Peptide sequencing by mass spectrometry

Abstract

Dipeptides were concerted to the N-acetyl-O-n-butyl derivatives and individual mass spectra were recorded. A library of more than 50 of these spectra were placed on magnetic tape. Dipeptide derivative mixtures were prepared and separated by probe distillation (microdistillation) and were identified by matching mass spectral data to standard spectra on the tape. The partial mass spectra of individual dipeptide derivatives were obtained by computer plotting of elimination curves for all prominent ions observed during the microdistillation. Curve parameters were obtained by fitting an empirical equation to the data through non-linear regression analysis. Ions having similar curve parameters were grouped and relative intensities were calculated for each ion in the group. The masses and relative intensities were then used to search the magnetic tape using a least-squares program. Successful identifications were made on dipeptide derivatives in simple mixtures. Sequence work was accomplished using the enzyme dipeptidyl transferase (DAPI), E. C. No. 3.4.4.9, which liberates dipeptides from an oligopeptide's N-terminus. A different dipeptide mixture was similarly obtained from the same oligopeptide after the N-terminal amino acid was removed by Edman degradation. An octapeptide was sequenced in this manner. Dipeptides were also identified from DAP I hydrolysis mixtures of a hexapeptide and the A chain of insulin, containing 21 amino acids.