ADP-ribosylation is a technically challenging PTM which has just emerged into the field of PTM-specific proteomics. But this fragile modifier requires special treatment on both a data acquisition and data processing level: it is highly labile under higher-energy collisional dissociation (HCD), and the degree of lability can depend on the site it modifies. Its behavior thus violates some assumptions on which proteomics algorithms are based. Here we present nonlocalized ADPr searching: a simple principle for maximizing sensitivity toward ADP-ribosylation when searching conventional HCD data. By scoring the strong fragment ions generally observed in ADPr spectra rather than the weak and often absent localization-dependent ions, nonlocalized searches are more sensitive. They also run significantly faster, due to reduced search space, and require no assumptions about which amino acids can be modified. We illustrate implementation in three search systems: Morpheus, MaxQuant, and MASCOT, and we also present a means of rapidly finding and extracting ADP-ribosylated peptide spectra from large datasets for more focused searching. This approach both improves identification of ADP-ribosylated peptides and avoids mis-localization of the modification sites.
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This work was funded by the Deutsche Forschungsgemeinschaft (Cellular Stress Responses in Aging-Associated Diseases) (grant EXC 229 to I.M.) and the European Union’s Horizon 2020 research and innovation program (Marie Skłodowska-Curie grant agreement 657501 to J.J.B. and I.M.). Very special thanks to Craig Wenger for adding neutral loss searching features to the Morpheus system. Thanks as well to Dr. Ilian Atanassov for useful discussions.
Villén J, Beausoleil SA, Gygi SP (2009) Evaluation of the utility of neutral-loss-dependent MS3 strategies in large-scale phosphorylation analysis. Proteomics 8(21):4444–4452CrossRefGoogle Scholar
Neuhauser N, Michalski A, Cox J, Mann M (2012) Expert system for computer-assisted annotation of MS/MS spectra. Mol Cell Proteomics 11(11):1500–1509CrossRefGoogle Scholar
Myers SA, Daou S, Affar EB, Burlingame AL (2013) Electron transfer dissociation (ETD): the mass spectrometric breakthrough essential for O-GlcNAc protein site assignments – a study of the O-GlcNAcylated protein host cell factor C1. Proteomics 13(6):982–991CrossRefGoogle Scholar
Cox J, Mann M (2008) MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat Biotechnol 26:1367–1372CrossRefGoogle Scholar
Cox J, Neuhauser N, Michalski A, Scheltema RA, Olsen JV, Mann M (2011) Andromeda: a peptide search engine integrated into the MaxQuant environment. J Proteome Res 10:1794–1805CrossRefGoogle Scholar
Wenger CD, Coon JJ (2013) A proteomics search algorithm specifically designed for high-resolution tandem mass spectra. J Proteome Res 12(3):1377–1386CrossRefGoogle Scholar