Sequence determination of N‐terminal and C‐terminal blocked peptides containing N‐alkylated amino acids and structure determination of these amino acid constituents by using fast‐atom‐bombardment/tandem mass spectrometry

Peptides, blocked either at the N or C terminus, and thus unsuited for Edman degradation, and those containing N‐alkylated amino acids, which are not detectable when using conventional amino acid analysis, can be easily sequenced by applying a method in which fast atom bombardment (FAB) is combined with tandem mass spectrometry (MSMS). Moreover, the structure ofthe N‐alkylated amino acid constituents is provided by this approach. A widely applicable strategy will be presented, and to demonstrate its scope and limitations eithteen analogues of seqences related to the C terminus of substance P, a biologically active neuropeptide, were investigated. The power and reliability of the approach will be demonstrated by analyzing an ‘unknown’ peptide. Moreover, the detection and structure elucidation of N‐alkylated amino acids which usually escape amino acid analysis will be described, as will be the unequivocal differentiation and identification of isomeric MeLeu/Melle. The influence of the N‐alkylation on the mass spectrometric fragmentation behaviour will be discussed. Furthermore, the sequencing of two adipokinetic hormones by using the combined FAB‐MSMS approach is described. Analysis of peptides can be achieved with sample sizes less than 0.1 μmol and be completed within 2–4 h.