An evaluation of high-field asymmetric-waveform ion mobility spectrometry coupled to electron-transfer/higher-energy collision dissociation for ADP-ribosylation proteomics

Objective ADP-ribosylation plays an important role in many cellular processes. Our previous work implemented field asymmetric-waveform ion mobility spectrometry (FAIMS) and in-source collision-induced dissociation (CID) on the quadrupole-Orbitrap to increase ADP-ribosyl peptide yield and acceptor site confidence of higher-energy collisional dissociation (HCD)-dependent ADP-ribosyl peptide identifications. In this study, we evaluated whether FAIMS on the quadrupole-ion trap-Orbitrap also improves electron-transfer/higher-energy collision dissociation (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 than those that used 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, 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). Conclusion Although FAIMS increases the 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 because this dissociation method works optimally with highly charged peptides, non-ADP-ribosyl and ADP-ribosyl alike.