Gas-Phase Ion/Ion Chemistry as a Probe for the Presence of Carboxylate Groups in Polypeptide Cations

  • Anthony M. Pitts-McCoy
  • Christopher P. Harrilal
  • Scott A. McLuckeyEmail author
Research Article


The reactivity of 1-hydroxybenzoyl triazole (HOBt) esters with the carboxylate functionality present in peptides is demonstrated in the gas phase with a doubly deprotonated dianion. The reaction forms an anhydride linkage at the carboxylate site. Upon ion trap collisional-induced dissociation (CID) of the modified peptide, the resulting spectrum shows a nominal loss of the mass of the reagent and a water molecule. Analogous phenomenology was also noted for model peptide cations that likely contain zwitterionic/salt-bridged motifs in reactions with a negatively charged HOBt ester. Control experiments indicate that a carboxylate group is the likely reactive site, rather than other possible nucleophilic sites present in the peptide. These observations suggest that HOBt ester chemistry may be used as a chemical probe for the presence and location of carboxylate groups in net positively charged polypeptide ions. As an illustration, deprotonated sulfobenzoyl HOBt was reacted with the [M+7H]7+ ion of ubiquitin. The ion was shown to react with the reagent and CID of the covalent reaction product yielded an abundant [M+6H-H2O]6+ ion. Comparison of the CID product ion spectrum of this ion with that of the water loss product generated from CID of the unmodified [M+6H]6+ ion revealed the glutamic acid at residue 64 as a reactive site, suggesting that it is present in the deprotonated form.

Graphical Abstract


Ion/ion reactions Zwitterion Protonated peptide Carboxylates Triazole ester 



This work was supported by the National Institutes of Health (NIH) under Grant GM R37-45372.

Supplementary material

13361_2018_2079_MOESM1_ESM.docx (796 kb)
ESM 1 (DOCX 785 kb)


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Copyright information

© American Society for Mass Spectrometry 2018

Authors and Affiliations

  1. 1.Department of ChemistryPurdue UniversityWest LafayetteUSA

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