Unbiased identification of cysteine S-nitrosylation sites on proteins

Abstract

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...

Structural profiling of endogenous S-nitrosocysteine residues reveals unique features that accommodate diverse mechanisms for protein S-nitrosylation

“…This false positive rate compares favorably with the greater than 90% specificity reported for immobilized metal affinity chromatography used for the enrichment of phosphopeptides from mouse liver (14). Therefore, pretreatment with UV served as negative control throughout based on previous studies that documented the elimination of S-nitrosocysteine without affecting other cysteine modifications (15,16). Collectively, these experiments showed that the reaction of protein Snitrosocysteine with the phenylmercury compounds is specific and efficient and achieved selective identification of the modified residue, the ultimate qualifier for the unambiguous assignment of Snitrosylated proteins through the inclusion of negative controls.…”

Site-Mapping of In Vitro S-nitrosation in Cardiac Mitochondria: Implications for Cardioprotection

“…Detection of S-Nitrosated Proteins by Biotin Switch Assay-SNOmodifications were detected in GSNO treated mitochondrial lysates using the biotin switch assay described in (21) (discussed in (17,19,21,26)), see Captured proteins were eluted with 40 L of elution buffer containing 100 mmol/L DTT, mixed with 15 L of 4ϫ LDS sample buffer, boiled, separated by SDS-PAGE (4 -12%) and silver stained according to the protocol described in (27).…”

Identification and Quantification of S-Nitrosylation by Cysteine Reactive Tandem Mass Tag Switch Assay

“…6 Switch Assay-SNO-modifications were detected in GSNO treated HPAEC lysates using the biotin switch assay (12,14) (discussed in (14,16,25), see Fig. 1A for reaction schema).…”