Investigation of several unique tandem mass spectrometric fragmentation patterns of NFDEIDR, an orcokinin analog, and its N-terminal dimethylated form

Orcokinins are a family of myotropic neuropeptides widely present in various decapod crustaceans and insect species. The majority of the orcokinins identified to date share a conserved sequence of NFDEIDR at their N-termini. Electrospray ionization quadrupole time-of-flight tandem mass spectrometric (ESI-QTOF-MS/MS) analysis of doubly charged orcokinin precursor ions reveals the presence of a y((n-1)) + 10 peak, which is more intense than that for the y((n-1)) ion. To elucidate the identity of this novel fragment ion and understand the mechanism underlying this fragmentation, we employed a combined approach involving the use of isotopic N-terminal dimethylation, methyl esterification, and isotope-encoded NFDEIDR. Comparison of the fragmentation patterns of these chemically modified orcokinin analogs allowed the determination of the structure of the y((n-1)) + 10 ion as y((n-1)) + CO-H2O. The y(x) + CO-H2O ions, along with the y(x) + CO and y(x) + CONH3 ions, are also present in the MS/MS spectra of NFDEIDR and several other peptides. Additionally, we report two other unusual fragmentation ions in the MS/MS spectra of N-terminal dimethyl NFDEIDR (2+), which yields the novel fragment ions of the y((n-1)) + 38 ion and the [M+2H-59](2+) ion. These two ion series involve the neutral loss of the asparagine side chain. The same sets of ions are also present in other peptides with dimethyl-modified asparagines at the N-terminus. The competition between the side-chain loss and loss of dimethylamine is described. The loss of the side chain of N-terminal dimethyl Asp(1) is reported as well. We also report for the first time the neutral loss of ammonia from the N-terminal amino group of Asn(1) and the loss Of CO2 from the side chain of aspartic acid. Copyright (c) 2006 John Wiley & Sons, Ltd.