A Combined Gas-Phase Separation Strategy for ADP-ribosylated Peptides

[Image: see text] ADP-ribosylation (ADPr) is a post-translational modification that is best studied using mass spectrometry. Method developments that are permissive with low inputs or baseline levels of protein ribosylation represent the next frontier in the field. High-field asymmetric waveform ion mobility spectrometry (FAIMS) reduces peptide complexity in the gas phase, providing a means to achieve maximal ADPr peptide sequencing depth. We therefore investigated the extent to which FAIMS with or without traditional gas-phase fractionation–separation (GPS) can increase the number of ADPr peptides. We examined ADPr peptides enriched from mouse spleens. We gleaned additional insight by also reporting findings from the corresponding non-ADPr peptide contaminants and the peptide inputs for ADPr peptide enrichment. At increasingly higher negative compensation voltages, ADPr peptides were more stable, whereas the non-ADPr peptides were filtered out. A combination of 3 GPS survey scans, each with 8 compensation voltages, resulted in 790 high-confidence ADPr peptides, compared to 90 with GPS alone. A simplified acquisition strategy requiring only two injections corresponding to two MS1 scan ranges coupled to optimized compensation voltage settings provided 402 ADPr peptides corresponding to 234 ADPr proteins. We conclude that our combined GPS strategy is a valuable addition to any ADP-ribosylome workflow. The data are available via ProteomeXchange with identifier PXD040898.