Identification of S-nitrosated mitochondrial proteins by S-nitrosothiol difference in gel electrophoresis (SNO-DIGE): implications for the regulation of mitochondrial function by reversible S-nitrosation.

TitleIdentification of S-nitrosated mitochondrial proteins by S-nitrosothiol difference in gel electrophoresis (SNO-DIGE): implications for the regulation of mitochondrial function by reversible S-nitrosation.
Publication TypeJournal Article
Year of Publication2010
AuthorsChouchani, ET, Hurd, TR, Nadtochiy, SM, Brookes, PS, Fearnley, IM, Lilley, KS, Smith, RAJ, Murphy, MP
JournalBiochem J
Volume430
Issue1
Pagination49-59
Date Published2010 Aug 15
ISSN1470-8728
KeywordsAnimals, Electrophoresis, Gel, Two-Dimensional, In Vitro Techniques, Male, Membrane Potential, Mitochondrial, Mice, Mice, Inbred C57BL, Mitochondria, Mitochondrial Proteins, Myocardial Reperfusion Injury, Myocardium, Oxidation-Reduction, Proteomics, Rats, S-Nitrosothiols, Superoxides
Abstract

The S-nitrosation of mitochondrial proteins as a consequence of NO metabolism is of physiological and pathological significance. We previously developed a MitoSNO (mitochondria-targeted S-nitrosothiol) that selectively S-nitrosates mitochondrial proteins. To identify these S-nitrosated proteins, here we have developed a selective proteomic methodology, SNO-DIGE (S-nitrosothiol difference in gel electrophoresis). Protein thiols in control and MitoSNO-treated samples were blocked, then incubated with copper(II) and ascorbate to selectively reduce S-nitrosothiols. The samples were then treated with thiol-reactive Cy3 (indocarbocyanine) or Cy5 (indodicarbocyanine) fluorescent tags, mixed together and individual protein spots were resolved by 2D (two-dimensional) gel electrophoresis. Fluorescent scanning of these gels revealed S-nitrosated proteins by an increase in Cy5 red fluorescence, allowing for their identification by MS. Parallel analysis by Redox-DIGE enabled us to distinguish S-nitrosated thiol proteins from those which became oxidized due to NO metabolism. We identified 13 S-nitrosated mitochondrial proteins, and a further four that were oxidized, probably due to evanescent S-nitrosation relaxing to a reversible thiol modification. We investigated the consequences of S-nitrosation for three of the enzymes identified using SNO-DIGE (aconitase, mitochondrial aldehyde dehydrogenase and alpha-ketoglutarate dehydrogenase) and found that their activity was selectively and reversibly inhibited by S-nitrosation. We conclude that the reversible regulation of enzyme activity by S-nitrosation modifies enzymes central to mitochondrial metabolism, whereas identification and functional characterization of these novel targets provides mechanistic insight into the potential physiological and pathological roles played by this modification. More generally, the development of SNO-DIGE facilitates robust investigation of protein S-nitrosation across the proteome.

DOI10.1042/BJ20100633
Alternate JournalBiochem. J.
Citation Key10.1042/BJ20100633
PubMed ID20533907
PubMed Central IDPMC2911678
Grant ListHL-071158 / HL / NHLBI NIH HHS / United States
MC_U105663142 / / Medical Research Council / United Kingdom
R01 HL071158 / HL / NHLBI NIH HHS / United States
/ / Medical Research Council / United Kingdom