title: Optimized Fragmentation Regime for Diazirine Photo-Cross-Linked Peptides.
abstract: Cross-linking/mass spectrometry has evolved into a robust technology that reveals structural insights into proteins and protein complexes. We leverage a new tribrid instrument with improved fragmentation capacities in a systematic comparison to identify which fragmentation method would be best for the identification of cross-linked peptides. Specifically, we explored three fragmentation methods and two combinations: collision-induced dissociation (CID), beam-type CID (HCD), electron-transfer dissociation (ETD), ETciD, and EThcD. Trypsin-digested, SDA-cross-linked human serum albumin (HSA) served as a test sample, yielding over all methods and in triplicate analysis in total 2602 matched PSMs and 1390 linked residue pairs at 5% false discovery rate, as confirmed by the crystal structure. HCD wins in number of matched peptide-spectrum-matches (958 PSMs) and identified links (446). CID is most complementary, increasing the number of identified links by 13% (58 links). HCD wins together with EThcD in cross-link site calling precision, with approximately 62% of sites having adjacent backbone cleavages that unambiguously locate the link in both peptides, without assuming any cross-linker preference for amino acids. Overall quality of spectra, as judged by sequence coverage of both peptides, is best for EThcD for the majority of peptides. Sequence coverage might be of particular importance for complex samples, for which we propose a data dependent decision tree, else HCD is the method of choice. The mass spectrometric raw data has been deposited in PRIDE (PXD003737).
authors.affiliation: Chair of Bioanalytics, Institute of Biotechnology, Technische Universität Berlin , 13355 Berlin, Germany.|Wellcome Trust Centre for Cell Biology, University of Edinburgh , Edinburgh EH9 3BF, United Kingdom.
authors.forename: Sven H
authors.initials: SH
authors.lastname: Giese
authors.affiliation: Wellcome Trust Centre for Cell Biology, University of Edinburgh , Edinburgh EH9 3BF, United Kingdom.
authors.forename: Adam
authors.initials: A
authors.lastname: Belsom
authors.affiliation: Chair of Bioanalytics, Institute of Biotechnology, Technische Universität Berlin , 13355 Berlin, Germany.|Wellcome Trust Centre for Cell Biology, University of Edinburgh , Edinburgh EH9 3BF, United Kingdom.
authors.forename: Juri
authors.initials: J
authors.lastname: Rappsilber
mesh_terms: D003432:Cross-Linking Reagents / Q000737:chemistry*; D003978:Diazomethane / Q000737:chemistry*; D006801:Humans; D010455:Peptides / Q000737:chemistry*; D012709:Serum Albumin / Q000737:chemistry / Q000378:metabolism; D053719:Tandem Mass Spectrometry; D014466:Ultraviolet Rays
chemical_list: D003432:Cross-Linking Reagents; D010455:Peptides; D012709:Serum Albumin; D003978:Diazomethane