Effect of N-terminal glutamic acid and glutamine on fragmentation of peptide ions |
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Authors: | Bhaskar Godugu Pedatsur Neta Yamil Simón-Manso Stephen E Stein |
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Institution: | (1) Chemical and Biochemical Reference Data Division, National Institute of Standards and Technology, Gaithersburg, MD 20899, USA; |
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Abstract: | A prominent dissociation path for electrospray generated tryptic peptide ions is the dissociation of the peptide bond linking
the second and third residues from the ammo-terminus. The formation of the resulting b2 and y
n−2 fragments has been rationalized by specific facile mechanisms. An examination of spectral libraries shows that this path
predominates in diprotonated peptides composed of 12 or fewer residues, with the notable exception of peptides containing
glutamine or glutamic acid at the N-terminus. To elucidate the mechanism by which these amino acids affect peptide fragmentation,
we synthesized peptides of varying size and composition and examined their MS/MS spectra as a function of collision voltage
in a triple quadrupole mass spectrometer. Loss of water from N-terminal glutamic acid and glutamine is observed at a lower
voltage than any other fragmentation, leading to cyclization of the terminal residue. This cyclization results in the conversion
of the terminal amine group to an imide, which has a lower proton affinity. As a result, the second proton is not localized
at the N-terminus but is readily transferred to other sites, leading to fragmentation near the center of the peptide. Further
confirmation was obtained by examining peptides with N-terminal pyroglutamic acid and N-acetyl peptides. Peptides with N-terminal
proline maintain the trend of forming b2 and y
n−2 because their ring contains an imine rather than imide and has sufficient proton affinity to retain the proton at the N-terminus. |
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