Abstract
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 (MS^3^) experiments. Additional mechanistic information was obtained by exchanging the labile hydrogens for deuterium. Protonated H—Gln—Gly—OH fragments by loss of NH~3~ and loss of H~2~O in metastable ion fragmentation; under collision‐induced dissociation (CID) conditions loss of H—Gly—OH + CO from the [MH − NH~3~]^+^ ion forms the base peak C~4~H~6~NO^+^ (m/z 84). Protonated dipeptides with an α‐linkage, H—Glu—Xxx—OH, are characterized by elimination of H~2~O 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 γ‐linkage, H—Glu(Xxx—OH)—OH, do not show elimination of H~2~O or formation of m/z 102 but rather show elimination of NH~3~, particularly in metastable ion fragmentation, and elimination of H—Xxx—OH to form m/z 130. Both the α‐ and γ‐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 H~2~O and formation of m/z 102 are observed for the protonated α‐tripeptide H—Glu—Gly—Phe—OH whereas the protonated γ‐tripeptide H—Glu(Gly—Gly—OH)—OH shows loss of NH~3~ and formation of m/z 130 as observed for dipeptides with the γ‐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 H~2~O whereas those of structure H—Xxx—Gln—OH show abundant elimination of NH~3~. 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~1~ ion or the y~1~″ ion. Particularly when Xxx = Gly, under CID conditions the initial loss of NH~3~ from the glutamine containing dipeptide is followed by elimination of a second NH~3~ while the initial loss of H~2~O from the glutamic acid dipeptide is followed by elimination of NH~3~. 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 NH~3~ 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 © 2003 John Wiley & Sons, Ltd.