Measurement of S-nitrosylation occupancy in the myocardium with cysteine-reactive tandem mass tags: Short communication

Mark J. Kohr, Angel Aponte, Junhui Sun, Marjan Gucek, Charles Steenbergen, Elizabeth Murphy

Research output: Contribution to journalArticle

Abstract

RATIONALE: S-nitrosylation (SNO) is a reversible, thiol-based protein modification that plays an important role in the myocardium by protecting critical cysteine residues from oxidation. However, little is known with regard to the percentage of a given protein that is modified by SNO (ie, SNO occupancy). Current methods allow for the relative quantification of SNO levels, but not for the determination of SNO occupancy. OBJECTIVE: To develop a method for the measurement of SNO occupancy, and apply this methodology to determine SNO occupancy in the myocardium. METHODS AND RESULTS: We developed a differential cysteinereactive tandem mass tag (cysTMT) labeling procedure for the measurement of SNO occupancy. To validate this cysTMT labeling method, we treated whole-heart homogenates with the S-nitrosylating agent S-nitrosoglutathione and determined maximal SNO occupancy. We also examined SNO occupancy under more physiological conditions and observed that SNO occupancy is low for most protein targets at baseline. Following ischemic preconditioning, SNO occupancy increased to an intermediate level compared to baseline and Snitrosoglutathione treatment, and this is consistent with the ability of SNO to protect against cysteine oxidation. CONCLUSIONS: This novel cysTMT labeling approach provides a method for examining SNO occupancy in the myocardium. Using this approach, we demonstrated that IPC-induced SNO occupancy levels are sufficient to protect against oxidation.

Original languageEnglish (US)
Pages (from-to)1308-1312
Number of pages5
JournalCirculation research
Volume111
Issue number10
DOIs
StatePublished - Oct 26 2012

Keywords

  • S-nitrosylation
  • ischemic preconditioning
  • occupancy
  • oxidation

ASJC Scopus subject areas

  • Physiology
  • Cardiology and Cardiovascular Medicine

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