Assignment of disulfide bonds in proteins by partial acid hydrolysis and mass spectrometry.
A new method is described for locating disulfide bonds in proteins which cannot be cleaved between half-cystinyl residues by enzymic methods, as is often the case for tightly coiled proteins, or for proteins in which half-cystinyl residues are not separated by residues required for enzymic cleavage. Partial acid hydrolysis of a model protein, hen egg-white lysozyme, produces a mixture of disulfide-containing peptides from which the disulfide connections may be deduced. The usefulness of a combination of HPLC, fast atom bombardment mass spectrometry, and computer-assisted analysis to identify disulfide-containing peptides present in the partial acid hydrolysate of the model protein is demonstrated. Chromatographic fractions of the hydrolysate were analyzed by mass spectrometry before and after chemical reduction of the disulfide bonds to determine the molecular weights of disulfide-containing peptides. Computer-assisted analysis was then used to relate the molecular weights of these peptides to specific segments of the protein from which the disulfide connectivities could be determined. Partial acid hydrolysis of proteins, which is attractive because it proceeds relatively independent of the amino acid sequence and structure, and because disulfide interchange is unlikely to occur in dilute acid, has become practical because disulfide-containing peptides present in complex mixtures can be identified rapidly and definitively by this method.
Zhou
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ZR
Department of Medicinal Chemistry and Pharmacognosy, School of Pharmacy and Pharmacal Sciences, Purdue University, West Lafayette, Indiana 47907.
Smith
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DL
D000143:Acids; D000595:Amino Acid Sequence; D000818:Animals; D002645:Chickens; D004220:Disulfides / Q000032:analysis*; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009113:Muramidase / Q000737:chemistry; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry*; D012984:Software; D016339:Spectrometry, Mass, Fast Atom Bombardment
D000143:Acids; D004220:Disulfides; D011506:Proteins; D009113:Muramidase