Monitoring reactions of nitric oxide with peptides and proteins by electrospray ionization-mass spectrometry.
Recent studies have demonstrated the biological importance of the interaction of nitric oxide (NO) with proteins. Protein-associated targets of NO include heme, Cys, and Tyr. Electrospray ionization-mass spectrometry was used to monitor the results of exposure of model peptides and an enzyme to NO under different conditions and thus addressed aspects of NO-protein interactions. The molecular mass of a decapeptide containing a single Cys residue increased by 29 Da upon treatment with NO under aerobic and acidic conditions, consistent with the substitution of one NO moiety. The mass of reduced somatostatin, a peptide containing two Cys residues, increased by 58 Da, consistent with the substitution of two NO moieties. These substitutions were prevented by pretreatment of the peptides with N-ethylmaleimide. The strength of the nitrosothiol bond was examined by varying the amount of energy applied to the peptide ions and indicated a labile species. Cys residues were very rapidly nitrosated, while other reactions were observed to occur at much slower rates. These include the further oxidation of nitrosothiol to sulfonic acid and nitration of Tyr. Peptides treated with NO at physiological pH were observed to undergo dimerization as well as nitrosation. These studies were extended to the enzyme p21ras, whose activity has been postulated to be modulated by nitrosothiol formation, and revealed the formation of a single nitrosothiol on p21ras upon NO treatment. These data suggest that electrospray ionization-mass spectrometry allows for quantitation and characterization of nitrosothiol bonds in peptides and proteins.
Mirza
U A
UA
Rockefeller University, New York, New York 10021, USA.
Chait
B T
BT
Lander
H M
HM
D000595:Amino Acid Sequence; D007700:Kinetics; D013058:Mass Spectrometry; D008623:Mercaptoethanol; D008969:Molecular Sequence Data; D009569:Nitric Oxide / Q000378:metabolism*; D009603:Nitroso Compounds / Q000737:chemistry*; D011506:Proteins / Q000378:metabolism*; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism; D026403:S-Nitrosothiols; D013004:Somatostatin / Q000378:metabolism
D009603:Nitroso Compounds; D011506:Proteins; D026403:S-Nitrosothiols; D009569:Nitric Oxide; D013004:Somatostatin; D008623:Mercaptoethanol; C032635:S-nitrosomercaptoethanol; D016283:Proto-Oncogene Proteins p21(ras)