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 of the N-alkylated amino acid constituents is provided by this approach. A widely applicable strategy will be presented, and to demonstrate its scope and limitations eighteen analogues of sequences 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/MeIle. 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 mumol and be completed within 2-4 h.
K
K
Eckart
H
H
Schwarz
M
M
Chorev
C
C
Gilon
D000478:Alkylation; D000595:Amino Acid Sequence; D013058:Mass Spectrometry / Q000379:methods; D010455:Peptides / Q000032:analysis*; D011487:Protein Conformation
D010455:Peptides