Nonenzymatic Posttranslational Modifications and Peptide Cleavages Observed in Peptide Epimers

Connor C. Long, Aleksandra Antevska, David H. Mast, Samuel Okyem, Jonathan V. Sweedler, Thanh D. Do

Research output: Contribution to journalArticlepeer-review


Posttranslational modifications (PTMs) play vital roles in cellular homeostasis and are implicated in various pathological conditions. This work uses two ion mobility spectrometry-mass spectrometry (IMS-MS) modalities, drift-tube IMS (DT-IMS) and trapped IMS (TIMS), to characterize three important nonenzymatic PTMs that induce no mass loss: l/d isomerization, aspartate/isoaspartate isomerization, and cis/trans proline isomerization. These PTMs are assessed in a single peptide system, the recently discovered pleurin peptides, Plrn2, from Aplysia californica. We determine that the DT-IMS-MS/MS can capture and locate asparagine deamidation into aspartate and its subsequent isomerization to isoaspartate, a key biomarker for age-related diseases. Additionally, nonenzymatic peptide cleavage via in-source fragmentation is evaluated for differences in the intensities and patterns of fragment peaks between these PTMs. Peptide fragments resulting from in-source fragmentation, preceded by peptide denaturation by liquid chromatography (LC) mobile phase, exhibited cis/trans proline isomerization. Finally, the effects of differing the fragmentation voltage at the source and solution-based denaturation conditions on in-source fragmentation profiles are evaluated, confirming that LC denaturation and in-source fragmentation profoundly impact N-terminal peptide bond cleavages of Plrn2 and the structures of their fragment ions. With that, LC-IMS-MS/MS coupled with in-source fragmentation could be a robust method to identify three important posttranslational modifications: l/d isomerization, Asn-deamidation leading to Asp/IsoAsp isomerization, and cis/trans proline isomerization.

Original languageEnglish (US)
Pages (from-to)1898-1907
Number of pages10
JournalJournal of the American Society for Mass Spectrometry
Issue number9
StatePublished - Sep 6 2023


  • IsoAsp formation
  • asparagine deamidation
  • cis/trans proline isomerization
  • mass spectrometry
  • posttranslational modifications

ASJC Scopus subject areas

  • Structural Biology
  • Spectroscopy


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