Polypeptide sequencing by a gas chromatograph mass spectrometer computer system: II—characterization of complex mixtures of oligopeptides as trimethylsilylated polyamino alcohols

Abstract

Abstract-Complex mixtures of 0-trimethylsilylated polyamino alcohols, which have been generated by either acid or enzymatic hydrolysis of polypeptides and subsequent derivatization, are completely characterized by a gas chromatograph mass spectrometer computer system. These peptide derivatives possess excellent gas chromatographic properties; a wide range of derivatives from di-to hexapeptides may be separated in a single chromatographic experiment. The identification of these compounds, either manually or with the assistance of the computer, is based on three sets of data which are automatically generated after the g.c.m.s. computer experiment : (1) mass spectra, which exhibit sequence-determining ions of high abundance ; (2) selected ion records, which allow efficient location of peptide derivatives in the gas chromatogram as well as resolution of incompletely separated fractions ; (3) retention indices. which can be calculated from values which have been assigned to each amino acid residue

Amino acid sequence determination of the blocked N‐terminal tryptic peptide of Neurospora tyrosinase by mass spectrometry

“…The gas chromatographic fraction eluting with a retention index of 1904 exhibited the mass spectrum shown in fig.2 which clearly indicates the sequence of N-Ac-Ser-Thr. The abundant ion m/e 162 indicates a peptide which N-acetyl-Ser at the amino-terminus (for the sequence ions and retention indices of N-acetylated and I.iAlD4 -reduced peptides see [ 191). Also the retention index measured for this GC-peak (1904) is in agreement with the index predicted for the derivative of iV-acetyl-Ser-Thr on a SE-30 column.…”

Four decades of structure determination by mass spectrometry: From alkaloids to heparin

“…For that work, the protein had to be subjected to partial acid hydrolysis to generate the complex mixture of many overlapping di-to hexapeptides. By that time we had continuously improved the methodology through the replacement of acetylation with trifluoroacetylation [34] (second step of Scheme 3) and O-trimethylsilylation [35] to increase volatility, which extended applicability to these larger peptides. Direct coupling of the gas chromatograph with the mass spectrometer [27] made it possible to identify 61 peptides in the hydrolyzate of monellin-I in a single experiment.…”

Structure Determination of Natural Products by Mass Spectrometry

“…These derivatives also were sufficiently volatile to be amenable to gas chromatography (GC) (12), thus allowing the separation of the complex mixtures expected from partial hydrolyses of proteins. Over time, the chemical procedure was improved with the use of trifluoroacetylation (13) and silylation (14), which allowed its extension to penta-or even hexapeptides. With the interfacing of the GC to the MS (15) and to an IBM 1800 computer (16), a powerful system was created for determining, finally, the first primary structure of a protein, subunit I of monellin, entirely by mass spectrometry (17).…”