title: Mass spectrometry-based quantitative proteomics for dissecting multiplexed redox cysteine modifications in nitric oxide-protected cardiomyocyte under hypoxia.
abstract: OBJECTIVE
  Distinctive states of redox-dependent cysteine (Cys) modifications are known to regulate signaling homeostasis under various pathophysiological conditions, including myocardial injury or protection in response to ischemic stress. Recent evidence further implicates a dynamic interplay among these modified forms following changes in cellular redox environment. However, a precise delineation of multiplexed Cys modifications in a cellular context remains technically challenging. To this end, we have now developed a mass spectrometry (MS)-based quantitative approach using a set of novel iodoacetyl-based Cys-reactive isobaric tags (irreversible isobaric iodoacetyl Cys-reactive tandem mass tag [iodoTMT]) endowed with unique irreversible Cys-reactivities.
  
  
  RESULTS
  We have established a sequential iodoTMT-switch procedure coupled with efficient immunoenrichment and advanced shotgun liquid chromatography-MS/MS analysis. This workflow allows us to differentially quantify the multiple redox-modified forms of a Cys site in the original cellular context. In one single analysis, we have identified over 260 Cys sites showing quantitative differences in multiplexed redox modifications from the total lysates of H9c2 cardiomyocytes experiencing hypoxia in the absence and presence of S-nitrosoglutathione (GSNO), indicative of a distinct pattern of individual susceptibility to S-nitrosylation or S-glutathionylation. Among those most significantly affected are proteins functionally implicated in hypoxic damage from which we showed that GSNO would protect.
  
  
  METHODS
  We demonstrate for the first time how quantitative analysis of various Cys-redox modifications occurring in biological samples can be performed precisely and simultaneously at proteomic levels.
  
  
  CONCLUSIONS
  We have not only developed a new approach to map global Cys-redoxomic regulation in vivo, but also provided new evidences implicating Cys-redox modifications of key molecules in NO-mediated ischemic cardioprotection.
authors.affiliation: 1 Institute of Biochemical Sciences, National Taiwan University , Taipei, Taiwan .
authors.forename: Kuan-Ting
authors.initials: KT
authors.lastname: Pan
authors.forename: Yi-Yun
authors.initials: YY
authors.lastname: Chen
authors.forename: Tsung-Hsien
authors.initials: TH
authors.lastname: Pu
authors.forename: Yu-Shu
authors.initials: YS
authors.lastname: Chao
authors.forename: Chun-Yi
authors.initials: CY
authors.lastname: Yang
authors.forename: Ryan D
authors.initials: RD
authors.lastname: Bomgarden
authors.forename: John C
authors.initials: JC
authors.lastname: Rogers
authors.forename: Tzu-Ching
authors.initials: TC
authors.lastname: Meng
authors.forename: Kay-Hooi
authors.initials: KH
authors.lastname: Khoo
mesh_terms: D000818:Animals; D015687:Cell Hypoxia; D002460:Cell Line; D003545:Cysteine / Q000378:metabolism*; D019803:Glutathione Disulfide / Q000378:metabolism; D013058:Mass Spectrometry; D032383:Myocytes, Cardiac / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism*; D010084:Oxidation-Reduction; D040901:Proteomics / Q000379:methods; D051381:Rats; D026422:S-Nitrosoglutathione / Q000378:metabolism
chemical_list: D009569:Nitric Oxide; D026422:S-Nitrosoglutathione; D003545:Cysteine; D019803:Glutathione Disulfide