Fragmentation reactions of protonated peptides containing glutamine or glutamic acid

A variety of protonated dipeptides and tripeptides containing glutamic acid or glutamine were prepared by electrospray ionization or by fast atom bombardment ionization and their fragmentation pathways elucidated using metastable ion studies, energy-resolved mass spectrometry and triple-stage mass spectrometry MS3) experiments. Additional mechanistic information was obtained by exchanging the labile hydrogens for deuterium. Protonated H-Gln-Gly-OH fragments by loss of NH3 and loss of H2O in metastable ion fragmentation; under collision-induced dissociation (CID) conditions loss of H - Gly - OH + CO from the [MH - NH3](+) ion forms the base peak C4H6NO+ (m/z 84). Protonated dipeptides with an alpha-linkage, H-Glu-Xxx-OH, are characterized by elimination of H2O and by elimination of H-Xxx-OH plus CO to form the glutamic acid immonium ion of m/z 102. By contrast, protonated dipeptides with a gamma-linkage, H-Glu(Xxx-OH)-OH, do not show elimination of H2O or formation of m/z 102 but rather show elimination of NH3, particularly in metastable ion fragmentation, and elimination of H-Xxx-OH to form m/z 130. Both the alpha- and gamma-dipeptides show formation of [H-Xxx-OH]H+, with this reaction channel increasing in importance as the proton affinity (PA) of H-Xxx-OH increases. The characteristic loss of H2O and formation of m/z 102 are observed for the protonated alpha-tripeptide H - Glu - Gly - Phe - OH whereas the protonated gamma-tripeptide H - Glu(Gly - Gly - OH) - OH shows loss of NH3 and formation of m/z 130 as observed for dipeptides with the gamma-linkage. Both tripeptides show abundant formation of the y(2)" ion under CID conditions, presumably because a stable anhydride neutral structure can be formed. Under metastable ion conditions protonated dipeptides of structure H-Xxx-Glu-OH show abundant elimination of H2O whereas those of structure H-Xxx-Gln-OH show abundant elimination of NH3. The importance of these reaction channels is much reduced under CID conditions, the major fragmentation mode being cleavage of the amide bond to form either the a, ion or the y(1)" ion. Particularly when Xxx = Gly, under CID conditions the initial loss of NH3 from the glutamine containing dipeptide is followed by elimination of a second NH3 while the initial loss of H2O from the glutamic acid dipeptide is followed by elimination of NH3. Isotopic labelling shows that predominantly labile hydrogens are lost in both steps. Although both [H-Gly-Glu-Gly-OH]H+ and [H-Gly-Gln-Gly-OH]H+ fragment mainly to form b(2) and a(2) ions, the latter also shows elimination of NH3 plus a glycine residue and formation of protonated glycinamide. Isotopic labelling shows extensive mixing of labile and carbon-bonded hydrogens in the formation of protonated glycinamide. Copyright (C) 2003 John Wiley Sons, Ltd.