Conclusions
In-source CID converts ADP-ribosyl to phosphoribosyl-H2O peptides that are more amenable to HCD-dependent peptide sequencing, providing an alternative method for acceptor site determination when ETD-based methods are not available.
Methods
ADP-ribosyl (ADPr) peptides derived from the human macrophage-like cell line THP-1 were analyzed on a quadrupole-Orbitrap. We monitored the dissociation of ADPr (+ 541.061 Da) to phosphoribosyl-H2O (+ 193.997 Da) peptides while varying the source and high-field asymmetric waveform ion mobility mass spectrometry (FAIMS) compensation voltages. Xcorr and ptmRS were used to evaluate peptide sequencing and acceptor site confidence, respectively. Phosphoribosyl-H2O acceptor sites were compared with those determined by electron-transfer higher-energy collision dissociation (EThcD), performed on a quadrupole-ion trap-Orbitrap.
Results
In-source CID of ADPr peptides to their phosphoribosyl-H2O derivatives increased with increasing source voltage (up to 50 V), as judged by monitoring the corresponding modification loss ([adenosine monophosphate/AMP]+) and the number of identified phosphoribosyl-H2O peptide identifications. The average Xcorr increased from 1.36 (ADPr) to 2.26 (phosphoribosyl-H2O), similar to that achieved with EThcD for ADPr peptides (2.29). The number of high-confidence acceptor sites (> 95%) also increased, from 31% (ADPr) to 70% (phosphoribosyl-H2O), which was comparable to EThcD (70%). Conclusions: In-source CID converts ADP-ribosyl to phosphoribosyl-H2O peptides that are more amenable to HCD-dependent peptide sequencing, providing an alternative method for acceptor site determination when ETD-based methods are not available.