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Comprehensive identification of nitroxyl-reactive cysteines...
Comprehensive identification of nitroxyl-reactive cysteines in human platelet proteins by quantitative… Lin, Liwen 2011
Title Comprehensive identification of nitroxyl-reactive cysteines in human platelet proteins by quantitative mass spectrometry
Creator Lin, Liwen
Description HNO, a small compound which can be produced upon hydrolysis of Angeli's Salt, is known to be able to inhibit platelet aggregation. An important step to elucidate the mechanism of the antiaggregatory effect of HNO and define the drug's targets is the identification of HNO-reactive proteins in platelets. The sulfhydryl group is considered the major target of HNO. It can be modified into either sulfinamide or disulfide. The sulfinamide modification is directly detectable by mass spectrometry; however, cysteines converted into disulfide groups by HNO cannot be monitored this way, because the origin of the other cysteine it reacts with is unknown. The goal of this thesis was to identify HNO-reactive cysteines in human platelet proteins regardless of the eventual modification states and discover cysteines which might play key roles in platelet inhibition by HNO. In the first series of studies, a differential alkylation method was developed, evaluated on a protein model and applied on human platelets to identify HNO-reactive cysteines. The results show that 32 HNO-reactive cysteines from 18 proteins were identified. Moreover, utilizing the detectable mass shift derived from sulfinamide, the modification states (i.e., sulfinamide or disulfide) of the 32 HNO-reactive cysteines were further investigated. In the second series of studies, isotope-coded affinity tag enrichment was combined with the differential alkylation method to increase the number of HNO-reactive cysteines discovered. The results show that 159 HNO-reactive cysteines from 78 proteins were identified. Studies have shown that platelet inhibition by HNO was time-dependent; the effect was not evident after 60 minutes of incubation with HNO. Therefore, a time-dependent study was performed to identify cysteines whose HNO-induced modifications were reversed after 60 minutes of incubation. The results show that the modifications of 83 cysteines out of the 159 HNO-reactive cysteines were reversible. Based on the reactivity toward HNO, the reversibility of HNO-induced modifications and the biological functions, talin, filamin, α-actinin and integrin αIIbβ3 are proposed as potential drug targets that may play key roles in platelet inhibition by HNO.
Rights Attribution-NonCommercial-NoDerivs 3.0 Unported
Degree Master of Science - MSc
Program Chemistry
Affiliation Science, Faculty of
24-ubc_2011_fall_lin_liwen.pdf [ 1.46MB ]
COMPREHENSIVE IDENTIFICATION OF NITROXYL-REACTIVE CYSTEINES IN HUMAN PLATELET PROTEINS BY QUANTITATIVE MASS SPECTROMETRY  by  LIWEN LIN  B.Sc, Tsinghua University, 2008   A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE  in  The Faculty of Graduate Studies (Chemistry)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  August 2011  © Liwen Lin, 2011 ii  Abstract HNO, a small compound which can be produced upon hydrolysis of Angeli's Salt, is known to be able to inhibit platelet aggregation. An important step to elucidate the mechanism of the antiaggregatory effect of HNO and define the drug's targets is the identification of HNO-reactive proteins in platelets. The sulfhydryl group is considered the major target of HNO. It can be modified into either sulfinamide or disulfide. The sulfinamide modification is directly detectable by mass spectrometry; however, cysteines converted into disulfide groups by HNO cannot be monitored this way, because the origin of the other cysteine it reacts with is unknown. The goal of this thesis was to identify HNO-reactive cysteines in human platelet proteins regardless of the eventual modification states and discover cysteines which might play key roles in platelet inhibition by HNO.  In the first series of studies, a differential alkylation method was developed, evaluated on a protein model and applied on human platelets to identify HNO-reactive cysteines. The results show that 32 HNO-reactive cysteines from 18 proteins were identified. Moreover, utilizing the detectable mass shift derived from sulfinamide, the modification states (i.e., sulfinamide or disulfide) of the 32 HNO-reactive cysteines were further investigated.  In the second series of studies, isotope-coded affinity tag enrichment was combined with the differential alkylation method to increase the number of HNO-reactive cysteines discovered. The results show that 159 HNO-reactive cysteines from 78 proteins were identified. Studies have shown that platelet inhibition by HNO was time-dependent; the effect was not evident after 60 minutes of incubation with HNO. Therefore, a time-dependent study was performed to identify cysteines whose HNO-induced modifications were reversed after 60 minutes of incubation. The results show that the modifications of 83 cysteines out of the 159 HNO-reactive cysteines were iii  reversible. Based on the reactivity toward HNO, the reversibility of HNO-induced modifications and the biological functions, talin, filamin, α-actinin and integrin αIIbβ3 are proposed as potential drug targets that may play key roles in platelet inhibition by HNO. iv  Preface Ethics approval for this study was granted by UBC Clinical Research Ethics Board and informed consent was granted by donors. The Ethics Certificate Number is H07-01943.  Two papers based on Chapter 2 and Chapter 3 will be submitted for publication respectively. The anticipated authors are: • first paper (Chapter 2) Liwen Lin, Cordula Klockenbusch, Geraldine Walsh, Ru Li, Juergen Kast • second paper (Chapter 2) Liwen Lin, Cordula Klockenbusch, Ru Li, Juergen Kast  I am responsible for the following work: • identified research questions with help from the supervisor • performed all the experiments except preparation of platelets from human blood and treatment of platelets with Angeli's Salt, which was done by Dr. Geraldine Walsh and Dr. Cordula Klockenbusch • performed all the data analysis • prepared all the figures and tables except Figure 3.4, which was prepared with the help of Ru Li  v  Table of Contents Abstract .......................................................................................................................... ii  Preface ........................................................................................................................... iv  Table of Contents ........................................................................................................... v  List of Tables ............................................................................................................. viii  List of Figures ............................................................................................................... ix  Acknowledgements ........................................................................................................ x  Dedication ..................................................................................................................... xi  1  Introduction ............................................................................................................. 1  1.1  Hemostasis and thrombosis ........................................................................... 1  1.2  Platelet activation responses ......................................................................... 1  1.3  Inhibition of platelet aggregation .................................................................. 4  1.4  The role of thiol/disulfide in platelet function .............................................. 5  1.5  Nitroxyl (HNO) ............................................................................................. 6  1.5.1  Chemical properties of HNO ............................................................. 6  1.5.2  Pharmacological properties of HNO .................................................. 9  1.6  Mass spectrometry ...................................................................................... 10  1.6.1  Ion sources ....................................................................................... 10  1.6.1.1 ESI.................................................................................................... 10  1.6.1.2 MALDI ............................................................................................ 11  1.6.2  Mass analyzers ................................................................................. 11  1.6.2.1 TOF .................................................................................................. 11  1.6.2.2 Quadrupole ....................................................................................... 12  1.7  MS-based proteomics.................................................................................. 13  1.7.1  Peptide identification by MS/MS spectra ........................................ 13  1.7.2  Post-translational modifications ....................................................... 14  1.7.3  Isotope-coded affinity tag (ICAT) ................................................... 14  vi  1.8  Thesis overview .......................................................................................... 15  2  Mapping and Distinguishing HNO-induced Modifications on Cysteines in Platelet Proteins ........................................................................................................... 17  2.1  Introduction ................................................................................................. 17  2.2  Experimental section ................................................................................... 18  2.2.1  Reagents ........................................................................................... 18  2.2.2  Sample preparation .......................................................................... 19  2.2.3  LC-MS ............................................................................................. 20  2.2.4  Data acquisition and analysis ........................................................... 21  2.3  Results and discussion ................................................................................ 22  2.3.1  Principle of the differential alkylation strategy ............................... 22  2.3.2  Validation of the differential alkylation strategy on a protein model  ……………………………………………………………………..24  2.3.3  Application of the differential alkylation strategy to platelet proteins  ……………………………………………………………………..28  2.3.4  Evaluation of HNO-reactive proteins that may have functions in platelet inhibition by HNO........................................................................... 32  2.3.5  Distinction between the HNO-induced modifications: sulfinamide and disulfide ................................................................................................. 36  2.3.6  Conclusions ...................................................................................... 40  3  Identification of HNO-reactive Cysteines in Platelet Proteins by Isotope-coded Affinity Tag Enrichment and Differential Alkylation ................................................. 41  3.1  Introduction ................................................................................................. 41  3.2  Experimental section ................................................................................... 42  3.2.1  Reagents ........................................................................................... 42  3.2.2  Platelet preparation and AS treatment ............................................. 42  3.2.3  ICAT treatment ................................................................................ 43  3.2.4  LC-MS ............................................................................................. 43  vii  3.2.5  Data acquisition and analysis ........................................................... 43  3.3  Results and discussion ................................................................................ 44  3.3.1  Identification of HNO-reactive cysteines ........................................ 44  3.3.2  Identification of cysteines whose HNO-induced modifications were reversed after 60 min of incubation ............................................................. 49  3.3.3  Evaluation of HNO-reactive proteins with reversible modifications that may have functions in platelet inhibition by HNO ............................... 54  3.3.4  Conclusions ...................................................................................... 58  4  Conclusions ........................................................................................................... 60  4.1  Summary ..................................................................................................... 60  4.2  Future directions ......................................................................................... 61  References .................................................................................................................... 63  Appendices ................................................................................................................... 68  A.  Protein functions of the 18 HNO-reactive proteins identified by using IAA/NEM as alkylation reagents ......................................................................... 68  B.  Reversibility of sulfinamide by TCEP ........................................................ 71  C.  ICATL%-changes of the 82 HNO-unreactive cysteine-containing peptides among the overlapping 241 cysteines .................................................................. 73  D.  ICATL%-changes of the 159 HNO-reactive cysteine-containing peptides among the overlapping 241 cysteines .................................................................. 77  E.  Protein functions of the HNO-reactive proteins identified by using ICAT as alkylation reagents ............................................................................................... 84   viii  List of Tables Table 2.1 The modification states of the 32 HNO-reactive cysteines identified by using IAA/NEM as alkylation reagents. ...................................................... 39  Table 2.2 The seven sulfinamide-modified peptides identified by the mass shift-based method but not by the differential alkylation method. .............. 40  ix  List of Figures Figure 1.1 Stages in platelet plug formation. ......................................................... 2  Figure 1.2 Pathways that support platelet activation. ............................................ 3  Figure 1.3 A brief overview of proteins in inside-out signaling and outside-in signaling during platelet activation. ............................................................... 4  Figure 1.4 HNO-thiol reaction. .............................................................................. 7  Figure 2.1 Differential alkylation strategy for detection of HNO-reactive cysteines. ...................................................................................................................... 23  Figure 2.2 Extracted ion chromatograms of the triply charged peptide VNPCIGGVILFHETLYQK of aldolase from a) the control and b) HNO-treated model. ..................................................................................... 26  Figure 2.3 AS dose responses of the five cysteines from aldolase. ..................... 28  Figure 2.4 Cell components distribution of the 18 HNO-reactive proteins identified by using IAA/NEM as alkylation reagents. ................................. 29  Figure 2.5 AS dose responses of the 32 HNO-reactive cysteines. ....................... 30  Figure 2.7 MS/MS spectrum of the sulfinic acid-modified peptide DIC*NDVLSLLEK in protein 14-3-3 zeta/delta. ........................................ 37  Figure 3.1 Differential alkylation strategy using ICAT as alkylation reagents. ... 45  Figure 3.2 Venn diagram of peptides in the four sets of cysteines with ICATL%-changes. ........................................................................................ 47  Figure 3.3 Comparison of the numbers of HNO-reactive proteins and peptides identified by using the ICAT and IAA/NEM approaches. .......................... 48  Figure 3.4 Cell components distribution of the 78 HNO-reactive proteins identified by using ICAT as alkylation reagents. ......................................... 49  Figure 3.5 Representatives of peptides in Group A-E. ........................................ 53  Figure 3.6 Top 5 canonical pathways relevant to the 55 proteins with reversible HNO-induced modifications. ....................................................................... 54  x  Acknowledgements In the first place I would like to thank my supervisor Dr. Juergen Kast for his supervision, advice, and guidance from the initial to the final level of this research. Above all and the most needed, he provided me great encouragement and support in various ways, and enlarged my vision of science. I have also benefited by advice and guidance from Dr. Geraldine Walsh who always kindly granted me her time for answering my questions. I am much indebted to Jason Rogalski for his valuable advice in science discussion, supervision in mass spectrometry and furthermore, using his precious time to read this thesis and giving his critical comments about it. I would also like to thank Dr. Cordula Klockenbusch and Davin Carter for reading my thesis and giving me their precious opinions. Many thanks go to Shujun Lin and the rest of lab members for their support during my studies. I would also like to thank Nick Stoynov for ensuring that the FT-ICR and Orbitrap were always performing way above specifications.  I would also like to thank my friends for all of their endless encouragement and optimism. Finally, to my parents, I sincerely thank you for all of your support throughout my years of education, both morally and financially. xi  Dedication  To my parents, 1  1 Introduction 1.1 Hemostasis and thrombosis Platelets are small, smooth- and discoid-shaped anucleated blood cells, generally 2-4 µm in diameter that are produced by megakaryocytes in the bone marrow1. A healthy adult produces around 1×1011 platelets every day and the lifespan of circulating platelets is 5 to 9 days. Platelets play a fundamental role in hemostasis and thrombosis. Hemostasis is the formation of thrombi upon the damage to the endothelium of blood vessels. Conversely, thrombosis is the unwanted formation of a thrombus, which can result in events such as stroke and myocardial infarction2-4. It is important to maintain a balance between blood fluidity and rapid thrombus formation in response to injury. Thus understanding the role of platelets in both benign and pathological responses is essential, especially in designing new approaches and therapies for diseases.  1.2 Platelet activation responses The formation of a hemostatic thrombus is a complex process and involves three successive and closely integrated stages: initiation, extension and stabilization (Fig. 1.1). Upon vessel injury, extracellular matrix constituents such as von Willebrand factor (vWF) and collagen are exposed to the bloodstream. The formation of a platelet plug is initiated by collagen-vWF complex or by thrombin. Signals from these interactions act through a network of signaling pathways to enhance the adhesive and procoagulant properties of platelets. This leads to tethering of platelets at the site of vessel injury and formation of a monolayer. Extension occurs when additional platelets adhere to the monolayer and become activated, leading to the release of agonists including thrombin, thromboxane A2 (TxA2) and adenosine diphosphate (ADP).  2   Figure 1.1 Stages in platelet plug formation. 1. Initiation. Vascular injury exposes sub-endothelium and releases signaling molecules. Platelets are captured and activated via collagen-VWF complex or thrombin. Platelets adhere and spread, forming a monolayer. 2. Extension. Additional platelets are activated via the release of platelet agonists and stick to each other via fibrinogen-, fibrin-, or vWF-integrin αIIbβ3 interactions. 3. Stabilization. The platelet plug is stabilized by close contacts between platelets, along with a fibrin meshwork (shown in blue).  Interactions between platelet agonists and receptors on the platelet membrane lead to activation of phospholipase C isoforms (PLC) (Fig.1.2). The active PLC hydrolyzes phosphatidylinositol-4,5-bisphosphate (PIP2), forming inosito-1,4,5-triphosphate (IP3) and diacylglycerol (DAG). IP3 increases the cytosolic Ca2+ concentration leading to activation of phospholipase A2 (PLA2) and generation of TxA2 through cyclooxygenase (COX) and thromboxane synthase (TS)5. DAG activates protein kinase C (PKC), initiating protein phosphorylation events. In response to Ca2+ and DAG, CalDAG-GEFI, a guanine nucleotide exchange factor, activates Rap1, leading to the binding of talin to integrin αIIbβ3, the major target of these activation events. These initial platelet responses leading to the activation of integrin αIIbβ3 are known as inside-out signaling. It leads to conformational changes in extracellular domains through the action on integrin cytoplasmic domains, further increases its binding affinity to ligands, including fibrinogen, fibronectin and vWF. The binding between integrins and ligands emanates outside-in signals, triggering a number of post-ligand-binding events that include cytoskeletal reorganization, shape changes, spreading and formation of platelet aggregates and blood clots. Regarding the essential role of integrin αIIbβ3 in platelet activation, proteins positioned upstream and downstream of integrin αIIbβ3 are shown in Fig. 1.3. 3   Stabilization is the later events of platelet plug formation. At this stage, the outside-in signaling through integrin αIIbβ3 is amplified, and close contacts between adjacent platelets are formed to stabilize the platelet aggregates and prevent premature disaggregation.   Figure 1.2 Pathways that support platelet activation. Abbreviations: ADP, adenosine diphosphate; ATP, adenosine triphosphate; AA, arachidonic acid; COX, cyclooxygenase; DAG, diacylglycerol; IP3, inosito-1,4,5-triphosphate; PIP2, phosphatidylinositol-4,5-bisphosphate; PL, phospholipids; PF4, platelet factor 4; PGH2, prostaglandin H2; PAR, protease-activated receptor; PKC, protein kinase C; TxA2, thromboxane A2; TP, thromboxane; TS, thromboxane synthase; vWF, von Willebrand factor. This figure was adapted from Dr. Geraldine Walsh and modified with permission.  4   Figure 1.3 A brief overview of proteins in inside-out signaling and outside-in signaling during platelet activation. Abbreviations: ILK, integrin linked kinase; LIMS1, LIM and senescent cell antigen-like-containing domain protein 1; FAK, focal adhesion kinase; Arp2/3, actin-related protein 2/3; VASP, vasodilator-stimulated phosphoprotein.  1.3 Inhibition of platelet aggregation In vivo, endothelial cells can synthesize platelet inhibitors which primarily include nitric oxide (NO), prostacyclin (PGI2) and CD396. These inhibitors limit the size and growth of the hemostatic plug or thrombus during the initiation, extension and stabilization of platelets.  In vitro, HNO, which can be produced by Angeli's Salt (AS) upon hydrolysis at physiological conditions, has been shown to inhibit platelet aggregation in a time- and concentration-dependent manner7. Bermejo et al. showed that after two minutes of incubation of platelets with AS, the platelet aggregation was significantly inhibited with a threshold concentration of 1 µM; however, this effect was not evident when platelets were preincubated with AS for 60 minutes. The inhibition appears to be independent of the receptors involved in the platelet response. While HNO does not alter platelet glycoprotein expression (i.e., CD42b, CD41, CD61, CD29 and CD36), it does significantly reduce the expression of platelet activation markers (i.e., CD62p, CD63 and PAC-1)7. Sodium nitroprusside (SNP), a NO donor, has also been shown to 5  inhibit platelet aggregation7. Moreover, studies showed that L-cysteine reduced the antiaggregatory effect of HNO while it increased the effect of NO. This indicates a difference between HNO and NO in the mechanisms of platelet inhibition.  1.4 The role of thiol/disulfide in platelet function Cysteine is an amino acid containing a sulfhydryl group in the side-chain. It can undergo numerous reactions, such as forming disulfide bonds in proteins secreted to the extracellular medium, and binding to metals especially in enzymes, like zinc in alcohol dehydrogenase and copper in the blue copper proteins8, 9. The oxidation of cysteine can result in a number of post-translational modifications including S-nitrosylation, sulfenic acid, disulfide bond, S-glutathionylation, sulfinic acid and sulfonic acid.  The modifications on cysteines are involved in a variety of important protein activities and signaling events. The thiol-mediated modification has proved to be particularly important in the regulation of platelet responses, especially regarding the activation status of integrin αIIbβ3. It has been reported that integrin αIIbβ3 contains a redox site comprised of several unpaired cysteines, and dithiothreitiol (DTT) reduces two disulfide bonds within the cysteine-rich domain of integrin αIIbβ3, leading to a conformational change and the opening of ligand-binding sites10. Protein disulfide isomerase (PDI) is an enzyme that can catalyse isomerisation of disulfide bonds including thiol-disulfide exchange, reduction and oxidation reactions. Integrin αIIbβ3 has been predicted to have an endogenous thiol isomerase activity as a result of the presence of nine CXXC repeats in the β3 subunit, which are contained in the active site of PDI11. However, the exact role of thiol/disulfide in the regulation of integrin αIIbβ3 activation is not fully known.  Moreover, studies show that micromolar concentrations of glutathione (GSH) in the presence of agonists cause platelet aggregation; however, GSH alone in the absence of 6  agonists does not cause the aggregation. Conversely, millimolar concentrations of GSH inhibit platelet aggregation12. Platelet aggregation can also be induced by millimmolar concentrations of DTT in the absence of any agonists12. Therefore, the integrity of platelets and part of its biological functions are dependent on thiol groups, although their exact roles have yet to be determined.  1.5 Nitroxyl (HNO) Nitroxyl (HNO) has received intense attention due to its potential as a therapeutic agent. The biologically relevant chemistry and pharmacological properties of HNO are briefly described here.  1.5.1 Chemical properties of HNO From the earliest report, it was noted that the primary species of HNO at physiological pH in solution was the corresponding anion, NO−, due to deprotonation as seen  in Reaction 1: - 2 3HNO H O NO H O ++ +R                                                       (1) However, later studies showed that HNO is actually a poor acid with pKa>11, thus at physiological pH the contribution of NO− from HNO is predicted to be minimal13. The proton transfer between HNO and NO− is forbidden, as a result of the different ground states14: singlet for HNO and triplet for NO−. Therefore, HNO is likely the exclusive species present in the acid-base equilibrium of HNO/NO− in biological systems. HNO can be produced by one-electron reduction of NO, however studies have shown that the formation of HNO from NO is unlikely to happen due to NO's low reduction potential (lower than -0.4 V vs. normal hydrogen electrode)15.  The chemistry of HNO is complicated by its rapid self-consumption as described by Reaction 216, 17. 7  2 22HNO HON NOH N O H O→ = → +                                            (2) The rate constant of this reaction is approximately 8×106 M-1 S-1.13 In vivo, concentrations of HNO are unlikely to exceed low micromolar levels. At such concentrations, dimerization will be substantially inhibited as HNO favors other reactivity such as the reaction with thiols18, 19.  As shown in Fig. 1.4, the reaction of HNO with a cysteine (0) leads to an initial formation of N-hydroxysulfenamide (1) via attack of the nucleophilic sulfur atom on the electrophilic nitrogen atom of HNO. The intermediate (1) can eliminate a hydroxyl ion, followed by hydration and deprotonation to form sulfinamide (4). The sulfinamide (4) has a 31 Da mass shift from the cysteine (0), making it directly detectable by MS. The intermediate (1) can also react with a second sulfhydryl to yield a disulfide linkage (5). Due to the other cysteine residue (6) in the reaction being of unknown origin, the disulfide product will be unpredictable and heterogeneous, and therefore the cysteine (0) converted into the disulfide group (5) cannot be monitored by MS.  N OH S H R R S+ NH S+ OH R NH2 S O R NH2 RSH+HNO R'SH NH2OH + RSSR' (disulfide) 0 Da (cysteine) +31 Da (sulfinamide) OH- H2O H+ 2 3 41 5 0 6  Figure 1.4 HNO-thiol reaction.  Recent studies showed that the formation of disulfides or sulfinamides is dependent on the hydrophobicity of the environment, the availability of a local base, and the identity of the thiol substituent: in a hydrophobic environment, the formation of disulfides would become favorable; if a base is present in the local environment and with stronger 8  electron withdrawing substituents, the formation of sulfinamides would be favored20. The disulfide can be converted back to the starting thiol, as it is able to exchange with free thiols or be reduced in a biological system. The reversion of the sulfinamide has been predicted to be possible which may require enzymatic intervention21.These can lead to the reversion of thiol structures and regeneration of protein function.  Various proteins have been shown to react with HNO. The cytoplasmic concentration of GSH varies between 1-10 mM depending on the cell type22. The GSH/GSSG (i.e., glutathione disulfide) ratio is important in regulating the general redox status of a cell. High concentration (1-3 mM) of HNO has been shown to deplete GSH completely in cells23. At low concentrations, HNO is expected to be scavenged by GSH due to the high concentration of GSH in a biological system; however, recent research shows that HNO can inhibit thiol proteins even at low concentration without significantly altering the GSH/GSSG ratio24. It indicates that HNO is more reactive towards certain thiols even in the presence of a high concentration of GSH. Moreover, HNO targets proteins with different reactivity25, 26. For example, the rate constants of reactions of bovine serum albumin (BSA) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) with HNO are 6×106  M-1 S-1 and 1×109  M-1 S-1 respectively, greater than that of GSH which is 2×106  M-1 S-1.20  Among several HNO donors, Angeli's salt (sodium trioxodinitrate, AS) is the best studied and most widely used. At 4<pH<8.5, AS decomposes to produce HNO and nitrite, initiated by protonation of the dianion as shown in Reaction 3: 2 2 3 2N O H HNO NO − + −+ → +                                                       (3) It has been shown that the decay of AS decreases with elevated pH, thus stock solutions of AS are relatively stable at high pH27. At pH<4, AS converts to an NO donor, thus the acid-base conditions must be considered when using AS28. 9  1.5.2 Pharmacological properties of HNO One of the first findings revealing the potency of HNO as a drug is the anti-alcoholism ability of cyanamide, a HNO donor. The HNO generated by cyanamide was found to modify the active site cysteine thiolate and in turn inhibit the enzyme aldehyde dehydrogenase29.  HNO's effects on cardiac function have been widely reported and its potential use as a treatment for cardiac failure is promising. HNO has been shown to exert a positive, load-independent inotropic action in both normal and failing hearts30. This effect is independent and additive to β-adrenergic stimulation, strikingly different from the effect of NO, which blunts adrenergic signaling30. HNO can also stimulate the release of calcitonin gene-related peptide (CGRP), which has a positive inotropic activity; while NO does not increase CGRP31. These observations suggest that HNO and NO participate in distinct cardiovascular signaling pathways.  Exposure to the HNO donor, AS, immediately prior to ischemia exacerbates the tissue damage in isolated rat heart32. However, administration of HNO before induced ischemia protectively preconditioned the heart against the subsequent reperfusion injury to a greater extent than NO donors33. It suggests that the interactions between HNO and its biotargets are highly condition-dependent.  Despite the potential as a pharmacological treatment, HNO also exhibits toxicity such as depleting cellular GSH23. In vitro, HNO can react with O2 to generate potent oxidizing species capable of breaking DNA strands, thereby augmenting the oxidative damage34. However, whether administration of HNO at the doses needed as a clinical agent will induce such toxicity remains to be determined, as studies to date suggest that the toxicity is attributed only to high concentrations of HNO. 10  1.6 Mass spectrometry Mass spectrometry (MS) is an established analytical technology in many fields. The principle of MS is the measurement of the mass-to-charge ( /m z ) ratio of gas-phase ions. MS consists of an ion source that ionizes analyte molecules, a mass analyzer that measures /m z  ratios, and a detector that registers the number of ions at each /m z ratio value.  1.6.1 Ion sources Among many types of ion sources such as electron impact, chemical ionization, field desorption and so on, electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) are used most for protein characterization. They are categorized as "soft" ionization sources, because analyte molecules are desorbed and ionized to gas phase without dissociation.  1.6.1.1 ESI In ESI, ions in solution are liberated from the solvent matrix. Generally, sample solutions elute from a reverse-phase liquid chromatography column and then pass through a capillary at high voltage with respect to the entrance of the mass analyzer. A Taylor cone is formed at the tip of the capillary due to the high density of positive charges. The charged droplets are dispersed and move toward the counter electrode. As the solvent evaporates, the size of the droplet decreases and the charge density increases. The droplet breaks up into smaller droplets until the surface coulombic force exceed the surface tension force, the Rayleigh limit35. This process continues until each droplet contains only one analyte ion on average.  11  1.6.1.2 MALDI For MALDI, samples are prepared by mixing the analyte with a large excess of ultraviolet light-absorbing matrices. The most commonly used matrices are α-cyano-4-hydroxy-cinnamic acid (CHCA, for peptides) and sinapinic acid (for proteins). The analyte/matrix mixture is deposited onto a metallic plate, and the solvent is dried allowing the matrix and the analyte to co-crystallise. On irradiation with a focused laser beam, the energy is absorbed by the matrix and subsequently transferred to analyte molecules, which are desorbed into the gas phase. A charge transfer occurs between the matrix and analyte molecules via proton transfer.  1.6.2 Mass analyzers There are many different types of mass analyzers used in modern mass spectrometers, such as time-of-flight (TOF), quadrupole, ion trap and Fourier transform ion cyclotron resonance analyzers. They are different from each other in design and performance, each with its own strengths and weaknesses. Mass analyzers can be used alone, or put together in sequence to create a tandem mass spectrometer to take advantage of the strengths of each, such as triple quadrupoles, tandem TOFs, quadrupole TOFs and quadrupole traps.  1.6.2.1 TOF Following ion formation, ions are accelerated away from the ion source by an electric potential, pass through a field-free drift region and reach the detector. The principle of TOF is to measure the flight time of ions passing through the drift region. It is related to the /m z  value of ions as following, 1/2/( ) 2 m zt L eU = 12  where t  is the flight time, L  is the length of the drift region, m  is the ionic mass, z is the charge state, U  is the electrostatic potential and e  is the unit of charge. Thus, ions will be separated according to the /m z  ratio.  There are two main developments that improve the resolving power of TOF by compensating for energy difference of ions after ionization. The first development is accomplished by a mass reflectron. Instead of being detected after a single pass through the field-free drift region, ions are reflected back into the drift region by an electric field and then detected in the drift region. The more energetic ion penetrates deeper in the reflectron and takes a slightly longer path to the detector than the ion with the same /m z  but less kinetic energy. Thus, ions with the same /m z  can be focused at the detector and the resolution is enhanced. The second one is accomplished by delayed ion extraction. A delay is introduced between the end of the ionization pulse and the application of the extraction pulse, thus ions dissipate prior to being drawn from the ion source. When the extraction electric potential is applied, ions further away from the drift region are accelerated at higher potential than ions with the same /m z  but closer to the drift region. Thus, ions with the same /m z  will arrive at the detector at the same time.  1.6.2.2 Quadrupole The quadrupole mass analyser consists of four electronically conducting cylindrical rods, with the opposite rods being electrically connected. A combination of direct current (DC) and radio frequency (RF) voltage is applied to the rods to modulate the trajectory of ions entering the quadrupole fields. The potential values are set such that only the ion of interest has a stable trajectory through the quadrupole and other ions with lower or higher /m z  values are unstable and are ejected from the quadrupole. The quadrupole mass filter can be set to transmit a single type of ion or to scan over a wide /m z  range by stepping through many individual /m z  values. 13  1.7 MS-based proteomics A typical MS-based proteomics experiment usually consists of five steps. First, the proteins to be analyzed are isolated from cells or tissues by biochemical fractionation or affinity selection. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is often used to separate proteins with different masses. Second, the proteins are degraded into peptides by enzymes, usually by trypsin. The intact proteins can also be subjected to MS analysis without digestion. Third, the peptides are separated using single dimension or multiple dimensions of peptide separation such as high-performance liquid chromatography (HPLC). Fourth, the analyte is ionized by ESI or MALDI into small multiply or singly charged ions in the gas phase and analyzed by mass analyzers. A tandem mass spectrometric analysis (MS/MS) is usually carried out to obtain sufficient information for sequencing peptides: a precursor ion of interest is selected by the first mass analyzer; it collides with the inert gas in a collision cell, during which the kinetic energy of the precursor ion is converted into internal energy; the ion becomes excited to an unstable state and then fragments; the fragment ions are detected by the second mass analyzer and MS/MS spectra are recorded. Fifth, mass spectra are interpreted manually or by database search to identify peptides and proteins, followed by analysis of quantitative information and interpretation of protein bioinformation.  1.7.1 Peptide identification by MS/MS spectra As large numbers of MS/MS spectra are generated in proteomics experiments, manual interpretation is impractical. Database search for protein identification is a widespread tool for high throughput proteomics nowadays, which relies on the precursor mass information and fragment mass information. To make an identification theoretical MS/MS spectra of a list of known peptides which have the same mass as the precursor ion are generated from a database, and these theoretical fragmentation patterns are then compared to experimental spectra to identify peptides. Several database search 14  algorithms have been described in the literature, including SEQUEST, Mascot and X!Tandem. The major differences among them are the spectra comparison method and the scoring scheme. SEQUEST uses cross-correlation methods that correlate experimental spectra with theoretical spectra, and Xcorr is used as the score function36. Mascot matches the experimental fragments to the predicted fragments, starting with the most intense ions. The probability that the matches are random is calculated and its negative logarithm is given as a score37. One of X!Tandem's strengths is its automatic search for modified peptides, but only on proteins it has otherwise identified. X!Tandem uses hyperscore and E-value calculation as the score function38.  1.7.2 Post-translational modifications Post-translational modification (PTM) is the chemical or enzymatic modification of a protein after its translation. During PTM, chemical groups such as phosphate, carbohydrate and acetate are attached to one or more amino acids, or disulfide bonds are formed. Researches show that PTMs not only regulate individual protein structure and function, but also modulate protein-protein interaction39. For example, phosphorylation can activate a kinase or provide docking sites for the binding partner; ubiquitylation affects protein degradation; disulfide-bond formation stabilizes protein structure and is often involved in redox processes. As a PTM adds or subtracts a specific mass to/from a protein, it makes the mapping, identification and characterization of the PTM achievable by MS. By comparing the MS/MS spectra of the modified peptide with that of the original peptide, the exact modification site can therefore be determined.  1.7.3 Isotope-coded affinity tag (ICAT) The ICAT reagent consists of three components (Fig. 1.4): 1) a protein reactive group (i.e., iodoacetamide) that reacts with free cysteine residues; 2) an isotopically labeled tag in which stable isotopes are incorporated; and 3) an affinity tag, biotin, that enables 15  selective isolation of ICAT reagent-labeled peptides. The cleavable ICAT reagent has an acid-labile linker situated between the biotin and isotopic tag. The removal of the biotin tag reduces the mass of the remaining tag attached to the peptide and increases the fragmentation efficiency and ultimately the success rate of peptide identification.  Figure 1.4 Structure of the acid-cleavable ICAT reagent.  The isotopic tags contain nine either "light" (12C) or "heavy" (13C) carbon atoms. Isotopic peptides are chemically identical except a mass difference of 9 Da, therefore, they elute as pairs from a reverse-phase column. In addition, because the iodoacetamide moiety of ICAT reagents is thiol-specific, the complexity of the original peptide mixture is greatly reduced after enrichment. Mass spectrometric comparison of peptides labeled with heavy and light reagents provides a ratio of the concentration of the proteins in the original sample.  1.8 Thesis overview Thiol reactions are an important component of the discrete biochemistry of HNO. Thiols play a critical role in protein structure and function as well as in the redox balance of the cell. Therefore, platelet inhibition by HNO is much likely due to HNO-induced modifications on cysteines. It is important to identify HNO-reactive proteins that may have functions in platelet inhibition to further understand the mechanism of the antiaggregatory affect of HNO. The thiol can be modified into either sulfinamide or disulfide by HNO. So far, only the sulfinamide modification in human platelet proteins has been detected by MS, as it has a mass shift from the sulfhydryl40. 16  The current experimental challenge includes identification of the cysteine converted into disulfide, as with the other cysteine residue in the reaction being of unknown origin, the disulfide product is unpredictable and heterogeneous.  The goals of this thesis are: a) identification of HNO-reactive cysteines regardless of eventual modification states; b) distinction between the two products (sulfinamide or disulfide); c) determination of the most likely targets causing HNO-induced platelet inhibition. To achieve these goals, a differential alkylation method was developed, evaluated on a protein model and applied on platelets to identify HNO-reactive cysteines in Chapter 2. Furthermore, a mass shift-based method was utilized for identification of the sulfinamide modification. Combination of these two methods could distinguish the two products. In Chapter 3, an ICAT approach was combined with the differential alkylation method to enrich cysteines from platelet proteins and to map as many HNO-reactive cysteines as possible. A time-dependent study was also performed to identify cysteines whose HNO-induced modifications were reversed after 60 minutes of incubation with HNO for further identification of proteins that may have key functions in platelet inhibition by HNO. 17  2 Mapping and Distinguishing HNO-induced Modifications on Cysteines in Platelet Proteins 2.1 Introduction HNO has received intense attention from the research and medical communities because of its effects on many biological functions, including its ability to modulate contractility, induce vasodilatation and prevent atherosclerosis and vascular thrombosis41-43. It has been also shown that a small amount of HNO can precondition the heart against ischemia reperfusion injury33. Other pharmacological studies have shown that HNO can inhibit the thiol enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and the enzyme aldehyde dehydrogenase, such that it can be used as an anti-alcoholism treatment24, 29. HNO can also afford neuroprotection from excitotoxic assault by downregulating N-methyl-D-aspartate (NMDA) receptors44. At high concentrations, HNO was found to deplete cellular glutathione (GSH) and induce double-stranded DNA breaks, revealing the cytotoxicity of HNO23, 34. Whether the dose of HNO needed as a clinical agent will induce such toxicity remains to be determined, however, studies to date suggest that the toxicity is attributed only to high concentrations of HNO. HNO's potential as a therapeutic agent in the cardiovascular system is therefore promising.  Furthermore, there has been interest in the inhibition of platelet aggregation by Angeli's Salt (sodium trioxodinitrate, AS), which can produce HNO upon hydrolysis. It was shown that HNO inhibits the aggregation in a concentration- and time-dependent manner, and the inhibition is independent of the receptors involved in platelet aggregation. HNO does not alter platelet glycoprotein expression, but it significantly reduces the expression of platelet activation markers7. However, the exact mechanism of the inhibition is still unknown. Lack of knowledge regarding which platelet proteins 18  are reactive toward HNO hampers the study of the mechanism of platelet inhibition by HNO.  Among several HNO donors, AS is the best studied and most widely used. It releases HNO at 4<pH<8.5 and forms only NO at pH<427, 28. HNO is the one-electron reduction product of NO and a conjugate acid of NO-. It has been shown that the HNO-mediated effects are different from those of NO, probably because HNO tends to be much more thiophilic19, 30, 45, 46. Sulfhydryl groups including cysteine residues in proteins are considered the major targets of HNO19. They are modified into sulfinamide or disulfide groups. The sulfinamide modification is directly detectable by mass spectrometry; it appears as a mass shift of 31 Da from the sulfhydryl. However, cysteines converted into disulfide groups through reaction with HNO cannot be monitored in this way because the origin of the other cysteine it reacts with is unknown40. Here, we developed a differential alkylation strategy to identify HNO-reactive cysteines regardless of eventual modification state, and evaluated it on a protein model. We then applied the method to platelet proteins. The mass shift-based method was used to identify cysteines modified into sulfinamides. The combination of these two methods thus allows us to distinguish the two modification products. Investigation to identify HNO-modified cysteine targets sheds some light on the reactivity and specificity of platelet proteins towards HNO, and provides a method that can be utilized to study the mechanism of platelet inhibition by HNO.  2.2 Experimental section 2.2.1 Reagents Standard proteins including aldolase (rabbit) and lysozyme (chicken) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Angeli's salt (AS, Na2[ONNO2]) was purchased from Cayman Chemicals (Ann Arbor, MI, USA). Sequencing grade trypsin was from Promega (Madison, WI, USA). Formic acid and acetonitrile were from Fisher 19  Scientific (Waltham, MA). Sodium hydroxide (NaOH) was from MERCK KGaA (Darmstadt, Germany). Reagents for BCA Protein Assay were form Thermo Scentific (Rochford, IL, USA). All other reagents including ammonium bicarbonate (NH4HCO3), N-ethylmaleimide (NEM), DTT, L-cysteine and iodoacetamide (IAA) were from Sigma-Aldrich (St. Louis, MO, USA).  2.2.2 Sample preparation Aldolase (containing 8 free cysteines) and lysozyme (containing 4 disulfides) were combined as a protein model. Typically, 1.6 µl aldolase from a stock of 25 µg/µl aldolase in H2O and 0.18 µl lysozyme from a stock of 20 µg/µl in H2O were added to 50 µl buffer (1 M Tris pH 7.4, 0.05 M Guanidine·HCl). The mixture was treated with either AS (1-, 5-, 10-, 20- and 40-fold molar ratios of AS per cysteine per protein) from a stock of 0.1 M AS in 10 mM NaOH, or a vehicle control for 30 min at 25℃. Then the mixture was alkylated with 100-fold molar ratio of NEM for 1 h at 37℃ (reagent excess here refers to molar excess over the number of cysteines per protein). The sample was incubated with 400-fold molar ratio of L-cysteine for 1h at 37℃ to quench excess NEM. Subsequently the sample was reduced by 1000-fold molar ratio of DTT for 1h at 56℃ and alkylated with 3100-fold molar ratio of IAA for 1 h at 37℃. Finally, a tryptic in-solution digestion (30:1 (w/w) protein:trypsin) was performed overnight at 37℃.  Whole blood was drawn from the antecubital vein of healthy human volunteers into ACD buffer (15% (v/v) acid-citrate-dextrose anticoagulant, pH 6.0). Platelets were isolated by centrifugation, washed twice in CGSA buffer (10 mM trisodium citrate, 30 mM dextrose, 1 IU/ml apyrase, pH 6.5), and resuspended at physiological concentration (200-350x109/L) in ETS buffer (Tris buffer, pH 7.4, 5 mM EDTA). Platelets were treated with either AS (1 mM and 10 mM) from a stock of 1 M AS in 10 mM NaOH or a vehicle control for 2 min at room temperature. Samples were spun at 1800 rpm for 2 min at room temperature to pellet platelets. Platelet pellets were resuspended in 400 µl lysis buffer (50 mM NH4HCO3 and 1% sodium deoxycholate, 20  pH 8.0) and immediately boiled at 98°C for 5 min. Samples were spun at 13,000 rpm for 5 min at room temperature to pellet debris, and supernatants were removed and stored frozen. The concentration of platelet proteins was determined by bicinchoninic acid assay (BCA Assay).  Regarding the differential alkylation method, platelet proteins were reacted with equal amounts of NEM (1:1 w/w) for 1 h at 37℃, L-cysteine (4-fold molar ratio of NEM) for 1 h at 37℃, DTT (2.5-fold molar ratio of L-cysteine) for 1h at 56℃ and IAA (3.1-fold molar ratio of DTT) for 1 h at 37℃ successively. The platelet proteins were then digested with trypsin (30:1 (w/w) protein:trypsin) overnight at 37℃. Regarding the mass shift-based method, platelet proteins were not reacted with the alkylation reagents but were directly digested with trypsin (30:1 (w/w) protein:trypsin) overnight at 37℃ after AS treatment.  2.2.3 LC-MS Tryptic peptide mixtures of the protein model were purified using OMIX® C18 tips and reconstituted in 2% acetonitrile/5% formic acid. The peptides were separated on an Ultimate nanocapillary HPLC system equipped with a 15 cm long, 75 µm inner diameter (I.D.), 3 µm particle size reverse phase C18 column (LC Packings) using H2O: acetonitrile: formic acid as the mobile phase with a gradient elution. The peptides were then eluted into a hybrid quadrupole-TOF LC/MS/MS mass spectrometer (QStar XL; Applied Biosystems Sciex/MDS, Concord, Ont., Canada) for mass spectrometric analysis.  Tryptic peptide mixtures of the platelet proteins were purified using OMIX® C18 tips and reconstituted in 3% acetonitrile/1% trifluoroacetic acid/0.5% acetic acid. The peptides were separated on an Agilent 1100 Series nanoflow HPLC system equipped with a 15 cm long, 75 µm I.D., 3 µm particle size reverse phase (C18, Dr. Maisch, Germany) using H2O: acetonitrile: acetic acid as the mobile phase with a gradient 21  elution. The peptides were then eluted into a linear ion trap-FT-ICR (LTQ-FT Mass Spectrometer, ThermoFisher Scientific) for mass spectrometric analysis. 2.2.4 Data acquisition and analysis The raw data from the QStar XL were processed with the Mascot script of Analyst QS version 1.6b24 (Matrix Science, London, U.K.) to generate Mascot Generic Format (MGF) peak lists. Database searches were performed using the Mascot search engine (Matrix Science) against Swiss Prot Database. The following search criteria were used: trypsin cleavage specificity with up to one missed cleavage site, no fixed modifications, variable modifications of carbamidomethyl, NEM-modified cysteine and oxidized methione, ±0.15 Da peptide tolerance and ±0.4 Da MS/MS tolerance. The Quantitation Wizard of Analyst QS was used to extract ion chromatograms of specific peptides and integrate peak areas. The Analyst QS smoothing algorithm was applied to chromatograms to remove local variations, with 0.5, 1 and 0.5 as the weighting values for the preceding point, the current point and the following data point respectively.  Identification of peptides from platelet proteins was performed by extracting MGF files from the LTQ-FT data using DTASuperCharge (version 1.37) and searching against the human Swiss Prot Database using Mascot. The following search criteria were used: trypsin cleavage specificity with up to one missed cleavage site, no fixed modifications, variable modifications of carbamidomethyl, NEM-modified cysteine and oxidized methione, sulfinamide, deoxidized cysteine (sulfinic acid), ±20 ppm peptide tolerance and ±0.6 Da MS/MS tolerance. Peak areas of specific peptides were collected from the extracted ion chromatograms manually to allow for relative quantitation of different modifications. Information of protein functions was obtained from UniProt (http://www.uniprot.org/). 22  2.3 Results and discussion 2.3.1 Principle of the differential alkylation strategy To determine the specific cysteine targets of HNO, a differential alkylation strategy was developed, and is outlined in Fig. 2.1. In the HNO-treated sample (the right panel), initially the HNO-reactive cysteines are modified into either sulfinamides or exogenous disulfides (RSSR', i.e., disulfides induced by HNO). The HNO-unreactive cysteines remain as free sulfhydryl groups and are then blocked upon NEM addition. Excess NEM is quenched by L-cysteine. Addition of DTT following L-cysteine reduces both sulfinamides and exogenous disulfides to free sulfhydryls40, and all endogenous disulfides are also reduced. Addition of the second alkylation reagent, IAA, labels all the reduced cysteines (whether initially as sulfinamides, exogenous disulfides or endogenous disulfides), while the NEM-modified cysteines remain. The control sample is not treated with AS, therefore all of the sulfhydryl groups, regardless of their reactivity toward HNO, are blocked by NEM. Addition of DTT reduces all endogenous disulfides which are then labeled by IAA, while the NEM-modified cysteines remain.  23  SH SHSS SH SHSS SH SHSS SH SONH2SS -HNO S SS SONH2SS +NEM +NEM N O O SHHS SHHS SH SC H 2 CO NH 2 SC H 2 CO NH 2 +IAA +IAA SC H 2 CO NH 2 SC H 2 CO NH 2 SC H 2 CO NH 2 +DTT +DTT SH SH S N O O S N O O S N O O S N O O S N O O S N O O S N O O S N O O S N O O S N O O S N O O SH SSR' SSR' SH SC H 2 CO NH 2 Control HNO-treated +HNO  Figure 2.1 Differential alkylation strategy for detection of HNO-reactive cysteines. The proteins are treated with NEM to block free thiols, DTT to reduce sulfinamides and disulfides, and subsequently IAA to label the reduced free thiols. In the ultimate products, only HNO-reactive cysteines appear differentially labeled between the control and the HNO-treated sample. The horizontal lines indicate protein amino acid sequences.  Comparing the ultimate products of the control and the HNO-treated samples, there are three possible scenarios. First, a cysteine residue is modified by NEM in the control and by IAA in the HNO-treated sample. This indicates that the cysteine is reactive toward HNO; it is converted into sulfinamide or disulfide by HNO initially, and is reduced by DTT and alkylated by IAA subsequently. Second, a cysteine residue is modified by NEM in both samples. This indicates that the cysteine is not reactive toward HNO; it is not modified by HNO initially, and is then blocked by NEM. Third, a cysteine residue is modified by IAA in both samples. This indicates that the cysteine is involved in an endogenous disulfide bond; it is reduced upon addition of DTT and then alkylated by IAA. Only the HNO-reactive cysteine appears to be differently modified between the 24  control and the HNO-treated sample, regardless of whether it is initially modified into a sulfinamide or a disulfide by HNO. This difference is used to determine the reactivity of a cysteine towards HNO.  The peak area of a cysteine with a modification (i.e., IAA modification or NEM modification) was calculated as follows: the ion chromatograms of the cysteine-containing peptide with different charge states in the full MS scan were extracted. The peptides with up to one missed tryptic cleavage that contain this specific cysteine were all considered and their peak areas were summed up as the total peak area of this specific cysteine. To quantitatively assess the HNO-induced modification extent, its IAA-modification percentage was calculated as follows. peak area of IAA modification IAA-modfication percentage 100 peak area of IAA modification + peak area of  NEM modification  = × Theoretically the IAA-modifcation percentage for a HNO-reactive cysteine should be 0% in the control sample and 100% in the HNO-treated sample, assuming it is initially available to react quantitatively with HNO. The more reactive a cysteine is, the higher its IAA-modification percentage will be.  2.3.2 Validation of the differential alkylation strategy on a protein model The differential alkylation strategy was applied to the protein model (aldolase and lysozyme) for evaluation. Aldolase contains 8 cysteine residues, all of which are in the free sulfhydryl form in aldolase's native state; lysozyme contains 8 cysteine residues, each of which is bound into a disulfide bond in lysozyme's native state. The combination of these two proteins has both free sulfhydryls and disulfides, and therefore can be used as a good model for evaluating the strategy.  The protein model was treated with either AS (1-, 5-, 10-, 20- and 40-fold molar ratio of AS per cysteine per protein) or a vehicle control as described above. The differential 25  alkylation strategy was then applied to the samples, followed by tryptic digestion and LC-MS/MS analysis.  An AS dose response analysis of the protein model was carried out. The results show that for lysozyme at all AS concentrations, 7 cysteine-containing peptides were sequenced by MS: peptides 33-51, 40-51, 80-91, 92-114, 92-115 and 133-143, which together covered 6 cysteine residues: Cys-48, Cys-82, Cys-94, Cys-98, Cys-112 and Cys-133. In both the control and the HNO-treated samples, these cysteines were modified by IAA, because they were all involved in disulfide bonds in the protein and would therefore neither react with HNO nor NEM. Upon addition of DTT, they were reduced to sulfhydryls and then alkylated by IAA. The mass to charge ratios (m/z) of the corresponding NEM-modified peptides were searched in the full MS scan, but none were found to have ion abundances above background. This ruled out the possibility that the NEM-modified peptides were present, but not sequenced due to low abundance.  For aldolase, 11 cysteine-containing peptides were sequenced at all AS concentrations: peptides 61-87, 70-87, 174-200, 174-201, 202-208, 202-215, 260-304, 290-304, 331-342, 332-342 and 332-364, which together covered 5 cysteine residues: Cys-73, Cys-178, Cys-202, Cys-290 and Cys-339. The lack of coverage of the other cysteines (Cys-135, Cys-150 and Cys-240 of aldolase, Cys-24 and Cys-145 of lysozyme) likely resulted from the fact that the corresponding cysteine-containing tryptic peptides were not readily amenable to MS analysis due to their extreme low or high molecular weights. The ion chromatograms of the corresponding sequenced peptides were extracted and the peak areas of the IAA- and NEM-modified peptides were integrated as described before. Representative extracted ion chromatograms of the triply charged cysteine-containing peptide 70-87 (VNPCIGGVILFHETLYQK) are shown in Fig. 2.2. The peptide eluting at 102 min corresponded to peptide 70-87 with IAA-modification, whereas the peptide eluting at 108 min corresponded to the same peptide but with 26  NEM-modification. In the control, the majority (93%) of peptide 70-87 was modified by NEM (Fig. 2.2a), whereas in the HNO-treated sample (40-fold molar ratio of AS), the majority (97%) of it was modified by IAA (Fig. 2.2b). This indicates that Cys-73 was in the sulfhydryl form initially and thus available to react with HNO.  There were trace levels of IAA-modification and NEM-modification observed in the control and the HNO-treated sample respectively. The concentrations of NEM and IAA used were 100- and 3100-fold molar ratio of the number of cysteines per protein respectively, and the reaction times were both 1 h; therefore, these conditions should allow the reaction between sulfhydryl and IAA/NEM to undergo as complete as possible. The trace level of IAA-modification in the control could be due to oxidation during the experimental process: the free thiol in aldolase may become partially oxidized (e.g., into a disulfide) during the experiments, and thus later reduced by DTT and alkylated by IAA. The trace level of NEM-modification in the HNO-treated sample could be due to the incomplete reaction between HNO and free thiols, depending on the reactivity of thiols to HNO. This led to the NEM-modification of the remaining free thiols.  20 40 60 80 100 120 140 Time, min 0 2.0e3 in te ns ity  co un ts IAA-modification NEM-modification b) 20 40 60 80 100 120 140 Time, min 0 2.1e3 in te ns ity  co un ts IAA-modification NEM-modification a)  Figure 2.2 Extracted ion chromatograms of the triply charged peptide VNPCIGGVILFHETLYQK of aldolase from a) the control and b) HNO-treated model.  27  In the similar fashion, peak areas of other IAA- and NEM-modified peptides were calculated and the IAA-modification percentages of the corresponding cysteines are shown in Fig. 2.3. In the control, the IAA-modification percentages were low (12%-29%) as the cysteines were initially in the free sulfhydryl forms and thus reacted with NEM. The difference of the IAA-modification percentages among the 5 cysteines in the control was probably due to different oxidation extents of these 5 cysteines during the experimental process. Upon AS treatment, the IAA-modification percentage increased dramatically (93%-97%) as sulfhydryls were converted into either sulfinamide or disulfide groups by HNO initially, and then reduced by DTT and alkylated by IAA subsequently. There were no noticeable differences in the IAA-modification percentages among different AS doses, suggesting that the reaction between sulfhydryl and HNO was saturated at the low AS dose (i.e., equal molar ratio of AS per cysteine per protein). In HNO-treated samples, the percentages did not increase to 100% as in theory, probably due to the incomplete reaction between sulfhydryl and HNO, even at the high AS dose.  The application of the differential alkylation strategy on the protein model validated it as a method to identify HNO-reactive cysteines, becasue the IAA-modification percentages of the free cysteines from aldolase increased upon AS treatment. The method was then used to study HNO-induced modifications in platelets.  28  0% 20% 40% 60% 80% 100% 0 1 5 10 20 40 AS dose (fold molar ratio of per Cys per protein) IA A -m od ifi ca tio n Pe rc en ta ge Cys73 Cys178 Cys202 Cys290 Cys339 ≈ ≈ ≈ ≈  Figure 2.3 AS dose responses of the five cysteines from aldolase. Peptides 61-87, 70-87, 174-200, 174-201, 202-208, 202-215, 260-304, 290-304, 331-342, 332-342 and 332-364 of aldolase were sequenced, covering Cys-73, Cys-178, Cys-202, Cys-290 and Cys-339. Data are mean ± standard error of the mean (SEM) from n=3 repeats.  2.3.3 Application of the differential alkylation strategy to platelet proteins Platelets were isolated from whole blood and treated with either AS or a vehicle control, and the differential alkylation strategy was then applied. The platelet proteins were tryptic-digested overnight and analyzed by LC-MS/MS.  Peptides present with both IAA- and NEM-modifications which were MS/MS sequenced were chosen for further analysis. Peak areas of the IAA- and NEM-modified peptides at different AS doses (i.e., 0, 1 and 10 mM) were integrated and the corresponding IAA-modification percentages were calculated. The difference of the IAA-modification percentages between 0 mM and 10 mM AS doses was used to determine the modification extent on a cysteine. This led to the identification of 32 HNO-reactive cysteines from 18 proteins (Appendix A).  29  Assignment of the HNO-reactive proteins to cell component was based on UniProt and Gene Ontology. The number of proteins in each cell component category was counted and the categories were listed (Fig. 2.4). Categories were non-exclusive: the same protein may be listed in more than one category depending on its gene ontology. There are 5 categories with proteins more than two, and cytosol is the cell component with the highest number of HNO-reactive proteins. Most of the 18 proteins are abundant proteins in platelets, as MS-based proteomics is biased toward highly abundant proteins if no enrichment step is performed.  3 10 4 3 4 0 2 4 6 8 10 12 N um be r of  p ro te in s actin cytoskeleton cytosol extracellular region focal adhesion platelet alpha granule lumen  Figure 2.4 Cell components distribution of the 18 HNO-reactive proteins identified by using IAA/NEM as alkylation reagents. Categories with proteins less than 3 were not listed.  The dose response curves of the various peptides were grouped together based on their similar response trends. Cysteines in Fig. 2.5a showed a dramatic increase of IAA-modification percentage upon AS treatment; the percentage was 0%-40% at 0 mM and it increased to 100% at 1 mM, within which concentration the platelet inhibition by HNO has been shown to occur. Cysteines in Fig. 2.5b also showed a dramatic increase upon AS treatment; however, the percentage increased to 100% only at the higher dose (i.e., 10 mM). The increase of the modification extent between 1 mM and 10 mM may be related to the cyototoxic effects of AS, known to occur at high mM concentrations, and therefore may not be physiologically relevant19. Cysteines in Fig. 2.5c-d showed a 30  moderate increase of IAA-modification percentage upon AS treatment; the percentage was 40%-75% at 0 mM, increasing to 100% at 1 mM (Fig. 2.5c) or 10 mM (Fig. 2.5d). The IAA-modification percentages of cysteines in Fig. 2.5e did not increase to 100% even at the higher dose (10 mM) and were grouped together. Cysteines in Fig. 2.5f showed a slight increase of IAA-modification percentage upon AS treatment; the percentage started at 75%-92% at 0 mM, increasing to 100% at 10 mM.  0% 50% 100% 0 1 10 AS(mM) (c) TLN1(722-741) TLN1(1920-1933) FLNA(810-828) FLNA(1720-1725) VINC(1047-1055) UBE2N(86-92) 0% 50% 100% 0 1 10 AS (mM) (a) TLN1(944-957) FLNA(468-484) FLNA(1152-1162) HSP7C(602-609) 0% 50% 100% 0 1 10 AS(mM) (b) FLNA(2368-2387) GELS(669-675) FIBG(365-382) 0% 50% 100% 0 1 10 AS(mM) (e) FLNA(1008-1019) VINC(319-326) TPIS(207-219) 0% 50% 100% 0 1 10 AS(mM) (d) FLNA(1247-1272) G3P(235-248) ACTN1(479-492) ACTN1(773-794) 1433Z(92-103) HSP7C(574-583) HBB(84-96) 0% 50% 100% 0 1 10 AS(mM) (f) MYH9(566-576) ACTN1(148-156) ACTN1(332-344) URP2(114-129) COF1(35-44) CXCL7(104-112) FLNA(2150-2165) FIBB(314-328) ITA2B(156-170)  Figure 2.5 AS dose responses of the 32 HNO-reactive cysteines. IAA-modification percentages are given as mean ± SEM from n=3 technical repeats.  IA A -m od ifi ca tio n pe rc en ta ge  IA A -m od ifi ca tio n pe rc en ta ge  IA A -m od ifi ca tio n pe rc en ta ge  31  Theoretically, the IAA-modification percentage for a HNO-reactive cysteine should be 0% at 0 mM. However, for some cysteines in Fig. 2.5, the IAA-modification percentages at 0 mM were not 0%. This is likely due to oxidation (e.g., as disulfide bond) either in platelets in vivo or during the experimental process. If a free cysteine is partially oxidized initially, it cannot be blocked by NEM but would then be reduced by DTT and alkylated by IAA, leading to a non-0% IAA-modification percentage at 0 mM. According to the scheme, as long as the cysteine is HNO-reactive, the IAA-modification percentage will increase upon AS treatment, even though it is partially oxidized initially. Therefore, the difference of the IAA-modification percentages was used to estimate the modification extent of a cysteine.  The results show that the 18 HNO-reactive proteins had different responses to HNO. HNO was released when AS was added to platelets. As HNO penetrated the membrane, a concentration gradient was likely formed. The extracellular proteins encountered HNO relatively early at a higher gradient, thus they would be more likely to be modified at low AS dose. However, this study showed that integrin αIIbβ3, a membrane protein, had a lower modification extent than some intracellular proteins (e.g., gelsolin and filamin). This indicates that the relative reactivity of a protein towards HNO needs to be considered besides the protein location. Although the intracellular protein encounters HNO at a lower gradient, it may have a higher modification extent if it is more reactive to HNO. Moreover, as a protein may distribute in more than one compartment in the cell, it is possible that only the protein in some specific compartment is involved in the HNO response. Therefore, the modification extent of a HNO-reactive cysteine in compartments involved in the HNO response could be masked by the same cysteine in compartments not involved in the HNO response. The increase of IAA-modification percentage upon AS treatment is an average of the modification extent on a cysteine of a protein in the whole cell. Thus, cysteines grouped as slightly-reactive ones also need to be considered for further analysis. 32  It is noteworthy that within one protein (i.e., talin-1, filamin-A, vinculin, α-actinin-1), cysteines in different positions showed various responses to HNO. For example, the difference of the IAA-modification percentages of Cys-732, Cys-956 and Cys-1927 in talin-1 between 0 mM and 1 mM is 0.68, 0.36 and 0.5 respectively, indicating different HNO-modification extents. The different responses of cysteines within one protein may be related to the surface accessibility of cysteines in the protein and their relative reactivity towards HNO.  2.3.4 Evaluation of HNO-reactive proteins that may have functions in platelet inhibition by HNO 32 HNO-reactive cysteines were identified, however, cysteines that have specific functions in platelet inhibition by HNO can only account for a small fraction. As mentioned before, integrin αIIbβ3 plays essential roles in platelet activation. Therefore, the roles of the HNO-reactive proteins which are involved in integrin αIIbβ3 signaling pathway were discussed to map proteins that may play key roles in the antiaggregatory effect of HNO.  Integrin αIIbβ3 is a cysteine-rich protein. It was initially thought that it had no free sulfydryls and all cysteines were disulfide-bonded, but several groups have recently shown that free sulfhydryls are indeed present in integrin αIIbβ347-49. Our results show that the IAA-modification percentage of Cys-159 in integrin αIIb increased from 88% at 0 mM to 100% at 10 mM. This indicates that most of it was already disulfide-bonded and the free sulfhydryl form of it got modified. For Cys-504 and Cys-718 in integrin αIIb, only their IAA-modified forms were found, either in the control or the AS treated samples. The corresponding m/z ratios of their NEM-modified peptides were searched in the full MS scan, but none were found to have ion abundances above background. It indicates that Cys-504 and Cys-718 were initially disulfide-bonded. Research has shown that integrin αIIbβ3 activation involves disulfide bond alternation and the pattern 33  of sulfhydryls in integrin αIIbβ3 varies depending on the integrin activation state, with more free cysteines in activated αIIbβ3 than in resting αIIbβ348.  Studies have shown that the integrin β tail is directly accessible to some intracellular proteins including talin, kindlin, α-actinin, myosin and filamin. Cys-732, Cys-956 and Cys-1927 of talin-1 were HNO-reactive. Talin is a major actin-binding protein, involved in connection of major cytoskeletal structures to plasma membrane. Talin binds to the integrin β3 tail via a variant of the classical PTB domain (phosphotyrosine-binding domain)-NPxY interaction, disrupting the interaction of integrin αIIb and β3. This binding of talin was shown to be the final common step in integrin αIIbβ3 activation, irrespective of the platelet-activating stimulus and signaling pathways. Mutations in either talin or integrin β3 tail were shown to disrupt the complex formation, and thus inhibit integrin activation. In the absence of talin, megakaryocytes do not activate integrins in response to agonist stimulation50, 51.  The IAA-percentage of Cys-128 in kindlin-3 increased from 77% at 0 mM to 100% at 10 mM, and it was grouped as a slightly HNO-reactive cysteine. Kindlin-3 is restricted to hematopoietic cells and is particularly abundant in megakaryocytes and platelets. The PTB-like subdomain within the kindlin FERM (four-point-one, ezrin, radixin, moesin) domain has high homology with that of talin. Kindlin binds to the second NPxY motif in integrin β3 tail whereas talin binds to the first motif52, 53. Studies have shown that inhibition of the binding between kindlin and integrin inhibited integrin activation, and platelets lacking kindlin-3 cannot activate integrins despite normal talin expression. Kindlin-3 is also required for integrin αIIbβ3-dependent outside-in signaling.  The IAA-percentage of Cys-480 and Cys-774 in α-actinin-1 increased from 43% and 59% at 0 mM to 97% and 100% at 10 mM respectively. They were grouped as moderately HNO-reactive cysteines. The IAA-percentage of Cys-154 and Cys-332 in 34  α-actinin-1 increased from 78% and 81% at 0 mM to 100% at 10 mM. They were grouped as slightly HNO-reactive cysteines. α-actinin is well known to function in integrin outside-in signaling events: it co-localizes with integrin in focal adhesions and is found to be localized along stress fibers. Interestingly, a recent study showed that the interaction between α-actinin and integrin β2 could modulate integrin affinity54. Moreover, Tomiyama et al. recently showed that α-actinin was associated with resting αIIbβ3 in platelets; α-actinin dissociated from integrin αIIbβ3 when the integrin was activated, whereas, α-actinin reassociated with integrin αIIbβ3 when the integrin activation signaling dwindled. It suggested that α-actinin may play a potential role as a negative regulator of integrin αIIbβ3 in resting platelets and in regulating αIIbβ3 through inside-out signaling55.  Cys-478, Cys-483, Cys-810, Cys-1018, Cys-1157, Cys-1260, Cys-1723, Cys-2160 and Cys-2378 in filamin-A showed various responses to HNO. Filamin promotes branching of actin filaments, links actin filaments to membrane glycoproteins and also acts as a mechanical link between integrins and the cytoskeleton. Filamin can also bind to the integrin β3 tail, and the binding was found to integrate integrin signaling and reorganization of the actin cytoskeleton. Studies also revealed that filamin- and talin-binding sites overlap in the integrin β3 tail and the two proteins compete for binding to integrin. It suggests that filamin-integrin interactions may impact talin-dependent integrin activation.  The IAA-percentage of Cys-569 in myosin-9 increased from 79% at 0 mM to 100% at 10 mM, and it was grouped as a slightly HNO-reactive cysteine. Myosin comprises a family of ATP-dependent motor proteins and is responsible for part of the actin-based motility. It also binds directly to the integrin β3 tail, but this interaction was found to be dependent on the phosphorylation of the tyrosin resiudes in the integrin β3 tail following platelet aggregation. Vinculin also contained HNO-reactive cysteines as shown in Fig. 2.5. Vinculin is a membrane-cytoskeletal protein in focal adhesion 35  plaques that is involved in the linkage of integrin adhesion molecules to the actin cytoskeleton. The amino-terminus of vinculin binds to talin and the carboxy-terminal end can bind to actin. The IAA-modification percentage of Cys-365 in fibrinogen γ chain increased from 0% at 0 mM to 93% and 100% at 1 mM and 10 mM respectively, thus Cys-365 was highly responsive towards HNO. However, the modification extent of Cys-316 in fibrinogen β chain did not increase much upon AS treatment: the difference of the IAA-modification percentage is 10% between 0 and 10 mM. Fibrinogen is the primary ligand of integrin. It binds to activated intergrin αIIbβ3 on the platelet surface, leading to the formation of platelet aggregates.  Bermejo et al. showed that HNO does not affect the platelet membrane glycoprotein expression, but it inhibits the expression of the platelet activation markers (i.e., CD62, CD63 and PAC-1). This suggests that the inhibition by HNO may be mediated through a signaling pathway that inactivated integrin αIIbβ3, as suggested by other groups48, 56. Therefore, proteins positioned downstream of integrin αIIbβ3 including myosin and vinculin are unlikely to function as the targets of HNO in the platelet inhibition, albeit they may contribute to or even amplify the effect. Fibrinogen is also unlikely to be relevant in HNO-induced platelet inhibition, as it is stored in platelets and released upon activation, which is a result of the outside-in signaling events.  Talin is positioned upstream of integrin αIIbβ3. Although the HNO-reactive cysteines in talin (i.e., Cys-732, Cys-956 and Cys-1927) do not lie in the FERM domain (86-403), their HNO-induced modifications may alter the confirmation of talin, and further interfere with the binding site exposure and the interaction with integrin β3 tail, as thiols/disulfides have been shown to play key roles in protein structure. Studies have shown that talin is uniformly distributed throughout the cytoplasm in resting platelets, while in activated platelets talin is concentrated at the cytoplasmic face of the plasma membrane57. The disulfide formation with other proteins may immobilize talin and hamper its movement to the membrane to bind integrin αIIbβ3, thereby influencing the 36  downstream effectors. Therefore, based on the reactivity toward HNO and the role in integrin αIIbβ3 activation, talin is considered as the most likely protein candidate to function in the HNO-induced platelet inhibition. Kindlin, filamin, α-actinin and integrin αIIb contain HNO-reactive cysteines; kindlin and filamin can bind to integrin β3 and can be involved in inside-out signaling, and α-actinin has also been suggested to play a potential role in regulating integrin αIIbβ3 by inside-out signaling. Therefore, these 4 proteins should also be considered as potential candidates.  2.3.5 Distinction between the HNO-induced modifications: sulfinamide and disulfide The differential alkylation method alone cannot distinguish between sulfinamide and disulfide formed by HNO, because both of them are reducible by DTT and appear as IAA-modifications in ultimate products. The sulfinamide has a 31 Da mass shift from the sulfhydryl. In the previous work in our lab, complete conversion of sulfinamide to sulfinic acid was observed (Fig. 2.6), leading to a neutral loss (NL) of 66 Da (HS(O)OH)40. Therefore, the 32 Da mass shift was utilized to determine the sulfinic acid modification in the present study, thereby identifying the sulfinamide-modified cysteines in platelets. To achieve this aim, platelet proteins treated with or without AS were tryptic-digested overnight without application of the differential alkylation method and were analyzed by LC-MS/MS.  Figure 2.6 Conversion of sulfinamide to sulfinic acid.  Among the 32 HNO-reactive cysteines identified by the differential alkylation method, four were determined to be modified into sulfinamides by the mass shift-based method 37  (Cys-732 in talin-1, Cys-1157 in filamin-A, Cys-94 in 14-3-3 zeta/delta and Cys-94 in hemoglobin β subunit). A representative MS/MS spectrum of peptide DIC*NDVLSLLEK with sulfinic acid modification in protein 14-3-3 zeta/delta is shown in Fig. 2.7. The doubly-charged precursor ion was fragmented to produce b-ions and y-ions on both sides of the cysteine (b2/b3 and y9/y10 in Fig. 2.7), with a mass difference of 135.15 Da between b-ions (b2/b3) and 135.12 Da between y-ions (y9/y10), which corresponds to the mass of a sulfinic acid-modified cysteine. A neutral loss of 66 Da (HS(O)OH) from the sulfic acid-modification on the precursor ion was also observed. Hence, this cysteine was confirmed to be sulfinamide-modified by HNO initially.  200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 m/z 0 2300 In te ns ity b2 b3 b62+ b11 y10 b11-H2O b10-H2O y9 y10-H2O b8 y8 b8-H2O y7 y5 y4 y3 y2 [M+2H-NL-H2O]2+ [M+2H-2H2O]2+ b9-H2O D I C* N D V L S L L E K b2 b3 b8 b9b10 b11 y2y3y4y5y7y8y9y10 Cys-S(O)OH b62+  Figure 2.7 MS/MS spectrum of the sulfinic acid-modified peptide DIC*NDVLSLLEK in protein 14-3-3 zeta/delta.  The other 28 cysteines that were not confirmed by MS/MS as being sulfinamide-modified were more likely disulfide-modified. However, it is possible that the sulfinic acid modifications of these 28 cysteines existed but were not sequenced by MS/MS due to low abundance. Therefore, the corresponding m/z ratios of the sulfinic acid-modified peptides were searched in the full MS scan. For the 19 cysteines (Table 38  2.1), no peaks corresponding to the sulfinic acid modifications were found to have ion abundances above background, thus they were considered as being disulfide-modified. For the other 9 cysteines, peaks that may correspond to the sulfinic acid modifications were observed in MS level, thus they may be sulfinic acid-modified but further targeted MS/MS fragmentation is needed to confirm this. 39  Table 2.1 The modification states of the 32 HNO-reactive cysteines identified by using IAA/NEM as alkylation reagents. Modification state Peptide sulfinamide 1433Z_DICNDVLSLLEK (92-103) FLNA_AHVVPCFDASK (1152-1162) HBB_GTFATLSELHCDK (84-96) TLN1_VVAPTISSPVCQEQLVEAGR (722-741) disulfide ACTN1_CQLEINFNTLQTK (332-344) ACTN1_ICDQWDNLGALTQK (479-492) CXCL7_ICLDPDAPR (104-112) FIBB_NYCGLPGEYWLGNDK (314-328) FIBG_CHAGHLNGVYYQGGTYSK (365-382) FLNA_SPYTVTVGQACNPSACR (468-484) FLNA_CAPGVVGPAEADIDFDIIR (810-828) FLNA_IVGPSGAAVPCK (1008-1019) FLNA_YVICVR (1720-1725) FLNA_VHSPSGALEECYVTEIDQDK (2368-2387) GELS_LFACSNK (669-675) HSP7C_CNEIINWLDK (574-583) HSP7C_VCNPIITK (602-609) ITA2B_TPVGSCFLAQPESGR (156-170) MYH9_ADFCIIHYAGK (566-576) TLN1_VQELGHGCAALVTK (1920 - 1933) TPIS_IIYGGSVTGATCK (207-219) UBE2N_ICLDILK (86-92) VINC_TNLLQVCER (1047-1055) cannot be confirmed ACTN1_EGLLLWCQR (148-156) ACTN1_ACLISLGYDIGNDPQGEAEFAR (773-794) COF1_AVLFCLSEDK (35-44) FLNA_LQVEPAVDTSGVQCYGPGIEGQGVFR (1247-1272) FLNA_APSVANVGSHCDLSLK (2150-2165) G3P_VPTANVSVVDLTCR (235-248) TLN1_ASAGPQPLLVQSCK (944-957) URP2_ASFSQPLFQAVAAICR (114-129) VINC_REILGTCK (319-326)  Based on the mass shift-based method, besides the four cysteines (Cys-732 in talin-1, Cys-1157 in filamin-A, Cys-94 in 14-3-3 zeta/delta and Cys-94 in hemoglobin β subunit), there were seven other cysteines identified as being sulfinamide-modified (Table 2.2). However, they were not determined as HNO-reactive cysteines by the 40  differential alkylation method: because the IAA- and/or NEM-modifications of these peptides were not identified by MS/MS sequencing due to low abundance, the IAA-modification percentage cannot be calculated and the reactivity of these cysteines toward HNO cannot be determined.  Table 2.2 The seven sulfinamide-modified peptides identified by the mass shift-based method but not by the differential alkylation method. Protein Sequence TLN1 AGALQCSPSDAYTK (1934-1947) QELAVFCSPEPPAK (2155-2168) FLNA VQVQDNEGCPVEALVK (709-724) HBB LLGNVLVCVLAHHFGK (106-121) LDHA VIGSGCNLDSAR (158-169) MYH9 VEDMAELTCLNEASVLHNLK (83-102) TBA4A TIGGGDDSFTTFFCETGAGK (41-60)  2.3.6 Conclusions To identify HNO-reactive cysteines regardless of the eventual modification states, a differential alkylation method was developed, evaluated on a protein model and applied on platelets. The results show that 32 HNO-reactive cysteines from 18 proteins were identified. Furthermore, the mass shift-based method was utilized to identify the sulfinamide modification. The results show that 4 out of the 32 cysteines were modified into sulfinamides and 19 cysteines were modified into disulfides. The modification states of the remaining 9 cysteines cannot be confirmed due to the lack of corresponding MS/MS fragmentations. Based on the reactivity toward HNO and the biological functions, talin, kindlin, filamin, α-actinin and integrin αIIbβ3 are proposed as the potential candidates that may play key roles in platelet inhibition by HNO. 41  3 Identification of HNO-reactive Cysteines in Platelet Proteins by Isotope-coded Affinity Tag Enrichment and Differential Alkylation 3.1 Introduction HNO has received intense attention because of its effects on many biological functions, including its ability to inhibit platelet aggregation7. Cysteines in proteins are the major targets of HNO, which can be modified into either sulfinamide or disulfide groups. In order to elucidate the mechanism of HNO's effects on platelet aggregation, an important step is the identification of HNO-reactive cysteines in platelet proteins. A differential alkylation strategy was developed previously to identify HNO-reactive cysteines and quantify the modification extent.  In this chapter, an isotope-coded affinity tag (ICAT) approach was applied. The advantage of ICAT is that the enrichment of cysteine-containing peptides and the co-elution of light and heavy isotopic ICAT labeled peptides decrease the dynamic range and increase the number of HNO-reactive cysteines identified, especially those in low abundant proteins. Studies have shown that after 2 min of incubation, Angeli's Salt (AS) inhibits platelet aggregation in a dose-dependent manner, with a threshold concentration of 1 μM7. Therefore, a dose study was performed with 3 μM and 1 mM AS, which were lower than the concentrations used in Chapter 2 (i.e., 1 mM and 10 mM). Moreover, the inhibition effects have been shown to be not evident after 60 min of incubation with 3 μM AS7. Therefore, a time-dependent study was performed to identify cysteines whose HNO-induced modifications could be reversed after 60 min. This approach helped to discover proteins which may be involved in platelet inhibition by HNO. 42  3.2 Experimental section 3.2.1 Reagents The ICAT reagent kit was obtained from Applied Biosystems Sciex (Foster City, CA, USA). Angeli's salt (AS, Na2[ONNO2]) was purchased from Cayman Chemicals (Ann Arbor, MI, USA). Formic acid, methanol (MeOH), acetic acid and acetonitrile (ACN) were from Fisher Scientific (Waltham, MA, USA). Sodium hydroxide (NaOH) was from MERCK KGaA (Darmstadt, Germany). Reagents for BCA Protein Assay were form Thermo Scientific (Rochford, IL, USA). All other reagents including ammonium bicarbonate (NH4HCO3), L-cysteine and α-cyano-4-hydroxycinnamic acid  (CHCA) were from Sigma-Aldrich (St. Louis, MO, USA). Amicon Ultra-0.5 mL centrifugal filters were from Millipore (Billerica, MA, USA).  3.2.2 Platelet preparation and AS treatment Whole blood was drawn from the antecubital vein of healthy human volunteers into ACD buffer (15% (v/v) acid-citrate-dextrose anticoagulant, pH 6.0). Platelets were isolated by centrifugation, washed twice in physiological buffer (10 mM trisodium citrate, 30 mM dextrose, 1 IU/ml apyrase, pH 6.5), and resuspended at physiological concentration (3.5x108/ ml) in ETS buffer (Tris buffer, pH 7.4, 5 mM EDTA). Platelets were preincubated for 8 min at 37°C and were treated with either AS (3 µM and 1 mM) from a stock of 1 M AS in 10 mM NaOH or a vehicle control for 2 min or 60 min at 37°C. Samples were spun at 3000 rpm for 2 min at room temperature to pellet platelets. Platelet pellets were resuspended in 200 µl lysis buffer (50 mM NH4HCO3, 1% sodium deoxycholate, pH 8.0) and immediately incubated at 99°C for 5 min. Samples were spun at 14,000 rpm for 5 min at 4°C to pellet debris, and supernatants were removed and stored frozen at -80 °C. The concentration of platelet proteins in the sample solution was determined by BCA Protein Assay. 43  3.2.3 ICAT treatment Platelet proteins (100 µg) were treated with heavy isotopic ICAT reagent (ICATH, 0.2 µmol) for 2 h at 37℃. The sample was then incubated with L-cysteine (0.4 µmol) for 40 min at 37℃  to quench ICATH. Centrifugal filters were used to remove excess L-cysteine. Subsequently the sample was reduced by tris(2-carboxyethyl)phosphine (TCEP, 0.1 µmol) for 2 min at 99℃ and alkylated with light isotopic ICAT reagent (ICATL, 0.2 µmol) for 2 h at 37℃. The sample was then incubated with L-cysteine (0.4 µmol) for 40 min at 37℃ to quench ICATL, and was digested with trypsin (30:1 (w/w) protein:trypsin) for 15 h at 37°C. Peptides were purified by a cation exchange cartridge and ICAT-labeled peptides were isolated by an avidin affinity cartridge. The sample was dried, resuspended in the cleavage reagent and incubated for 2 h at 37°C to remove the biotin tag. The acid-cleaved peptides were then dried and suspended in 5% formic acid for LC-MS/MS.  3.2.4 LC-MS Peptide samples were purified by solid phase extraction on C18 stage tips. Peptides were separated on an Agilent 1200 Series nanoflow HPLC system equipped with a 2 cm long, 100 µm I.D., 3 µm beads packed trap column (Aqua C18, Phenomenex, CA, USA), a 50 µm I.D., 3 µm beads packed analytical column (C18, Dr. Maisch, Germany) and a 20 μm I.D. fused silica gold coated spray tip, using H2O: ACN: acetic acid as the mobile phase with a gradient elution. The peptides were then eluted into a linear-trapping quadrupole-Orbitrap mass spectrometer (LTQ-Orbitrap Velos, ThermoFisher Scientific) for mass spectrometric analysis.  3.2.5 Data acquisition and analysis The raw data from the LTQ-Orbitrap were processed with Proteome Discoverer to generate MGF files. The MGF files were searched against the human Swiss Prot Database using Mascot. The following search criteria were used: trypsin cleavage 44  specificity with up to one missed cleavage site, no fixed modifications, variable modifications of oxidized methionine, ICATL- and ICATH-labeled cysteines, ±20 ppm peptide tolerance and ±0.6 Da MS/MS tolerance. The database search result file was associated with the corresponding .RAW data file in MSQuant (v1.4) (http://msquant. sourceforge.net/) for peptide quantitation in an automated fashion. Information of protein functions was obtained from UniProt (http://www.uniprot.org/).  3.3 Results and discussion 3.3.1 Identification of HNO-reactive cysteines As mentioned before, the incubation of platelets with AS for 2 min significantly inhibited platelet aggregation7. It was proposed that HNO-induced modifications on cysteines might alter the conformation of key proteins and disulfide formations with other proteins might immobilize key proteins, thereby hampering protein interaction and signal transduction. However, the inhibition effect has been shown to be not evident when platelets were incubated with 3 μM AS for 60 min7. It is proposed that the HNO-induced modifications on cysteines disappear after 60 min of incubation. Sulfinamides can convert to sulfinic acids upon hydrolysis, and studies have shown that oxidation of certain protein thiols to sulfinic acids can be reversed by specific ATP-dependent enzymes21. Therefore, the possibility exists that sulfinamides could be recycled to the initial thiols following hydrolysis to sulfinic acids. Moreover, disulfides can also be reversed to free thiols by proteins such as protein disulfide isomerase (PDI), thioredoxin (TRX) and NADPH-dependent reductase58.  In this series of studies, platelets were first treated with 0 μM, 3 μM or 1 mM AS for 2 min or 60 min respectively. The differential alkylation method was then applied using ICAT as the alkylation reagents (Fig. 3.1): the samples are treated with ICATH to block free thiols, TCEP to reduce sulfinamides and disulfides (Appendix B), and ICATL to label the reduced free thiols subsequently. After 2 min of incubation with AS, a 45  HNO-reactive cysteine is modified into either sulfinamide or disulfide and is ultimately ICATL-labeled; while in the control, it is ultimately ICATH-labeled. After 60 min of incubation, a cysteine whose HNO-induced modifications are reversible will be recycled to the initial thiol and is ultimately ICATH-labeled; while in the control, it is also ICATH-labeled. Therefore, the modification extents at 3 μM-2 min and 1 mM-2 min were used to identify HNO-reactive cysteines, and the modification extents at 3 μM-60 min and 1 mM-60 min were used to further identify cysteines whose HNO-induced modifications were reversed.   Figure 3.1 Differential alkylation strategy using ICAT as alkylation reagents. The proteins are treated with ICATH to block free thiols, TCEP to reduce sulfinamides and disulfides, and ICATL to label the reduced free thiols. The horizontal lines indicate protein amino acid sequences. It is assumed that the HNO-induced modifications on this cysteine are reversed after 60 min of incubation with HNO.  MSQuant was used to integrate peak areas of ICATL- and ICATH-labeled peptides and determine ICATH:ICATL ratios. The ratio was converted to the ICATL-modification percentage (ICATL%) as follows, similar to the calculation of the IAA-modification percentage in Chapter 2. 46  L H L 1ICAT %= ICAT1+ ICAT  Six datasets of cysteines with ICATL% were produced from the six experimental points (0 μM, 3 μM and 1 mM at 2 min and 60 min respectively). As discussed before, the difference of ICATL-modification percentage (ICATL%-change) between the AS treated sample and the control sample represents the modification extent. Therefore, four sets of cysteines with ICATL%-changes can be generated from the six datasets of cysteines with ICATL% as follows. L L L L L L L L L ICAT %-change at 3 μM-2 min=ICAT % at 3 μM-2 min - ICAT % at 0 μM-2 min ICAT %-change at1 mM-2 min=ICAT % at 1 mM-2 min - ICAT % at 0 μM-2 min ICAT %-change at 3 μM-60 min=ICAT % at 3 μM-60 min - ICAT % L L L  at 0 μM-60 min ICAT %-change at1 mM-60 min=ICAT % at 1 mM-60 min - ICAT % at 0 μM-60 min  Some ICAT-labeled peptides were not MS/MS sequenced in both the AS treated sample and the control sample, due to the variability from experiment to experiment. Thus, the ICATL% of these peptides can be calculated at one experimental point but not the other, and the ICATL%-change between AS treatment and the control is not available. As shown in Fig. 3.2, the numbers of cysteines with ICATL%-changes in the four sets are 471, 337, 414 and 433 at 3 μM-2 min, 1 mM-2 min, 3 μM-60 min and 1 mM-60 min respectively. 241 cysteines are found in all four sets.  47   Figure 3.2 Venn diagram of peptides in the four sets of cysteines with ICATL%-changes.  The sequences of these 241 cysteines and their ICATL%-changes at 3 μM-2 min, 1 mM-2 min, 3 μM-60 min and 1 mM-60 min are listed (Appendix C and D). HNO-reactive cysteines were then identified based on the ICATL%-changes at 3 μM-2 min and 1 mM-2 min.  According to Fig. 3.1, the ICATL% of a HNO-reactive cysteine at 3 μM-2 min and 1 mM-2 min should increase compared to the control, thus the ICATL%-change should be above 0. Moreover, the ICATL%-change of a HNO-reactive cysteine at 1 mM-2 min should be higher than that at 3 μM-2 min due to the higher AS dose. Among the 241 cysteines, there were 58 cysteines whose ICATL%-changes at 3 μM-2 min were below 0, 11 cysteines whose ICATL%-changes at 1 mM-2 min were below 0, 10 cysteines whose ICATL%-changes at 1 mM-2 min were smaller than that at 3 μM-2 min, and 3 cysteines whose ICATL%-changes at both 3 μM-2 min and 1 mM-2 min were 0 (Appendix C). Therefore, these 82 cysteines were not HNO-reactive cysteines by considering the magnitude of ICATL%-change.  For the other 159 cysteines, the ICATL%-changes at 3 μM-2 min and 1 mM-2 min were above 0, and the ICATL%-changes at 1 mM-2 min were higher than that at 3 μM-2 min 48  (Appendix D). Therefore, these 159 cysteines were identified as HNO-reactive cysteines, representing 78 proteins. These proteins showed various responses to HNO, likely due to the cellular location and the relative reactivity to HNO. Different cysteines within one protein also responded differently to HNO, likely due to the surface accessibility and the relative reactivity towards HNO.  The numbers of HNO-reactive cysteines and proteins identified in the present chapter were compared with those in Chapter 2. There were 32 HNO-reactive cysteines and 18 proteins identified in Chapter 2 by using IAA/NEM as alkylation reagents, and 159 HNO-reactive cysteines and 78 proteins identified in this chapter by using ICAT as alkylation reagents (Fig. 3.3). The numbers of HNO-reactive cysteines and proteins were significantly increased by the ICAT enrichment approach. The results show that there were 11 HNO-reactive cysteines and 2 proteins which were identified by the IAA/NEM approach but not by the ICAT approach. The reason for this is that these cysteine-containing peptides were not MS/MS sequenced in all the 6 experimental points and not all the corresponding ICATL%-changes were available, thus they were not found within the 241 overlapping cysteines in the four sets of cysteines.   Figure 3.3 Comparison of the numbers of HNO-reactive proteins and peptides identified by using the ICAT and IAA/NEM approaches.  The functions of these 78 HNO-reactive proteins were obtained from UniProt (Appendix E), covering a wider range of platelet functions compared to the results in Chapter 2 (Appendix A). Assignment of the 78 proteins to cell components was 49  performed as in Chapter 2 and the number of proteins in each category is shown in Fig. 3.4. There are 25 categories with proteins more than three, which cover a wider range of cell components compared with the 5 categories in Chapter 2. Cytosol, cytoplasm, plasma membrane and platelet alpha granule lumen are the top 4 cell components with high numbers of HNO-reactive proteins.  6 3 12 7 26 4 3 9 7 3 5 3 6 3 3 4 3 1111 4 3 3 3 3 4 0 5 10 15 20 25 30 N um be r of  p ro te in s actin cytoskeleton cell surface cytoplasm cytoskeleton cytosol endoplasmic reticulum lumen external side of plasma membrane extracellular region extracellular space fibrinogen complex focal adhesion integral to membrane integral to plasma membrane integrin complex melanosome membrane fraction perinuclear region of cytoplasm plasma membrane platelet alpha granule lumen platelet alpha granule membrane protein complex proteinaceous extracellular matrix ribonucleoprotein complex soluble fraction stress fiber  Figure 3.4 Cell components distribution of the 78 HNO-reactive proteins identified by using ICAT as alkylation reagents. Categories with proteins less than three were not listed.  3.3.2 Identification of cysteines whose HNO-induced modifications were reversed after 60 min of incubation 159 HNO-reactive cysteines were identified based on their ICATL%-changes at 3 μM-2 min and 1 mM-2 min. As the inhibition effect by HNO was not evident after 60 min of 50  incubation, the HNO-induced modifications on some of the HNO-reactive cysteines were thought to be reversed. Therefore, the ICATL%-changes at 3 μM-60 min and 1 mM-60 min of these 159 HNO-reactive cysteines were then further analyzed to determine cysteines with reversed modifications.  Theoretically for a cysteine whose HNO-induced modifications were reversed after 60 min, the ICATL% at 60 min should be the same compared to the control and the ICATL%-change should be 0. Based on the modification extents at 60 min, the 159 HNO-reactive cysteines were assorted into 5 groups (i.e., Group A-E). Only 2 peptides in each group were chosen as representatives for illustration due to the large number of cysteines (Fig. 3.5). The ICATL%-changes of all the 159 cysteines are listed and the groups they belong to are noted (Appendix D).  There are 10 cysteines in Group A. For each of them, the ICATL%-change at 3 μM-60 min and 1 mM-60 min is smaller than that at 3 μM-2 min and 1 mM-2 min. Therefore, the cysteines in Group A were modified to a less extent at 60 min than at 2 min, indicating the reversion of HNO-induced modifications. Moreover, the ICATL%-change at 1 mM-60 min is smaller than that at 3 μM-60 min, indicating that the modification was more reversed at the higher dose. Representatives of Group A are shown in Fig. 3.4a: peptide LCNSPSPQMNGKPCEGEAR of thrombospondin-1 shows an ICATL%-change equal to 0 at both 3 μM-60 min and 1 mM-60 min. This indicates that after 60 min this HNO-modified cysteine was fully reversed to the initial thiol. The ICATL%-changes of peptide DCVGDVTENQICNK of thrombospondin-1 at 3 μM-60 min and 1 mM-60 min are reduced but not 0, indicating that the HNO-induced modification was partially reversed after 60 min.  There are 7, 23 and 43 cysteines in Group B, C and D respectively. For each cysteine in these 3 groups, the ICATL%-change at 3 μM-60 min is smaller than that at 3 μM-2 min, indicating that the HNO-induced modifications were reversed after 60 min of 51  incubation with 3 μM AS. The difference among Group B, C and D lies in the magnitude of the ICATL%-change at 1 mM-60 min. In Group B, the ICATL%-change at 1 mM-60 min was smaller than that at 3 μM-2 min but higher than that at 3 μM-60 min, indicating that the modification was less reversed at the higher dose at 60 min; in Group C, it was higher than that at 3 μM-2 min but lower than at 1 mM-2 min; in Group D, it was the highest compared to that at 3 μM-2 min, 1 mM-2 min and 1 mM-60 min. Representatives of Group B, C and D are shown in Fig. 3.5b-d respectively.  Theoretically, for a cysteine whose HNO-induced modification is reversible, the ICATL%-change at 1 mM-60 min should be lower than those at 2 min. The reason for the higher modification extent at 1 mM-60 min than those at 2 min in Group C and D is unknown. It might be due to additional effects at high AS doses (1-10 mM) such as toxicity23. It is noteworthy that in Bermejo et al.'s study the AS dose used to test the reversibility of HNO's antiaggregatory effect was 3 μM rather than 1 mM7. Therefore, as long as the ICATL%-change of a cysteine at 3 μM-60 min is smaller than that at 3 μM-2 min, the cysteine can be considered as a candidate to be involved in platelet inhibition by HNO. Thus, cysteines in Group A-D are all potential candidates for further analysis, representing 83 cysteines and 55 proteins.  For some peptides in Group A-D, the ICATL%-change at 3 μM-60 min or 1 mM-60 min is smaller than 0, indicating that the ICATL% decreased upon AS treatment compared to the control at 60 min. As discussed in Chapter 2, a part of the free cysteine can be initially oxidized (e.g., as disulfide bond) either in platelets in vivo or during the experimental process, and the rest of it are then modified into either sulfinamides or disulfides upon AS addition. After 60 min of incubation with AS, the HNO-modified part of the cysteine are reversed to the initial thiols, and the initially oxidized part can also be reversed; while in the control, the initially oxidized part are not reversed without AS treatment. Therefore, the ICATL% at 3 μM-60 min or 1 mM-60 min can be smaller than that in the control, and the ICATL%-change can be below 0. 52   There are 76 cysteines in Group E. For each of them, the ICATL%-change at 3 μM-60 min is higher than that at 3 μM-2 min and the ICATL%-change at 1 mM-60 min is higher than that at 3 μM-60 min. This indicates that the HNO-induced modifications were not removed after 60 min. Therefore, these cysteines were unlikely to play key roles in platelet inhibition by HNO although they were HNO-reactive. Representatives of Group E are shown in Fig. 3.5e. 53  0% 10% 20% 30% 40% 50% 60% TSP1_DCVGDVTENQICNK TSP1_LCNSPSPQMNGKPCEGEAR IC A T L% -c ha ng e a) Group A 3uM-2min 1mM-2min 3uM-60min 1mM-60min -5% 0% 5% 10% 15% 20% 25% IGKC_VYACEVTHQGLSSPVTK LEGL_VEILCEHPR IC A T L% -c ha ng e b) Group B 3uM-2min 1mM-2min 3uM-60min 1mM-60min -5% 0% 5% 10% 15% 20% 25% 30% 1433F_NCNDFQYESK CLIC1_LHIVQVVCK IC A T L% -c ha ng e c) Group C 3uM-2min 1mM-2min 3uM-60min 1mM-60min -5% 0% 5% 10% 15% 20% 25% 30% 35% 40% CCS_LACGIIAR FLNA_APSVANVGSHCDLSLK IC A T L% -c ha ng e d) Group D 3uM-2min 1mM-2min 3uM-60min 1mM-60min 0% 5% 10% 15% 20% 25% 30% 35% 40% ALBU_ALVLIAFAQYLQQCPFEDHVK CAP1_NSLDCEIVSAK IC A T L% -c ha ng e e) Group E 3uM-2min 1mM-2min 3uM-60min 1mM-60min  Figure 3.5 Representatives of peptides in Group A-E. Abbreviations: 1433Z, 14-3-3 protein zeta/delta; ALBU, serum albumin; CAP1, adenylyl cyclase-associated protein 1, CCS, copper chaperone for superoxide dismutase; CLIC1, chloride intracellular channel protein 1; FLNA, filamin-A; IGKC, Ig kappa chain C region; LEGL, galectin-related protein; TSP1, thrombospondin-1. 54  3.3.3 Evaluation of HNO-reactive proteins with reversible modifications that may have functions in platelet inhibition by HNO The 83 cysteines in Group A-D were HNO-reactive and their HNO-induced modifications were reversible. Therefore, these cysteines may play key roles in platelet inhibition by HNO. The represented 55 proteins in Group A-D were analyzed by Ingenuity Pathway Analysis (IPA, Ingenuity® Systems, www.ingenuity.com) to identify the most significant canonical pathways relevant to the data. The significance of the association between the proteins and the canonical pathway was measured in a ratio of the number of molecules from the data set that map to the pathway divided by the total number of molecules in the IPA library that map to the canonical pathway, and a p-value determining the probability that the association between the proteins in the dataset and the pathway is explained by chance alone. Fig. 3.6 shows the top 5 canonical pathways relevant to the 55 proteins.  0.00 0.05 0.10 0.0 2.5 5.0 7.5 Integrin Signaling ILK Signaling Germ Cell-Sertoli Cell Junction Signaling Caveolar-mediated Endocytosis Signaling Actin Cytoskeleton Signaling R atio -l og (p -v al u e) -log(p-value) Ratio  Figure 3.6 Top 5 canonical pathways relevant to the 55 proteins with reversible HNO-induced modifications.  Among the top 5 canonical pathways, integrin signaling, ILK signaling (integrin-linked kinase signaling) and actin cytoskeleton signaling are relevant to platelet activation. IPA analysis shows that among the 55 proteins with reversible HNO-induced modifications, there are 12 proteins involved in these 3 canonical pathways (Table 3.1). 55  Table 3.1 The HNO-reactive cysteines of the 14 proteins associated with integrin signaling. Protein Peptide Group Canonical Pathway ACTB LCYVALDFEQEMATAASSSSLEK (216-238) A  Integrin Signaling, ILK Signaling, Actin Cytoskeleton Signaling ACTN1 DGLGFCALIHR (175-185) A  Integrin Signaling, ILK Signaling, Actin Cytoskeleton Signaling ACTN4 ACLISLGYDVENDR (792-805) B  Integrin Signaling, ILK Signaling, Actin Cytoskeleton Signaling COF1 AVLFCLSEDK (35-44) D  ILK Signaling, Actin Cytoskeleton Signaling FLNA AHVVPCFDASK (1152-1162) C  ILK Signaling APSVANVGSHCDLSLK (2150-2165) D LQVEPAVDTSGVQCYGPGIEGQGVFR (1247-1272) C THEAEIVEGENHTYCIR (2185-2201) D VQVQDNEGCPVEALVK (709-924) C ITA2B HSPICHTTMAFLR (572-584) C  Integrin Signaling TPVGSCFLAQPESGR (157-170) A ITB3 LAGIVQPNDGQCHVGSDNHYSASTTMDYPSLGLMTEK (288-324) B  Integrin Signaling, ILK Signaling YCRDEIESVK (660-669) C LIMS1 CDLCQEVLADIGFVK (97-111) B  Integrin Signaling CHAIIDEQPLIFK (138-150) B MYH9 ADFCIIHYAGK (566-576) D  ILK Signaling, Actin Cytoskeleton Signaling KMEDSVGCLETAEEVK (1372-1387) C VEDMAELTCLNEASVLHNLK (83-120) D RHOA HFCPNVPIILVGNK (105-118) A  Integrin Signaling, ILK Signaling, Actin Cytoskeleton Signaling IGAFGYMECSAK (151-162) D TLN1 AGALQCSPSDAYTK (1934-1947) D  Integrin Signaling ASAGPQPLLVQSCK (994-957) D 56  Protein Peptide Group Canonical Pathway TLN1 CVSCLPGQR (1199-1207) D  Integrin Signaling ZYX CNTCGEPITDR (444-454) C  Integrin Signaling CSVCSEPIMPEPGRDETVR (504-522) D 57  These 12 proteins are likely to play a role in platelet inhibition by HNO based on their reactivity to HNO and the pathways in which they are involved. Their biological functions are discussed below to determine the most likely proteins which may play key roles in the antiaggregatory effect of HNO.  Actin (ACTB), cofilin (COF1), RhoA (RHOA), LIM and senescent cell antigen-like-containing domain protein 1 (LIMS1) and zyxin (ZYX) contain HNO-reactive cysteines whose modifications were reversed after 60 min of incubation with 3 μM AS. The platelet cytoskeleton is made up of long cytoplasmic actin filaments, and the membrane skeleton is composed of short submembranous actin filaments59. Binding of integrin αIIbβ3 to its ligands triggers the reorganization of the actin cytoskeleton that underlies filopodia and lamellopodia formation, which are the essence of platelet shape change. Cofilin is an actin-binding protein which causes depolymerization at the minus end of filament, thereby preventing actin reassembly. RhoA is a GTPase that regulates certain actin rearrangements and transcription events. Studies have shown that RhoA is not involved in regulating the ligand binding function of integrin αIIbβ3, whereas it is involved in outside-in signaling responses that may be dependent on specific actin rearrangements controlled by RhoA60. LIM and senescent cell antigen-like-containing domain protein 1 is an effector of integrin signaling, coupling surface receptors to downstream signaling molecules involved in the regulation of cell survival, cell proliferation and cell differentiation. It is likely involved in integrin signaling through its interaction with integrin-linked kinase (ILK, Fig. 1.3). Zyxin is a zinc-binding phosphoprotein and is important for the formation of actin-rich structures. It may modulate the cytoskeletal organization of actin bundles (Fig. 1.3). These proteins (ACTB, COF1, RHOA, LIMS1 and ZYX) are positioned downstream of integrin αIIbβ3, therefore, they are unlikely to function as the targets of HNO in platelet inhibition, albeit they may contribute to or even amplify the effect.  58  The biological functions of integrin αIIbβ3, talin, kindlin, α-actinin, filamin and myosin have been discussed in Chapter 2. The present results show that myosin contains HNO-reactive cysteines whose modifications were reversible. However, it reacts with integrin following platelet aggregation, thus it is excluded as candidates. In the case of kindlin, Cys-128 was determined as a HNO-reactive cysteine both in Chapter 2 and the present chapter. However, the results of the time-dependent study show that the modification on Cys-128 was not reversed after 60 min of incubation (Appendix D). Thus, kindlin is unlikely to be a functional protein in the HNO-induced platelet inhibition.  Talin, α-actinin, filamin and integrin αIIbβ3 contain HNO-reactive cysteines whose modifications were reversed after 60 min of incubation with 3 μM AS. Based on the reactivity toward HNO, the reversibility of the HNO-induced modifications and the role in platelet activation, talin is proposed to be the most likely protein candidate to function in the HNO-induced platelet inhibition, and α-actinin, filamin and integrin αIIbβ3 should also be considered as potential candidates.  3.3.4 Conclusions An ICAT enrichment approach was combined with the differential alkylation method to identify more HNO-reactive cysteines. The results show that 159 HNO-reactive cysteines were determined, representing 78 proteins. The numbers were significantly increased compared with the results in Chapter 2. A time-dependent study was also performed to discover cysteines whose HNO-induced modifications were reversed after 60 min of incubation. The results show that among the 159 cysteines, the modifications of 83 of them were reversed after 60 min of incubation with 3 μM AS. Based on the reactivity toward HNO, the reversibility of HNO-induced modifications 59  and the biological functions, talin, α-actinin, filamin and integrin αIIbβ3 are proposed as potential candidates that may play key roles in platelet inhibition by HNO. 60  4 Conclusions 4.1 Summary The overall goal of this thesis was to identify HNO-reactive cysteines in platelet proteins regardless of eventual modification states (i.e., sulfinamide or disulfide) and to map cysteines whose HNO-induced modifications were reversed after 60 min of incubation with AS. This study helps to discover proteins which may play key roles in platelet inhibition by HNO.  In Chapter 2, a differential alkylation method was developed using IAA/NEM as alkylation reagents, evaluated on a protein model and applied on human platelets. 32 HNO-reactive cysteines from 18 proteins were identified. Combining these results with the mass-shift based method, we further distinguished the HNO-induced products of these cysteines as either sulfinamides or disulfides: 4 out of the 32 cysteines were confirmed to be modified into sulfinamides by MS/MS; 19 cysteines were considered as being modified into disulfides; 9 cysteines may be sulfinamide-modified but further targeted MS/MS fragmentation is needed to confirm this.  In Chapter 3, ICAT was used as alkylation reagents to enrich cysteine-containing peptides. 159 HNO-reactive cysteines were identified, representing 78 proteins. The numbers of HNO-reactive cysteines and proteins identified were highly enhanced compared to the IAA/NEM approach. The modification extents of the 159 HNO-reactive cysteines at 60 min were then used to identify cysteines with reversible HNO-induced modifications. The results show that the modifications of 83 out of the 159 cysteines were reversed after 60 min of incubation with 3 μM AS; the modifications of the other 76 cysteines were not removed.  61  Based on the reactivity of cysteines to HNO, the reversibility of HNO-induced modifications and the role in platelet activation, talin is proposed to be the most likely candidate to play a role in HNO-induced platelet inhibition, and α-actinin, filamin and integrin αIIbβ3 may also be considered as potential candidates.  4.2 Future directions Generally, this study proposes a strategy that can be utilized to identify drug targets: as long as the drug-induced modifications are known (e.g. sulfinamide and disulfide), the drug targets can be discovered by determining the modifications directly (e.g., the mass shift-based method) or indirectly (e.g., the differential alkylation method). Besides Angeli's Salt, NO is also a known drug to inhibit platelet aggregation, which reacts with thiols to form S-nitrosothiols (often referred to as RSNOs). Further studies can be carried out to identify the targets of NO in platelet inhibition and compare them with those of HNO. This could help to more deeply understand the mechanisms of platelet inhibition by drugs.  In Chapter 2, there were 9 HNO-reactive cysteines whose modification states could not be confirmed as either sulfinamides or disulfides, because the precursor ions of these peptides corresponding to the sulfinamide modificaitons were found in MS level but they were not MS/MS fragmented. Targeted MS/MS fragmentation could be performed in the future to confirm their modification states.  Although cysteines were enriched by the ICAT approach in Chapter 3, the presence of high-abundance proteins in platelets (e.g., talin, filamin and actin) limits the detection of medium- and low-abundance proteins. Future work could use ProteoMiner protein enrichment technology to reduce the dynamic range. ProteoMiner technology is based on the treatment of complex protein samples with a combinatorial ligand library. Each unique ligand binds to a unique protein sequence and there is one ligand per bead. 62  Limited bead capability allows maximum concentration of low-abundance proteins while diluting high-abundance species, thereby decreasing the dynamic range of proteins in the sample.  Less complex samples are preferred in mass spectrometric analysis, thereby a high-resolution cation-exchange column could be used for fractionation to yield individual samples containing fewer peptides before biotinylated peptides purification. This could also be achieved by separating the purified and cleaved ICAT reagent-labeled peptides by capillary reversed-phase HPLC before MS analysis. 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Plow, E.; Qin, J.; Byzova, T., Kindling the flame of integrin activation and function with kindlins. Curr. Opin. Hematol. 2009, 16, (5), 323-328. [53]. Moser, M.; Nieswandt, B.; Ussar, S.; Pozgajova, M.; Fässler, R., Kindlin-3 is essential for integrin activation and platelet aggregation. Nat. Med. 2008, 14, (3), 325-330. [54]. Smith, A.; Carrasco, Y. R.; Stanley, P.; Kieffer, N.; Batista, F. D.; Hogg, N., A talin-dependent LFA-1 focal zone is formed by rapidly migrating T lymphocytes. J. Cell. Biol. 2005, 170, (1), 141-151. [55]. Tadokoro, S.; Nakazawa, T.; Kamae, T.; Kiyomizu, K.; Kashiwagi, H.; Honda, S.; Kanakura, Y.; Tomiyama, Y., A potential role for α-actinin in inside-out αIIbβ3 signaling. Blood 2011, 117, (1), 250-258. 67  [56]. Keh, D.; Thieme, A.; Kürer, I.; Falke, K.; Gerlach, H., Inactivation of platelet glycoprotein IIb/IIIa receptor by nitric oxide donor 3-morpholino-sydnonimine. Blood Coagul. Fibrinolysis 2003, 14, (4), 327-334. [57]. Beckerle, M. C.; Miller, D. E.; Bertagnolli, M. E.; Locke, S. J., Activation-dependent redistribution of the adhesion plaque protein, talin, in intact human platelets. J. Cell. Biol. 1989, 109, (6), 3333-3346. [58]. Spadaro, D.; Yun, B. W.; Spoel, S. H.; Chu, C.; Wang, Y. Q.; Loake, G. J., The redox switch: dynamic regulation of protein function by cysteine modifications. Physiol. Plant 2010, 138, (4), 360-371. [59]. Michelson, A. D., Platelets. second ed.; Elsevier Inc. : 2007. [60]. Leng, L.; Kashiwagi, H.; Ren, X.; Shattil, S., RhoA and the function of platelet integrin aIIbb3. Blood 1998, 91, (11), 4206-4215.  68  Appendices A. Protein functions of the 18 HNO-reactive proteins identified by using IAA/NEM as alkylation reagents SwissProt Accession # Entry name Protein Function P63104 1433Z_HUMAN 14-3-3 protein zeta/delta Adapter protein implicated in the regulation of a large spectrum of both general and specialized signaling pathways. Binds to a large number of partners, usually by recognition of a phosphoserine or phosphothreonine motif. Binding generally results in the modulation of the activity of the binding partner. P12814 ACTN1_HUMAN Alpha-actinin-1 F-actin cross-linking protein which is thought to anchor actin to a variety of intracellular structures. This is a bundling protein. P23528 COF1_HUMAN Cofilin-1 Controls reversibly actin polymerization and depolymerization in a pH-sensitive manner. It has the ability to bind G- and F-actin in a 1:1 ratio of cofilin to actin. It is the major component of intranuclear and cytoplasmic actin rods. P02775 CXCL7_HUMAN Platelet basic protein A platelet basic protein precusor that is processed to produce different smaller proteins including LA-PF4, TC-2, Beta-TG,NAP-2 and etc. P02675 FIBB_HUMAN Fibrinogen beta chain Fibrinogen has a double function: yielding monomers that polymerize into fibrin and acting as a cofactor in platelet aggregation. P02679 FIBG_HUMAN Fibrinogen gamma chain Fibrinogen has a double function: yielding monomers that polymerize into fibrin and acting as a cofactor in platelet aggregation. P21333 FLNA_HUMAN Filamin-A Promotes orthogonal branching of actin filaments and links actin filaments to membrane glycoproteins. Anchors various transmembrane proteins to the actin cytoskeleton and serves as a scaffold for a wide range of cytoplasmic signaling proteins. Interaction with FLNA may allow neuroblast migration from the ventricular zone into the cortical plate. Tethers cell surface-localized furin, modulates its rate of 69  SwissProt Accession # Entry name Protein Function internalization and directs its intracellular trafficking. P04406 G3P_HUMAN Glyceraldehyde-3-phosp hate dehydrogenase Independent of its glycolytic activity it is also involved in membrane trafficking in the early secretory pathway. P06396 GELS_HUMAN Gelsolin Calcium-regulated, actin-modulating protein that binds to the plus (or barbed) ends of actin monomers or filaments, preventing monomer exchange (end-blocking or capping). It can promote the assembly of monomers into filaments (nucleation) as well as sever filaments already formed. P68871 HBB_HUMAN Hemoglobin subunit beta Involved in oxygen transport from the lung to the various peripheral tissues.LVV-hemorphin-7 potentiates the activity of bradykinin, causing a decrease in blood pressure. P11142 HSP7C_HUMAN Heat shock cognate 71 kDa protein Acts as a repressor of transcriptional activation. Inhibits the transcriptional coactivator activity of CITED1 on Smad-mediated transcription. P08514 ITA2B_HUMAN Integrin alpha-IIb Integrin alpha-IIb/beta-3 is a receptor for fibronectin, fibrinogen, plasminogen, prothrombin, thrombospondin and vitronectin. It recognizes the sequence R-G-D in a wide array of ligands. It recognizes the sequence H-H-L-G-G-G-A-K-Q-A-G-D-V in fibrinogen gamma chain. Following activation integrin alpha-IIb/beta-3 brings about platelet/platelet interaction through binding of soluble fibrinogen. This step leads to rapid platelet aggregation which physically plugs ruptured endothelial cell surface. P35579 MYH9_HUMAN Myosin-9 Cellular myosin that appears to play a role in cytokinesis, cell shape, and specialized functions such as secretion and capping. Q9Y490 TLN1_HUMAN Talin-1 Probably involved in connections of major cytoskeletal structures to the plasma membrane. High molecular weight cytoskeletal protein concentrated at regions of cell-substratum contact and, in lymphocytes, at cell-cell contacts P60174 TPIS_HUMAN Triosephosphate isomerase Triose-phosphate isomerase activity 70  SwissProt Accession # Entry name Protein Function P61088 UBE2N_HUMAN Ubiquitin-conjugating enzyme E2 N The UBE2V2/UBE2N heterodimer catalyzes the synthesis of non-canonical poly-ubiquitin chains that are linked through 'Lys-63'. This type of poly-ubiquitination does not lead to protein degradation by the proteasome. Mediates transcriptional activation of target genes. Plays a role in the control of progress through the cell cycle and differentiation. Plays a role in the error-free DNA repair pathway and contributes to the survival of cells after DNA damage. Acts together with the E3 ligases, HLTF and SHPRH, in the 'Lys-63'-linked poly-ubiquitination of PCNA upon genotoxic stress, which is required for DNA repair. Q86UX7 URP2_HUMAN Kindlin-3 Plays a central role in cell adhesion in hematopoietic cells. Acts by activating the integrin beta-1-3 (ITGB1, ITGB2 and ITGB3). Required for integrin-mediated platelet adhesion and leukocyte adhesion to endothelial cells. Required for activation of integrin beta-2 (ITGB2) in polymorphonuclear granulocytes (PMNs) P18206 VINC_HUMAN Vinculin Involved in cell adhesion. May be involved in the attachment of the actin-based microfilaments to the plasma membrane. May also play important roles in cell morphology and locomotion.  71  B. Reversibility of sulfinamide by TCEP The peptide MHRQETVDCLK-NH2 was provided as a gift from Phil Owen of the Biomedical Research Centre. 15 µl peptide MHRQETVDCLK-NH2 from a stock solution of 200 µM in H2O was dissolved in 35 µl Tris buffer (0.05 M, pH 7.4), and was treated with 2 µl AS (7.5 µg/µl). The samples was then treated with 1, 2, 10 mM tris(2-carboxyethyl)phosphine (TCEP) respectively for 2 min at 99°C. CHCA was used as the MALDI matrix at a concentration of 33 mM in 1:1 (v/v) ACN/MeOH. Matrix and the standard peptide were then mixed in a 1:1 (v/v) ratio and spotted onto a MALDI plate using the dried-droplet method and analyzed by MALDI-TOF (4700 Proteomics Analyzer) controlled by 4700 Explorer v2.0 software (Applied Biosystems, Foster City, CA).  The results show that upon AS treatment, peptide MHRQETVDCLK-NH2 was mainly sulfinamide-modified, and the disulfide modification was also observed (Fig. A1a). The sulfinamide was partially reduced by treatment with 1 mM and 2 mM TCEP (Fig. A1b-c). The disulfide was partially reduced upon 1 mM TCEP addition and was completely loss at 2 mM (Fig. A1b-c). At 10 mM TCEP, the sulfinamide modification was fully reversed. 72  799.0 1342.2 1885.4 2428.6 2971.8 3515.0 Mass (m/z) 100 0 %  In te ns ity Unmodified peptide 1358.7 Sulfinamide 1389.7 Disulfide 2714.4 799.0 1342.2 1885.4 2428.6 2971.8 3515.0 Mass (m/z) 100 0 %  In te ns ity Unmodified peptide 1358.7 Sulfinamide 1389.7 Disulfide 2714.4 799.0 1342.2 1885.4 2428.6 2971.8 3515.0 Mass (m/z) 100 0 %  In te ns ity Unmodified peptide 1358.7 Sulfinamide 1389.7 799.0 1342.2 1885.4 2428.6 2971.8 3515.0 Mass (m/z) 100 0 %  In te ns ity Unmodified peptide 1358.7 0 mM TCEP 1 mM TCEP 2 mM TCEP 10 mM TCEP a) b) c) d)  Figure B. Reduction of the sulfinamide and disulfide modification by TCEP. The peptide MHRQETVDCLK-NH2 was modified by HNO following treatment with Angeli's salt. Treatment with a) no TCEP, b) 1 mM TCEP, c) 2 mM TCEP and d) 10 mM TCEP for 2 min at 99°C led to reduction of both the sulfinamide and disulfide modification, with complete loss of sulfinamide at 10 mM TECP and disulfide at 2 mM TCEP. 73  C. ICATL%-changes of the 82 HNO-unreactive cysteine-containing peptides among the overlapping 241 cysteines Protein Peptide ICATL%-changea 3μM-2mina 1mM-2mina 3μM-60mina 1mM-60mina 1433T YDDMATCMK (19-27) -0.9% 24.0% 2.5% 6.8% 1B35 MYGCDLGPDGR (122-132) -2.8% 20.0% -0.9% 6.7% A4 STNLHDYGMLLPCGIDK (162-180) -11.6% 18.3% 1.6% 30.3% ALBU AACLLPK (199-205) -0.3% 1.3% 10.0% 34.4% AAFTECCQAADK (187-198) -1.9% -2.0% 11.8% 28.2% ADDKETCFAEEGK (585-597) -0.8% 6.5% 5.6% 36.0% SHCIAEVENDEMPADLPSLAADFVESK (311-337) -1.9% 18.2% 6.2% 27.5% VHTECCHGDLLECADDR (265-281) -1.5% -18.5% 4.5% 37.7% ETYGEMADCCAK (106-117) 0.0% -5.6% 5.1% 16.1% NECFLQHKDDNPNLPR (123-138) 2.7% 0.6% 0.6% 16.3% YICENQDSISSK (287-298) 11.3% 8.7% 9.6% 26.8% CCAAADPHECYAK (384-396) 0.0% 0.0% 1.0% 35.7% B2MG SNFLNCYVSGFHPSDIEVDLLK (40-61) -8.1% 10.5% 4.9% 17.8% BIN2 IGCYVTIFQNISNLR (203-217) -9.3% 18.5% 10.2% 32.1% CLUS EILSVDCSTNNPSQAK (307-322) -6.6% 7.1% 7.0% 18.8% CNN2 CASQSGMTAYGTR (175-187) 4.6% -7.7% 6.0% 17.0% CASQVGMTAPGTR (215-227) 1.3% -0.8% 6.4% 20.8% ENOA VNQIGSVTESLQACK (344-358) 3.4% 3.2% 9.0% 47.1% FIBG FGSYCPTTCGIADFLSTYQTK (41-61) -11.5% 0.0% 6.7% 62.9% FKB1A RGQTCVVHYTGMLEDGK (19-35) 5.8% 1.8% 5.3% 26.4% FLNA AHEPTYFTVDCAEAGQGDVSIGIK (786 - 809) -6.7% 18.3% 10.1% 44.1% CSYQPTMEGVHTVHVTFAGVPIPR (444-467) -0.6% 29.2% 5.7% 39.7% 74  Protein Peptide ICATL%-changea 3μM-2mina 1mM-2mina 3μM-60mina 1mM-60mina GP1BA AMTSNVASVQCDNSDKFPVYK (254-274) -3.8% 20.2% 6.0% 52.9% LTQLNLDRCELTK (73-85) 4.7% 4.1% 5.0% 39.7% HSP7C GPAVGIDLGTTYSCVGVFQHGK (4-25) -0.6% 31.1% 3.4% 33.7% ITA2B AEGGQCPSLLFDLR (91-104) -4.6% 11.9% 2.9% 18.0% SCVLPQTK (503-510) -3.6% 14.4% 1.8% 9.0% TPVSCFNIQMCVGATGHNIPQK (511-532) -2.3% 6.6% 2.9% 13.7% ITB1 KGCPPDDIENPR (73-84) 0.9% -1.6% 9.1% 9.1% ITB3 TTCLPMFGYK (208-217) -2.2% 7.2% 43.1% 33.7% YCECDDFSCVR (546-556) -5.5% 10.2% 5.9% 27.2% SILYVVEEPECPK (703-715) 0.0% -0.1% 7.8% 33.8% TDTCMSSNGLLCSGR (590-604) 0.0% -66.3% 3.3% 22.2% LIMS1 FVEFDMKPVCK (291-301) 3.2% 0.9% 1.9% -1.9% LRRF1 CMVEVPQELETSTGHSLEK (456-474) -0.8% 9.6% 2.4% 10.4% LTBP1 CEYCDSGYR (981-989) -0.3% 3.7% 3.1% 9.8% CVDIDECTQVQHLCSQGR (913-930) -46.1% 0.0% -3.2% 1.0% DQCEDIDECQHR (1116-1127) -15.3% 2.9% -0.1% 3.5% DSDDYAQLCNIPVTGR (1569-1584) -1.4% 4.4% 0.0% 0.0% EICPGGMGYTVSGVHR (727-742) -0.7% 7.7% -2.7% -5.4% NGFCLNTRPGYECYCK (1438-1453) -0.5% 1.4% 0.2% 1.8% YTCICYEGYR (896-905) 0.7% -23.7% 0.8% 6.8% VVICHLPCMNGGQCSSR (400-416) 0.0% 0.0% 0.3% 5.9% LYAM3 CAEGFMLR (416-423) -1.6% 3.0% 1.5% 9.9% CPLNPHSHLGTYGVFTNAAFDPSP (807-830) -0.5% 22.1% -5.7% 11.2% FECQPGYR (352-359) -2.2% 1.4% -4.9% 19.3% 75  Protein Peptide ICATL%-changea 3μM-2mina 1mM-2mina 3μM-60mina 1mM-60mina LYAM3 LEGPNNVECTTSGR (608-621) -2.9% 3.3% 0.5% 12.2% NNEDCVEIYIK (127-137) -0.8% 5.3% 0.2% 23.0% WSATPPTCK (622-630) -1.7% 5.5% 2.6% 29.6% WTDSPPMCEAIK (560-571) -53.6% 7.4% -1.1% 38.2% HALCYTASCQDMSCSK (155-170) 0.0% 0.0% 8.6% 42.6% ML12A NAFACFDEEATGTIQEDYLR (104-123) -79.5% -19.8% 3.4% 56.6% MMRN1 GECEDMLSK (590-598) -3.6% 9.8% 11.1% 62.4% NID1 AECLNPSQPSR (1133-1143) -0.6% 1.7% 10.2% 50.8% PARK7 DVVICPDASLEDAKK (49-63) 40.9% 20.8% 4.9% 33.8% PDLI1 CGTGIVGVFVK (263-273) 5.2% 4.1% 5.5% 21.3% PPGB DLECVTNLQEVAR (253-265) -2.8% 0.0% 8.3% 36.1% RINI LGDVGMAELCPGLLHPSSR (239-257) 2.1% -1.7% 3.4% 16.8% SODC DGVADVSIEDSVISLSGDHCIIGR (93-116) -1.3% 18.4% 1.4% 11.7% SPRC APLIPMEHCTTR (257-268) -0.1% 17.2% -0.1% 13.0% FFETCDLDNDK (269-279) -3.6% 9.4% 6.2% 20.5% LHLDYIGPCK (141-150) -0.4% 16.3% 4.8% -11.7% TFDSSCHFFATK (118-129) -1.7% -2.3% 6.8% 47.4% YIPPCLDSELTEFPLR (151-166) -0.9% 18.7% 11.1% 35.1% SRGN CNPDSNSANCLEEK (40-53) -4.7% 7.3% 15.8% 38.4% TBB1 NTMAACDLR (298-306) -2.8% 14.9% 5.6% 43.4% TEBP HLNEIDLFHCIDPNDSK (49-65) -0.3% 10.8% 2.6% 41.4% THAS LYGPLCGYYLGR (73-84) -5.1% 12.7% 9.2% 25.5% TLN1 ACEFAGFQCQIQFGPHNEQK (235-254) -0.8% 13.3% 3.4% 41.6% TPIS IIYGGSVTGATCK (207-219) -2.4% 18.9% -3.8% 41.9% 76  Protein Peptide ICATL%-changea 3μM-2mina 1mM-2mina 3μM-60mina 1mM-60mina TRML1 FLPEGCQPLVSSAVDR (54-69) 3.1% -10.9% 4.7% 32.8% VLVCSKPVTYATVIFPGGNK (272-291) 7.7% 1.8% 1.5% 12.4% TSP1 AQLYIDCEK (165-173) -0.5% 22.3% 5.6% 3.1% DDFDHDSVPDIDDICPENVDISETDFR (932-958) -1.9% 6.8% 17.4% 48.6% DGIGDACDDDDDNDKIPDDR (732-751) -1.3% 13.1% 3.4% 44.9% DTDMDGVGDQCDNCPLEHNPDQLDSDSDR (823-851) -9.7% 0.0% -0.8% 5.4% KDNCPNLPNSGQEDYDK (715-731) -0.4% 14.5% -2.7% 16.8% EVPDACFNHNGEHR (594-607) 2.4% 0.2% 0.3% 25.5% TXND5 IAEVDCTAER (376-385) -0.7% 6.7% 5.2% 5.9% VINC CDRVDQLTAQLADLAAR (545-561) 1.6% -0.1% 2.8% 25.8% VWF SGFTYVLHEGECCGR (2479-2493) -7.6% 7.1% -1.7% 4.1% VAQCSQKPCEDSCR (2465-2478) 0.0% -3.5% -0.5% 1.6%  a: The ICATL%-changes at 3 μM-2 min, 1 mM-2 min, 3 μM-60 min and 1 mM-60 min are the differences of ICATL% between AS treatment (i.e., 3 μM and 1 mM) and the control (i.e., 0 μM) after 2 min and 60 min of incubation respectively. 77  D. ICATL%-changes of the 159 HNO-reactive cysteine-containing peptides among the overlapping 241 cysteines Protein Peptide ICATL%-changea Groupb 3μM-2mina 1mM-2mina 3μM-60mina 1mM-60mina 1433F ELETVCNDVLSLLDK (92-106) 3.7% 21.1% -0.7% -6.2% A NCNDFQYESK (111-120) 4.5% 26.4% 1.6% 15.3% C 1433Z DICNDVLSLLEK (92-103) 3.2% 28.9% 2.3% 12.7% C YDDMAACMK (19-27) 1.2% 11.7% -0.2% 8.0% C A4 CLVGEFVSDALLVPDK (117-132) 2.7% 14.4% -15.0% 0.0% A ACTB CDVDIR (285-290) 0.2% 17.8% 2.0% 15.6% E CPEALFQPSFLGMESCGIHETTFNSIMK (257-284) 0.8% 18.4% 0.8% 27.0% E LCYVALDFEQEMATAASSSSLEK (216-238) 2.6% 15.8% 1.0% 0.2% A ACTN1 DGLGFCALIHR (175-185) 7.0% 16.9% 0.0% 0.0% A ACTN4 ACLISLGYDVENDR (792-805) 3.9% 18.2% 2.4% 3.4% B ALBU ALVLIAFAQYLQQCPFEDHVK (45-65) 1.7% 26.9% 8.0% 28.8% E CCTESLVNR (500-508) 3.0% 5.1% 5.8% 37.5% E LCTVATLR (98-105) 1.4% 10.6% -16.4% 23.4% D LKECCEKPLLEK (299-310) 0.3% 2.8% 2.5% 41.1% E LVRPEVDVMCTAFHDNEETFLK (139-160) 0.6% 6.2% 6.6% 35.3% E MPCAEDYLSVVLNQLCVLHEK (470-490) 0.9% 0.9% 5.2% 17.1% E QNCELFEQLGEYK (414-426) 2.5% 22.7% 2.0% 32.7% D RPCFSALEVDETYVPK (509-524) 1.4% 9.1% 1.8% 59.7% E TCVADESAENCDK (76-88) 0.6% 2.2% 3.4% 16.5% E ALDOA ALANSLACQGK (332-342) 3.1% 19.2% -0.5% 16.5% C YASICQQNGIVPIVEPEILPDGDHDLKR (174-201) 10.7% 16.4% 5.6% 19.2% D ARP3 DYEEIGPSICR (399-409) 6.4% 16.0% 10.5% 16.2% E 78  Protein Peptide ICATL%-changea Groupb 3μM-2mina 1mM-2mina 3μM-60mina 1mM-60mina ARP3 TLTGTVIDSGDGVTHVIPVAEGYVIGSCIK (162-191) 7.9% 13.1% 8.4% 24.4% E YSYVCPDLVK (231-240) 4.8% 19.3% 6.5% 17.4% E CALR HEQNIDCGGGYVK (99-111) 2.4% 11.8% 1.1% 25.1% D CAP1 NSLDCEIVSAK (423-433) 1.8% 13.9% 24.5% 34.1% E CCS LACGIIAR (225-232) 5.7% 9.0% -0.4% 11.7% D CD36 SQVLQFFSSDICR (261-273) 0.1% 11.4% 2.8% 28.2% E CD68 LQAAQLPHTGVFGQSFSCPSDR (297-318) 4.8% 20.9% 10.0% 40.0% E CF115 CANLFEALVGTLK (39-51) 5.6% 24.2% 1.1% 8.9% C CH60 CEFQDAYVLLSEK (237-249) 0.3% 20.7% 0.9% 8.6% E CLC1B YYGDSCYGFFR (108-118) 3.7% 7.9% 3.0% 32.7% D CLIC1 IGNCPFSQR (21-29) 3.3% 15.1% 1.8% 5.1% C LHIVQVVCK (184-192) 6.6% 23.5% -2.2% 10.8% C CNN2 DGTILCTLMNK (56-66) 6.2% 22.7% 5.7% 28.1% D COF1 AVLFCLSEDK (35-44) 4.5% 12.3% -2.6% 14.7% D HELQANCYEEVKDR (133-146) 5.7% 5.8% 9.2% 33.1% E CPNS1 TDGFGIDTCR (136-145) 6.4% 42.4% 7.2% 22.6% E CSRP1 NLDSTTVAVHGEEIYCK (43-59) 5.2% 20.1% 3.7% 20.5% D CXCL7 GTHCNQVEVIATLK (86-99) 2.1% 19.5% 6.5% 40.1% E ICLDPDAPR (104-112) 0.4% 6.0% 8.7% 65.8% E DBNL AEEDVEPECIMEK (119-131) 3.4% 26.3% 8.0% 37.3% E GACASHVSTMASFLK (95-109) 10.2% 20.6% 9.4% 50.9% D FHL1 CLHPLANETFVAK (71-83) 4.6% 16.9% 6.6% 46.3% E FDCHYCR (5-11) 5.7% 12.3% 5.1% 46.5% D FIBB LESDVSAQMEYCR (212-224) 0.1% 19.0% 3.7% 7.6% E 79  Protein Peptide ICATL%-changea Groupb 3μM-2mina 1mM-2mina 3μM-60mina 1mM-60mina FIBG CHAGHLNGVYYQGGTYSK (365-382) 5.6% 31.2% 6.7% 61.7% E VAQLEAQCQEPCKDTVQIHDITGK (154-177) 4.9% 20.5% 10.9% 64.3% E FLNA AHVVPCFDASK (1152-1162) 7.5% 51.4% 7.1% 21.5% C APSVANVGSHCDLSLK (2150-2165) 14.0% 32.2% 6.4% 37.8% D ATCAPQHGAPGPGPADASK (2541-2559) 5.5% 33.4% 8.3% 40.3% E CAPGVVGPAEADIDFDIIR (810-828) 9.8% 36.6% 10.3% 40.1% E CSGPGLSPGMVR (1453-1464) 4.5% 26.4% 6.1% 44.9% E IECDDKGDGSCDVR (621-634) 2.3% 2.4% 6.2% 34.7% E IVGPSGAAVPCK (1008-1019) 3.8% 27.4% 6.6% 45.5% E LQVEPAVDTSGVQCYGPGIEGQGVFR (1247-1272) 7.9% 28.9% 3.7% 27.9% C MDCQECPEGYR (2474-2484) 6.3% 17.7% 6.6% 36.1% E RAPSVANVGSHCDLSLK (2149-2165) 2.4% 25.3% 3.2% 26.4% E THEAEIVEGENHTYCIR (2185-2201) 7.9% 11.4% 5.7% 24.7% D VDINTEDLEDGTCR (2090-2103) 6.1% 7.2% 8.0% 27.3% E VHSPSGALEECYVTEIDQDK (2368-2387) 1.0% 16.5% 1.0% 15.1% E VQVQDNEGCPVEALVK (709-724) 6.9% 27.6% 3.0% 13.5% C VTYCPTEPGNYIINIK (2104-2119) 5.8% 18.9% 8.2% 36.3% E YVICVR (1720-1725) 5.0% 25.8% 6.6% 54.2% E G3P VPTANVSVVDLTCR (235-548) 8.3% 19.6% 6.9% 53.4% D G6B VNLSCGGVSHPIR (31-43) 5.3% 13.6% 34.4% 37.8% E GP1BA VASHLEVNCDK (25-35) 4.8% 20.0% -7.8% 30.0% D GPIX ALETMGLWVDCR (28-39) 1.8% 8.4% 5.6% 34.3% E CASPSLAAHGPLGR (113-126) 1.0% 9.3% 4.8% 26.0% E GSTP1 ASCLYGQLPK (46-55) 0.9% 21.1% 4.5% 25.6% E 80  Protein Peptide ICATL%-changea Groupb 3μM-2mina 1mM-2mina 3μM-60mina 1mM-60mina HBB GTFATLSELHCDK (84-96) 10.4% 28.3% 3.5% 21.5% C LLGNVLVCVLAHHFGK (106-121) 5.3% 15.1% 4.1% 23.8% D HBD GTFSQLSELHCDK (84-96) 10.8% 27.7% 5.4% 32.2% D LLGNVLVCVLAR (106-117) 7.3% 21.7% 5.0% 28.6% D HSP7C VCNPIITK (602-609) 6.7% 22.2% 2.4% 8.7% C IF5A1 KYEDICPSTHNMDVPNIK (68-85) 1.6% 12.7% 1.3% 8.1% C IGKC VYACEVTHQGLSSPVTK (83-99) 11.8% 19.2% 0.6% 6.8% B ITA2B AEGGQCPSLLFDLRDETR (91-108) 1.2% 6.0% 3.9% 14.7% E HSPICHTTMAFLR (572-584) 1.9% 11.1% -5.7% 10.1% C TPVGSCFLAQPESGR (157-170) 1.9% 22.6% 0.0% 0.0% A VVLCELGNPMK (715-725) 0.1% 7.1% 6.1% 16.3% E ITB1 CDDLEALK (64-71) 2.2% 9.3% 5.7% 41.1% E DKLPQPVQPDPVSHCK (677-692) 1.8% 9.1% 6.0% -14.5% E ITB3 CPTCPDACTFKK (627-638) 3.4% 3.4% 21.9% 34.3% E DNCAPESIEFPVSEAR (73-88) 2.3% 9.2% 12.8% 12.8% E LAGIVQPNDGQCHVGSDNHYSASTTMDYPSLGLMTEK (288-324) 7.5% 24.2% -11.7% 3.6% B YCRDEIESVK (660-669) 0.6% 7.7% -1.5% 1.5% C KPYM NTGIICTIGPASR (44-56) 6.3% 20.8% 0.3% 19.2% C LAC SYSCQVTHEGSTVEK (83-97) 10.0% 13.4% 6.0% 36.9% D LEGL LIVPFCGHIK (36-45) 8.3% 17.8% 1.5% 24.1% D VEILCEHPR (126-134) 4.7% 16.0% -3.6% 4.7% B LIMS1 CDLCQEVLADIGFVK (97-111) 2.2% 6.2% -0.3% 2.2% B CHAIIDEQPLIFK (138-150) 7.6% 10.1% 0.1% 5.5% B 81  Protein Peptide ICATL%-changea Groupb 3μM-2mina 1mM-2mina 3μM-60mina 1mM-60mina LRRF1 CEMSEHPSQTVR (750-761) 3.1% 16.0% 1.2% 3.2% C LTBP1 ASGLGDHCEDINECLEDK (1152-1169) 15.6% 15.6% -0.3% 21.8% D CPLPGTAAFK (717-726) 0.5% 3.7% -0.1% 9.4% D KPSYHGYNQMMECLPGYK (600-617) 8.1% 13.8% 1.9% 5.8% B LCQIPVHGASVPK (429-441) 2.2% 5.9% -4.2% 40.2% D VVFTPSICK (354-362) 0.5% 1.4% 6.0% 3.9% E MMRN1 CTSDMETILTFIPQFHR (765-781) 6.8% 21.4% 7.8% 40.9% E MTPN HHITPLLSAVYEGHVSCVK (67-85) 6.7% 16.5% 0.9% 23.2% D MYH9 ADFCIIHYAGK (566-576) 6.1% 8.8% 0.1% 27.7% D KLEEEQIILEDQNCK (975-989) 3.4% 25.5% 7.0% 46.2% E KMEDSVGCLETAEEVK (1372-1387) 5.1% 34.1% -1.7% 8.8% C KQELEEICHDLEAR (910-923) 6.4% 40.1% 6.5% 30.8% E VEDMAELTCLNEASVLHNLK (83-120) 9.3% 15.4% 4.4% 34.6% D MYL9 NAFACFDEEASGFIHEDHLR (105-124) 4.2% 12.5% 9.5% 33.6% E NDUS6 VIACDGGGGALGHPK (84-98) 3.3% 15.9% 0.3% 34.9% D NEXN GSAASTCILTIESK (661-674) 6.4% 27.8% 0.7% 12.6% C PARK7 GLIAAICAGPTALLAHEIGFGSK (100-122) 2.7% 11.2% 7.9% 10.4% E VTVAGLAGKDPVQCSR (33-48) 5.1% 14.6% 8.1% 35.8% E PDLI1 AALANLCIGDVITAIDGENTSNMTHLEAQNR (39-69) 5.9% 18.7% 6.9% 34.4% E GCTDNLTLTVAR (72-83) 5.2% 25.0% 5.2% 19.6% E GHFFVEDQIYCEK (297-309) 2.9% 17.9% 5.6% 20.4% E PECA1 CTIQVTHLAQEFPEIIIQK (256-274) 2.7% 6.2% 6.9% 8.2% E PGK1 ACANPAAGSVILLENLR (107-123) 4.4% 17.0% 5.8% 19.1% E PLF4 AGPHCPTAQLIATLK (63-77) 4.2% 21.4% 6.8% 26.2% E 82  Protein Peptide ICATL%-changea Groupb 3μM-2mina 1mM-2mina 3μM-60mina 1mM-60mina PLF4 ICLDLQAPLYK (82-92) 2.2% 13.4% 8.1% 25.0% E PPIA HTGPGILSMANAGPNTNGSQFFICTAK (92-118) 4.5% 9.3% 13.5% 31.5% E IIPGFMCQGGDFTR (56-69) 5.9% 9.1% 5.7% 31.0% D KITIADCGQLE (155-165) 2.3% 12.5% 48.4% 46.9% E PROF1 CYEMASHLR (128-136) 3.4% 20.3% 5.2% 28.8% E RHOA HFCPNVPIILVGNK (105-118) 6.7% 15.3% 2.3% 2.3% A IGAFGYMECSAK (151-162) 1.7% 7.3% -36.6% 28.1% D LVIVGDGACGK (8-18) 0.2% 1.4% 1.9% 2.0% E TCLLIVFSK (19-27) 1.0% 1.6% 8.6% 20.6% E RS12 LVEALCAEHQINLIK (64-78) 2.0% 15.3% -3.9% 16.4% D RTN4 YSNSALGHVNCTIK (1091-1104) 6.0% 28.7% 3.6% 21.9% C SODC GLTEGLHGFHVHEFGDNTAGCTSAGPHFNPLSR (38-70) 6.8% 8.5% -0.3% 21.7% D STIP1 ALSVGNIDDALQCYSEAIK (14-32) 9.2% 34.9% 7.8% 38.5% D TAGL2 DGTVLCELINALYPEGQAPVK (58-78) 16.7% 26.4% 7.4% 35.9% D TBB1 EIVHIQIGQCGNQIGAK (3-19) 4.7% 18.0% -76.5% -26.6% A TBB2A LTTPTYGDLNHLVSATMSGVTTCLR (217-241) 54.3% 55.0% 3.9% -8.6% A TAVCDIPPR (351-359) 5.3% 18.0% 6.6% 29.4% E TEBP LTFSCLGGSDNFK (36-48) 8.8% 22.5% 2.7% 40.1% D THAS DVFSSTGCKPNPSR (302-315) 10.3% 26.7% 9.2% 31.6% D TLN1 AGALQCSPSDAYTK (1934-1947) 5.5% 21.6% 3.3% 32.0% D ASAGPQPLLVQSCK (944-957) 12.6% 24.3% 7.3% 47.9% D CVSCLPGQR (1199-1207) 21.4% 23.0% 7.8% 27.3% D MVAAATNNLCEAANAAVQGHASQEK (2399-2423) 6.7% 31.7% 10.5% 40.6% E NCGQMSEIEAK (285-295) 3.8% 25.2% 5.7% 26.5% E 83  Protein Peptide ICATL%-changea Groupb 3μM-2mina 1mM-2mina 3μM-60mina 1mM-60mina TLN1 VQELGHGCAALVTK (1920-1933) 10.2% 23.8% 12.5% 42.6% E TPIS IAVAAQNCYK (60-69) 4.2% 9.5% 5.8% 24.4% E VAHALAEGLGVIACIGEK (114-131) 2.7% 11.5% -2.9% 5.7% C TPM4 EENVGLHQTLDQTLNELNCI (229-248) 7.8% 34.2% 5.6% 21.1% C TSP1 CNYLGHYSDPMYR (663-675) 3.1% 5.7% 2.6% 42.9% D DCVGDVTENQICNK (530-543) 12.8% 27.8% 3.9% 2.2% A DNCPFHYNPAQYDYDR (752-767) 0.4% 50.2% 25.3% 25.3% E GDACDHDDDNDGIPDDK (891-907) 0.1% 23.8% 2.0% 13.5% E GDACKDDFDHDSVPDIDDICPENVDISETDFR (927-958) 2.7% 22.8% 0.4% 28.9% D LCNNPTPQFGGK (518-529) 0.5% 0.9% -5.4% 3.6% D LCNSPSPQMNGKPCEGEAR (461-479) 48.7% 52.4% 0.0% 0.0% A RPPLCYHNGVQYR (314-326) 1.0% 5.1% 2.2% 12.5% E URP2 ASFSQPLFQAVAAICR (114-129) 3.8% 24.3% 5.4% 48.2% E VINC TNLLQVCER (1047-1055) 3.0% 22.8% 5.7% 41.4% E VWF GLRPSCPNSQSPVK (1922-1935) 5.6% 17.7% 3.2% 28.5% D LVCPADNLR (774-782) 0.0% 6.7% 3.2% 10.1% E ZYX CNTCGEPITDR (444-454) 1.8% 15.7% 1.1% 10.6% C CSVCSEPIMPEPGRDETVR (504-522) 6.5% 13.9% 2.6% 14.5% D  a: The ICATL%-changes at 3 μM-2 min, 1 mM-2 min, 3 μM-60 min and 1 mM-60 min are the differences of ICATL% between AS treatment (i.e.,3 μM and 1 mM) and the control (0 μM) after 2 min and 60 min respectively. b: The 159 cysteines were assorted in Group A-E according to the magnitude of ICATL%-change at 60 min. 84  E. Protein functions of the HNO-reactive proteins identified by using ICAT as alkylation reagents Functions of 1433Z, ACTN1, COF1, CXCL7, FIBB, FIBG, FLNA, G3P, HBB, HSP7C, ITA2B, MYH9, TLN1, TPIS, URP2 and VINC were listed in Appendix A. SwissProt Accession # Entry name Protein Function Q04917 1433F_HUMAN 14-3-3 protein eta Adapter protein implicated in the regulation of a large spectrum of both general and specialized signaling pathways. Binds to a large number of partners, usually by recognition of a phosphoserine or phosphothreonine motif. Binding generally results in the modulation of the activity of the binding partner. P05067 A4_HUMAN Amyloid beta A4 protein Functions as a cell surface receptor and performs physiological functions on the surface of neurons relevant to neurite growth, neuronal adhesion and axonogenesis. Involved in cell mobility and transcription regulation through protein-protein interactions. P60709  ACTB_HUMAN actin Actins are highly conserved proteins that are involved in various types of cell motility and are ubiquitously expressed in all eukaryotic cells. O43707 ACTN4_HUMAN Alpha-actinin-4 F-actin cross-linking protein which is thought to anchor actin to a variety of intracellular structures. This is a bundling protein. Probably involved in vesicular trafficking via its association with the CART complex. The CART complex is necessary for efficient transferrin receptor recycling but not for EGFR degradation. P02768 ALBU_HUMAN Serum albumin Serum albumin, the main protein of plasma, has a good binding capacity for water, Ca2+, Na+, K+, fatty acids, hormones, bilirubin and drugs. Its main function is the regulation of the colloidal osmotic pressure of blood. Major zinc transporter in plasma, typically binds about 80% of all plasma zinc. P04075 ALDOA_HUMAN Fructose-bisphosphate aldolase A An enzyme that catalyses a reverse aldol reaction. P61158 ARP3_HUMAN Actin-related protein 3 Functions as ATP-binding component of the Arp2/3 complex which is involved in regulation of actin polymerization and together with an activating nucleation-promoting factor (NPF) mediates the formation 85  SwissProt Accession # Entry name Protein Function of branched actin networks. P27797 CALR_HUMAN Calreticulin Molecular calcium-binding chaperone promoting folding, oligomeric assembly and quality control in the ER via the calreticulin/calnexin cycle. This lectin interacts transiently with almost all of the monoglucosylated glycoproteins that are synthesized in the ER. Interacts with the DNA-binding domain of NR3C1 and mediates its nuclear export. Q01518  CAP1_HUMAN Adenylyl cyclase-associated protein 1 Directly regulates filament dynamics and has been implicated in a number of complex developmental and morphological processes, including mRNA localization and the establishment of cell polarity. O14618 CCS_HUMAN Copper chaperone for superoxide dismutase Delivers copper to copper zinc superoxide dismutase (SOD1). P16671 CD36_HUMAN Platelet glycoprotein 4 Seems to have numerous potential physiological functions. Binds to collagen, thrombospondin, anionic phospholipids and oxidized LDL. May function as a cell adhesion molecule. Directly mediates cytoadherence of Plasmodium falciparum parasitized erythrocytes. Binds long chain fatty acids and may function in the transport and/or as a regulator of fatty acid transport. P34810 CD68_HUMAN Macrosialin Could play a role in phagocytic activities of tissue macrophages, both in intracellular lysosomal metabolism and extracellular cell-cell and cell-pathogen interactions. Binds to tissue- and organ-specific lectins or selectins, allowing homing of macrophage subsets to particular sites. Rapid recirculation of CD68 from endosomes and lysosomes to the plasma membrane may allow macrophages to crawl over selectin-bearing substrates or other cells. Q9P1F3 CF115_HUMAN Costars family protein C6orf115 Unknown 86  SwissProt Accession # Entry name Protein Function P10809 CH60_HUMAN 60 kDa heat shock protein Implicated in mitochondrial protein import and macromolecular assembly. May facilitate the correct folding of imported proteins. May also prevent misfolding and promote the refolding and proper assembly of unfolded polypeptides generated under stress conditions in the mitochondrial matrix. Q9P126 CLC1B_HUMAN C-type lectin domain family 1 member B Acts as a receptor for the platelet-aggregating snake venom protein rhodocytin. Rhodocytin binding leads to tyrosine phosphorylation and this promotes the binding of spleen tyrosine kinase (Syk) and initiation of downstream tyrosine phosphorylation events and activation of PLC-gamma-2. Acts as an attachment factor for human immunodeficiency virus type 1 (HIV-1) and facilitates its capture by platelets. O00299 CLIC1_HUMAN Chloride intracellular channel protein 1 Can insert into membranes and form chloride ion channels. Channel activity depends on the pH. Membrane insertion seems to be redox-regulated and may occur only under oxidizing conditions. Involved in regulation of the cell cycle. Q99439 CNN2_HUMAN Calponin-2 Thin filament-associated protein that is implicated in the regulation and modulation of smooth muscle contraction. It is capable of binding to actin, calmodulin, troponin C and tropomyosin. The interaction of calponin with actin inhibits the actomyosin Mg-ATPase activity. P04632 CPNS1_HUMAN Calpain small subunit 1 Regulatory subunit of the calcium-regulated non-lysosomal thiol-protease which catalyzes limited proteolysis of substrates involved in cytoskeletal remodeling and signal transduction. P21291 CSRP1_HUMAN Cysteine and glycine-rich protein 1 Could play a role in neuronal development. Q9UJU6 DBNL_HUMAN Drebrin-like protein Actin-binding adapter protein. May act as a common effector of antigen receptor-signaling pathways in leukocytes. Its association with dynamin suggests that it may also connect the actin cytoskeleton to endocytic function. Acts as a key component of the immunological synapse that regulates T-cell activation by bridging TCRs and the actin cytoskeleton to gene activation and endocytic processes. Binds to F-actin but is not involved in actin polymerization, capping or bundling. Does not bind G-actin. 87  SwissProt Accession # Entry name Protein Function Q13642 FHL1_HUMAN Four and a half LIM domains protein 1 May have an involvement in muscle development or hypertrophy. O95866 G6B_HUMAN Protein G6b Inhibits platelet aggregation and activation by agonists such as ADP and collagen-related peptide. Appears to operate in a calcium-independent manner. Isoform B is a putative inhibitory receptor. Isoform A may be (continued) its activating counterpart. P07359  GP1BA_HUMAN Platelet glycoprotein Ib alpha chain GP-Ib, a surface membrane protein of platelets, participates in the formation of platelet plugs by binding to the A1 domain of vWF, which is already bound to the subendothelium. P14770 GPIX_HUMAN Platelet glycoprotein IX The GPIb-V-IX complex functions as the vWF receptor and mediates vWF-dependent platelet adhesion to blood vessels. The adhesion of platelets to injured vascular surfaces in the arterial circulation is a critical initiating event in hemostasis. GP-IX may provide for membrane insertion and orientation of GP-Ib. P09211 GSTP1_HUMAN Glutathione S-transferase P Conjugation of reduced glutathione to a wide number of exogenous and endogenous hydrophobic electrophiles. P02042 HBD_HUMAN Hemoglobin subunit delta Involved in oxygen transport from the lung to the various peripheral tissues. P63241  IF5A1_HUMAN Eukaryotic translation initiation factor 5A-1 mRNA-binding protein involved in translation elongation. Has an important function at the level of mRNA turnover, probably acting downstream of decapping. Involved in actin dynamics and cell cycle progression, mRNA decay and probably in a pathway involved in stress response and maintenance of cell wall integrity. P01834 IGKC_HUMAN Ig kappa chain C region Unknown P05556 ITB1_HUMAN Integrin beta-1 Beta-1 integrins recognize the sequence R-G-D in a wide array of ligands. Isoform beta-1B interferes with isoform beta-1A resulting in a dominant negative effect on cell adhesion and migration (in vitro). Receptors for collagen, fibronectin, fibrinogen, laminin and etc. P05106 ITB3_HUMAN Integrin beta-3 Integrins alpha-IIb/beta-3 and alpha-V/beta-3 recognize the sequence R-G-D in a wide array of ligands. Receptors for fibronectin, laminin, thrombospondin, vWF and etc. 88  SwissProt Accession # Entry name Protein Function P14618 KPYM_HUMAN Pyruvate kinase isozymes M1/M2 Glycolytic enzyme that catalyzes the transfer of a phosphoryl group from phosphoenolpyruvate (PEP) to ADP, generating ATP. Stimulates POU5F1-mediated transcriptional activation. Plays a general role in caspase independent cell death of tumor cells. The ratio betwween the highly active tetrameric form and nearly inactive dimeric form determines whether glucose carbons are channeled to biosynthetic processes (continued) or used for glycolytic ATP production. The transition between the two forms contributes to the control of glycolysis and is important for tumor cell proliferation and survival. P0CG04 LAC1_HUMAN Ig lambda-1 chain C regions Unknown Q3ZCW2 LEGL_HUMAN Galectin-related protein Does not bind lactose, and may not bind carbohydrates. P48059 LIMS1_HUMAN LIM and senescent cell antigen-like-containing domain protein 1 Effector of integrin and growth factor signaling, coupling surface receptors to downstream signaling molecules involved in the regulation of cell survival, cell proliferation and cell differentiation. Focal adhesion protein, part of the complex ILK-PINCH. This complex is considered to be one of the convergence points of integrin- and growth factor-signaling pathway. Q32MZ4 LRRF1_HUMAN Leucine-rich repeat flightless-interacting protein 1 Transcriptional repressor which preferentially binds to the GC-rich consensus sequence (5'-AGCCCCCGGCG-3') and may regulate expression of TNF, EGFR and PDGFA. May control smooth muscle cell proliferation following artery injury through PDGFA repression. May also bind double-stranded RNA. Q14766 LTBP1_HUMAN Latent-transforming growth factor beta-binding protein 1 May be involved in the assembly, secretion and targeting of TGFB1 to sites at which it is stored and/or activated. May play critical roles in controlling and directing the activity of TGFB1. May have a structural role in the extra cellular matrix (ECM). Q13201 MMRN1_HUMAN Multimerin-1 Carrier protein for platelet (but not plasma) factor V/Va. Plays a role in the storage and stabilization of factor V in platelets. Upon release following platelet activation, may limit platelet and plasma factor Va-dependent thrombin generation. Ligand for integrin alpha-IIb/beta-3 and integrin alpha-V/beta-3 on 89  SwissProt Accession # Entry name Protein Function activated platelets, and may function as an extracellular matrix or adhesive protein. P58546 MTPN_HUMAN Myotrophin Potential role in cerebellar morphogenesis. May function in differentiation of cerebellar neurons, particularly of granule cells. Seems to be associated with cardiac hypertrophy. P24844 MYL9_HUMAN Myosin regulatory light polypeptide 9 Myosin regulatory subunit that plays an important role in regulation of both smooth muscle and nonmuscle (continued) cell contractile activity via its phosphorylation. Implicated in cytokinesis, receptor capping, and cell locomotion. O75380 NDUS6_HUMAN NADH dehydrogenase [ubiquinone] iron-sulfur protein 6, mitochondrial Accessory subunit of the mitochondrial membrane respiratory chain NADH dehydrogenase (Complex I), that is believed not to be involved in catalysis. Complex I functions in the transfer of electrons from NADH to the respiratory chain. The immediate electron acceptor for the enzyme is believed to be ubiquinone. Q0ZGT2 NEXN_HUMAN Nexilin Involved in regulating cell migration through association with the actin cytoskeleton. Has an essential role in the maintenance of Z line and sarcomere integrity. Q99497 PARK7_HUMAN Protein DJ-1 Protects cells against oxidative stress and cell death. O00151 PDLI1_HUMAN PDZ and LIM domain protein 1 Cytoskeletal protein that may act as an adapter that brings other proteins (like kinases) to the cytoskeleton. P16284 PECA1_HUMAN Platelet endothelial cell adhesion molecule Induces susceptibility to atherosclerosis. Cell adhesion molecule which is required for leukocyte transendothelial migration (TEM) under most inflammatory conditions. P00558 PGK1_HUMAN Phosphoglycerate kinase 1 In addition to its role as a glycolytic enzyme, it seems that PGK-1 acts as a polymerase alpha cofactor protein (primer recognition protein). P02776 PLF4_HUMAN Platelet factor 4 Released during platelet aggregation. Neutralizes the anticoagulant effect of heparin because it binds more strongly to heparin than to the chondroitin-4-sulfate chains of the carrier molecule. Chemotactic for neutrophils and monocytes. Inhibits endothelial cell proliferation, the short form is a more potent inhibitor than the longer form. 90  SwissProt Accession # Entry name Protein Function P62937 PPIA_HUMAN Peptidyl-prolyl cis-trans isomerase A PPIases accelerate the folding of proteins. It catalyzes the cis-trans isomerization of proline imidic peptide bonds in oligopeptides." P07737 PROF1_HUMAN Profilin-1 Binds to actin and affects the structure of the cytoskeleton. At high concentrations, profilin prevents the polymerization of actin, whereas it enhances it at low concentrations. By binding to PIP2, it inhibits the formation of IP3 and DG. P61586 RHOA_HUMAN Transforming protein RhoA Regulates a signal transduction pathway linking plasma membrane receptors to the assembly of focal adhesions and actin stress fibers. P25398 RS12_HUMAN 40S ribosomal protein S12 Structural constituent of ribosome. Q9NQC3 RTN4_HUMAN Reticulon-4 Developmental neurite growth regulatory factor with a role as a negative regulator of axon-axon adhesion and growth, and as a facilitator of neurite branching. Regulates neurite fasciculation, branching and extension in the developing nervous system. Involved in down-regulation of growth, stabilization of wiring and restriction of plasticity in the adult CNS. P00441 SODC_HUMAN Superoxide dismutase [Cu-Zn] Destroys radicals which are normally produced within the cells and which are toxic to biological systems. P31948 STIP1_HUMAN Stress-induced-phosphopr otein 1 Mediates the association of the molecular chaperones HSC70 and HSP90 (HSPCA and HSPCB). Q8H278 TAGL2_HUMAN Transgelin-2 A homolog of the protein transgelin, which is one of the earliest markers of differentiated smooth muscle. The function of this protein has not yet been determined. Q9H4B7 TBB1_HUMAN Tubulin beta-1 chain Tubulin is the major constituent of microtubules. It binds two moles of GTP, one at an exchangeable site on the beta chain and one at a non-exchangeable site on the alpha-chain Q13885 TBB2A_HUMAN Tubulin beta-2A chain Tubulin is the major constituent of microtubules. It binds two moles of GTP, one at an exchangeable site on the beta chain and one at a non-exchangeable site on the alpha-chain 91  SwissProt Accession # Entry name Protein Function Q15185 TEBP_HUMAN Prostaglandin E synthase 3 Molecular chaperone that localizes to genomic response elements in a hormone-dependent manner and disrupts receptor-mediated transcriptional activation, by promoting disassembly of transcriptional regulatory complexes. P24557 THAS_HUMAN Thromboxane-A synthase An enzyme that catalyses Thromboxane A2 formation. P67936 TPM4_HUMAN Tropomyosin alpha-4 chain Binds to actin filaments in muscle and non-muscle cells. Plays a central role, in association with the troponin complex, in the calcium dependent regulation of vertebrate striated muscle contraction. Smooth muscle contraction is regulated by interaction with caldesmon. In non-muscle cells is implicated in stabilizing cytoskeleton actin filaments. Binds calcium. P07996 TSP1_HUMAN Thrombospondin-1 Adhesive glycoprotein that mediates cell-to-cell and cell-to-matrix interactions. P04275 VWF_HUMAN von Willebrand factor Important in the maintenance of hemostasis, it promotes adhesion of platelets to the sites of vascular injury by forming a molecular bridge between sub-endothelial collagen matrix and platelet-surface receptor complex GPIb-IX-V. Q15942 ZYX_HUMAN Zyxin Adhesion plaque protein. Binds alpha-actinin and the CRP protein. Important for targeting TES and ENA/VASP family members to focal adhesions and for the formation of actin-rich structures. May be a component of a signal transduction pathway that mediates adhesion-stimulated changes in gene expression.