title: Measurement of S-nitrosylation occupancy in the myocardium with cysteine-reactive tandem mass tags: short communication.
abstract: BACKGROUND
  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.
  
  
  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.
authors.affiliation: Laboratory of Cardiac Physiology, Systems Biology Center, National Heart Lung and Blood Institute, Bethesda, MD 20892, USA.
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Kohr
authors.forename: Angel
authors.initials: A
authors.lastname: Aponte
authors.forename: Junhui
authors.initials: J
authors.lastname: Sun
authors.forename: Marjan
authors.initials: M
authors.lastname: Gucek
authors.forename: Charles
authors.initials: C
authors.lastname: Steenbergen
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Murphy
mesh_terms: D000818:Animals; D003545:Cysteine / Q000032:analysis / Q000378:metabolism; D019194:Ischemic Preconditioning / Q000379:methods*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D015428:Myocardial Reperfusion Injury / Q000378:metabolism*; D009206:Myocardium / Q000737:chemistry / Q000378:metabolism*; D010084:Oxidation-Reduction; D010477:Perfusion; D026422:S-Nitrosoglutathione / Q000032:analysis / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000032:analysis / Q000378:metabolism; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D013438:Sulfhydryl Compounds; D026422:S-Nitrosoglutathione; D003545:Cysteine