Assignment of disulfide bonds in proteins by fast atom bombardment mass spectrometry.
A rapid and sensitive method for assignment of disulfide bonds using fast atom bombardment mass spectrometry is described for hen egg white lysozyme and bovine ribonuclease A. The protein is initially digested to a mixture of peptides using chemical and enzymatic methods under conditions which minimize disulfide bond reduction and exchange. The digested sample is analyzed directly by fast atom bombardment mass spectrometry before and after chemical reduction of cystine residues. An important feature of the method is that it is not necessary to completely resolve the peptides in the digest chromatographically prior to analysis. The disulfide-containing peptides are also characterized directly by prolonged exposure of the sample to the high energy xenon atom beam which results in the reduction of cystine residues. Intra- as well as interchain disulfide bond assignments are made on the basis of the mass difference between the molecular ions (MH+) of the oxidized and reduced peptides. Confirmation of the mass assignments may be obtained from the mass spectra of the digests after one cycle of manual Edman degradation. Although the quantity of protein required to unambiguously assign all of the disulfide linkages will depend on the ease with which the appropriate peptide fragments can be formed, results from these studies indicate that approximately 1 nmol of protein is usually sufficient.
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002645:Chickens; D002851:Chromatography, High Pressure Liquid; D003488:Cyanogen Bromide / Q000494:pharmacology; D004220:Disulfides / Q000032:analysis*; D004529:Egg White; D009113:Muramidase; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000032:analysis*; D012259:Ribonuclease, Pancreatic; D013054:Spectrophotometry, Atomic; D014357:Trypsin / Q000378:metabolism
D004220:Disulfides; D011506:Proteins; D012259:Ribonuclease, Pancreatic; D009113:Muramidase; D014357:Trypsin; D003488:Cyanogen Bromide