Amino acid sequence determination by g.l.c.--mass spectrometry of permethylated peptides. Optimization of the formation of chemical derivatives at the 2-10 nmol level.
A new technique is described that permits the permethylation of acylated peptides at the 2-10 nmol level. The presence of up to 400 micrograms of sodium dodecyl sulphate per sample does not affect the reaction yields. The technique, which is a miniaturization of the widely used methyl iodide/dimethylsulphinyl carbanion procedure, employs a layer of hexane to exclude moisture and oxygen from the reaction mixture. Analysis of the peptide derivatives by combined g.l.c.--mass spectrometry permits amino acid sequence information to be obtained. In addition to studies of digests of a model substrate (glucagon), the new permethylation technique has been used to identify a peptide of interest from a digest of a cytochrome and to define the N-termini of two proteins at the 5 nmol level.
D000107:Acetylation; D000595:Amino Acid Sequence; D004121:Dimethyl Sulfoxide / Q000138:chemical synthesis; D008401:Gas Chromatography-Mass Spectrometry; D005934:Glucagon; D007371:Interferon-gamma; D008722:Methods; D008745:Methylation; D010446:Peptide Fragments
D010446:Peptide Fragments; D007371:Interferon-gamma; D005934:Glucagon; D004121:Dimethyl Sulfoxide