Determination of interchain crosslinkages in insulin B-chain dimers by fast atom bombardment mass spectrometry.
New methodology for identifying and locating crosslinkages in peptides is described. Pepsin is used to cleave insulin and B-chain dimers of insulin into fragments under conditions which retain the original peptide crosslinkages. After partial separation by HPLC, the peptides are analyzed by fast atom bombardment mass spectrometry (FABMS) to determine their molecular weights. The molecular weights of peptide fragments expected from the pepsin digests of human insulin and related model compounds are calculated from the amino acid sequence of the intact peptide. Digestion products are identified by matching their molecular weights, as determined by FABMS, with calculated molecular weights. Locations of interchain crosslinkages are deduced after the peptide fragments have been assigned to specific segments of the parent peptide. The validity of the method has been established by correctly identifying structurally important products in the pepsin digests of model compounds such as human, bovine, and porcine insulins. Procedures developed with the model compounds were used to identify crosslinkages in peptides of unknown structure isolated from an insulin A-chain/B-chain combination reaction mixture. Evidence is presented for formation of three different types of crosslinkages, disulfide, lanthionine, and sulfoxide.
Toren
P
P
Department of Medicinal Chemistry and Pharmacognosy, School of Pharmacy and Pharmacal Sciences, Purdue University, West Lafayette, Indiana 47907.
Smith
D
D
Chance
R
R
Hoffman
J
J
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D007328:Insulin / Q000032:analysis*; D013058:Mass Spectrometry / Q000379:methods; D008956:Models, Chemical; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis*
D000596:Amino Acids; D007328:Insulin; D010446:Peptide Fragments