S-nitrosoproteome in endothelial cells revealed by a modified biotin switch approach coupled with western blot-based two-dimensional gel electrophoresis

Bin Huang, Chung Ling Liao, Ya Ping Lin, Shih Chung Chen, Danny Ling Wang

Research output: Contribution to journalArticle

16 Citations (Scopus)

Abstract

NO-mediated S-nitrosation of cysteine residues has been recognized as a fundamental post-translational modification. S-Nitrosation of endothelial cell (EC) proteins can alter function and affect vascular homeostasis. Trace amounts of S-nitrosoproteins in endothelial cells (ECs) in vivo coupled with lability of the S-nitroso bond have hindered a comprehensive characterization. We demonstrate a convenient and reliable method, requiring minimal sample, for the screening and identification of S-nitrosoproteins. ECs treated with the NO donor S-nitroso-N-acetylpenicillamine (SNAP) were subjected to the biotin switch method of labeling, then detected by analytical Western blot-based two-dimensional gel electrophoresis (2-DE). More than 89 SNAP-increased S-nitrosoproteins were detected and 28 of these were successfully excised from preparative 2-DE gel and identified by LC-MS/MS. Moreover, the nitrosocysteine residue for each protein (HSPA9/368, β-actin/16, TMP3/170, vimentin/328) was also determined, and the relative ratio of S-nitrosation/non-S-nitrosation for Cys328 of vimentin was estimated using MASIC software. By the combination of the biotin switch method with 2-DE and Western blot analysis, S-nitrosoproteins can be screened and characterized by MS, providing a basis for further study of the physiological significance of each S-nitrosoproteins.

Original languageEnglish
Pages (from-to)4835-4843
Number of pages9
JournalJournal of Proteome Research
Volume8
Issue number10
DOIs
Publication statusPublished - 2009

Keywords

  • Biotin switch
  • Endothelial cell
  • Mass spectrometry
  • MS
  • Nitric oxide
  • S-nitrosation

ASJC Scopus subject areas

  • Biochemistry
  • Chemistry(all)

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