 QCR(K) Putative disintegrin and (K)GyNPPYCQPKQGAFGS C:Carbamidomethylation 6 3 10.03 73.6 2.94E+07 3100.46 Q6NVV9 metalloproteinase domain-containing IDDGHLVPPTER(S) y:Phosphorylated Y protein 5 (K)KEESEESDDDMGFGLF 7 2 9.16 92.4 9.41E+06 1949.76 P05386 60S acidic ribosomal protein P1 D(-) (K)GIEGVQVIPLIPGAGEII ATP-binding cassette sub-family D 8 3 9 100 2.85E+06 2542.49 P28288 IADNIIK(F) member 3 (K)LLVSNLDFGVSDADIQ 9 3 8.65 72 7.00E+06 2841.46 Q86V81 THO complex subunit 4 ELFAEFGTLK(K) (R)LYVGNIPFGITEEAMM 10 3 8.58 77.1 1.91E+07 2794.3 P26368 Splicing factor U2AF 65 kDa subunit DFFNAQMR(L) (K)ITGMLLEIDNSELLHM 11 3 8.22 94 2.05E+08 2740.39 P11940 Polyadenylate-binding protein 1 LESPESLR(S) (R)AGTLTVEELGATLTSL 12 3 8.16 81.7 1.67E+07 2513.36 P58107 Epiplakin LAQAQAQAR(A) (K)MssGGEKVVLKPNDVS Rap guanine nucleotide exchange factor 13 3 7.96 74.9 3.43E+06 s:Phosphorylated S 2749.46 Q8WZA2 VFTTLTINGR(L) 4 (R)GsYGDLGGPIITTQVTI Heterogeneous nuclear 14 2 7.89 95.5 2.80E+06 s:Phosphorylated S 1917.03 P61978 PK(D) ribonucleoprotein K (K)MPSFGVSAPGKSMEAS 15 3 7.43 84.3 7.20E+06 2353.14 Q8IVF2 Protein AHNAK2 VDVSELK(A) (R)HQEIWSILESVWITIyQ 16 3 7.42 72.9 1.41E+07 y:Phosphorylated Y 3092.55 Q5T5J6 Transcriptional protein SWT1 NSTDVFQR(L) (K)FFLEEIQLGEELLAQG Mitochondrial import receptor subunit 17 3 7.34 89.6 2.77E+06 2385.19 Q15388 EYEK(G) TOM20 homolog

129

(R)ECGVGVIVTPEQIEEAV 18 3 7.16 91.5 5.55E+06 C:Carbamidomethylation 2483.25 P47897 Glutaminyl-tRNA synthetase EAAINR(H) (R)LMEPIYLVEIQCPEQV 19 3 7 100 2.60E+06 C:Carbamidomethylation 2989.55 P13639 Elongation factor 2 VGGIYGVLNR(K) (R)GsGAGEKPYECADCAK C:Carbamidomethylation 20 3 6.53 74.7 3.99E+06 3093.43 Q96EG3 Zinc finger protein 837 AFGLFsHLVEHR(R) s:Phosphorylated S (R)GLPFQANAQDIINFFAP 21 3 6.5 100 1.20E+07 2456.35 Q12849 G-rich sequence factor 1 LKPVR(I) C:Carbamidomethylation (K)tVTSSAVCVSPDHDIyL t:Phosphorylated Kelch repeat and BTB domain- 22 3 6.44 72.2 6.68E+06 2387.17 Q8WVZ9 AAQPR(K) T
y:Phosphorylated containing protein 7 Y (K)QSVQGAGTsyRNVLQA y:Phosphorylated CASP8 and FADD-like apoptosis 23 3 6.4 81 3.95E+06 2119.13 O15519 AIQK(S) Y
s:Phosphorylated S regulator (K)LTLGLPTPKTDSYLQL 24 3 6.36 80.3 3.54E+06 2267.28 Q6NUN7 Uncharacterized protein C11orf63 HNKK(R) (R)GsQPPDMASALTDRTS 25 3 6.34 80.9 3.89E+06 s:Phosphorylated S 3170.53 Q86UC2 Radial spoke head protein 3 homolog RAPSTYTYTSRPR(A) (K)EGPGHMGRsLDQTSPC C:Carbamidomethylation 26 3 6.17 84.3 1.46E+07 2406.2 Q6XZF7 Dynamin-binding protein PLVLVR(I) s:Phosphorylated S

Table 7: Proteins identified in Fraction 2 of of cells grown with only As(III), fraction isolated based on Size Exclusion

Chromatography (SEC).

130

Peptide Total Matched Accession No Charge Score Sequence Modifications Entry name Intensity Intensity parent mass number (K)SPYLYPLYGLGELPQGF 1 2 17.78 91.9 1.06E+07 2141.11 P31150 Rab GDP dissociation inhibitor alpha AR(L) (R)YGINTTDIFQTVDLWEG 2 2 16.51 90.7 1.91E+07 2100.03 P37802 Transgelin-2 K(N) Alpha-enolase OS=Homo sapiens 3 2 11.5 100 3.41E+06 (K)VVIGMDVAASEFFR(S) 1540.78 P06733 GN=ENO1 PE=1 SV=2 (R)NIQVDEANLLTWQGLI 4 3 11.01 76.9 7.18E+06 2852.45 P68036 Ubiquitin-conjugating enzyme E2 L3 VPDNPPYDK(G) (K)SsANAAGGANSGGGSSG 5 3 10.76 66.1 3.79E+08 s:Phosphorylated S 2651.16 P41225 Transcription factor SOX-3 GASGGGGGTDQDRVK(R) (K)TLVLSNLSYSATEETLQ 6 3 10.3 80.1 8.80E+06 2501.27 P19338 Nucleolin EVFEK(A) (R)KGAAPTPPGKtGPsAAQ t:Phosphorylated 7 3 9.19 60.9 1.11E+07 1791.97 Q13428 Treacle protein AGK(Q) T
s:Phosphorylated S (K)TEFLSFMNTELAAFTK( 8 2 9.09 91.4 3.34E+06 1849.9 P31949 Protein S100-A11 N) (R)sGSPSALRVLAEVQEGR Retinoic acid receptor responder 9 3 9.07 69.5 2.18E+08 s:Phosphorylated S 2465.33 P49788 AWINPK(E) protein 1 (K)AAGSTRQPIRGyVQPAD 10 3 8.83 65.9 1.25E+09 y:Phosphorylated Y 2382.23 O95425 Supervillin TGHTAK(L) (R)LtSLPPKGGTsNGYAKT t:Phosphorylated 11 3 8.8 73 1.93E+08 2377.25 Q9UMD9 Collagen alpha-1(XVII) chain ASLGGGSR(L) T
s:Phosphorylated S C:Carbamidomethylation (R)RELDIGIsAtYCGAHSVP 12 3 8.65 76.1 2.44E+07 t:Phosphorylated 2074.04 Q96NU7 Probable imidazolonepropionase K(G) T
s:Phosphorylated S (R)HPGLAVELELEPALPAE 13 3 8.65 73 3.46E+08 s:Phosphorylated S 2228.19 Q96CD0 F-box/LRR-repeat protein 8 sVTR(V) (R)SSVAAMHWMDGsVVTR 14 3 8.62 65.9 1.34E+09 s:Phosphorylated S 2156.01 P22891 Vitamin K-dependent protein Z EHR(G) C:Carbamidomethylation 15 3 8.3 71.9 5.29E+07 (R)CRVVAQDsHFsIQTIK(E) 1888.97 Q8TDY2 RB1-inducible coiled-coil protein 1 s:Phosphorylated S (R)DVYLSPRDDGYSTKDSY Heterogeneous nuclear 16 3 8.26 65.5 1.40E+09 2311.05 P38159 SSR(D) ribonucleoprotein G (K)MTVCLETEKKSPLSWI 17 3 8 61.7 3.31E+08 C:Carbamidomethylation 2308.16 P53004 Biliverdin reductase A EEK(G) (R)GPPGPQGPPGSPGRAG 18 3 7.98 65.4 1.73E+08 t:Phosphorylated T 2153.09 Q96A84 EMI domain-containing protein 1 AVGtPGER(G)

131

(R)AGAsPGSRLLPGsPSLLL 19 3 7.9 66 2.11E+07 s:Phosphorylated S 2375.32 Q495Z4 Uncharacterized protein C17orf65 PAATWR(T) (R)RQssVLSQASTAGGDHE Uncharacterized protein C15orf62, 20 3 7.87 66.4 7.32E+08 s:Phosphorylated S 2379.09 A8K5M9 EYSNR(E) mitochondrial (R)SLSKSDSDLLTCSPTED C:Carbamidomethylation Ankyrin repeat and SAM domain- 21 3 7.57 63.5 7.61E+07 2458.11 Q92625 ATMGsR(S) s:Phosphorylated S containing protein 1A (K)GSAALGGALALAERSsR 22 3 7.55 69.1 8.38E+06 s:Phosphorylated S 2231.15 Q6NZ67 Mitotic-spindle organizing protein 2B EGSSQR(M) (R)LPsDSAGIPQAGGEAEP 23 3 7.39 60.8 2.68E+08 s:Phosphorylated S 2232.09 Q99592 Zinc finger protein 238 HATAAGK(T) (R)AKGsVsLEQILPAEEEV 24 3 7.39 68 4.77E+06 s:Phosphorylated S 1955.04 Q13115 Dual specificity protein phosphatase 4 R(A) (K)ENsALAKGSPSSQSIPEK 25 3 7.37 63.1 5.93E+07 s:Phosphorylated S 2159.09 Q9H1H9 Kinesin-like protein KIF13A NSK(S) (K)SSSEEVLERDLGMGDQ 26 3 7.32 71.8 5.44E+06 2309.1 Q96T83 Sodium/hydrogen exchanger 7 KVSSR(G) (K)TRSSAGVTGTTGLSAKS t:Phosphorylated 27 3 7.31 64.9 1.06E+08 2379.25 Q7Z5P9 Mucin-19 GtsIPSAGK(T) T
s:Phosphorylated S (K)NtQsNEDLKQEKSELEE t:Phosphorylated 28 3 7.28 62.6 7.79E+07 2149.03 Q08379 Golgin subfamily A member 2 K(L) T
s:Phosphorylated S 29 2 7.25 93.9 4.46E+06 (R)DSLLQDGEFSMDLR(T) 1625.75 P07737 Profilin-1

(K)tPEEDVKEVEVDRSETS 30 3 7.25 66 1.28E+09 t:Phosphorylated T 2378.16 Q99715 Collagen alpha-1(XII) chain TSLK(D) C:Carbamidomethylation (R)sPAPGLCPIyKPPETRPA 31 3 7.18 61.4 2.69E+08 y:Phosphorylated 2079.11 A2VEC9 SCO-spondin K(W) Y
s:Phosphorylated S (K)sIFSKAQVEYLSISEDPK( 32 3 7.09 62.8 4.14E+08 s:Phosphorylated S 2041.05 Q6ZRQ5 Protein MMS22-like K) (R)VsALNsVHCEHVEDEGE C:Carbamidomethylation 33 3 7.03 79.2 1.65E+09 2153.95 Q13085 Acetyl-CoA carboxylase 1 SR(Y) s:Phosphorylated S (K)sDLEtQISSLNEKLANLN t:Phosphorylated Uveal autoantigen with coiled-coil 34 3 7.02 65.8 9.56E+06 2145.12 Q9BZF9 R(K) T
s:Phosphorylated S domains and ankyrin repeats (R)stDAPSQSTGDRKTGSV t:Phosphorylated Trinucleotide repeat-containing gene 6B 35 3 6.97 74.9 1.47E+08 2379.13 Q9UPQ9 GSWGAAR(G) T
s:Phosphorylated S protein (R)LDVPPEGRCASAPARPA 36 3 6.94 73.7 1.20E+08 C:Carbamidomethylation 2456.28 Q9H3T2 Semaphorin-6C LSAPAPR(L) (R)TIPPELQEQLKTVKtLA 37 3 6.94 61.1 1.29E+07 t:Phosphorylated T 2037.2 Q9H0J4 Glutamine-rich protein 2 K(E) (K)SNIDIssGLEDEEPKRPL Membrane-associated 38 3 6.8 68.8 1.72E+08 s:Phosphorylated S 2380.25 Q9BZ71 PRK(Q) phosphatidylinositol transfer protein 3 132

(K)tRVTGDHVDLTTCPLAA C:Carbamidomethylation 39 3 6.79 66.4 9.28E+06 2468.22 Q15828 Cystatin-M GAQQEK(L) t:Phosphorylated T (R)CRRVsEAPsLPVVFIDGH C:Carbamidomethylation Glutaredoxin domain-containing 40 3 6.76 65.4 7.89E+06 2757.41 A8MXD5 YLGGAEK(I) s:Phosphorylated S cysteine-rich protein 1 (K)GEDGEMVKLENLFEAL 41 3 6.67 90 4.28E+06 2150.04 P31944 Caspase-14 NNK(N) (K)HVPGEIGLNYVLMADV 42 3 6.57 63.3 1.19E+08 2311.25 Q13797 Integrin alpha-9 AKKEK(G) (K)FIGFGtDSWVyPNISIPE t:Phosphorylated Cyclic nucleotide-gated cation channel 43 3 6.56 66 7.13E+06 2392.17 Q16281 HGR(L) T
y:Phosphorylated Y alpha-3 (MGKGTVsGLVQVVDAET Putative uncharacterized protein 44 3 6.53 62 4.29E+07 s:Phosphorylated S 1875.98 Q8IVU5 GK(V) C3orf46 C:Carbamidomethylation (R)APtFSYGPDGNGFSLGCs Probable palmitoyltransferase 45 3 6.53 69 1.15E+08 t:Phosphorylated 1961.87 Q5W0Z9 K(N) ZDHHC20 T
s:Phosphorylated S (R)KLLSAEERISQTVEILK( 46 3 6.28 64.4 1.50E+08 1957.13 Q99719 Septin-5 H) (K)sDLEtQISSLNEKLANLN t:Phosphorylated Uveal autoantigen with coiled-coil 47 3 6.27 65.3 4.54E+08 2145.12 Q9BZF9 R(K) T
s:Phosphorylated S domains and ankyrin repeats (R)AHKGSTLSQWSLGNGT Endoplasmic reticulum 48 3 6.26 73.5 1.42E+08 s:Phosphorylated S 2156.11 Q7Z2K6 PVTsK(G) metallopeptidase 1 (R)VAVFGKDYGGyLsTYIL y:Phosphorylated Dipeptidyl aminopeptidase-like protein 49 3 6.21 75.1 3.76E+07 2162.15 P42658 PAK(G) Y
s:Phosphorylated S 6 (K)LTVEEAVRMGIVGPEF 50 3 6.21 63.4 9.28E+07 2118.13 Q15149 Plectin KDK(L) (R)KVHVsTVNPNyAGGEPK y:Phosphorylated 51 3 6.16 66.1 9.65E+07 1953.03 P17483 Homeobox protein Hox-B4 R(S) Y
s:Phosphorylated S (R)VYLEGtCVEWLRRYLE C:Carbamidomethylation HLA class I histocompatibility antigen, 52 3 6.12 68.8 2.08E+07 2385.2 P30455 NGK(E) t:Phosphorylated T A-36 alpha chain (R)ADAtGATGVRLKEGGNI 53 3 6.12 64.7 1.09E+07 t:Phosphorylated T 1871.99 Q96L93 Kinesin-like protein KIF16B NK(S) (R)sLAGMKYHVMANHNSL 54 3 6.09 72.2 2.81E+07 s:Phosphorylated S 2224.17 Q96ME7 Zinc finger protein 512 PILK(A) (R)EtPsEEEQAQKQSGMEQ t:Phosphorylated 55 3 6.04 76.8 2.67E+08 2148.95 Q6ZUT6 Uncharacterized protein C15orf52 GR(L) T
s:Phosphorylated S

133

Table 8: Proteins identified in fraction 3 of of cells grown with only As(III), fraction isolated based on Size Exclusion

Chromatography (SEC).

Peptide Total N No Charge Score Sequence Modifications Accession number Entry name Intensity Intensity 1 2 12.3 96.1 3.41E+06 (K)LPIDVTEGEVISLGLP P26599 Polypyrimidine tract-binding protein 1 FGK(V) 2 2 10 100 7.68E+06 (K)ILLANFLAQTEALMR( P06744 Glucose-6-phosphate isomerase G) 3 2 9.73 94.4 1.21E+07 (R)DSLLQDGEFSMDLR(T P07737 Profilin-1 ) 4 2 8.81 70.6 2.13E+07 (K)EVSVRTGGLADKsSR( s:Phosphorylated S P52732 Kinesin-like protein KIF11 K) 5 3 8.76 65.4 1.58E+06 (R)GKAyLRNAVVVItGAT t:Phosphorylated Q6IAN0 Dehydrogenase/reductase SDR family SGLGK(E) Ts:Phosphorylate d S 10 3 7.87 65.8 8.18E+06 (K)RGITSKVLPLQLENIF Q03001 Dystonin YK(L) 11 3 7.77 64.6 7.53E+06 (R)IQSLEATIEKLLSSEsK( s:Phosphorylated S Q86TI0 TBC1 domain family member 1 L) 12 3 7.71 64.5 9.54E+06 (K)SLRFIDVEFSEPTIILF Q9Y4K1 Absent in melanoma 1 protein ER(E) 13 3 7.59 62.8 7.22E+07 (K)KMEEEGVSVSEMEAT Q96BT3 Centromere protein T GAQGPSR(V) 14 3 7.57 62.6 1.34E+07 (K)NGHANGHLNIGVDIP s:Phosphorylated S Q86VZ5 Phosphatidylcholine:ceramide TPDGSFsIK(I) cholinephosphotransferase 1 15 3 7.56 61.9 1.19E+07 (K)GKFLINLEGGDIREES s:Phosphorylated S Q9NSE4 Isoleucyl-tRNA synthetase, mitochondrial sYK(V) 16 2 7.5 100 1.18E+07 (R)DSLLQDGEFSMDLR(T P07737 Profilin-1 134

) 17 3 7.42 61.5 7.21E+07 (K)GYTGANQSRMAVSKt t:Phosphorylated T Q5VTH9 WD repeat-containing protein 78 VLIPPELK(T) 18 3 7.4 67.2 6.88E+06 (R)YKASLFtEEEAEQYKQ t:Phosphorylated T O75132 Zinc finger BED domain-containing protein 4 DLIR(E) 19 3 7.3 71 4.07E+06 (R)ATNTDLLLAYANLML P49792 E3 SUMO-protein ligase RanBP2 LTLSTR(D) 20 3 7.29 63.6 2.39E+07 (R)QLNVIDNQRTLSQMS Q8WZA2 Rap guanine nucleotide exchange factor 4 HRLEPR(R) 21 3 7.23 60.3 1.47E+07 (K)SQGSEWRyVLVSTVR C:Carbamidomethylat Q9BYK8 Peroxisomal proliferator-activated receptor TCAK(S) ion y:Phosphorylated A-interacting complex 285 kDa protein Y 22 3 7.15 64.6 3.22E+06 (K)sDLEtQISSLNEKLANL t:Phosphorylated Q9BZF9 Uveal autoantigen with coiled-coil domains NR(K) T
s:Phosphorylate and ankyrin repeats d S 23 3 7.05 65.6 4.40E+06 (R)SLSKSDSDLLTCSPTE C:Carbamidomethylat Q92625 Ankyrin repeat and SAM domain-containing DATMGsR(S) ion s:Phosphorylated protein 1A S 24 3 7.05 60.1 3.44E+06 (K)RREYQSPSEEESEPEA Q9Y468 Lethal(3)malignant brain tumor-like protein 1 MEK(Q) 25 3 7.04 67.7 6.12E+06 (K)MsALKQsTSEASVLGE s:Phosphorylated S Q8NCK3 Zinc finger protein 485 R(T) 26 3 6.82 66.2 2.92E+06 (R)tPVNLSAASRPVCLPH C:Carbamidomethylat Q5K4E3 Polyserase-2 PEHYFLPGSR(C) ion t:Phosphorylated T 27 3 6.81 74 4.43E+06 (R)KVRLEPVLPCVAALsS C:Carbamidomethylat Q96P50 Arf-GAP with coiled-coil, ANK repeat and VGtLDR(K) ion t:Phosphorylated PH domain-containing protein 3 T
s:Phosphorylate d S 28 3 6.73 70.9 9.06E+06 (R)StDAPSQsTGDRKTGS t:Phosphorylated Q9UPQ9 Trinucleotide repeat-containing gene 6B VGSWGAAR(G) T
s:Phosphorylate protein d S 29 3 6.61 63 6.14E+07 (K)RGITSKVLPLQLENIF Q03001 Dystonin YK(L) 30 3 6.5 64.6 2.79E+07 (R)LMVSRPFSVSQDGAS Q9Y238 Deleted in lung and esophageal cancer protein QDHR(A) 1 31 3 6.42 60.8 4.99E+06 (K)EVVESCRGKNLFFST C:Carbamidomethylat O60701 UDP-glucose 6-dehydrogenase NIDDAIK(E) ion 32 3 6.28 64.2 2.25E+06 (K)MsQQVAELGREtEEL t:Phosphorylated Q8NF91 Nesprin-1 RQMIK(I) T
s:Phosphorylate d S

135

33 3 6.19 72 3.43E+07 (K)ANGVKPStVHIACTPQ C:Carbamidomethylat Q96FA3 Protein pellino homolog 1 AAKAISNK(D) ion t:Phosphorylated T 34 3 6.06 74 4.98E+06 (R)VSEPSGDSSAAGQPLG Q96HA7 Tonsoku-like protein PAPPPPIRVR(V) 35 3 6.01 76 6.22E+06 (K)ENDFKGLTsLyGLILN y:Phosphorylated Q9BXN1 Asporin NNK(L) Y
s:Phosphorylate d S Table 9: Proteins identified in fraction 4 of of cells grown with only As(III), fraction isolated based on Size Exclusion

Chromatography (SEC).

Peptide Total Matched Accession No Charge Score Sequence Modifications Entry name Intensity Intensity parent mass number (R)VTPVDYLLGVADLTGELM 1 2 22.91 98.6 5.08E+06 2062.089 Q99598 Translin-associated protein X R(M)

2 2 20.39 98 6.58E+06 (R)DLVEAVAHILGIR(D) 1405.816 P49327 Fatty acid synthase

(K)IEAELQDICNDVLELLDK(Y 3 2 19.91 98 1.27E+07 C:Carbamidomethylation 2130.064 P31946 14-3-3 protein beta/alpha ) (K)ITVVGVGQVGMACAISILG L-lactate dehydrogenase B 4 2 19.84 96.2 1.82E+07 C:Carbamidomethylation 1973.092 P07195 K(S) chain 5 2 18.65 94.6 2.57E+07 (R)LISQIVSSITASLR(F) 1487.879 P68363 Tubulin alpha-1B chain

(R)TLLEGSGLESIISIIHSSLAE 6 3 17.83 95.1 8.31E+06 2422.319 P49327 Fatty acid synthase PR(V) (R)LGFSEVELVQMVVDGVK( 7 3 17.66 89.8 8.68E+06 1848.978 P12277 Creatine kinase B-type L) (R)YVEPIEDVPCGNIVGLVGV 8 3 17.39 86.1 3.32E+07 C:Carbamidomethylation 2759.432 P13639 Elongation factor 2 DQFLVK(T) (K)DAQVVQVVLDGLSNILK(M 9 2 16.95 98.8 4.18E+06 1811.027 O00629 Importin subunit alpha-4 ) (K)KPSETQELVQQVLSLATQ 10 3 16.86 84.6 1.18E+07 2911.464 P63010 AP-2 complex subunit beta DSDNPDLR(D)

136

(K)LGCEVLGVSVDSQFTHLA 11 3 16.64 73.4 7.91E+06 C:Carbamidomethylation 2699.361 P32119 Peroxiredoxin-2 WINTPR(K) (R)IYDDDFFQNLDGVANALDN Ubiquitin-like modifier- 12 3 16.55 84.6 6.35E+07 2600.19 P22314 VDAR(M) activating enzyme 1 T-complex protein 1 subunit 13 2 16.37 96 2.76E+07 (R)AQLGVQAFADALLIIPK(V) 1768.037 P40227 zeta Heterogeneous nuclear 14 2 15.69 86.8 7.60E+06 (R)ILSISADIETIGEILK(K) 1714.984 P61978 ribonucleoprotein K ATP synthase subunit beta, 15 2 15 91.5 4.28E+06 (R)DQEGQDVLLFIDNIFR(F) 1921.965 P06576 mitochondrial (R)NDFQLIGIQDGYLSLLQDS Eukaryotic translation 16 3 14.64 80.3 3.63E+07 2580.294 P63241 GEVR(E) initiation factor 5A-1 L-lactate dehydrogenase A-like 17 2 14.5 84.5 1.03E+07 (K)LIIVSNPVDILTYVAWK(L) 1944.121 Q9BYZ2 6B (R)TLLEGSGLESIISIIHSSLAE 18 3 14.43 97.5 1.60E+07 2422.319 P49327 Fatty acid synthase PR(V) (R)ALDLFSDNAPPPELLEIINE 19 3 14.35 83.1 1.33E+07 2637.366 Q15084 Protein disulfide-isomerase A6 DIAK(R) T-complex protein 1 subunit 20 2 14.11 88.1 2.46E+07 (R)AQLGVQAFADALLIIPK(V) 1768.037 P40227 zeta (K)DGPLNMILDDGGDLTNLIH 21 3 13.1 80.2 8.51E+07 2252.123 P23526 Adenosylhomocysteinase TK(Y) 22 2 12.58 93 8.62E+06 (K)ETPFELIEALLK(Y) 1402.783 Q08211 ATP-dependent RNA helicase A

Pyruvate kinase isozymes 23 3 12.53 92.1 2.94E+06 (K)FGVEQDVDMVFASFIR(K) 1859.9 P14618 M1/M2 (R)ECGVGVIVTPEQIEEAVEA 24 3 12.52 79 5.59E+06 C:Carbamidomethylation 2483.245 P47897 Glutaminyl-tRNA synthetase AINR(H) (K)DAFDRNPELQNLLLDDFFK 6-phosphogluconate 25 3 12.48 92.7 2.57E+07 2310.14 P52209 (S) dehydrogenase, decarboxylating KH domain-containing, RNA- (K)DSLDPSFTHAMQLLTAEIE 26 3 12.36 96.9 1.07E+07 2246.101 Q07666 binding, signal transduction- K(I) associated protein 1 27 2 11.64 77.6 6.19E+06 (R)LISQIVSSITASLR(F) 1487.879 P68363 Tubulin alpha-1B chain

(K)SLQDIIAILGMDELSEEDKL ATP synthase subunit beta, 28 3 11.47 81.3 1.16E+07 2675.381 P06576 TVSR(A) mitochondrial (K)ITVVGVGQVGMACAISILG L-lactate dehydrogenase B 29 2 11.26 83.9 1.38E+07 C:Carbamidomethylation 1973.092 P07195 K(S) chain

137

(R)NDFQLIGIQDGYLSLLQDS Eukaryotic translation 30 3 11.05 83.7 3.05E+07 2580.294 P63241 GEVR(E) initiation factor 5A-1 31 2 10.96 97.9 3.91E+06 (K)SLELLPIILTALATK(K) 1595.998 Q9NVI1 Fanconi anemia group I protein

(R)KFDVNTSAVQVLIEHIGNL 32 3 10.93 73.8 1.34E+07 2368.262 Q00610 Clathrin heavy chain 1 DR(A) (K)DGPLNMILDDGGDLTNLIH 33 3 10.84 80.5 2.92E+07 2252.123 P23526 Adenosylhomocysteinase TK(Y)

(K)QtPLSATAAPQTPDSDIFTF t:Phosphorylated Rab GTPase-activating protein 34 3 10.68 62.6 7.23E+06 2864.456 Q9Y3P9 SVsLEIK(E) T
s:Phosphorylated S 1

C:Carbamidomethylation (K)sVDECKLHKGGyNGLNQC 35 3 10.51 78.1 6.49E+06 y:Phosphorylated 2864.349 Q8TD23 Zinc finger protein 675 LPTMQSK(M) Y
s:Phosphorylated S

(K)FFEGPVTGIFSGYVNSMLQ 36 3 10.47 70.3 7.67E+06 2584.243 P55060 Exportin-2 EYAK(N) (K)KPTETQELVQQVLSLATQ 37 3 10.14 79.9 6.51E+06 2925.48 Q10567 AP-1 complex subunit beta-1 DSDNPDLR(D) (R)IVAFADAAVEPIDFPIAPVY Acetyl-CoA acetyltransferase, 38 3 10.03 83.3 2.53E+07 2818.51 P24752 AASMVLK(D) mitochondrial (K)HFADLLPGFLQAVNDSCY 39 3 9.85 80.3 9.78E+06 C:Carbamidomethylation 2966.399 O00410 Importin-5 QNDDSVLK(S) (R)RLNGLSsSVEYNIMELEQE 40 3 9.48 99.4 3.03E+07 s:Phosphorylated S 3136.619 Q8WVP7 Limb region 1 protein homolog LENVKTLK(T) (K)YLEsKEDVADALLQTDQSL Interferon-induced guanylate- 41 3 9.47 69.4 1.17E+07 s:Phosphorylated S 2482.22 P32456 sEK(E) binding protein 2 (K)EQPLDEELKDAFQNAYLE Sodium/potassium-transporting 42 3 9.39 84.7 9.26E+06 2834.384 P05023 LGGLGER(V) ATPase subunit alpha-1 L-lactate dehydrogenase B 43 2 9.35 78.3 8.22E+06 (K)SLADELALVDVLEDK(L) 1629.858 P07195 chain

(K)GLDYEGGGCRFLRyDCVIS C:Carbamidomethylation Procollagen-lysine,2- 44 3 9.11 80.8 1.16E+08 2577.197 O60568 SPR(K) y:Phosphorylated Y oxoglutarate 5-dioxygenase 3

(R)LGFSEVELVQMVVDGVK( 45 2 8.97 87.1 9.65E+06 1848.978 P12277 Creatine kinase B-type L)

138

(R)IVAFADAAVEPIDFPIAPVY Acetyl-CoA acetyltransferase, 46 3 8.67 75.6 2.55E+07 2818.51 P24752 AASMVLK(D) mitochondrial (R)NDFQLIGIQDGYLSLLQDS Eukaryotic translation 47 3 8.61 78.1 7.05E+06 2580.294 P63241 GEVR(E) initiation factor 5A-1 (K)DAQVVQVVLDGLSNILK(M 48 3 8.53 85.9 2.07E+06 1811.027 O00629 Importin subunit alpha-4 ) 49 2 8.33 83.7 2.74E+07 (K)MAVTFIGNSTAIQELFK(R) 1869.978 P07437 Tubulin beta chain

(R)tCEGTEPWSRTTSLGDSLN C:Carbamidomethylation 50 3 8.26 63.2 1.60E+07 2805.274 Q13796 Protein Shroom2 AHSAAEK(A) t:Phosphorylated T

L-lactate dehydrogenase A-like 51 3 8.23 94.1 3.25E+06 (K)LIIVSNPVDILTYVAWK(L) 1944.121 Q9BYZ2 6B (R)TLLEGSGLESIISIIHSSLAE 52 3 8.16 90.1 4.74E+06 2422.319 P49327 Fatty acid synthase PR(V) (K)SLQDIIAILGMDELSEEDKL ATP synthase subunit beta, 53 3 8.04 72.9 1.18E+07 2675.381 P06576 TVSR(A) mitochondrial Uncharacterized protein 54 3 7.84 81.5 4.48E+06 (K)EMSPFSssSTTHLFSK(C) s:Phosphorylated S 1772.816 Q8N8D9 C5orf56 (R)ANLTILFDKYLPTCLDTLR( 55 3 7.83 62.9 2.52E+07 C:Carbamidomethylation 2267.21 Q9NYC9 Dynein heavy chain 9, axonemal T) (K)STAISLFYELSENDLNFIK( 56 3 7.74 86.6 3.94E+06 2204.112 P13639 Elongation factor 2 Q) (K)KISFNFSEIMAsTGWNSEL Uncharacterized protein 57 3 7.73 63.4 1.64E+07 s:Phosphorylated S 2289.122 Q8TAL5 K(L) C9orf43 Eukaryotic translation 58 2 7.73 89.2 1.08E+07 (R)HLVFPLLEFLSVK(E) 1541.909 P60228 initiation factor 3 subunit E (K)ISSNEQPPAAWPPRQDMGs 59 3 7.68 63 9.66E+06 s:Phosphorylated S 2726.31 Q8N944 Protein FAM123C GLFGQR(W) Leucine-rich repeat and (R)LPLKLSHVQSQtNGGPSPT 60 3 7.67 64.4 1.94E+07 t:Phosphorylated T 2186.193 Q6PJG9 fibronectin type-III domain- PK(A) containing protein 4 (R)FDPTQFQDCIIQGLTETGT Basic leucine zipper and W2 61 3 7.61 79.1 3.95E+06 C:Carbamidomethylation 2897.387 Q7L1Q6 DLEAVAK(F) domain-containing protein 1

(K)VAyVQLAHGQtFTFPDLFP t:Phosphorylated 39S ribosomal protein L23, 62 3 7.53 63.9 8.08E+06 2408.229 Q16540 EK(D) T
y:Phosphorylated Y mitochondrial

T-complex protein 1 subunit 63 2 7.5 100 6.81E+06 (R)AQLGVQAFADALLIIPK(V) 1768.037 P40227 zeta

139

64 2 7.48 88.3 3.04E+07 (K)MAVTFIGNSTAIQELFK(R) 1869.978 P07437 Tubulin beta chain

(R)ITRSQPNHTPAGPPGPSSNP t:Phosphorylated 65 3 7.44 63.2 6.62E+06 2883.446 Q9BQ15 SOSS complex subunit B1 VsNGKEtR(R) T
s:Phosphorylated S

C:Carbamidomethylation Protein kinase C-binding 66 2 7.37 62.6 1.06E+07 (R)ANCINLPGWyHCECR(D) 1949.82 Q99435 y:Phosphorylated Y protein NELL2

T-complex protein 1 subunit 67 2 7.3 80.2 6.36E+06 (R)AQLGVQAFADALLIIPK(V) 1768.037 P40227 zeta (R)TTFtLQQLEALEAVFAQtH Dorsal root ganglia homeobox 68 3 7.26 82.9 6.64E+06 t:Phosphorylated T 3026.526 A6NNA5 YPDVFTR(E) protein 69 2 7.22 93.3 3.41E+07 (K)MAVTFIGNSTAIQELFK(R) 1869.978 P07437 Tubulin beta chain

(R)INALTAASEAACLIVSVDET 70 3 7.12 77.7 2.50E+07 C:Carbamidomethylation 2289.201 Q99832 T-complex protein 1 subunit eta IK(N) Fructose-bisphosphate aldolase (R)TVPPAVTGITFLSGGQSEE 71 3 7.06 91.1 2.19E+06 3057.574 P04075 A OS=Homo sapiens EASINLNAINK(C) GN=ALDOA PE=1 SV=2 (R)KLEGDSTDLSDQIAELQAQ 72 3 7.06 83.4 2.34E+07 2615.341 P35579 Myosin-9 IAELK(M)

(K)ICEEETGSTSIQAADSTAVN C:Carbamidomethylation Putative oxidoreductase 73 3 7.06 86.5 6.78E+06 3010.416 Q49A26 GSITPtDKK(I) t:Phosphorylated T GLYR1

y:Phosphorylated Anaphase-promoting complex 74 2 7.01 64.2 4.26E+06 (R)LLEsMKAQyVAGNGFR(K) 1783.916 Q9UJX5 Y
s:Phosphorylated S subunit 4

(K)tHGCPEWREECSFELPPGA C:Carbamidomethylation Rab11 family-interacting 75 3 6.96 61.9 2.67E+07 2926.361 Q9BXF6 LDGLLR(A) t:Phosphorylated T protein 5

(R)ILADLEDYLNELWEDKEG Eukaryotic translation 76 3 6.94 96 1.40E+07 2293.123 Q99613 K(K) initiation factor 3 subunit C (R)TLQSGLALLYGFLPDFDW 77 3 6.93 78.7 2.54E+07 2312.233 Q8TE99 Acid phosphatase-like protein 2 KK(I) (R)AMERGGTVPADLEAAAAS Mitotic spindle assembly 78 3 6.85 76.7 2.77E+06 s:Phosphorylated S 2898.488 Q9Y6D9 LPssKEVAELK(K) checkpoint protein MAD1 (K)LESEKLNVAEVtQSEIAQK 79 3 6.81 92.6 4.33E+06 t:Phosphorylated T 2372.267 Q9NVD7 Alpha-parvin QK(L)

140

C:Carbamidomethylation (K)DFLTLCEKtSTGEKLSEFN 80 3 6.76 82.8 3.82E+06 t:Phosphorylated 2691.282 Q9BUY5 Zinc finger protein 426 QsEK(I) T
s:Phosphorylated S

(R)LYKSNELFGSVIFKLPsNR( ATP-binding cassette sub- 81 3 6.66 79.4 5.35E+06 s:Phosphorylated S 2212.213 Q86UK0 S) family A member 12

t:Phosphorylated 82 2 6.61 79.1 4.72E+06 (R)NTsLSETSRGGQPSVTtK(S) 1849.925 Q12955 Ankyrin-3 T
s:Phosphorylated S

(K)ITVVGVGQVGMACAISILG L-lactate dehydrogenase B 83 2 6.57 88.3 8.98E+06 C:Carbamidomethylation 1973.092 P07195 K(S) chain Methylmalonic aciduria and (R)VVNPKAFSTAGSSGSDEsH C:Carbamidomethylation 84 3 6.56 74.7 4.90E+06 3014.427 Q9H3L0 homocystinuria type D protein, VAAAPPDICsR(T) s:Phosphorylated S mitochondrial (K)TASMLWLLQQEMVtWRL Nuclear pore complex protein 85 3 6.55 70.4 3.28E+06 t:Phosphorylated T 3080.617 P57740 LASLYRDR(I) Nup107 (R)AVSIFNKEGCLEIVLKYLS HEAT repeat-containing 86 3 6.5 100 5.16E+06 C:Carbamidomethylation 2339.279 Q7Z4Q2 R(F) protein 3 (R)TLLEGSGLESIISIIHSSLAE 87 3 6.5 100 4.97E+06 2422.319 P49327 Fatty acid synthase PR(V)

C:Carbamidomethylation (K)LLtVCKPFDLHASPHAsIK( 88 3 6.46 80.7 1.21E+07 t:Phosphorylated 2134.148 Q3B820 Protein FAM161A R) T
s:Phosphorylated S

C:Carbamidomethylation (R)KCsELLYVFQtQLALKLLQ 89 3 6.46 82.2 3.55E+06 t:Phosphorylated 2696.488 Q86VV8 Rotatin CLK(V) T
s:Phosphorylated S

(R)IETLEQKLtCLELIQNTHSQ C:Carbamidomethylation Histone-lysine N- 90 3 6.37 89.5 9.10E+06 2886.47 Q9BYW2 SCLK(S) t:Phosphorylated T methyltransferase SETD2

(R)DyLEANIDAIHRSTDHIEEs y:Phosphorylated Cas scaffolding protein family 91 3 6.3 70.5 3.47E+06 2583.243 Q9NQ75 VR(E) Y
s:Phosphorylated S member 4

(R)IWLKDKHLALQFIDWVLR( 92 3 6.29 85.7 1.38E+07 2294.317 Q15849 Urea transporter 2 G)

141

(R)EVAAFAQFGSDLDAATQQ ATP synthase subunit alpha, 93 3 6.29 94 5.63E+06 2338.167 P25705 LLSR(G) mitochondrial (R)VLGYEVLYWTDDsKEsMIG 94 3 6.25 69.4 2.69E+07 s:Phosphorylated S 2602.322 Q9UQ52 Contactin-6 KIR(V) (R)KsQLIISPHSTTELPVLFYPS Putative uncharacterized 95 3 6.24 67.2 1.49E+07 s:Phosphorylated S 2754.519 A6PW82 ALGR(A) protein CXorf30

(K)QCGsCWAFsATGALEGQM C:Carbamidomethylation 96 3 6.21 65.1 5.98E+06 2678.227 O60911 Cathepsin L2 FRKTGK(L) s:Phosphorylated S

(R)MEAMEKQIASLtGLVQSAL SRC kinase signaling inhibitor 97 3 6.19 65.2 1.23E+07 t:Phosphorylated T 2289.231 Q9C0H9 LR(G) 1 98 2 6.17 89.6 6.54E+06 (R)ASVRIRSsVEILR(V) s:Phosphorylated S 1485.886 Q8N960 Centrosomal protein of 120 kDa (R)SGTGSGGSTPHISGPGPGR 99 3 6.08 78.8 3.23E+06 2407.171 Q9H165 B-cell lymphoma/leukemia 11A PSSKEGR(R) 100 2 6.08 71.3 7.30E+06 (K)TQHGVLsQQFVELINK(C) s:Phosphorylated S 1840.992 Q12846 Syntaxin-4 Table 10: Proteins identified in fraction 1 of cells grown with SeMet, fraction isolated based on Size Exclusion Chromatography

(SEC).

Matched Peptide Total Accession No Charge Score Sequence Modifications parent Entry name Intensity Intensity number mass Polyadenylate-binding 1 3 21.32 97.5 3.79E+08 (K)ITGMLLEIDNSELLHMLESPESLR(S) 2740.39 P11940 protein 1 2 2 21.25 97.5 8.82E+07 (K)VVIGMDVAASEFFR(S) 1540.783 P06733 Alpha-enolase

U5 small nuclear 3 2 17.83 85.6 5.18E+06 (R)ETYEVLLSFIQAALGDQPR(D) 2150.113 O75643 ribonucleoprotein 200

kDa helicase Elongation factor 1-alpha 4 3 15.51 64.9 1.68E+07 (K)NMITGTSQADCAVLIVAAGVGEFEAGISK(N) C:Carbamidomethylation 2909.438 P68104 1 5 3 13.34 96.6 9.87E+06 (K)NLEVLNFFNNQIEELPTQISSLQK(L) 2818.462 Q15404 Ras suppressor protein 1

142

6 2 11.99 92.1 5.36E+06 (R)GPGTSFEFALAIVEALNGK(E) 1921.007 Q99497 Protein DJ-1

7 3 11 100 1.77E+07 (K)EALAQCVLAEIPQQVVGYFNTYK(L) C:Carbamidomethylation 2641.333 O75131 Copine-3 Isochorismatase domain- 8 3 10.67 75.1 1.62E+07 (R)QSGAFLSTSEGLILQLVGDAVHPQFK(E) 2742.446 Q96AB3 containing protein 2,

mitochondrial 60S acidic ribosomal 9 2 9.96 89.2 1.51E+07 (K)KEESEESDDDMGFGLFD(-) 1949.759 P05386 protein P1 1-phosphatidylinositol-3- 10 3 9.13 72.8 2.45E+07 (K)NSDPFAHSKDAsSTsSGQSGSKNEGDEER(G) s:Phosphorylated S 3011.285 Q9Y2I7 phosphate 5-kinase Poly(rC)-binding protein 11 3 9 100 2.62E+06 (R)QVTITGSAASISLAQYLINVR(L) 2205.224 Q15366 2 Elongation factor 1-alpha 12 3 8.74 90.6 5.99E+06 (K)NMITGTSQADCAVLIVAAGVGEFEAGISK(N) C:Carbamidomethylation 2909.438 P68104 1 Basic leucine zipper and 13 3 8.59 75.4 7.17E+06 (R)FDPTQFQDCIIQGLTETGTDLEAVAK(F) C:Carbamidomethylation 2897.387 Q7L1Q6 W2 domain-containing protein 1 14 2 8.45 84.8 4.96E+06 (R)GPGTSFEFALAIVEALNGK(E) 1921.007 Q99497 Protein DJ-1

t:Phosphorylated Tubulin polymerization- 15 2 8.27 83 3.93E+06 (K)VADGKSVtGTDVDIVFsK(V) T
s:Phosphorylated 1837.954 Q9BW30 promoting protein family S member 3 TBCC domain-containing 16 3 8.12 65.5 1.10E+07 (K)IYPLHELALWQPLHADsGFsKISK(T) s:Phosphorylated S 2750.466 Q9NVR7 protein 1 Protein disulfide- 17 3 8.09 93.3 1.05E+07 (R)ALDLFSDNAPPPELLEIINEDIAK(R) 2637.366 Q15084 isomerase A6 Nucleoside diphosphate 18 3 7.6 67 3.17E+06 (-)MANLERtFIAIKPDGVQRGLVGEIIK(R) t:Phosphorylated T 2868.613 P22392 kinase B Ras GTPase-activating 19 2 7.5 100 2.29E+06 (K)SSsPAPADIAQTVQEDLR(T) s:Phosphorylated S 1884.93 Q13283 protein-binding protein 1 Heterogeneous nuclear 20 2 7.44 86.5 3.70E+06 (R)ILSISADIETIGEILK(K) 1714.984 P61978 ribonucleoprotein K 21 3 7.25 81.9 1.70E+07 (K)EALAQCVLAEIPQQVVGYFNTYK(L) C:Carbamidomethylation 2641.333 O75131 Copine-3 Complement component 22 3 7.14 70.3 8.20E+06 (K)sIIQEKTSNFNAAIsLK(F) s:Phosphorylated S 1864.017 P02748 C9 Beta-1,3- 23 3 7 81.8 9.58E+06 (R)IFLLGLsIKLNGYLQRAILEEsR(Q) s:Phosphorylated S 2646.534 O43825 galactosyltransferase 2 24 2 6.85 78.7 6.39E+06 (R)DSLLQDGEFSMDLR(T) 1625.748 P07737 Profilin-1

L-lactate dehydrogenase 25 2 6.85 89.3 2.83E+06 (K)GYTSWAIGLSVADLAESIMK(N) 2112.068 P00338 A chain 26 3 6.79 91.9 1.00E+07 (R)TLLEGSGLESIISIIHSSLAEPR(V) 2422.319 P49327 Fatty acid synthase

143

27 3 6.77 79.6 7.14E+06 (K)MADALLFGNFGVQNITAAIQLYESLAK(E) 2898.507 Q5TEA6 Protein sel-1 homolog 2

y:Phosphorylated Rab proteins 28 3 6.5 100 3.17E+06 (R)FNIDLVSKLLysQGLLIDLLIK(S) Y
s:Phosphorylated 2518.49 P26374 geranylgeranyltransferase S component A 2 Cytoplasmic dynein 2 29 3 6.41 80 5.05E+06 (K)LQNLLSELEAGLGIVLRRSDtNLtK(L) t:Phosphorylated T 2753.552 Q8NCM8 heavy chain 1 30 3 6.39 93.2 8.09E+06 (R)GLPFQANAQDIINFFAPLKPVR(I) 2456.345 Q12849 G-rich sequence factor 1

y:Phosphorylated ATP-binding cassette 31 3 6.39 94.4 8.96E+06 (R)FQKGRIEFENVHFsyADGR(E) Y
s:Phosphorylated 2300.121 Q9NP58 sub-family B member 6, S mitochondrial Polyadenylate-binding 32 3 6.38 86.8 3.64E+08 (K)ITGMLLEIDNSELLHMLESPESLR(S) 2740.39 P11940 protein 1 C:Carbamidomethylation 33 3 6.28 72.3 6.37E+07 (K)VLGSSIHMECKVSGSLPIsAQWFK(D) 2661.353 Q8WZ42 Titin s:Phosphorylated S Origin recognition 34 3 6.2 67.9 4.52E+06 (K)DQNYVEIMGRDVQESLKNGSAtGGGNK(V) t:Phosphorylated T 2867.359 Q13416 complex subunit 2 X-ray repair cross- 35 3 6.2 83.8 6.18E+06 (K)QLNHFWEIVVQDGITLITK(E) 2254.223 P13010 complementing protein 5 Synaptotagmin-like 36 2 6.15 76.8 3.37E+06 (R)SAtRGEIITPKTDtGR(S) t:Phosphorylated T 1702.908 Q8TDW5 protein 5 Alanine aminotransferase 37 3 6.11 72 6.64E+06 (R)RDGGVPADPDNIyLTTGASDGISTILK(I) y:Phosphorylated Y 2746.389 Q8TD30 2 SWI/SNF complex 38 3 6.04 79.1 6.67E+06 (R)EWTEQETLLLLEALEMYKDDWNK(V) 2897.391 Q92922 subunit SMARCC1

Table 11: Proteins identified in fraction 2 of cells grown with SeMet, fraction isolated based on Size Exclusion Chromatography

(SEC).

144

Peptide Total Matched Accession No Charge Score Sequence Modifications Entry name Intensity Intensity parent mass number 1 2 17.34 97.9 8.15E+06 (K)DATNVGDEGGFAPNILENK(E) 1960.925 P06733 Alpha-enolase

Adenylyl cyclase- 2 3 16.59 89.3 9.96E+06 (K)AGAAPYVQAFDSLLAGPVAEYLK(I) 2351.228 Q01518 associated protein 1 Rab GDP 3 2 16.47 88.8 1.09E+07 (K)SPYLYPLYGLGELPQGFAR(L) 2141.107 P31150 dissociation

inhibitor alpha 4 2 10 100 1.17E+07 (R)YGINTTDIFQTVDLWEGK(N) 2100.028 P37802 Transgelin-2

5 3 9.9 79.6 4.84E+06 (K)TLVLSNLSYSATEETLQEVFEK(A) 2501.266 P19338 Nucleolin

6 3 9.44 86.3 7.12E+06 (R)YVEPIEDVPCGNIVGLVGVDQFLVK(T) C:Carbamidomethylation 2759.432 P13639 Elongation factor 2 C:Carbamidomethylation Folylpolyglutamate 7 3 8.82 61.1 4.94E+08 (K)GKGsTCAFTECILRSyGLK(T) y:Phosphorylated 2148.058 Q05932 synthase, Y
s:Phosphorylated S mitochondrial C:Carbamidomethylation 8 3 8.47 69.2 1.93E+07 (K)EDsCTASSKNYKNASGVVNSSPR(S) 2458.126 Q8N5G2 Macoilin s:Phosphorylated S Endoplasmic 9 3 8.42 63 1.95E+09 (R)AHKGSTLSQWSLGNGtPVTSK(G) t:Phosphorylated T 2156.11 Q7Z2K6 reticulum metallopeptidase 1 Leucine-rich 10 3 8.41 61.1 5.72E+07 (R)GLEELHLEGLFPQELAR(A) 1951.028 Q6NSJ5 repeat-containing

protein 8E Transmembrane 11 3 8.26 70.4 6.29E+07 (K)DVGAKVTGFsEGVVDsVK(G) s:Phosphorylated S 1793.928 O94876 and coiled-coil domains protein 1 Myosin 12 3 8.25 64.1 3.97E+08 (R)ELEKsQRSQIsSVNSDVEALR(R) s:Phosphorylated S 2375.216 Q6WCQ1 phosphatase Rho- interacting protein Collagen alpha- 13 3 8.18 61.5 8.42E+06 (R)GLPGPPGPQGESRTGPPGsTGsR(G) s:Phosphorylated S 2146.064 Q99715 1(XII) chain Laminin subunit 14 3 7.95 66.3 4.45E+08 (R)VKVMtDLGSGPITLLtDRR(Y) t:Phosphorylated T 2072.153 P25391 alpha-1 t:Phosphorylated 15 3 7.89 62.9 1.15E+08 (R)GPQGSVHFAAIRtSDAGRyR(C) 2146.09 Q8NDA2 Hemicentin-2 T
y:Phosphorylated Y Myotubularin- 16 3 7.62 62 4.99E+06 (K)HSGFSTSDNSIANtPQDYSGNMK(S) t:Phosphorylated T 2458.058 Q9Y216 related protein 7 t:Phosphorylated Bile salt export 17 3 7.58 66.5 1.66E+08 (K)ISAARFFQLLDRQPPISVyNtAGEK(W) 2821.499 O95342 T
y:Phosphorylated Y pump Zinc finger RNA- 18 3 7.57 61.6 3.18E+07 (K)VNPDLQVEVKPSIRARK(I) 1949.129 Q96KR1 binding protein

145

Serine/threonine- 19 3 7.45 63.4 5.10E+06 (R)sLLESVQKLDLEATIDKVVK(I) s:Phosphorylated S 2228.275 Q13535 protein kinase ATR C:Carbamidomethylation 20 3 7.41 69.6 1.02E+08 (K)ALyNCIHEDMKRLLPVVR(A) 2227.184 Q9NZJ4 Sacsin y:Phosphorylated Y 21 2 7.27 75.1 7.12E+06 (R)DSLLQDGEFSMDLR(T) 1625.748 P07737 Profilin-1

y:Phosphorylated 22 3 7.17 73.1 3.85E+06 (K)KAVKNFTEVHPDyGsHIQALLDK(Y) 2610.367 P04040 Catalase Y
s:Phosphorylated S Calcium- independent 23 3 7.14 60.2 1.23E+09 (K)LSTSAPKGLTKVNICMSR(I) C:Carbamidomethylation 1963.046 Q9NP80 phospholipase A2- gamma C:Carbamidomethylation Kinesin-like protein 24 3 6.94 63.5 4.88E+06 (K)SLIEGVIsGYNATVFAYGPTGCGK(T) 2461.207 Q2TAC6 s:Phosphorylated S KIF19 Ankyrin repeat and SAM domain- 25 3 6.89 67.9 2.79E+06 (-)MGKEQELLEAARTGHLPAVEK(L) 2307.213 Q92625 containing protein 1A t:Phosphorylated Alanine 26 3 6.88 68.7 7.03E+08 (R)DGGIPADPNNVFLsTGASDAIVtVLK(L) 2571.33 P24298 T
s:Phosphorylated S aminotransferase 1 C:Carbamidomethylation Keratin, type I 27 3 6.85 60.7 8.30E+07 (R)tVCVPRTVGMPCsPCPQGR(Y) t:Phosphorylated 2158.998 Q14532 cuticular Ha2 T
s:Phosphorylated S UDP-glucose 6- 28 3 6.79 65.5 9.39E+07 (R)FASRIIDSLFNtVtDKK(I) t:Phosphorylated T 1955.06 O60701 dehydrogenase G-rich sequence 29 3 6.79 89.8 3.97E+06 (R)GLPFQANAQDIINFFAPLKPVR(I) 2456.345 Q12849 factor 1 Procollagen C- C:Carbamidomethylation 30 3 6.78 75.9 6.62E+08 (R)DyPAGVTCVWHIVAPKNQLIELK(F) 2651.401 Q9UKZ9 endopeptidase y:Phosphorylated Y enhancer 2 t:Phosphorylated 31 3 6.78 60.3 3.62E+06 (K)QMNIPGGDRstPAAVGAMEDK(S) 2145.006 Q9UGI8 Testin T
s:Phosphorylated S Bromodomain and WD repeat- 32 3 6.69 63 5.46E+07 (K)RLRsSSSsLSSSGAPSPK(G) s:Phosphorylated S 1790.936 Q6RI45 containing protein 3 Casein kinase I 33 3 6.64 65.9 6.44E+06 (R)SsHGIRSSGTSSGVLMVGPNFR(V) s:Phosphorylated S 2233.114 P78368 isoform gamma-2 Zinc finger protein 34 3 6.62 71.6 3.26E+07 (R)SPSQGGsFSQVIFTNKSLGKR(D) s:Phosphorylated S 2225.167 Q8NF99 397 t:Phosphorylated Polyadenylate- 35 3 6.55 80.8 4.25E+08 (K)EFtNVyIKNFGEEVDDESLK(E) 2376.124 Q13310 T
y:Phosphorylated Y binding protein 4

146

36 3 6.5 68.7 1.01E+08 (K)AAMYsVEITVEKDKVTGETR(V) s:Phosphorylated S 2227.128 O75781 Paralemmin C:Carbamidomethylation Condensin complex 37 3 6.49 81.5 1.34E+08 (R)HLWNHSIIEEEFVSLVtGCCYR(L) 2749.286 Q15021 t:Phosphorylated T subunit 1 Glutathione 38 3 6.49 71 7.77E+07 (R)GHAAFTSDPKPtIEVSGKK(Y) t:Phosphorylated T 1970.034 P00390 reductase, mitochondrial Zinc finger CCCH 39 3 6.48 62.7 3.76E+07 (K)AsDPGAASTKSGKAsTLSR(R) s:Phosphorylated S 1791.92 Q86VM9 domain-containing protein 18 Uveal autoantigen t:Phosphorylated with coiled-coil 40 3 6.42 73.6 5.27E+06 (K)sDLEtQISSLNEKLANLNR(K) 2145.115 Q9BZF9 T
s:Phosphorylated S domains and ankyrin repeats Mitochondrial y:Phosphorylated 41 3 6.34 63.7 7.60E+08 (K)sPyLGLGPGHSYVSLFLADR(C) 2149.108 O15091 ribonuclease P Y
s:Phosphorylated S protein 3 Cation channel t:Phosphorylated sperm-associated 42 3 6.32 61.7 2.14E+08 (R)KtAIAsVSTLRNNEPNSQSK(F) 2145.126 Q9H7T0 T
s:Phosphorylated S protein subunit beta y:Phosphorylated Homeobox protein 43 3 6.29 66.1 2.77E+08 (R)KVHVsTVNPNyAGGEPKR(S) 1953.03 P17483 Y
s:Phosphorylated S Hox-B4 C:Carbamidomethylation Zinc finger protein 44 3 6.29 60.4 3.18E+06 (R)VsVFKCPSCPLLFAQKR(T) 2037.077 Q8N1G0 s:Phosphorylated S 687 Coiled-coil domain- C:Carbamidomethylation 45 3 6.17 68.1 5.40E+07 (K)LHCDsLETQIKSLHSENVK(L) 2238.118 Q5T0U0 containing protein s:Phosphorylated S 122 Biliverdin 46 3 6.06 65.7 4.81E+06 (K)MTVCLETEKKSPLSWIEEK(G) C:Carbamidomethylation 2308.156 P53004 reductase A Tetratricopeptide 47 3 6.06 60.1 2.52E+06 (R)GTMyRRLQEFDGAVEDFLK(V) y:Phosphorylated Y 2275.118 Q8NEE8 repeat protein 16 Transmembrane 7 48 3 6.05 71.6 1.72E+06 (R)KyVIIPTFWPTPKER(K) y:Phosphorylated Y 1875.053 Q9H295 superfamily member 4

Table 12: Proteins identified in fraction 3 of cells grown with SeMet, fraction isolated based on Size Exclusion Chromatography

(SEC). 147

Peptide Total Matched Accession No Charge Score Sequence Modifications Entry name Intensity Intensity parent mass number Glucose-6-phosphate 1 2 16.58 85.7 5.58E+07 (K)ILLANFLAQTEALMR(G) 1703.951 P06744 isomerase

2 2 12.54 89.9 4.68E+06 (R)YGINTTDIFQTVDLWEGK(N) 2100.028 P37802 Transgelin-2

3 3 9.98 68.5 1.04E+08 (R)RQREVtEITEIEEEYEISK(H) t:Phosphorylated T 2381.183 Q8WZ42 Titin

4 3 9.03 66.2 8.07E+07 (K)LTVEEAVRMGIVGPEFKDK(L) 2118.126 Q15149 Plectin

Pleckstrin homology 5 3 8.76 68 9.26E+06 (K)QsDIMMRDNLFEIVTTSR(T) s:Phosphorylated S 2156.048 Q9HB21 domain-containing family A member 1 (K)GYTSWSLLDKFEWEKGYsDR( Q6UWM 6 3 8.3 60.6 6.89E+07 s:Phosphorylated S 2467.157 Lactase-like protein Y) 7 Isoleucyl-tRNA 7 3 8.29 70.1 5.63E+07 (K)GKFLINLEGGDIREESsYK(V) s:Phosphorylated S 2155.103 Q9NSE4 synthetase, mitochondrial

8 3 8.27 63.1 1.90E+07 (R)GPGPKGPVGtVSEAQLARR(L) t:Phosphorylated T 1877.035 Q9NZM1 Myoferlin

(R)LRRAADHDVGSELPPEGVLGA 9 3 8.21 68.6 4.49E+08 2541.39 Q9UHG2 ProSAAS LLR(V) Glucose-6-phosphate 10 2 8.12 90.2 1.42E+07 (K)ILLANFLAQTEALMR(G) 1703.951 P06744 isomerase Uveal autoantigen with t:Phosphorylated 11 3 7.98 69.5 7.78E+06 (K)sDLEtQISSLNEKLANLNR(K) 2145.115 Q9BZF9 coiled-coil domains and T
s:Phosphorylated S ankyrin repeats Nucleolar and coiled- 12 2 7.97 86.9 2.29E+06 (R)VVPSDLYPLVLGFLR(D) 1687.978 Q14978 body phosphoprotein 1 Caspase recruitment 13 3 7.92 62.9 4.69E+06 (R)MNsNERVRIIsGSPLGSLAR(S) s:Phosphorylated S 2157.156 Q9BXL7 domain-containing protein 11 Lipid phosphate (R)LAKPVLCLGTLCTAFLTGLNR( 14 3 7.89 67.8 5.99E+06 C:Carbamidomethylation 2318.272 Q8TBJ4 phosphatase-related V) protein type 1

148

(K)HGAALQRSLTELDGLKIPSESG Oxysterol-binding 15 3 7.86 73.7 1.56E+07 2536.337 Q969R2 EK(L) protein 2 (K)AAsLPGKNGNPTFAAVTAGyD y:Phosphorylated 16 3 7.72 65.7 1.06E+09 2150.088 Q92545 K(S) Y
s:Phosphorylated S (R)LAGDQGSEEAPQRPPASSAtLR( 17 3 7.69 71.1 5.50E+06 t:Phosphorylated T 2238.111 Q9NPG4 Protocadherin-12 R) (R)GALsKGSESLtLMFSHEDQKK(I t:Phosphorylated Cardiomyopathy- 18 3 7.68 62.3 2.31E+07 2293.149 Q8N3K9 ) T
s:Phosphorylated S associated protein 5

19 3 7.63 64.2 7.14E+06 (R)SELRLDLVLPsGQsFRWR(E) s:Phosphorylated S 2159.172 O15527 N-glycosylase/DNA lyase

y:Phosphorylated WD repeat-containing 20 3 7.6 68.4 1.55E+06 (R)IsPGNQyIVSVSADGAILR(W) 1960.05 Q8N1V2 Y
s:Phosphorylated S protein 16 (K)IGTRSAEDPVSEVPAVsQHPR(T Adenomatous polyposis 21 3 7.53 63.8 3.15E+06 s:Phosphorylated S 2232.137 P25054 ) coli protein Secretory carrier- (K)tVQtAAANAASTAASSAAQNAF 22 3 7.53 67.7 2.68E+06 t:Phosphorylated T 2152.063 O15126 associated membrane K(G) protein 1 (K)HATENKVKNLTEEMAGLDETI 24 3 7.43 63.8 8.72E+06 2542.282 P12882 Myosin-1 AK(L) Dual specificity mitogen- C:Carbamidomethylation 25 3 7.29 71.5 3.20E+06 (R)DAGCRPYMAPERIDPsAsR(Q) 2148.991 P45985 activated protein kinase s:Phosphorylated S kinase 4 26 2 7.14 73.1 3.24E+07 (K)DVNLEVTAKPVPLNsGVR(F) s:Phosphorylated S 1908.055 Q13287 N-myc-interactor

(R)AGPAPKGSsLQVTFPSETLNLS Prostaglandin E2 27 3 7.03 82.2 1.01E+07 s:Phosphorylated S 2458.282 P35408 EK(C) receptor EP4 subtype C:Carbamidomethylation DNA replication 28 3 6.97 61.5 1.11E+08 (R)GDINVCIVGDPstAKSQFLK(H) t:Phosphorylated 2149.096 Q14566 licensing factor MCM6 T
s:Phosphorylated S NADH dehydrogenase (R)GLGMTLSYLFREPATINYPFEK 29 3 6.95 60.1 5.86E+07 2547.295 O00217 [ubiquinone] iron-sulfur (G) protein 8, mitochondrial 30 3 6.82 63.5 8.59E+06 (R)FGKyIDIyFNPSGVIEGAR(I) y:Phosphorylated Y 2146.097 Q6PIF6 Myosin-VIIb

ATP-dependent RNA 31 3 6.77 64.5 5.48E+07 (K)FDPNERYLHLAAKLLDAK(E) 2114.139 Q7Z478 helicase DHX29

149

t:Phosphorylated 32 3 6.71 68.3 5.58E+06 (R)EESTNLEDYEPNtVAsLLK(Q) 2152.029 Q15311 RalA-binding protein 1 T
s:Phosphorylated S (K)DSDDDsSDDEQEKKPEAPKLSK Splicing factor 3B 33 3 6.61 63.9 2.34E+07 s:Phosphorylated S 2463.101 Q13435 (K) subunit 2 C:Carbamidomethylation Electrogenic sodium 34 3 6.46 74 3.39E+07 (K)KKGSLDSDNDDSDCPysEK(V) y:Phosphorylated 2159.903 Q9Y6R1 bicarbonate Y
s:Phosphorylated S cotransporter 1 Golgi-specific brefeldin (R)ADAPDAGAQSDSELPSYHQND A-resistance guanine 35 3 6.45 65.6 1.40E+07 2758.219 Q92538 VSLDR(G) nucleotide exchange factor 1 Lethal(3)malignant 36 3 6.35 62.3 5.15E+06 (K)RREYQSPSEEESEPEAMEK(Q) 2311.014 Q9Y468 brain tumor-like protein

1 37 2 6.33 62.5 1.53E+07 (R)DSLLQDGEFSMDLR(T) 1625.748 P07737 Profilin-1

WD repeat-containing 38 3 6.23 69.7 3.06E+07 (K)HSTyKYGRPDEIIEERIQTK(A) y:Phosphorylated Y 2463.263 Q8NA23 protein 31 Chromodomain-helicase- 39 3 6.23 68.3 6.67E+06 (K)QEALQHLIRssGKLILLDK(L) s:Phosphorylated S 2162.266 O14646 DNA-binding protein 1 Glutamate (K)FWMTWKALGTLGLEERVNR( 40 3 6.09 64.3 4.16E+06 2307.207 Q6ZQY3 decarboxylase-like A) protein 1 (K)SFLLNHLLQGLPGLEsGEGGR 41 3 6.05 69.4 1.04E+07 s:Phosphorylated S 2447.315 Q9ULX5 RING finger protein 112 PR(G) (K)MREGDELCGQEEAFRTWAK( Nitric oxide synthase, 42 3 6.01 70.1 1.25E+08 C:Carbamidomethylation 2313.039 P29475 K) brain

Table 13: Proteins identified in fraction 4 of cells grown with SeMet, fraction isolated based on Size Exclusion Chromatography

(SEC).

150

151

152