Isotope edited product ion assignment by alpha-N labeling of peptides with [2H3(50%)]2,4-dinitrofluorobenzene.
An isotopic modification of Sanger's method for identifying peptide N-termini has been developed to assist peptide sequencing by tandem mass spectrometry. Tryptic peptides, such as Val-His-Leu-Thr-Pro-Val-Glu-Lys, are derivatized with an equimolar mixture of 2,4-dinitrofluorobenzene and [2H3]2,4-dinitrofluorobenzene. Under optimized derivatization conditions, the alpha-amino group could be derivatized while the epsilon-amine of the lysine side chain and the imidazole of histidine remained underivatized. The alpha-dinitrophenyl modified peptides were characterized by electrospray ionization-tandem mass spectrometry (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 employed where both isotopic [M + 2H]2+ ions were alternately subjected to collision-induced dissociation in the second quadrupole. Fragmentation in the ionization source generated identical product ion patterns that were observed during fragmentation in the second quadrupole. In the product ion mass spectra, 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 containing the N-terminus derivatized with a dinitrophenyl group are unequivocally distinguished from the product ions containing the C-terminus. The dinitrophenyl modification generally enhanced the production of a and b ions without diminishing y and z ion yields.
Chen
X
X
Department of Biochemistry, School of Medicine, Case Western Reserve University, Cleveland 44106-4935, USA.
Anderson
V E
VE
Chen
Y H
YH
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D004139:Dinitrofluorobenzene / Q000737:chemistry*; D007202:Indicators and Reagents / Q000737:chemistry; D013058:Mass Spectrometry; D010455:Peptides / Q000737:chemistry*; D014316:Tritium
D007202:Indicators and Reagents; D010455:Peptides; D014316:Tritium; D004139:Dinitrofluorobenzene