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

ABSTRACT: Objective: ADP-ribosylation is a post-translational modification that plays an important role in cellular processes. Our previous work implemented multiple gas-phase separation strategies (e.g., FAIMS) and in-source CID on the quadrupole-Orbitrap (Exploris 480) to increase the yield and acceptor site confidence scores of HCD-dependent ADP-ribosyl (ADPr) peptide identifications. We evaluated whether FAIMS coupled on the quadrupole-ion trap-Orbitrap (Fusion Lumos) also improves EThcD-dependent ADP-ribosyl peptide sequencing. Methods: ADP-ribosyl peptides derived from the human macrophage-like cell line THP-1 were analyzed on the Fusion Lumos fronted with a FAIMS Pro device. FAIMS plus/minus gas-phase segmentation (GPS) of the MS1 scan was applied to HCD and EThcD acquisitions. ADP-ribosyl peptide spectra were 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 3.2- and 3.8-fold more respectively for HCD, and 2.0- and 3.6-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, and that the proportion of ADP-ribosylated serine sites increased when THP-1 cells were activated with 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 non-ADP-ribosylated 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.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture, Macrophage

SUBMITTER: Sasha Singh  

LAB HEAD: Masanori Aikawa

PROVIDER: PXD068570 | Pride | 2025-12-26

REPOSITORIES: Pride