Isotope edited product ion assignment by alpha-N labeling of peptides with[H-2(3)(50%)]2,4-dinitrofluorobenzene

An isotopic modification of Sanger's method for identifying peptide N-termi ni has been developed to assist peptide sequencing by tandem mass spectrome try. Tryptic peptides, such as Val-His-Leu-Tkr-Pro-Val-Glu-Lys, are derivat ized with an equimolar mixture of 2,4-dinitrofluorobenzene and [H-2(3)]2,4- dinitrofluorobenzene. Under optimized derivatization conditions, the cr-ami no group could be derivatized while the E-amine of the lysine side chain an d the imidazole of histidine remained underivatized. The a-dinitrophenyl mo dified peptides were characterized by electrospray ionization-tandem mass s pectrometry (ESI-MS/MS) and liquid chromatography (LC)-ESI-MS. The [M + H]( +) ions showed a doublet pattern with a Delta m/z of 3 and the [M + 2H](2+) ions were recognized as doublets with a Delta m/z of 1.5. MS/MS was employ ed where both isotopic [M + 2H](2+) ions were alternately subjected to coll ision-induced dissociation in the second quadrupole. Fragmentation in the i onization source generated identical product ion patterns that were observe d during fragmentation in the second quadrupole. In the product ion mass sp ectra, the N-terminal a and b ions (no c ion observed) are doublets with a Delta m/z of 3 or 1.5, while the C-terminal y and z ions (no x ion observed ) are singlets appearing at identical masses. Thus, the product ions contai ning the N-terminus derivatized with a dinitrophenyl group are unequivocall y distinguished from the product ions containing the C-terminus. The dinitr ophenyl modification generally enhanced the production of a and b ions with out diminishing y and z ion yields. (C) 1999 American Society for Mass Spec trometry. (C) 1999 American Society for Mass Spectrometry.