Solid-phase capture for the detection and relative quantification of S-nitrosoproteins by mass spectrometry.
The proteomic analysis of S-nitrosylated protein (SNO-proteins) has long depended on the biotin switch technique (BST), which requires blocking of free thiols, ascorbate-based denitrosylation of SNO-Cys, biotinylation of nascent thiol and avidin-based affinity isolation. A more recent development is resin assisted-capture of SNO-proteins (SNO-RAC), which substitutes thiopropyl Sepharose (TPS) for biotin-avidin, thus reducing the number of steps required for enrichment of S-nitrosylated proteins. In addition, SNO-RAC facilitates on-resin proteolytic digestion following SNO-protein capture, greatly simplifying the purification of peptides containing sites of S-nitrosylation ("SNO-sites"). This resin-based approach has also now been applied to detection of alternative Cys-based modifications, including S-palmitoylation (Acyl-RAC) and S-oxidation (Ox-RAC). Here, we review the important steps to minimize false-positive identification of SNO-proteins, give detailed methods for processing of protein-bound TPS for mass spectrometry (MS) based analysis, and discuss the various quantitative MS methods that are compatible with SNO-RAC. We also discuss strategies to overcome the current limitations surrounding MS-based SNO-site localization in peptides containing more than one potential target Cys residue. This article therefore serves as a starting point and guide for the MS-focused exploration of SNO-proteomes by SNO-RAC.
Institute for Genome Sciences and Policy, Duke University Medical Center, Durham, NC 27710, United States.
J Will
JW
Thompson
Michael T
MT
Forrester
M Arthur
MA
Moseley
Matthew W
MW
Foster
D000818:Animals; D002846:Chromatography, Affinity; D006801:Humans; D011499:Protein Processing, Post-Translational; D020543:Proteome / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D026403:S-Nitrosothiols / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D052616:Solid Phase Extraction; D013194:Staining and Labeling; D053719:Tandem Mass Spectrometry / Q000379:methods
D020543:Proteome; D026403:S-Nitrosothiols