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Cellular ADP-ribosylome characterization with HCD and EThcD

ABSTRACT: Protein ADP-ribosylation is a post-translational modification involved in important physiological and pathological processes. Here, we present an optimized method using the Orbitrap Fusion Tribrid mass spectrometer, which drastically improves the overall ADP-ribosylation site localization score and boosts the identification of ADP-ribosylated peptides. We compared the HCD, ETcaD or EThcD fragmentation methods for the identification of ADP-ribose acceptor sites in a complex cellular sample and further combined them in a product dependent manner, exploiting the unique fragmentation properties of the ADP-ribose moiety as a trigger for a targeted fragmentation of modified peptides.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Prof. Michael O.Hottiger

PROVIDER: MSV000080008 | MassIVE | Thu Jul 28 06:38:00 BST 2016

SECONDARY ACCESSION(S): PXD004676

REPOSITORIES: MassIVE

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Publications

Combining Higher-Energy Collision Dissociation and Electron-Transfer/Higher-Energy Collision Dissociation Fragmentation in a Product-Dependent Manner Confidently Assigns Proteomewide ADP-Ribose Acceptor Sites.

Bilan Vera V Leutert Mario M Nanni Paolo P Panse Christian C Hottiger Michael O MO

Analytical chemistry 20170113 3

Protein adenosine diphosphate (ADP)-ribosylation is a physiologically and pathologically important post-translational modification. Recent technological advances have improved analysis of this complex modification and have led to the discovery of hundreds of ADP-ribosylated proteins in both cultured cells and mouse tissues. Nevertheless, accurate assignment of the ADP-ribose acceptor site(s) within the modified proteins identified has remained a challenging task. This is mainly due to poor fragm ...[more]

PMID: 28035797

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

Project description: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.

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Genome-wide analysis of ADP-ribosylation by ADPr-ChAP reveals its association with chromatin after oxidative stress

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