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Polypeptide sequencing by a gas chromatograph mass spectrometer computer system: I—generation and derivatization of complex mixtures of oligopeptides
Author(s) -
Kelley J. A.,
Nau H.,
Förster H.J.,
Biemann K.
Publication year - 1975
Publication title -
biomedical mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.475
H-Index - 121
eISSN - 1096-9888
pISSN - 0306-042X
DOI - 10.1002/bms.1200020607
Subject(s) - edman degradation , cyanogen bromide , chemistry , peptide , derivatization , chromatography , amino acid , hydrolysis , bovine pancreatic ribonuclease , peptide sequence , ribonuclease , aminopeptidase , mass spectrometry , leucine , organic chemistry , biochemistry , rna , gene
A generally applicable strategy for polypeptide sequencing has been developed which involves cleavage of a large peptide (for example, primary degradation peptides obtained by tryptic or cyanogen bromide cleavage of a protein) to a mixture of small peptides whose individual amino acid sequences are then determined without their prior isolation. This is accomplished by conversion of the peptide mixture into the corresponding mixture of O ‐trimethylsilylated polyamino alcohols through reduction of the N ‐acetylated peptide esters with lithium aluminum deuteride, followed by treatment with trimethylsilyldiethylamine. The conditions for the enzymatic or chemical cleavage were optimized to yield mixtures of peptides best suited for this technique and which represented complete overlap. Limited acid hydrolysis combined with a second experimen utilizing either an enzyme with broad specificity, a set of enzymes, or dipeptidyl aminopeptidase I on the original and/or Edman‐degraded molecule was found to be the best choice. This sequencing strategy was evaluated using 0.4 to 1.4 μmol of peptides with known structures (ribonuclease S ‐peptide, glucagon) and then applied to primary degradation peptides of rabbit skeletal muscle actin up to twenty amino acids long (0.4 to 1 μmol per experiment).