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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; Leutert, Mario; Nanni, Paolo; Panse, Christian; Hottiger, Michael O (2017). 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. Analytical Chemistry, 89(3):1523-1530.

Abstract

Protein 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 fragmentation of modified peptides. Here, using an Orbitrap Fusion Tribrid mass spectrometer, we present an optimized methodology that not only drastically improves the overall localization scores for the ADP-ribosylation acceptor sites, but also boosts ADP-ribosylated peptide identifications. First, we systematically compared the efficacy of HCD, ETcaD and EThcD fragmentation methods when determining ADP-ribose acceptor sites within complex cellular samples. We, then, tested the combination of HCD and EThcD fragmentation, which were employed in a product-dependent manner, and the unique fragmentation properties of the ADP-ribose moiety were used to trigger targeted fragmentation of only the modified peptides. The best results were obtained using a workflow that included initial fast, high energy HCD (Orbitrap, FT) scans, which produced intense ADP-ribose fragmentation ions. These potentially ADP-ribosylated precursors were then selected and analyzed via subsequent high-resolution HCD and EThcD fragmentation. Using these resulting high-quality spectra, we identified a xxxxxxKSxxxxx modification motif where lysine can serve as an ADP-ribose acceptor site. Due to the appearance of serine within this motif and its close presence to the lysine further analysis revealed that serine serves as new ADP-ribose acceptor site across the proteome.

Abstract

Protein 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 fragmentation of modified peptides. Here, using an Orbitrap Fusion Tribrid mass spectrometer, we present an optimized methodology that not only drastically improves the overall localization scores for the ADP-ribosylation acceptor sites, but also boosts ADP-ribosylated peptide identifications. First, we systematically compared the efficacy of HCD, ETcaD and EThcD fragmentation methods when determining ADP-ribose acceptor sites within complex cellular samples. We, then, tested the combination of HCD and EThcD fragmentation, which were employed in a product-dependent manner, and the unique fragmentation properties of the ADP-ribose moiety were used to trigger targeted fragmentation of only the modified peptides. The best results were obtained using a workflow that included initial fast, high energy HCD (Orbitrap, FT) scans, which produced intense ADP-ribose fragmentation ions. These potentially ADP-ribosylated precursors were then selected and analyzed via subsequent high-resolution HCD and EThcD fragmentation. Using these resulting high-quality spectra, we identified a xxxxxxKSxxxxx modification motif where lysine can serve as an ADP-ribose acceptor site. Due to the appearance of serine within this motif and its close presence to the lysine further analysis revealed that serine serves as new ADP-ribose acceptor site across the proteome.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > Functional Genomics Center Zurich
05 Vetsuisse Faculty > Department of Molecular Mechanisms of Disease
07 Faculty of Science > Department of Molecular Mechanisms of Disease
Dewey Decimal Classification:570 Life sciences; biology
610 Medicine & health
Language:English
Date:2017
Deposited On:23 Jan 2017 11:24
Last Modified:10 Feb 2017 09:12
Publisher:American Chemical Society (ACS)
ISSN:0003-2700
Publisher DOI:https://doi.org/10.1021/acs.analchem.6b03365
PubMed ID:28035797

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