FAIMS enhances the detection of PTM crosstalk sites

Kish R Adoni, Debbie L Cunningham, John K Heath, Aneika C Leney

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Protein post-translational modifications (PTMs) enable cells to rapidly change in response to biological stimuli. With hundreds of different PTMs, understanding these control mechanisms is complex. To date, efforts have focused on investigating the effect of a single PTM on protein function. Yet, many proteins contain multiple PTMs. Moreover, one PTM can alter the prevalence of another, a phenomenon termed PTM crosstalk. Understanding PTM crosstalk is critical; however, its detection is challenging since PTMs occur substoichiometrically. Here, we develop an enrichment-free, label-free proteomics method that utilizes high-field asymmetric ion mobility spectrometry (FAIMS) to enhance the detection of PTM crosstalk. We show that by searching for multiple combinations of dynamic PTMs on peptide sequences, a 6-fold increase in candidate PTM crosstalk sites is identified compared with that of standard liquid chromatography-tandem mass spectrometry (LC-MS/MS) workflows. Additionally, by cycling through FAIMS compensation voltages within a single LC-FAIMS-MS/MS run, we show that our LC-FAIMS-MS/MS workflow can increase multi-PTM-containing peptide identifications without additional increases in run times. With 159 novel candidate crosstalk sites identified, we envisage LC-FAIMS-MS/MS to play an important role in expanding the repertoire of multi-PTM identifications. Moreover, it is only by detecting PTM crosstalk that we can "see" the full picture of how proteins are regulated.

Original languageEnglish
Pages (from-to)930-939
Number of pages10
JournalJournal of Proteome Research
Issue number4
Early online date2 Mar 2022
Publication statusPublished - 1 Apr 2022

Bibliographical note

Funding Information:
The authors thank Adam Turner for creating FASTA files associated with multicombination PTM searches and Jeddidiah Bellamy-Carter for assistance with data analysis software. The authors thank the Cooper and Leney mass spectrometry groups at the University of Birmingham for helpful discussions. The authors acknowledge the Advanced Mass Spectrometry Facility (AMSF) at the University of Birmingham for the setup and maintenance of instruments. This work was supported by the Biotechnology and Biological Sciences Research Council (BBSRC) and University of Birmingham funded Midlands Integrative Biosciences Training Partnership (MIBTP2) (BB/M01116X/1). Technical support was funded by Wellcome Trust ISSF fund. The Eclipse mass spectrometer used in this research was funded by the BBSRC (BB/S019456/1).


  • post-translational modifications
  • crosstalk
  • proteomics
  • mass spectrometry


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