title: Optimization of the fragmentation in a frit-fast atom bombardment ion source for the sequencing of peptides at the picomole level.
abstract: Peptides derived from enzymatic digestions (cathepsin D and trypsin) were characterized and amino acid sequences determined by using their LC/MS spectra. A Frit-FAB interface that produces extensive peptide fragmentation and permits amino acid sequencing at the low picomole level is described for a model antigen, Staphylococcus aureus nuclease (Nase), and an enzyme of unknown structure, yeast aminopeptidase B. The amino acid sequences of peptides derived from digestion of Nase with cathepsin D (a relatively nonspecific endoprotease) were readily deduced and have provided insights into the nature of antigen processing. Frit-FAB LC/MS spectra of the Nase peptides contained a sufficient number of fragment ions to conclusively identify peptides with a mass below 2000 Da. Capillary LC/MS provided a means for the separation and identification of these enzymatically derived peptides in a fraction of the time that would have been required by gas-phase Edman sequence analysis. The optimized Frit-FAB experiment was consequently evaluated for the partial characterization of aminopeptidase B recently purified to homogeneity from Saccharomyces cerevisiae. Sequence-specific ions observed in the Frit-FAB mass spectra of these tryptic peptides were identical with those commonly observed in high-energy collision-induced dissociation (CID) spectra and included side-chain fragment ions that differentiated leucine from isoleucine. These fragment ions were used to deduce entire amino acid sequences for several of the tryptic peptides.
authors.affiliation: Department of Molecular Biology, Massachusetts General Hospital, Boston 02114.
authors.forename: D B
authors.initials: DB
authors.lastname: Kassel
authors.forename: K P
authors.initials: KP
authors.lastname: Williams
authors.forename: B D
authors.initials: BD
authors.lastname: Musselman
authors.forename: J A
authors.initials: JA
authors.lastname: Smith
mesh_terms: D000595:Amino Acid Sequence; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis*; D016339:Spectrometry, Mass, Fast Atom Bombardment
chemical_list: D010455:Peptides