Solid-phase capture for the detection and relative quantification of S-nitrosoproteins by mass spectrometry.

Journal Article (Journal Article;Review)

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.

Full Text

Duke Authors

Cited Authors

  • Thompson, JW; Forrester, MT; Moseley, MA; Foster, MW

Published Date

  • August 1, 2013

Published In

Volume / Issue

  • 62 / 2

Start / End Page

  • 130 - 137

PubMed ID

  • 23064468

Pubmed Central ID

  • PMC3568208

Electronic International Standard Serial Number (EISSN)

  • 1095-9130

Digital Object Identifier (DOI)

  • 10.1016/j.ymeth.2012.10.001


  • eng

Conference Location

  • United States