Structural profiling of endogenous S-nitrosocysteineresidues reveals unique features that accommodatediverse mechanisms for protein S-nitrosylationPaschalis-Thomas Douliasa,1, Jennifer L. Greenea,1, Todd M. Grecoa, Margarita Tenopouloua, Steve H. Seeholzera,Roland L. Dunbrackb, and Harry Ischiropoulosa,2

aChildren’s Hospital of Philadelphia Research Institute and Department of Pharmacology, Children’s Hospital of Philadelphia and University of Pennsylvania,Philadelphia, PA 19104; and bProgram in Molecular Medicine, Fox Chase Cancer Center, Philadelphia, PA 19111

Edited by Michael A. Marletta, University of California, Berkeley, CA, and approved August 19, 2010 (received for review June 9, 2010)

S-nitrosylation, the selective posttranslational modification of pro-tein cysteine residues to form S-nitrosocysteine, is one of themolecular mechanisms by which nitric oxide influences diversebiological functions. In this study, unique MS-based proteomicapproaches precisely pinpointed the site of S-nitrosylation in 328peptides in 192 proteins endogenously modified in WT mouse liver.Structural analyses revealed that S-nitrosylated cysteine residueswere equally distributed in hydrophobic and hydrophilic areas ofproteinswith an averagepredictedpKa of 10.01± 2.1. S-nitrosylationsites were over-represented in α-helices and under-represented incoils as comparedwith unmodified cysteine residues in the samepro-teins (χ2 test, P< 0.02). A quantile–quantile probability plot indicatedthat the distribution of S-nitrosocysteine residues was skewed to-ward larger surface accessible areas compared with the unmod-ified cysteine residues in the same proteins. Seventy percent of theS-nitrosylated cysteine residues were surrounded by negatively orpositively charged amino acids within a 6-Å distance. The location ofcysteine residues in α-helices and coils in highly accessible surfacesbordered by charged amino acids implies site directed S-nitrosylationmediatedbyprotein–proteinor smallmolecule interactions.Moreover,13 modified cysteine residues were coordinated with metals and15 metalloproteins were endogenously modified supporting metal-catalyzed S-nitrosylation mechanisms. Collectively, the endogenous S-nitrosoproteome in the liver has structural features that accommodatemultiple mechanisms for selective site-directed S-nitrosylation.

cysteine modification | nitric oxide | S-nitrosation | posttranslationalmodification | proteomics

Cysteine S-nitrosylation is a reversible and apparently selectiveposttranslational protein modification that regulates proteinactivity, localization, and stability within a variety of organs andcellular systems (1–6). Despite the considerable biological impor-tance of this posttranslational modification, significant gaps existregarding its in vivo specificity and origin. The identification ofin vivo S-nitrosylated proteins has indicated that not all reducedcysteine residues andnot all proteinswith reduced cysteine residuesare modified, implying a biased selection. Several biologicalchemistries have been proposed to account for the S-nitrosylationof proteins in vivo (1, 7, 8). Broadly, these include (i) oxidativeS-nitrosation byhigher oxides ofNO, (ii) transnitrosylationby smallmolecular weight NO carriers such as S-nitrosoglutathione ordinitrosyliron complexes, (iii) catalysis by metalloproteins, and (iv)protein-assisted transnitrosation, as elegantly documented for theS-nitrosylation of caspase-3 by S-nitrosothioredoxin (9, 10). Withthe exception of the protein-assisted transnitrosylation and metal-loprotein catalyzed S-nitrosylation, which we presume necessitatesprotein–protein interaction, the other proposed mechanisms arerather nondiscriminatory unless the microenvironment of selectivecysteine residues in proteins can specifically accommodate thesechemical modifications. Therefore, structural interrogation of en-dogenous S-nitrosylated proteins with site specific identification ofthe modified cysteine residues could provide valuable insights to

appreciate the biological selectivity of this posttranslational modi-fication. Attempts to investigate this very important biologicalquestion have not been possible largely because datasets of in vivomodified proteins are not available (1, 11). Previous structuralanalyses have beenattemptedusing limited data sets or by includingall sites of modification identified after exposing tissues or cells toS-nitrosylating agents (11).However, as the authorsof thesearticleshave indicated, these sites of modification represent putative sitesbut not necessarily those modified in vivo (11). We used organo-mercury reagents that react directly, efficiently, and specificallywith S-nitrosocysteine and thus enable the precise identificationof S-nitrosocysteine–containing peptides and independently S-nitrosylated proteins to assemble the in vivo S-nitrosocysteineproteome of the mouse liver. By using bioinformatic tools, we theninterrogated this enriched endogenous S-nitrosocysteine proteometo define the biochemical, biophysical, and structural environmentof the cysteine residues modified by S-nitrosylation, elements thatmight inform on how specificity of S-nitrosylation is achieved.

Results and DiscussionComplementary MS-Based Proteomics Identify the Endogenous LiverS-Nitrosocysteine Proteome. The reaction of phenylmercury com-pounds with S-nitrosocysteine results in the formation of a relativelystable thiol–mercurybond(12).Therefore,weusedanorganomercuryresin (MRC) synthesized by conjugation of ρ-amino-phenylmercuricacetate to N-hydroxysuccinimide–activated Affi-Gel 10 agarosebeads and a phenylmercury-polyethyleneglycol-biotin (mPEGb)compound to capture S-nitrosylated proteins and peptides (Fig.S1). The method consists of three steps: (i) blocking of reducedcysteine residues with methyl methanethiosulfonate (MMTS), (ii)capture and release of S-nitrosylated proteins or peptides, and (iii)liquid chromatography/tandemMSanalysis. β-Mercaptoethanol orperformic acid was used to release captured proteins or pep-tides, respectively. Mild performic acid was used to selectively andquantitatively release the bound peptides and more importantlyoxidize cysteine thiols to sulfonic acid, thereby creating a uniqueMS signature that permits site-specific identification of the modi-fied cysteine residues (13). Under the workflow used (Fig. S1),100% of cysteine-containing peptides were detected with the sul-fonic acid modification.

Author contributions: H.I. designed research; P.-T.D., J.L.G., M.T., and S.H.S. performedresearch; P.-T.D., T.M.G., and H.I. contributed new reagents/analytic tools; P.-T.D., J.L.G.,T.M.G., S.H.S., R.L.D., and H.I. analyzed data; and P.-T.D., J.L.G., T.M.G., and H.I. wrote thepaper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1P.-T.D. and J.L.G. contributed equally to this work.2To whom correspondence should be addressed. E-mail: ischirop@mail.med.upenn.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1008036107/-/DCSupplemental.

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To control for the inclusion of false-positive protein S-nitrosocysteine that may result from incomplete blocking of re-duced cysteine residues or nonspecific interactions with the resinsduring enrichment, pretreatment with UV, DTT, and copper-ascorbate (Cu-Asc) reduction or reaction with mercury chloridewere used to displace NO before the reaction with the phenyl-mercury compounds (Fig. 1A and Fig. S2A). For these experi-ments, we used mouse liver homogenates with 50 ± 10 pmolprotein S-nitrosocysteine per milligram of protein as quantified byreductive chemistries coupled to ozone-based chemiluminescence.Displacement of NO from S-nitrosocysteine residues by pre-treatment with UV, DTT, Cu-Asc, or mercury chloride decreasedprotein S-nitrosocysteine levels by greater than 99% as quantifiedby chemiluminescence (n = 3) and also eliminated reaction withthe phenylmercury compounds (Fig. 1A andFig. S2A).On average,only 3% of peptides (Fig. 1B) and 5% of proteins (Fig. S2B)were identified as false positives (present in untreated and UV-pretreated samples), demonstrating that the method maintainshigh specificity at each step. This false positive rate compares fa-vorably with the greater than 90% specificity reported for immo-bilized metal affinity chromatography used for the enrichment ofphosphopeptides from mouse liver (14). Therefore, pretreatmentwith UV served as negative control throughout based on previousstudies that documented the elimination of S-nitrosocysteinewithout affecting other cysteine modifications (15, 16). Collec-tively, these experiments showed that the reaction of protein S-

nitrosocysteine with the phenylmercury compounds is specific andefficient and achieved selective identification of the modified res-idue, the ultimate qualifier for the unambiguous assignment of S-nitrosylated proteins through the inclusion of negative controls.The ability of the method to reveal endogenous S-

nitrosoproteomes was assessed in WT mouse liver homogenates.Initially, three different mouse livers were analyzed independentlyfor protein and peptide capture by using both the MRC andmPEGb approach with 3 mg of starting material. From the threebiological replicates, sulfonic acid-containing peptides identifiedby SEQUEST database searches were pooled and those alsopresent in the UV-pretreated samples were removed. Similarly, allproteins identified by protein capture were pooled (those alsoidentified in the UV-pretreated samples were removed). The useof solid-phase and in-solution–based enrichment approaches waslargely complementary, as the number of shared protein identi-fications between protein capture methods was approximately50%, whereas each method individually contributed about thesame number of unique protein identifications (Table S1). As theMRC capture method allows for higher input, site-specific peptidecapture by this methodwas also performedwith the use of 30mg ofliver extract originating from three different mice. The inclusion ofthese additional biological replicates reduced biological varianceand improved the depth of the analysis while maintaining less than3% false identification rate. Overall by matching sulfonic acid-containing peptides with corresponding protein identifications, we

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Fig. 1. Site-specific identification and UV photolysis confirmation of S-nitrosocysteine. (A) Representative short (approximately 2 cm) colloidal blue-stainedprotein gel of mPEGb protein capture from WT mouse liver that was processed by GeLC-MS/MS analysis. Colloidal blue protein staining of bound fractionsfrom mPEGb protein capture of untreated liver homogenates showed protein enrichment. Pretreatment with UV (for 30 min), DTT (10 mM), Cu-Asc (5 mMAsc/copper 0.1 mM), or HgCl2 (20 mM) prevented this capture, demonstrating specificity for protein S-nitrosocysteine. (B) Representative base peak chro-matograms from mPEGb peptide capture demonstrates reduction in ion intensity after UV treatment (red) compared with the untreated sample (black). Forclarity, the chromatogram corresponding to UV treatment was plotted with a y-axis offset of 1E4. Inset: Average false-positive identification rate (FIR ± SD)from mPEGb and MRC represented the percentage of peptides that were identified in both the UV-treated and untreated samples across three independentbiological replicates. Also reported is the percent of noncysteinyl peptides identified (SI Materials and Methods). (C) Representative MS spectra of doublycharged sulfonic acid-containing tryptic peptides, YLLGTSLARPC97IAR (monoisotopic m/z, 791.4256; Top, Left) and FELTC132YSLAPQIK (monoisotopic m/z,780.8851; Bottom, Left), from argininosuccinate synthase. MS/MS spectra confirmed the sequence and site of sulfonic acid-containing peptides (C+48) fromargininosuccinate synthase identified in mouse liver (SI Materials and Methods). MS/MS spectra passed automatic and manual filter criteria (Fig. S2), and wereidentified with high SEQUEST cross correlation (Xc) scores at ppm mass error. Cys132 has been previously reported as a target of S-nitrosylation in humanargininosuccinate synthase (20), whereas the identification of Cys97 corresponded to a previously unidentified site of S-nitrosocysteine formation. (D) Col-loidal blue protein staining of bound fractions from MRC and mPEGb enrichment of S-nitrosylated proteins from WT and eNOS−/− mouse livers. UV photolysisdemonstrated specificity of the enrichment. The levels of protein S-nitrosocysteine were quantified by reductive chemistries coupled to chemiluminescent-based detection (41). The amount of S-nitrosylated protein captured by mPEGb from eNOS−/− livers (lane 8) was below the limit of detection by colloidal bluestaining. (E) Western blot analysis of bound and unbound fractions from WT mouse liver that were processed by MRC protein capture.

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precisely pinpointed 328 S-nitrosocysteine–containing peptides in192 proteins in untreated livers with a high level of confidence(complete list is provided in Table S1 and all of theMS/MS spectrafrom peptide capture can be viewed at http://www.research.chop.edu/tools/msms/spectra.pdf). The depth of this analysis representsa significant advancement versus present methodologies (17). Themajority of the proteins identified in the current study, 186, cor-responded to previously unidentified endogenous targets ofS-nitrosylation in the mouse liver, whereas six proteins (GAPDH,hemoglobin, β-tubulin, argininosuccinate synthase, alcohol de-hydrogenase, and catalase) have been previously identified as en-dogenously S-nitrosylated in hepatocytes and other organ systems(18–21). Proteins were also distributed across a wide range ofmolecular weights (13–272 kDa) and cellular localization includingmembrane-associated proteins, demonstrating the efficacy of themethod to identify S-nitrosocysteine independent of protein sizeand location. We selected very long chain specific acyl-CoA de-hydrogenase, heat shock cognate 71 protein, for which endogenousS-nitrosylation was not previously described, and GAPDH, whichhas been known to be S-nitrosylated and independently confirmedtheir selective enrichment by Western blot analysis after proteincapture (Fig. 1E).To further probe the biological specificity of our method

while demonstrating its utility for comparison of endogenousS-nitrosoproteomes, we analyzed livers from mice lacking endo-thelial NO synthase (eNOS−/−). Using chemiluminescence-basedquantification, a 10-fold decrease in protein S-nitrosocysteine lev-els of eNOS−/− livers was measured as compared with WT livers.Concomitantly, a reduced reactivity with phenylmercury com-pounds was observed (Fig. 1D). From the eNOS−/− livers, 36 sul-fonic acid-containing peptides in 26 proteinswere identified (TableS2), of which 24 were also identified in the WT liver. The data in-dicate that themajority of the endogenous liver S-nitrosoproteomeis dependent on eNOS-generated NO.

S-Nitrosylation Is Implicated in Multiple Metabolic Pathways. Pro-teomic experiments generate rich, diversedatasets that benefit fromcomputational analysis to extract biologically relevant and poten-tially novel information. Consequently, functional and ontologicalanalyses were conducted to assist in identifying cellular, molecular,and biological functions in which S-nitrosylation may play a role inthe liver. Sixty-five percent of S-nitrosylated proteins were localizedto the cytoplasm and mitochondrion, representing a significantenrichment comparedwith themouse genome(P=4.9e−34 andP=7.2e−22, respectively; Fig. S3A). A subset of S-nitrosylated proteinswere found to be present in cellmembranes, whereas the remainingproteins were distributed across nearly all cellular compartments(Fig. S3A). Gene ontology analysis revealed that 99 S-nitrosylatedproteins had catalytic activity largely composed of oxidoreductases(39%) and transferases (17%; Fig. S3B). These functions were alsofound to be significantly overrepresented (P= 1.28e−20 and 3.2e−3,respectively) in the liver S-nitrosoproteome compared with themouse genome (22). This is not surprising, as the molecular func-tions of S-nitrosylated proteins were assigned to diverse metabolicprocesses (i.e., amino acid synthesis, energy synthesis, lipid me-tabolism) that take place within the liver.Analysis of the data has also confirmed the presence of multiple

S-nitrosylated cysteine residues in nearly 45% (86 of 192) of theliver S-nitrosoproteome. Poly-S-nitrosylation is present in all top-ranking molecular functions, suggesting that multiple sites ofS-nitrosylation in vivo may regulate protein activity. This is in ac-cordance with other known posttranslational modifications suchas phosphorylation and lysine acetylation in which polyphos-phorylation and polyacetylation are considered regulators of pro-tein function and signaling (23, 24).To place these functional assignments into the context of bio-

chemical andmolecular signaling pathways, S-nitrosylatedproteinswere assembled into biological protein interaction and signaling

networks (Ingenuity Systems). The analysis was restricted to in-vestigate liver-related pathways. Sixteen S-nitrosylated proteinswere significantly clustered in a network that encompassed liverresponses to the hormone leptin (Fig. S3C). Leptin is an adipocyte-secreted hormone that primarily acts on the central nervous sys-tem to regulate energy homeostasis. Leptin also regulates livermetabolism, evident by the significant accumulation of lipids (fattyliver) inmice deficient in leptin (ob/ob) (25, 26) or leptin long-formreceptor (db/db) (27). The regulation of liver metabolism is at-tributed to the leptin-dependent repression of liver stearoyl-CoAdesaturase-1, the rate limiting step in monosaturated fat bio-synthesis (28). In addition, recent data indicate that leptin alsoregulates liver mitochondrial respiratory chain protein expression,mitochondrial function and structure (29), remarkably similar tothe previously recognized regulation of mitochondrial function byNO(30, 31). Interestingly, delivery of S-nitroso-N-acetylcysteinebygavage to ob/obmice prevented the development of fatty liver (32).Mice deficient in eNOS also experience abnormal fat deposition inthe liver (33), which was attributed in part to regulation of mito-chondrial fatty acid synthesis and activation of AMP-activatedprotein kinase (33). Moreover, 14 of the 16 proteins in the leptinnetwork (Fig. S3C) were absent in eNOS−/− liver S-nitrosocysteineproteome analysis, suggesting a potential relationship betweeneNOS-derived S-nitrosylation and leptin regulation of fatty acidmetabolism. Although it requires further experimentation, thedata indicate that S-nitrosylation may be a molecular link be-tween the actions of leptin and NO in liver fatty acid biosynthesisand mitochondrial metabolism. Overall, cellular localization andfunctional analyses revealed that the S-nitrosylated proteins iden-tified in the liver were largely cytosolic and mitochondrial enzymesthat function as oxidoreductases and transferases, which are criticalfor regulating amino acid, energy, and lipid biosynthesis, and maycoordinate the regulation of metabolic pathways by leptin and NO.

Biochemical, Biophysical, and Structural Properties of the ModifiedCysteine Residues. This enriched S-nitrosoproteome was in-terrogated for the structural properties of the modified cysteineresidues by using various bioinformatic tools and available crystalstructures of proteins. Table 1 provides the basic biochemical andbiophysical properties of the modified cysteine residues using re-duced unmodified cysteine residues in the same proteins as a com-parison group. Kyte-Doolittle hydropathy indices in 13-residuewindows were calculated to determine the influence of primarystructure of theproteinonmodifiedcysteine residues (Fig. 2A).Theaverage hydropathy index value was calculated to be −0.03 ± 0.69(n=309)which did not differ significantly when comparedwith themean value of the unmodified cysteines within the same proteins(0.10 ± 0.77, n= 1,382).Using crystal structures (Protein Data Bank) and the Propka

2.0 algorithm (34), the average predicted pKa of 142 cysteine res-idues S-nitrosylated in vivo was 10.0, which was not significantlydifferent from the average pKa of reduced unmodified cysteineresidues in the same proteins (pKa of 9.88). Only 15 modified cys-teine residues had predicted pKa values lower than 7.4 (5.66 ±1.24), indicating that these particular residues may be deproto-nated at physiological pH (Fig. 2B). Secondary structure analysisrevealed that S-nitrosocysteine residues were present in β-sheets(28%), helices (40%), and coils (32%). Unmodified cysteine resi-dues within the same proteins were localized primarily in coils(39%)andequally distributedacross helices and β-sheets (Table 1).Statistical analysis revealed an overrepresentation and under-representation of modified cysteines in helices and coils, respect-ively (P < 0.02).None of the cysteine residues found to be S-nitrosylated partic-

ipate in disulfide bonding in their known structures and none hasbeen reported to date to be modified by glutathiolation and alky-lating agents (www.uniprot.org). Furthermore, by using predictivealgorithms and literature searches, it was found that less than 20%

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of the cysteine residues were predicted to be sites of oxidation (35).These data and analysis indicate that themajority of the in vivo sitesof S-nitrosylation represent a unique population of cysteine resi-dues not chemically modified through other biological processes.

S-Nitrosocysteine Residues Are Equally Distributed in Hydrophobicand Hydrophilic Areas of the Proteins. The overall slightly nega-tive hydropathy index (Table 1) is not indicative of a trend forS-nitrosylated cysteine residues to localize in hydrophilic regions ofthe protein. Further inspection of the hydropathy indices ofmodified cysteine residues revealed that 139 of the 309 cysteineresidues reside nearly in hydrophobic regions, whereas 170 of the309 are located in hydrophilic regions. This observation led us tofurther examine whether S-nitrosylated cysteines belong to twodistinct populations regarding their hydrophobicity/hydrophilicity.Kernel density estimation revealed that hydropathy indices ex-

hibited a roughly normal distribution (Fig. 2A,Left), indicating thatthey corresponded to a single population of cysteine residues.Unmodified cysteine residues within the same proteins alsoshowed a roughly normal distribution as well (Fig. 2A, Right), in-dicating that there is no distinction between S-nitrosylated andunmodified cysteine residueswithin the sameproteins to localize inhydrophobic or hydrophilic areas of proteins. Previous studies haveindicated that S-nitrosylation is favored in hydrophobic regions ofthe proteins (36), presumably because of the increased localizedconcentration of NO-derived oxides, which may provide a suitablemicroenvironment for the S-nitrosylation of these cysteine resi-dues. The present data imply that, for a subset of proteins, hy-drophobicity may serve as a determinant for selective targeting ofcysteine residues for S-nitrosylation.

S-Nitrosylation Occurs on Cysteine Residues Adjacent to FlexibleRegions Within the Protein. To further explore the location ofS-nitrosylated cysteine residues in protein secondary structure, thefrequency of secondary structures for flanking residues (positions−10 to +10) was calculated and compared with the respectivefrequencies of the unmodified cysteine residues using the χ2 test. Ashift in secondary structure mainly from coils to helices was ob-served over the range of positions from −6 to 0 (P < 0.05), con-sistent with the presence of the majority of S-nitrosocysteineresidues in α-helices (Fig. 3A). Moreover, a 10% increase in thefrequency of coils from positions 0 to +3 (P < 0.001) concomitantwith a reduction in β-sheet frequency (P < 0.001) indicative ofa change in secondary structure was observed C-terminal from themodified cysteine residues. Unmodified cysteine residues withinthe same proteins were localized primarily in coils and with lowerfrequency in β-sheets and helices (Fig. S4A). Moreover, the fre-quency of coils did not change significantly across all flankingresidues (−10 to +10), indicating that shifts in secondary structurewere only between helices and β-sheets. Collectively, these datademonstrate that modified cysteine residues are predominantlypresent in secondary structures of proteins which may facilitatesite-directed S-nitrosylation by protein-protein interactions.

Surface Accessibility of S-Nitrosocysteine Residues. The relativeresidue surface accessibility (RSA) for all cysteines (modified andunmodified) within the S-nitrosoproteome was calculated withNaccess 2.1.1 using the radius of a water molecule (1.4 Å2) asa probe (37). Ninety-nine S-nitrosylated residues (71%) were cal-culated with an RSA of 10% or lower, indicating that those resi-dues were not accessible to the solvent, whereas the remaining 40modified cysteines (29%) had a relative RSA greater than 10%,meaning theywere solvent-accessible.Unmodified cysteines withinthe same proteins exhibited similar distribution between buried(77%) and solvent-accessible residues (23%). A quantile–quantileprobability plot was used to determine if there was enrichment forexposed or buried cysteines within the modified versus the un-

Table 1. Biochemical and biophysical properties of S-nitrosylated and unmodified cysteineresidues within the same proteins

Variable S-nitrosocysteine residues Unmodified cysteine residues

Hydropathy index 0.03 ± 0.69 (n = 309) 0.1 ± 0.77 (n = 382)Predicted pKa 10.0 ± 2.10 (n = 142) 9.88 ± 2.20 (n = 559)Helices, % 40* 29β-Sheets, % 28 32Coils, % 32† 39RSA‡ 71% buried (n = 99) 77% buried (n = 561)

29% exposed (n = 40) 23% exposed (n = 171)

*P < 0.02, †P < 0.01 using unmodified residues as control group.‡Residue surface accessibility (RSA) for cysteine residues was calculated by the accessible surface area normalizedby the accessible surface area of cysteine in the extended tripeptide Ala-Cys-Ala. A value of ≤10% was used ascutoff to denote a buried cysteine.

A

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5

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30

≥0 ≥1 ≥2 ≥3 ≥4 ≥5 ≥6 ≥7 ≥8 ≥9 ≥10 ≥11 ≥12 ≥13

senietsycdetalysortin-

S

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N = 635 N = 747N = 170 N = 139

ytisneD

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5.04.0

3.02.0

1.00.0

6.05.0

4.03.0

2.01.0

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-2 -1 0 1 2 3

Fig. 2. Hydropathy index and pKa values of S-nitrosylated residues. (A)Kernel density plot of hydropathy indices for S-nitrosocysteine (Left) andunmodified cysteine residues (Right). Hydropathy index was calculated for allS-nitrosylated (n = 170 hydrophilic, n = 139 hydrophobic) and unmodified (n =635 hydrophilic, n = 747 hydrophobic) cysteine residueswithin a 13 amino acidwindow, with a negative value indicating hydrophilicity. (B) Predicted pKavalue for each S-nitrosylated cysteine was calculated from the experimentalstructures and the distribution was represented as a histogram.

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modified group of cysteine residues (Fig. 3C). To produce this plot,the RSA values of modified and unmodified cysteine residues weresorted and values of RSA for each percentile were plotted againsteach other (i.e., RSA of smallest 1% of modified cysteine residuesvs. RSA of smallest 1% of unmodified cysteine residues, smallest2% vs. smallest 2%). The plot demonstrates that S-nitrosocysteineresidueshaveadistribution skewed toward larger surface-accessibleareas than unmodified cysteine residues within the same proteins.In addition, 70% of S-nitrosylated cysteine residues within 6 Åwere surrounded by negatively or positively charged amino acidsthat had their side chains pointed away from cysteine thiol groups.Although the presence of charged residues in the vicinity of themodified residues did not impact their predicted pKa, it may facil-itate site specific modification by accommodating protein or S-nitrosoglutathione association. This is in agreement withfindings ofMitchell et al., who demonstrated that charged residues near cys-teine 73 were required for interaction and transnitrosylation ofprocaspase-3 (38). Accordingly, the presence of cysteine residues inhighly exposed areas of proteins and in proximity to charged aminoacids suggests a protein or small molecule transnitrosation assistedmechanism of S-nitrosylation.

Metal Catalyzed S-Nitrosylation. Studies exploring the S-nitro-sylation of proteins in cells indicated that more than 50% of thecellular formation of protein S-nitrosocysteine is derived by dini-trosyliron complexes (7).Within the liver S-nitrosoproteome, 13 S-nitrosylated cysteine residues, which are directly involved in thechelation of metal ions, were identified (Table S3). Metal ligationmay provide site-directed modification of these residues. Alter-natively, dinitrosyliron complexes could be stabilized near cysteineresidues by interactionswith neighboring acidic residues.As stated,more than 70% of the modified cysteine residues are in closeproximity (

untreated liver S-nitrosoproteome (Table S1), 72 proteins were nolonger identified after Trx treatment (Table S4). Linear motifanalysis revealed two motifs in this subset of S-nitrosylated pro-teins. The highest-scoring motif had exclusively aspartic acidat position −1, whereas the second motif had exclusively thre-onine at position +5 (Fig. 3E). Notably, both motifs containedcharged amino acids at positions before and after the cysteineresidue, consistent with the idea that charged amino acids inS-nitrosocysteine–containing motifs may facilitate interactionwith Trx.In conclusion, byusingunique,highly specificMS-basedproteomic

methods, we identified an expanded endogenous S-nitrosoproteomefrom WT mouse liver. Despite that S-nitrosylated cysteine residueshad, in general, similar hydropathy distribution and predicted pKavalues as nonmodified cysteine residues in the same proteins, closerinterrogation of the surrounding primary and secondary structuresrevealed distinctions that direct site-specific S-nitrosylation of certaincysteine residues. The structural analysis of these proteins also un-covered structural features that can accommodate multiple mecha-nisms for S-nitrosylation in vivo. In addition, the data also revealedaputative linkamong leptin, eNOS, andproteinS-nitrosylation in theregulation of liver fatty acid metabolism. Overall, the use of globalproteomic methods enabled structural and functional characteriza-tion of the in vivo S-nitrosocysteine proteome and the formulation oftestable new hypotheses that can be explored in the future usingtargeted approaches.

Materials and MethodsChemicals and Reagents. All HPLC solvents were purchased from Burdick andJackson, and unless stated all other reagents were purchased from Sigma-Aldrich. Mercury/PEG/biotin compound was designed by the authors andsynthesized by SoluLink.

Capture of S-Nitrosylated Proteins Using MRC or mPEGb. Mercury resin syn-thesis, MRC solid-phase, and mPEGb compound-based protein and pep-tide enrichment are described in detail in the SI Materials and Methods andScheme S1.

Protein Sequence and Structural Analysis. A detailed description of proteinsequence and structural analysis exists in SI Materials and Methods.

Statistical Analyses. Graphs were constructed and statistical analyses wereperformed using GraphPad Prism 5 software (GraphPad). Statistical signifi-cance was determined by paired or unpaired nonparametric two-tailed t testsusing either the Mann-Whitney (unpaired) or Wilcoxon matched-pairs test.

ACKNOWLEDGMENTS. We thank the Protein Core at the Children’s Hospitalof Philadelphia Research Institute for their assistance with mass spectrome-try, Dr. Santosh S. Venkatesh for assistance with the χ2 test, Dr. DavidSchwartz (Solulink Biosciences, San Diego, CA) for the synthesis of organo-mercury-polyethyleneglycol-biotin, and Dr. Qi Fang for support with struc-tural analysis. This work was supported by National Institutes of HealthGrants AG13966 and HL054926, National Institute of Environmental HealthSciences Center of Excellence in Environmental Toxicology Grant ES013508(to H.I), and National Institute of General Medical Sciences AwardF31GM085903 (to J.L.G.). H.I. is the Gisela and Dennis Alter Research Pro-fessor of Pediatrics.

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