An ascorbate-dependent artifact that interferes with the interpretation of the biotin switch assay

As an example of an important redox-based protein posttranslational modification, protein S-nitrosation of specific cysteines is attracting more and more attention. The methods of detecting protein S-nitrosation in vitro or in vivo have been widely used in recent research, especially the biotin switch assay. An increase in band intensity in the presence of ascorbate is thought to be diagnostic for the presence of S-nitrosothiols. However, we found that this is a flawed assumption. In this study, bovine serum albumin (BSA) and even BSA prereduced by 20 mM 2-mercaptoethanol give false-positive signals for S-nitrosothiols (corresponding to a level of about 0.5-1% S-nitrosated BSA) when detected by the biotin switch assay. Higher blocking conditions could not diminish the signal, whereas omitting ascorbate in the step before biotinylation resulted in the disappearance of the signal. Further investigation of the mechanism showed that ascorbate increases the rate of the biotinylation reaction and accelerates the presence of the false-positive signal. Our results provide direct evidence that ascorbate could give rise to a significant false-positive signal in the biotin switch assay. Ascorbate treatment can interfere with the interpretation of the data. Hence, care should be taken when this method is used.     

Persistent S-nitrosation of complex I and other mitochondrial membrane proteins by S-nitrosothiols but not nitric oxide or peroxynitrite: implications for the interaction of nitric oxide with mitochondria

S-nitrosation of mitochondrial proteins has been proposed to contribute to the pathophysiological interactions of nitric oxide (NO) and its derivatives with mitochondria but has not been shown directly. Furthermore, little is known about the mechanism of formation or the fate of these putative S-nitrosothiols. Here we have determined whether mitochondrial membrane protein thiols can be S-nitrosated on exposure to free NO from 3,3-bis(aminoethyl)-1-hydroxy-2-oxo-1-triazene (DETA-NONOate) by interaction with S-nitrosoglutathione or S-nitroso-N-acetylpenicillamine (SNAP) and by the NO derivative peroxynitrite. S-Nitrosation of protein thiols was measured directly by chemiluminescence detection. S-Nitrosoglutathione and S-nitroso-N-acetylpenicillamine led to extensive protein thiol oxidation, with about 30% of the modified protein thiols persistently S-nitrosated. In contrast, there was no protein thiol oxidation or S-nitrosation on exposure to 3,3-bis (aminoethyl)-1-hydroxy-2-oxo-1-triazene. Peroxynitrite extensively oxidized protein thiols but produced negligible amounts of S-nitrosothiols. Therefore, mitochondrial membrane protein thiols are S-nitrosated by preformed S-nitrosothiols but not by NO or by peroxynitrite. These S-nitrosated protein thiols were readily reduced by glutathione, so S-nitrosation will only persist when the mitochondrial glutathione pool is oxidized. Respiratory chain complex I was S-nitrosated by S-nitrosothiols, consistent with it being an important target for S-nitrosation during nitrosative stress. The S-nitrosation of complex I correlated with a significant loss of activity that was reversed by thiol reductants. S-Nitrosation was also associated with increased superoxide production from complex I. These findings point to a significant role for complex I S-nitrosation and consequent dysfunction during nitrosative stress in disorders such as Parkinson disease and sepsis.     

S-Nitrosation of proteins during D-galactosamine-induced cell death in human hepatocytes

Nitric oxide (NO) participates in the cell death induced by d-Galactosamine (d-GalN) in hepatocytes, and NO-derived reactive oxygen intermediates are critical contributors to protein modification and hepatocellular injury. It is anticipated that S-nitrosation of proteins will participate in the mechanisms leading to cell death in d-GalN-treated human hepatocytes. In the present study, d-GalN-induced cell death was related to augmented levels of NO production and S-nitrosothiol (SNO) content. The biotin switch assay confirmed that d-GalN increased the levels of S-nitrosated proteins in human hepatocytes. S-nitrosocysteine (CSNO) enhanced protein S-nitrosation and altered cell death parameters that were related to S-nitrosation of the executioner caspase-3. Fifteen S-nitrosated proteins participating in metabolism, antioxidative defense and cellular homeostasis were identified in human hepatocytes treated with CSNO. Among them, seven were also identified in d-GalN-treated hepatocytes. The results here reported underline the importance of the alteration of SNO homeostasis during d-GalN-induced cell death in human hepatocytes.     

Metalloprotein-dependent decomposition of S-nitrosothiols: studies on the stabilization and measurement of S-nitrosothiols in tissues

The stabilization of S-nitrosothiols is critical for the development of assays to measure their concentration in tissues. Low-molecular-weight S-nitrosothiols are unstable in tissue homogenates, even in the presence of thiol blockers or metal-ion chelators. The aim of this study was to try and stabilize low-molecular-weight S-nitrosothiols in tissue and gain insight into the mechanisms leading to their decomposition. Rat tissues (liver, kidney, heart, and brain) were perfused and homogenized in the presence of a thiol-blocking agent (N-ethylmaleimide) and a metal-ion chelator (DTPA). Incubation of liver homogenate with low-molecular-weight S-nitrosothiols (L-CysNO, D-CysNO, and GSNO) resulted in their rapid decomposition in a temperature-dependent manner as measured by chemiluminescence. The decomposition of L-CysNO requires a cytoplasmic factor, with activity greatest in liver > kidney > heart > brain > plasma, and is inhibitable by enzymatic proteolysis or heating to 80 degrees C, suggesting that a protein catalyzes the decomposition of S-nitrosothiols. The ability of liver homogenate to catalyze the decomposition of L-CysNO is up-regulated during endotoxemia and is dependent on oxygen, with the major product being nitrate. Multiple agents were tested for their ability to block the decomposition of L-CysNO without success, with the exception of potassium ferricyanide, which completely blocked CysNO decomposition in liver homogenates. This suggests that a ferrous protein (or group of ferrous proteins) may be involved. We also show that homogenization of tissues in ferricyanide-containing buffers in the presence of N-ethylmaleimide and DTPA can stabilize both low- and high-molecular-weight S-nitrosothiols in tissues before the measurement of their concentration.     

Mechanistic studies of S-nitrosothiol formation by NO*/O2 and by NO*/methemoglobin

The mechanisms of formation of S-nitrosothiols under physiological conditions and, in particular, of generation of SNO-Hb (the hemoglobin form in which the cysteine residues beta93 are S-nitrosated) are still not completely understood. In this paper, we investigated whether, in the presence of O2, NO* is more efficient to nitrosate protein-bound thiols such as Cysbeta93 or low molecular weight thiols such as glutathione. Our results show that when substoichiometric amounts of NO* are mixed slowly with the protein solution, NO*, O2, and possibly NO2* and/or N2O3 accumulate in hydrophobic pockets of hemoglobin. Since the environment of the cysteine residue beta93 is rather hydrophobic, these conditions facilitate SNO-Hb production. Moreover, we show that S-nitrosation mediated by reaction of NO* with the iron(III) forms of Hb or Mb is significantly more effective when it can take place intramolecularly, as in metHb. Intermolecular reactions lead to lower S-nitrosothiol yields because of the concurring hydrolysis to nitrite.     

Ascorbic acid reduction of microtubule protein disulfides and its relevance to protein S-nitrosylation assays

The biotin switch assay was developed to aid in the identification of S-nitrosylated proteins in different cell types. However, our work with microtubule proteins including tubulin and its associated proteins tau and microtubule-associated protein-2 shows that ascorbic acid is not a selective reductant of protein S-nitrosothiols as described in the biotin switch assay. Herein we show that ascorbic acid reduces protein disulfides in tubulin, tau, and microtubule-associated protein-2 that are formed by peroxynitrite anion. Reduction of microtubule-associated protein disulfides by ascorbic acid following peroxynitrite treatment restores microtubule polymerization kinetics to control levels. We also show that ascorbic acid reduces the disulfide dithiobis(2-nitrobenzoic acid), a reagent commonly used to detect protein thiols. Not only do we describe a new reactivity of ascorbic acid with microtubule proteins but we expose an important limitation when using the biotin switch assay to detect protein S-nitrosylation.     

Mechanism of S-nitrosothiol formation and degradation mediated by copper ions.

Experimental evidence is presented supporting a mechanism of S-nitrosothiol formation and degradation mediated by copper ions using bovine serum albumin, human hemoglobin and glutathione as models. We found that Cu(2+), but not Fe(3+), induces in the presence of NO a fast S-nitrosation of bovine serum albumin and human hemoglobin, and the reaction is prevented by thiol blocking reagents. During the reaction, Cu(+) is accumulated and accounts for destabilization of the S-nitrosothiol formed. In contrast, glutathione rapidly dimerizes in the presence of Cu(2+), the reaction competing with S-nitrosation and therefore preventing the formation of S-nitrosoglutathione. We have combined the presented role of Cu(2+) in S-nitrosothiol formation with the known destabilizing effect of Cu(+), providing a unique simple picture where the redox state of copper determines either the NO release from S-nitrosothiols or the NO scavenging by thiol groups. The reactions described are fast, efficient, and may occur at micromolar concentration of all reactants. We propose that the mechanism presented may provide a general method for in vitro S-nitrosation.     

Identification of protein nitrosothiols using phosphine-mediated selective reduction

Regulation of protein function by S-nitrosation of critical cysteines is known to be an important mechanism for nitric oxide signaling. Evidence for this comes from several different experimental approaches including the ascorbate-based biotin switch method. However technical problems with specificity and sensitivity of ascorbate reduction of S-nitrosothiols limit its usefulness and reliability. In the current study we report the use of triphenylphosphine ester derivatives to selectively reduce SNO bonds in proteins. After triphenylphosphine ester reduction, thiols were tagged with biotin or fluorescently labeled maleimide reagents. Importantly we demonstrate that these compounds are specific reductants of SNO in complex biological samples and do not reduce protein disulfides or protein thiols modified by hydrogen peroxide. Reduction proceeds efficiently in cell extracts and in whole fixed cells. Application of this approach allowed us to demonstrate S-nitrosation of specific cellular proteins, label S-nitrosoproteins in whole fixed cells (especially the nuclear compartment) and demonstrate S-nitrosoprotein formation in cells expressing inducible nitric oxide synthase.     

Sinapinic acid can replace ascorbate in the biotin switch assay

Background

Protein S-nitrosation is an important post-translational modification altering protein function. Interaction of nitric oxide with thiols is an active area of research, and is one of the mechanisms by which NO exerts its biological effects. Biotin switch assay is the method, which has been developed to identify S-nitrosated proteins. The major concern with biotin switch assay includes reducing disulfide which may lead to false positives. We report a modification of the biotin switch assay where sinapinic acid is utilized instead of ascorbate to eliminate potential artifacts in the detection of S-nitrosated proteins.

Methods

The denitrosation ability of sinapinic acid was assessed by monitoring either the NO or NO₂^- released by chemiluminescent NO detection or by the griess assay, respectively. DTNB assay was used to compare disulfide reduction by ascorbate and sinapinic acid. Sinapinic acid and ascorbate were compared in the biotin switch detection of S-nitrosoproteins in RAW 264.7 cells ± S-nitrosocysteine (CysNO) exposure.

Results

We show that sinapinic acid has the ability to denitrosate S-nitrosothiols at pH 7.0 and denitrate plus denitrosate at pHs 8 and 8.5. Unlike ascorbate, sinapinic acid degrades S-nitrosothiols, but it does not reduce disulfide bridges.

Conclusions

Sinapinic acid denitrosate RSNO and does not reduce disulfides. Thus can readily replace ascorbate in detection of S-nitrosated proteins in biotin switch assay.

General significance

The work described is important in view of protein S-nitrosation. In this study we provide an important modification that eliminates artifacts in widely used technique for detecting the S-nitrosoproteome, the biotin switch assay.     

Quantitative proteomic analysis of S-nitrosated proteins in diabetic mouse liver with ICAT switch method

In this study we developed a quantitative proteomic method named ICAT switch by introducing isotope-coded affinity tag (ICAT) reagents into the biotin-switch method, and used it to investigate S-nitrosation in the liver of normal control C57BL/6J mice and type 2 diabetic KK-Ay mice. We got fifty-eight S-nitrosated peptides with quantitative information in our research, among which thirty-seven had changed S-nitrosation levels in diabetic mouse liver. The S-nitrosated peptides belonged to forty-eightproteins(twenty-eightwerenewS-nitrosated proteins), some of which were new targets of S-nitrosation and known to be related with diabetes. S-nitrosation patterns were different between diabetic and normal mice. Gene ontology enrichment results suggested that S-nitrosated proteins are more abundant in amino acid metabolic processes. The network constructed for S-nitrosated proteins by text-mining technology provided clues about the relationship between S-nitrosation and type 2 diabetes. Our work provides a new approach for quantifying S-nitrosated proteins and suggests that the integrative functions of S-nitrosation may take part in pathophysiological processes of type 2 diabetes.     

Characterization and application of the biotin-switch assay for the identification of S-nitrosated proteins

S-Nitrosation of protein cysteinyl residues has been suggested to be an important nitric oxide-dependent posttranslational modification. The so-called biotin-switch method has been developed to identify S-nitrosated proteins. This method relies on the selective reduction of S-nitrosothiols by ascorbate. In this study we have assessed the ability of ascorbate to reduce S-nitrosothiols and show that ascorbate is a very inefficient reducing agent. We show that higher concentrations of ascorbate and longer incubation times can significantly improve immunological detection of S-nitrosothiols. We have compared immunological detection of S-nitrosothiols with the level of intracellular S-nitrosothiols measured by tri-iodide chemiluminescence and show that the biotin-switch method is capable of detecting only high (nmol/mg protein) levels of intracellular S-nitrosothiols obtained after exposing cells to S-nitrosocysteine, but not the low levels observed during physiological nitric oxide formation. Preliminary proteomic analysis of protein S-nitrosothiols has identified elongation factor 2, heat shock protein 90 beta, and a 65-kDa macrophage protein homologous to human L-plastin as major nitrosation targets at high intracellular nitrosation levels in the murine macrophage-derived RAW 264.7 cell line. While the biotin-switch method may be a useful tool to aid in the positive identification of protein S-nitrosothiols, it cannot match the sensitivity of chemiluminescence-based methods and its use in proteomic studies likely suffers from selective detection of more easily reducible S-nitrosothiols.     

Mechanism of S-nitrosation of recombinant human brain calbindin D28K

Mass spectrometry and UV-vis absorption results support a mechanism for NO donation by S-nitrosoglutathione (GSNO) to recombinant human brain calbindin D(28K) (rHCaBP) that requires the presence of trace copper, added as either Cu,Zn-superoxide dismutase (CuZnSOD) or CuSO(4). The extent of copper-catalyzed rHCaBP S-nitrosation depends on the ratio of protein to GSNO and on the reaction time, and NO-transfer is prevented when copper chelators are present. CuZnSOD is an efficient catalyst of rHCaBP S-nitrosation, and the mechanism of CuZnSOD-catalyzed S-nitrosation involves reduction of the active-site Cu(II) by a number of the five free thiols in rHCaBP, giving rise to thiyl radicals. The Cu(I)ZnSOD formed catalyzes the reductive cleavage of GSNO present in solution to give GSH and release NO. rHCaBP thiyl radicals react with NO to yield the S-nitrosoprotein. Cu(II)ZnSOD is also reduced by GSH in a concentration-dependent manner up to 5 mM but not at higher GSH concentrations. However, unlike the rHCaBP thiyl radicals, GS(*) radicals dimerize to GSSG faster than their reaction with NO. The data presented here provide a biologically relevant mechanism for protein S-nitrosation by small S-nitrosothiols. S-nitrosation is rapidly gaining recognition as a major form of protein posttranslational modification, and the efficient S-nitrosation of CaBP by CuZnSOD/GSNO is speculated to be of neurochemical importance given that CaBP and CuZnSOD are abundant in neurons.     

S-nitrosation of Ca(2+)-loaded and Ca(2+)-free recombinant calbindin D(28K) from human brain

Calbindin D(28K) is noted for its abundance and specific distribution in mammalian brain and sensory neurons. It can bind three to five Ca(2+) ions and may act as a Ca(2+) buffer to maintain intracellular Ca(2+) homeostasis, but its exact role is still unknown. In the present study, mass spectrometric analysis reveals that the five cysteine residues in recombinant human brain calbindin D(28K) (rHCaBP) are derivatized with N-ethylmaleimide, consistent with the determination of 5.3 +/- 0.4 and 4.7 +/- 0.4 free thiols in the protein using the thiol-specific reagents 5,5'-dithiobis(2-nitrobenzoic acid) and 5-(octyldithio)-2-nitrobenzoic acid, respectively. The results of UV-vis and circular dichroism absorption, intrinsic fluorescence, and mass spectrometry measurements indicate that both Ca(2+)-loaded (holo) and Ca(2+)-free (apo) rHCaBP are S-nitrosated by S-nitrosocysteine (CysNO). The number of cysteine residues S-nitrosated in holorHCaBP and aporHCaBP are 2.6 +/- 0.05 and 3.4 +/- 0.09, respectively, as determined by the Saville assay. HolorHCaBP also undergoes S-nitrosation at one to three cysteine residues when exposed to S-nitrosoglutathione (GSNO), and Cys100 was found to be an S-nitrosation site by peptide mass mapping. Treatment of holorHCaBP with free NO resulted in a mass increase of 59 +/- 2 Da, corresponding to two NO adducts. Since up to four cysteine residues can be S-nitrosated in rHCaBP, it is proposed that the protein may act as a NO buffer or reservoir in the brain in a manner similar to serum albumin in blood. It is significant in this context that rHCaBP is found coexistent with nitric oxide synthase in cerebellum and that S-nitrosation varies with Ca(2+) binding, with S-nitrosation occurring to a greater extent in aporHCaBP than in the holoprotein. Furthermore, exposure of rHCaBP to either CysNO or GSNO also leads to rapid S-thiolation of Cys187. We demonstrate here for the first time that intrinsic protein fluorescence is a sensitive probe of protein S-nitrosation. This is due to efficient F?rster energy transfer (R(0) approximately 17 A) between tryptophan donors and S-nitrosothiol acceptors.     

Reduction and S-nitrosation of the neuropeptide oxytocin: implications for its biological function.

Oxytocin (OT; Cys-Tyr-Ile-Gln-Asn-Cys-Pro-leu-Gly), a posterior pituitary peptide hormone, is characterized by a Cys1-Cys6 disulfide bond in its stable, isolated state. This paper describes a simple, one-step method for the production of OT in its reduced, dithiol form (OT dithiol), free of reducing agent. The effects of temperature, pH, and metal-ion chelators on the autoxidation of OT dithiol were examined to establish if this form is likely to persist under biological conditions. It was found that OT dithiol has a half-life of 1.8h with respect to reformation of OT disulfide at 37 degrees C and pH 6.9 in the presence of the copper chelators, DTPA and neocuproine. S-Nitrosation of OT dithiol by acidified nitrite at pH 3.0 was examined by absorption spectroscopy and HPLC-UV-MS, which revealed that both singly and doubly S-nitrosated OT are formed. These results suggest novel chemical aspects to OT signaling, the biological implications of which are discussed here.     

An optimized strategy for ICAT quantification of membrane proteins

The work presented here focuses on the development of a method adapting isotope labeling of proteins with ICAT to the study of highly hydrophobic proteins. Conditions for the labeling of proteins were first established using two standard soluble proteins and iodoacetamidyl-3,6-dioxaoctanediamine biotin (PEO-iodoacetyl biotin). Results demonstrated the efficiency of the labeling in the presence of high concentrations of both SDS and urea. These conditions were then used to label a highly hydrophobic mitochondrial membrane protein, the adenine nucleotide translocator ANT-1, with PEO-iodoacetyl biotin and then with the cleavable ICAT reagent. The results presented here show that labeling of proteins with cleavable ICAT is possible and may even be improved in strong denaturing buffers containing both SDS at a concentration higher than 0.5% (w/v) and urea. These results open the possibility of applying the ICAT strategy to complex samples containing very hydrophobic proteins solubilized in urea-SDS buffers. The adaptability of the developed method is demonstrated here with preliminary results obtained during the study of membrane-enriched fractions prepared from murine embryonic stem cells.     

Cytochrome c-mediated formation of S-nitrosothiol in cells

S-nitrosothiols are products of nitric oxide (NO) metabolism that have been implicated in a plethora of signalling processes. However, mechanisms of S-nitrosothiol formation in biological systems are uncertain, and no efficient protein-mediated process has been identified. Recently, we observed that ferric cytochrome c can promote S-nitrosoglutathione formation from NO and glutathione by acting as an electron acceptor under anaerobic conditions. In the present study, we show that this mechanism is also robust under oxygenated conditions, that cytochrome c can promote protein S-nitrosation via a transnitrosation reaction and that cell lysate depleted of cytochrome c exhibits a lower capacity to synthesize S-nitrosothiols. Importantly, we also demonstrate that this mechanism is functional in living cells. Lower S-nitrosothiol synthesis activity, from donor and nitric oxide synthase-generated NO, was found in cytochrome c-deficient mouse embryonic cells as compared with wild-type controls. Taken together, these data point to cytochrome c as a biological mediator of protein S-nitrosation in cells. This is the most efficient and concerted mechanism of S-nitrosothiol formation reported so far.     

Site-mapping of in vitro S-nitrosation in cardiac mitochondria: implications for cardioprotection

S-nitrosation (SNO) of mitochondrial protein cysteines can be cardioprotective. Several targets have been implicated, yet the scope and identification of specific residues has not been fully assessed. To address this, a comprehensive assessment of mitochondrial SNO-modifiable cysteines was performed to determine nitric oxide (NO) susceptible pathways and identify novel mechanisms of oxidative cardioprotection. The biotin switch assay and mass spectrometry were used on rat cardiac mitochondrial lysates treated with the nitric oxide donor, S-nitrosoglutathione, and controls (n=3) to map 83 SNO-modified cysteine residues on 60 proteins. Of these, three sites have been reported, 30 sites are new to 21 proteins previously known to be S-nitrosated but which lacked site-specific information and 50 sites were found on 39 proteins not previously implicated in SNO pathways. The SNO-modifications occurred in only a subset of available cysteines, indicating a specific targeted effect. Functional annotation and site-specificity analysis revealed a twofold greater nitric oxide-susceptibility for proteins involved in transport; including regulators of mitochondrial permeability transition suggesting SNO-regulation and a possible protective mechanism. Additionally, we identified many novel SNO-modified proteins with cardioprotective potential involved in the electron transport chain, tricarboxylic acid cycle, oxidative stress defense, fatty acid and amino acid metabolism. These findings suggest that SNO-modification may represent a novel mechanism for the regulation of oxidative phosphorylation and/or cell death. S-nitrosation of mitochondrial permeability transition-associated proteins represents an intriguing potential link to cardioprotection.     

SNO spectral counting (SNOSC), a label-free proteomic method for quantification of changes in levels of protein S-nitrosation

Abstract

S-Nitrosation plays an important role in regulation of protein function and signal transduction. Discovering S-nitrosated targets is a prerequisite for further functional study. However, current proteomic methods used to quantify S-nitrosation are limited in their applicability to certain types of samples, or by the need for special reagents and complex procedures to obtain the results. Here we devised a label-free proteomic method for quantification of changes in the level of protein S-nitrosation on the basis of a spectral counting strategy, called S-nitrosothiol (SNO) spectral counting (SNOSC). With this method, samples can be from any source (cells, tissues); there is no need for labelling reagents or procedures, and the results yield quantitative information. Moreover, as it is based on the irreversible biotinylation procedure (IBP) for S-nitrosation protein enrichment, false positive targets caused by the interference of intermolecular disulphide bonds are ruled out. Using SNOSC we studied S-nitrosation in the cell line RAW264.7 induced exogenously with S-nitrosoglutathione (GSNO), or induced endogenously by lipopolysaccharides/interferon-gamma (LPS/IFN-γ). We detected a significant increase in S-nitrosation of 50 proteins after exogenous induction and 17 proteins after endogenous induction. We thus demonstrate that SNOSC is a widely applicable proteomic method for fast screening of SNO proteins.     

SNO spectral counting (SNOSC), a label-free proteomic method for quantification of changes in levels of protein S-nitrosation

S-Nitrosation plays an important role in regulation of protein function and signal transduction. Discovering S-nitrosated targets is a prerequisite for further functional study. However, current proteomic methods used to quantify S-nitrosation are limited in their applicability to certain types of samples, or by the need for special reagents and complex procedures to obtain the results. Here we devised a label-free proteomic method for quantification of changes in the level of protein S-nitrosation on the basis of a spectral counting strategy, called S-nitrosothiol (SNO) spectral counting (SNOSC). With this method, samples can be from any source (cells, tissues); there is no need for labelling reagents or procedures, and the results yield quantitative information. Moreover, as it is based on the irreversible biotinylation procedure (IBP) for S-nitrosation protein enrichment, false positive targets caused by the interference of intermolecular disulphide bonds are ruled out. Using SNOSC we studied S-nitrosation in the cell line RAW264.7 induced exogenously with S-nitrosoglutathione (GSNO), or induced endogenously by lipopolysaccharides/interferon-gamma (LPS/IFN-γ). We detected a significant increase in S-nitrosation of 50 proteins after exogenous induction and 17 proteins after endogenous induction. We thus demonstrate that SNOSC is a widely applicable proteomic method for fast screening of SNO proteins.