Reversible addition of NO to Cys-sulfur in proteins, a modification termed S-nitrosylation, has emerged as a ubiquitous signaling mechanism for regulating diverse cellular processes. A key first-step toward elucidating the mechanism by which S-nitrosylation modulates a protein's function is specification of the targeted Cys (SNO-Cys) residue. To date, S-nitrosylation site specification has been laboriously tackled on a protein-by-protein basis. Here we describe a high-throughput proteomic approach that enables simultaneous identification of SNO-Cys sites and their cognate proteins in complex biological mixtures. The approach, termed SNOSID (SNO Site Identification), is a modification of the biotin-swap technique [Jaffrey, S. R., Erdjument-Bromage, H., Ferris, C. D., Tempst, P. & Snyder, S. H. (2001) Nat. Cell. Biol. 3, 193–197], comprising biotinylation of protein SNO-Cys residues, trypsinolysis, affinity purification of biotinylated-peptides, and amino acid sequencing by liquid chromatography tandem MS. With this approach, 68 SNO-Cys sites were specified on 56 distinct proteins in S -nitrosoglutathione-treated (2–10 μM) rat cerebellum lysates. In addition to enumerating these S-nitrosylation sites, the method revealed endogenous SNO-Cys modification sites on cerebellum proteins, including α-tubulin, β-tubulin, GAPDH, and dihydropyrimidinase-related protein-2. Whereas these endogenous SNO proteins were previously recognized, we extend prior knowledge by specifying the SNO-Cys modification sites. Considering all 68 SNO-Cys sites identified, a machine learning approach failed to reveal a linear Cys-flanking motif that predicts stable transnitrosation by S -nitrosoglutathione under test conditions, suggesting that undefined 3D structural features determine S-nitrosylation specificity. SNOSID provides the first effective tool for unbiased elucidation of the SNO proteome, identifying Cys residues that undergo reversible S-nitrosylation.

Produced by the action of lightning in the atmosphere of the pre-biotic earth, nitric oxide (NO) is a free radical molecule that provided the major nitrogen source for development of life. Remarkably, when atmospheric sources of NO became restrictive, organisms evolved the capacity for NO biosynthesis and NO took on bioregulatory roles. We now recognize NO as an ancestral regulator of diverse and important biological functions, acting throughout the phylogenetic tree. In mammals, NO has been implicated as a pivotal regulator of virtually every major physiological system. The bioactivities of NO, and reactive species derived from NO, arise predominantly from their covalent addition to proteins. Importantly, S-nitrosylation of protein cysteine (Cys) residues has emerged as a preeminent effector of NO bioactivity. How and why NO selectively adds to particular Cys residues in proteins is poorly understood, yet fundamental to how NO communicates its bioactivities. Also, evolutionary pressures that have shaped S-nitrosylation as a biosignaling modality are obscure. Considering recently recognized NO signaling paradigms, we speculate on the origin of NO signaling in biological systems and the molecular adaptations that have endowed NO with the ability to selectively target a subset of protein Cys residues that mediate biosignaling.

Covalent addition of nitric oxide (NO) to Cys-sulfur in proteins, or S-nitrosylation, plays pervasive roles in the physiological and pathophysiological modulation of mammalian protein functions. Knowledge of the specific protein Cys residues that undergo NO addition in different biological settings is fundamental to understanding NO-mediated signal transduction. Here, we describe in detail an MS-based proteomic protocol for facile, high-throughput and unbiased discovery of SNO-Cys residues in proteins from complex biological samples. The approach, termed SNOSID (SNO-Cys site identification), can be used to identify endogenous and chemically induced S-nitrosylation sites in proteins from tissues or cells. Identified SNO-Cys sites may provide insights into novel mechanisms and proteins that mediate NO bioactivities in health and disease. SNOSID builds on the biotin-switch method for covalent addition of disulfide-linked biotin at S-nitrosylation sites on proteins. Biotinylated proteins are then subjected to trypsinolysis and the resulting biotin-tagged peptides are affinity-captured on streptavidin-agarose. After selective elution with b-mercaptoethanol, the peptides are sequenced using nanoflow liquid chromatography tandem mass spectrometry (nLC-MS/MS). Validation that identified peptide ions as originating from authentic NO-Cys-containing precursor proteins can be provided by establishing that these peptide ions are absent from control samples where S-NO bonds were subjected to prior photolysis, using a UV transilluminator. The protocol requires approximately 2 days for sample processing, including the incubation time for proteolysis. An additional 1-2 days is needed for sample analysis by nLC-MS/MS and data analysis/interpretation. INTRODUCTIONNO is a reactive radical species that is produced by NO synthases (NOSs), a family of enzymes encoded by three distinct genes in mammals. Remarkably, NO has been implicated in the physiological regulation of virtually every major mammalian system, including cardiovascular, respiratory, gastrointestinal, renal, neuronal, endocrine, reproductive and host defense. These different bioactivities result from covalent addition of NO to target proteins.An extensive literature has documented the pre-eminence of NO addition to Cys-sulfur (i.e., in terms of its ubiquity and the diversity of cell regulatory functions in which it is implicated) as a mediator of cell signaling 1 . It is worth noting that covalent attachment of NO to proteins is modulated by changes in the intracellular milieu, as well as by changes in the NO biosynthesis-this contrasts starkly with mechanisms of conventional signaling molecules, which are preformed, vesiculated and act by lock-and-key (non-covalent) binding to cell surface receptors 2 .Relative to O-phosphorylation, the discovery of S-nitrosylation sites on proteins has been quite challenging, primarily owing to the inherent chemical instability of the S-NO bond. S-NO bond instability can result in the failure to detect some authentic S-nitrosylation s...

Rationale Protein S-palmitoylation is the post-translational attachment of a saturated 16-carbon palmitic acid to a cysteine side chain via a thioester bond. Palmitoylation can affect protein localization, trafficking, stability, and function. The extent and roles of palmitoylation in endothelial cell (EC) biology is not well understood, in part due to technological limits on palmitoylprotein detection. Objective To develop a method using acyl-biotinyl exchange (ABE) technology coupled with mass spectrometry to globally isolate and identify palmitoylproteins in EC. Methods and Results More than 150 putative palmitoyl proteins were identified in EC using ABE and mass spectrometry. Among the novel palmitoylproteins identified is superoxide dismutase 1 (SOD1), an intensively studied enzyme that protects all cells from oxidative damage. Mutation of cysteine 6 prevents palmitoylation, leads to reduction in SOD1 activity in vivo and in vitro, and inhibits nuclear localization, thereby supporting a functional role for SOD1 palmitoylation. Moreover, we used ABE to search for substrates of particular protein acyl transferases in EC. We found that palmitoylation of the cell adhesion protein PECAM1 is dependent on the protein acyl transferase ZDHHC21. We show that knockdown of ZDHHC21 leads to reduced levels of PECAM1 at the cell surface. Conclusions Our data demonstrate the utility of EC palmitoylproteomics to reveal new insights into the role of this important post-translational lipid modification in EC biology.

Caveolae are organelles abundant in the plasma membrane of many specialized cells including endothelial cells (ECs), epithelial cells, and adipocytes, and in these cells, caveolin-1 (Cav-1) is the major coat protein essential for the formation of caveolae. To identify proteins that require Cav-1 for stable incorporation into membrane raft domains, a quantitative proteomics analysis using isobaric tagging for relative and absolute quantification was performed on rafts isolated from wild-type and Cav-1-deficient mice. In three independent experiments, 117 proteins were consistently identified in membrane rafts with the largest differences in the levels of Cav-2 and in the caveola regulatory proteins Cavin-1 and Cavin-2. Because the lung is highly enriched in ECs, we validated and characterized the role of the newly described protein Cavin-1 in several cardiovascular tissues and in ECs. Cavin-1 was highly expressed in ECs lining blood vessels and in cultured ECs. Knockdown of Cavin-1 reduced the levels of Cav-1 and -2 and weakly influenced the formation of high molecular weight oligomers containing Cav-1 and -2. Cavin-1 silencing enhanced basal nitric oxide release from ECs but blocked proangiogenic phenotypes such as EC proliferation, migration, and morphogenesis in vitro. Thus, these data support an important role of Cavin-1 as a regulator of caveola function in ECs. Molecular & Cellular Proteomics 9:2109 -2124, 2010.Caveolae have been extensively studied and shown to play an important role in the regulation of many cellular functions including cell signaling, vesicular transport, and lipid metabolism. Caveolae are especially abundant in adipocytes, endothelial cells (ECs), 1 fibroblasts, and smooth muscle cells (1-3), and there is evidence that the loss of caveola function may contribute to dyslipidemia, muscular dystrophy, cancer, diabetes, and cardiovascular diseases (1, 4 -7). Caveolae are a subset of lipid rafts characterized by a particularly high content of cholesterol and sphingolipids that contain the protein caveolin. There are three isoforms of caveolin, caveolin-1 (Cav-1), Cav-2, and Cav-3, that have been extensively studied (1, 4). Cav-1 and Cav-3 are required for caveola formation in different tissues (8 -10). Although Cav-2 is not required for caveola formation per se, (11) it regulates aspects of lung morphogenesis. Although the roles of caveolins in caveola formation are well known, the requirement of other proteins regulating caveolin function and thus caveola formation has been recently addressed (12)(13)(14).To identify proteins that stably interact and are regulated by Cav-1, we used an unbiased proteomics approach using isobaric tagging for relative and absolute quantification (iTRAQ). iTRAQ is a powerful quantitative proteomics tool for the relative quantification of proteins in complex mixtures by mass spectrometry (15), and this technology has been successfully used for global comparison of protein expression in many cells systems and in different tissues (16,17). As caveolae and Cav-1 ar...