Transition metal-peptide binding studied by metal-catalyzed oxidation reactions and mass spectrometry.
We have identified conditions that allow metal-catalyzed oxidation (MCO) reactions and mass spectrometry (MS) to correctly identify binding sites of first-row transition metal ions to model peptides. This work extends the applicability of the MCO/MS method to metals other than Cu(II). When the appropriate reducing agent (ascorbate, 10 mM) and oxidizing agent concentrations (1 mM persulfate, atmospheric O2, or both) are used, metal-bound amino acids can be sufficiently and specifically oxidized for clear identification by MS. The MCO reactions with Mn(II), Fe(II), Co(II), and Ni(II) occur to lesser extents than with Cu(II), but oxidation is still extensive enough to allow easy identification of the metal-bound residues. With the exception of aspartic acid, the known metal-binding amino acids of angiotensin I and bacitracin A are oxidized, while no oxidation is observed at nonbinding residues. Failure to oxidize aspartic acid is likely due to the relatively slow reactivity of its carboxylic acid side chain with reactive oxygen species, suggesting that the current MCO/MS protocol is transparent to such acidic residues. Overall, this study indicates that, just as is possible for Cu(II), the MCO/MS method should be suitable for determining the Mn(II)-, Fe(II)-, Co(II)-, and Ni(II)-binding sites of metalloproteins.
Bridgewater
Juma D
JD
Department of Chemistry, University of Massachusetts-Amherst, Amherst, Massachusetts 01003, USA.
Lim
Jihyeon
J
Vachet
Richard W
RW
D000595:Amino Acid Sequence; D000803:Angiotensin I / Q000737:chemistry / Q000378:metabolism; D001205:Ascorbic Acid / Q000494:pharmacology; D001414:Bacitracin / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D002264:Carboxylic Acids / Q000737:chemistry / Q000378:metabolism; D002384:Catalysis; D003300:Copper / Q000737:chemistry / Q000378:metabolism; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D017382:Reactive Oxygen Species / Q000737:chemistry / Q000378:metabolism; D013431:Sulfates / Q000494:pharmacology; D028561:Transition Elements / Q000737:chemistry* / Q000378:metabolism
D002264:Carboxylic Acids; D017382:Reactive Oxygen Species; D013431:Sulfates; D028561:Transition Elements; D001414:Bacitracin; D003300:Copper; D000803:Angiotensin I; C026461:bacitracin A; D001205:Ascorbic Acid