Detection and mapping of widespread intermolecular protein disulfide formation during cardiac oxidative stress using proteomics with diagonal electrophoresis.
Regulation of protein function by reversible cysteine-targeted oxidation can be achieved by multiple mechanisms, such as S-glutathiolation, S-nitrosylation, sulfenic acid, sulfinic acid, and sulfenyl amide formation, as well as intramolecular disulfide bonding of vicinal thiols. Another cysteine oxidation state with regulatory potential involves the formation of intermolecular protein disulfides. We utilized two-dimensional sequential non-reducing/reducing SDS-PAGE (diagonal electrophoresis) to investigate intermolecular protein disulfide formation in adult cardiac myocytes subjected to a series of interventions (hydrogen peroxide, S-nitroso-N-acetylpenicillamine, doxorubicin, simulated ischemia, or metabolic inhibition) that alter the redox status of the cell. More detailed experiments were undertaken with the thiol-specific oxidant diamide (5 mm), a concentration that induces a mild non-injurious oxidative stress. This increase in cellular oxidation potential caused global intermolecular protein disulfide formation in cytosolic, membrane, and myofilament/cytoskeletal compartments. A large number of proteins that undergo these associations were identified using liquid chromatography-mass spectrometry/mass spectrometry. These associations, which involve metabolic and antioxidant enzymes, structural proteins, signaling molecules, and molecular chaperones, were confirmed by assessing "shifts" on non-reducing immunoblots. The observation of widespread protein-protein disulfides indicates that these oxidative associations are likely to be fundamental in how cells respond to redox changes.
Department of Cardiology, Cardiovascular Division, King's College London, The Rayne Institute, St. Thomas' Hospital, London SE1 7EH, United Kingdom.
Jonathan P
JP
Brennan
Robin
R
Wait
Shajna
S
Begum
James R
JR
Bell
Michael J
MJ
Dunn
Philip
P
Eaton
D000818:Animals; D002478:Cells, Cultured; D004220:Disulfides / Q000302:isolation & purification*; D015180:Electrophoresis, Gel, Two-Dimensional; D006352:Heart Ventricles / Q000166:cytology; D015151:Immunoblotting; D008297:Male; D032383:Myocytes, Cardiac / Q000737:chemistry / Q000166:cytology / Q000378:metabolism*; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D011506:Proteins / Q000302:isolation & purification*; D040901:Proteomics / Q000379:methods*; D051381:Rats; D017208:Rats, Wistar
D004220:Disulfides; D011506:Proteins