Polypeptide sequencing by a gas chromatograph mass spectrometer computer system: I—generation and derivatization of complex mixtures of oligopeptides

Abstract

Abstract--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 0-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 experiment 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 I .4 pmol 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 pmol per experiment).

Mass spectrometric sequencing of peptides and proteins

“…13) has been improved over the years with respect to the chemical reactions involved and the instrumental techniques used (Ref. [6][7][8][9][10][11]. In its present form it consists of converting a mixture of peptides first to methyl esters which are then N-trifluoroacetylated.…”

“…The mass spectra of the O-TMS-polyamino alcohols are so simple and sequence specific that even only partially resolved peptide derivatives up to hexapeptides can be reliably identified (Ref. [6][7][8][9][10][11]. Peptide mixtures derived from partial degradation of polypeptides containing up to 50 amino acids have been successfully analyzed and this is just the size which one generally obtains upon tryptic or cyanogen bromide cleavage of proteins.…”

Sequencing of Peptide Mixtures by Edman Degradation and Field‐Desorption Mass Spectrometry

“…Nowadays, the former method can be successfully used for the determination of the partial or even almost complete sequences of polypeptides and proteins by cleavage of an amino acid residue from the N terminus in each degradation reaction. In contrast to the Edman procedure, gas chromatography/mass spectrometry [2,3] can be used for analysis of peptide mixtures prepared from polypeptides or proteins by partial hydrolysis, since the technique permits separation of the components and their identification in a single experiment. Although such prominent and ingenious procedures are available, more improved or different techniques are still desirable for the following reasons : first, it is desirable for chemical reactions to be as simple as possible and identification of the resulting components to be as accurate as possible; second, cross-checks by different methods are more desirable carboxypeptidase A (EC 3.4.17.1).…”

“…For amino acid sequencing of polypeptides and proteins the automated sequencer technique developed by Edman and Begg [I] and gas chromatography/mass spectrometry developed by Biemann et al [2] both have these good points. Nowadays, the former method can be successfully used for the determination of the partial or even almost complete sequences of polypeptides and proteins by cleavage of an amino acid residue from the N terminus in each degradation reaction.…”

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

“…The methyl e'sterified peptides were reduced by 1 ml 1 N LiAlD4 in dimethoxyethane at 85°C for 36 h [ 151. The amino alcohols were isolated by the extraction procedure, then trimethylsilylated as described [16,17]. Portions of 5-10% of the derivatized mixtures were injected 'on column' into a PerkinElmer F-22 gas chromatograph; a 1 meter 63.5 mm o.d.…”