In-source CID improves HCD-dependent fragmentation of ADP-ribosyl peptides

ABSTRACT: ADP-ribosylation is a posttranslational modification whose HCD products are dominated by complete or partial modification losses, complicating peptide sequencing and acceptor site localization efforts. We tested whether in-source CID performed on a quadrupole Orbitrap could convert ADPr to the smaller phosphoribose-H2O derivative to facilitate HCD-dependent peptide sequencing. Human macrophage like cell line THP-1-derived ADP-ribosyl (ADPr) peptides were analyzed on a quadrupole Orbitrap. We monitored the interconversion 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. In-source CID-HCD-derived phosphoribosyl-H2O acceptor sites were compared to those determined by EThcD, performed on a quadrupole ion trap Orbitrap. Interconversion of ADPr peptides to their phosphoribosyl-H2O derivatives increased with increasing source voltage (up to 50V), 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%). 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.

INSTRUMENT(S): Orbitrap Fusion Lumos, Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture, Macrophage

SUBMITTER: Sasha Singh  

LAB HEAD: Sasha A.

PROVIDER: PXD055586 | Pride | 2024-12-05

REPOSITORIES: Pride