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

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.