Identification of disulfide-containing peptides by performic acid oxidation and mass spectrometry.
In addition to reducing the analysis time, the direct examination of proteolytic digests by fast atom bombardment mass spectrometry (FABMS) greatly extends the information that is available from peptide mapping experiments. Mass spectral data are particularly useful for identifying post-translationally modified peptides. For example, the molecular weight of a disulfide-containing peptide may be used to locate the disulfide bond in the protein from which the peptide was derived. This paper describes a new procedure, which is useful for identifying disulfide-bonded peptides. Peptides are treated with performic acid to modify certain residues and thereby cause a characteristic change in the peptide molecular weight. This change in molecular weight is determined by FABMS and used to help identify peptides. Results for a series of small peptides demonstrate that Cys, Met, and Trp are the only residues that undergo a change in molecular weight under the conditions used here. Furthermore, these changes in molecular weight are diagnostic for each of the residues. Cysteinyl-containing peptides are of particular interest, because their identification is essential for locating disulfide bonds. The molecular weight of a peptide increases by 48 mu for each cysteinyl residue present. This approach is used to identify peptides that contain both cysteinyl and cystinyl residues in the peptic digest of bovine insulin. The method is extended to the analysis of a tryptic digest of cyanogen bromide-treated ribonuclease A. A computer-assisted analysis procedure is used to demonstrate the specificity with which peptide molecular weight is related to specific segments of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)
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Department of Medicinal Chemistry and Pharmacognosy, Purdue University, West Lafayette, Indiana 47907.
Smith
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D002851:Chromatography, High Pressure Liquid; D004220:Disulfides / Q000032:analysis*; D005561:Formates; D007328:Insulin; D013058:Mass Spectrometry; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000032:analysis; D010455:Peptides / Q000032:analysis*; D012259:Ribonuclease, Pancreatic
D004220:Disulfides; D005561:Formates; D007328:Insulin; D010446:Peptide Fragments; D010455:Peptides; C008817:peroxyformic acid; D012259:Ribonuclease, Pancreatic