BRIEF RESEARCH REPORT article
Front. Chem. Biol.
Sec. Quantitative and Analytical Techniques
This article is part of the Research TopicProteomics in AtherosclerosisView all 9 articles
An evaluation of high-field asymmetric-waveform ion mobility spectrometry coupled to electron-transfer/higher-energy collision dissociation for ADP-ribosylation proteomics
Provisionally accepted
- Brigham and Women's Hospital, Harvard Medical School, Boston, United States
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Objective: ADP-ribosylation plays an important role in many cellular processes. Our previous work implemented FAIMS and in-source CID on the quadrupole-Orbitrap to increase ADP-ribosyl peptide yield and acceptor site confidence of HCD-dependent ADP-ribosyl peptide identifications. In this study, we evaluated whether FAIMS on the quadrupole-ion trap-Orbitrap also improves EThcD-dependent ADP-ribosyl peptide sequencing. Methods: ADP-ribosyl peptides derived from THP-1 cells were analyzed on the Fusion Lumos fronted with a FAIMS Pro device. ADP-ribosyl peptides were sequenced using either HCD or EThcD, and annotated using the SEQUEST-HT algorithm and RiboMaP, an annotation tool specific for ADP-ribosylated peptide spectra. Results: HCD-dependent ADP-ribosyl peptide identifications were enriched at higher compensation voltages as compared to EThcD. The net number of unique ADP-ribosyl and non-ADP-ribosyl (contaminant) peptides across compensation voltages increased by 4.1-and 4.0-fold more respectively for HCD, and 2.0-and 4.1-fold respectively for EThcD, compared to no FAIMS. We also confirmed that while multiple injections of peptides employing distinct compensation voltages maximized the number of EThcD-dependent ADP-ribosyl peptide identifications, their associated XCorr and p-series scores decreased. The most frequent ADP-ribosyl acceptor site was lysine, followed by serine. The proportion of ADP-ribosylated serine sites increased when THP-1 cells were activated with interferon γ (IFN-γ). Conclusions: Although FAIMS increases EThcD-dependent sequencing depth of ADP-ribosyl peptides, the gains are less than when using HCD. The ability to filter out doubly charged contaminant peptides at increasingly higher negative compensation voltages benefits HCD, but not EThcD since this dissociation method works optimally with highly charged peptides, non-ADP-ribosyl and ADP-ribosyl alike.
Keywords: EThcD, FAIMS, posttranslational modification, tribrid mass spectrometer, macrophage
Received: 24 Sep 2025; Accepted: 27 Oct 2025.
Copyright: © 2025 Kasai, Shlayen, Nakamura, Aikawa and Singh. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence: Sasha A Singh, sasingh@bwh.harvard.edu
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