title: Radiolytic modification of sulfur-containing amino acid residues in model peptides: fundamental studies for protein footprinting.
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 gamma-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 gamma-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 approximately tryptophan > cystine is observed.
authors.affiliation: Center for Synchrotron Biosciences, Albert Einstein College of Medicine of Yeshiva University, 1300 Morris Park Avenue, Bronx, New York 10461, USA.
authors.forename: Guozhong
authors.initials: G
authors.lastname: Xu
authors.forename: Mark R
authors.initials: MR
authors.lastname: Chance
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D003545:Cysteine / Q000031:analogs & derivatives / Q000737:chemistry; D003553:Cystine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D005720:Gamma Rays; D008715:Methionine / Q000737:chemistry; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010455:Peptides / Q000737:chemistry* / Q000528:radiation effects*; D023302:Protein Footprinting / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D013455:Sulfur / Q000737:chemistry*; D053719:Tandem Mass Spectrometry / Q000379:methods; D014965:X-Rays
chemical_list: D000596:Amino Acids; D004220:Disulfides; D010455:Peptides; D003553:Cystine; D013455:Sulfur; D008715:Methionine; D003545:Cysteine; C013461:cysteine sulfinic acid