Radiolytic Modification of Sulfur-Containing Amino Acid Residues in Model Peptides: Fundamental Studies for Protein Footprinting
- 3 March 2005
- journal article
- Published by American Chemical Society (ACS) in Analytical Chemistry
- Vol. 77 (8), 2437-2449
- https://doi.org/10.1021/ac0484629
Abstract
Protein footprinting based on hydroxyl radical-mediated modification and quantitative mass spectroscopic analysis is a proven technique for examining protein structure, protein−ligand interactions, and structural allostery upon protein complex formation. The reactive and solvent-accessible amino acid side chains function as structural probes; however, correct structural analysis depends on the identification and quantification of all the relevant oxidative modifications within the protein sequence. Sulfur-containing amino acids are oxidized readily and the mechanisms of oxidation are particularly complex, although they have been extensively investigated by EPR and other spectroscopic methods. Here we have undertaken a detailed mass spectrometry study (using electrospray ionization mass spectrometry and tandem mass spectrometry) of model peptides containing cysteine (Cys-SH), cystine (disulfide bonded Cys), and methionine after oxidation using γ-rays or synchrotron X-rays and have compared these results to those expected from oxidation mechanisms proposed in the literature. Radiolysis of cysteine leads to cysteine sulfonic acid (+48 Da mass shift) and cystine as the major products; other minor products including cysteine sulfinic acid (+32 Da mass shift) and serine (−16 Da mass shift) are observed. Radiolysis of cystine results in the oxidative opening of the disulfide bond and generation of cysteine sulfonic acid and sulfinic acid; however, the rate of oxidation is significantly less than that for cysteine. Radiolysis of methionine gives rise primarily to methionine sulfoxide (+16 Da mass shift); this can be further oxidized to methionine sulfone (+32 Da mass shift) or another product with a −32 Da mass shift likely due to aldehyde formation at the γ-carbon. Due to the high reactivity of sulfur-containing amino acids, the extent of oxidation is easily influenced by secondary oxidation events or the presence of redox reagents used in standard proteolytic digestions; when these are accounted for, a reactivity order of cysteine > methionine ∼ tryptophan > cystine is observed.Keywords
This publication has 40 references indexed in Scilit:
- Radiolytic Modification of Acidic Amino Acid Residues in Peptides: Probes for Examining Protein−Protein InteractionsAnalytical Chemistry, 2004
- A Novel Database of Disulfide Patterns and its Application to the Discovery of Distantly Related HomologsJournal of Molecular Biology, 2004
- Sulfenic Acid Formation in Human Serum Albumin by Hydrogen Peroxide and PeroxynitriteBiochemistry, 2003
- Oxidation of methionyl residues in proteins: Tools, targets, and reversalFree Radical Biology & Medicine, 1995
- Critical Review of rate constants for reactions of hydrated electrons, hydrogen atoms and hydroxyl radicals (⋅OH/⋅O− in Aqueous SolutionJournal of Physical and Chemical Reference Data, 1988
- An ESR Investigation of the Reactions of Glutathione, Cysteine and Penicillamine Thiyl Radicals: Competitive Formation of RSO·, R·, RSSR, and RSS·International Journal of Radiation Biology, 1988
- Reaction mechanisms in the radiolysis of peptides, polypeptides, and proteinsChemical Reviews, 1987
- Pulse radiolysis study of sulfhydryl compounds in aqueous solutionThe Journal of Physical Chemistry, 1973
- Aqueous radiation chemistry of cysteine. I. Deaerated acidic solutionsThe Journal of Physical Chemistry, 1968
- Products of γ-Irradiation of Cysteine and Cystine1Journal of the American Chemical Society, 1960