Radiolytic Modification of Sulfur Containing Acidic Amino Residues in Model Peptides: Fundamental Studies for Protein Footprinting

Journal Article · Sat Jan 01 04:00:00 EST 2005 · Anal. Chem.

DOI:https://doi.org/10.1021/ac0484629· OSTI ID:913913

Xu, G; Chance, M

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 {approx} tryptophan > cystine is observed.

• Research Organization: Brookhaven National Laboratory (BNL) National Synchrotron Light Source
• Sponsoring Organization: Doe - Office Of Science
• DOE Contract Number: AC02-98CH10886
• OSTI ID: 913913
• Report Number(s): BNL--78481-2007-JA
• Journal Information: Anal. Chem., Journal Name: Anal. Chem. Journal Issue: 8 Vol. 77; ISSN 0003-2700; ISSN ANCHAM
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
AMINO ACIDS
CYSTEINE
CYSTINE
MASS SPECTROSCOPY
METHIONINE
MODIFICATIONS
ORGANIC SULFUR COMPOUNDS
OXIDATION
PEPTIDES
PROTEIN STRUCTURE
PROTEINS
RADIOLYSIS
RESIDUES
SULFONIC ACIDS
SULFUR
national synchrotron light source