Please use this identifier to cite or link to this item:
For citation please use:
Main Title: Optimized fragmentation regime for diazirine photo-cross-linked peptides
Author(s): Giese, Sven H.
Belsom, Adam
Rappsilber, Juri
Type: Article
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).
Subject(s): proteomics
Issue Date: 25-Jul-2016
Date Available: 11-Mar-2021
Is Part Of: 10.14279/depositonce-12031
Language Code: en
DDC Class: 570 Biowissenschaften; Biologie
Journal Title: Analytical Chemistry
Publisher: American Chemical Society (ACS)
Volume: 88
Issue: 16
Publisher DOI: 10.1021/acs.analchem.6b02082
Page Start: 8239
Page End: 8247
EISSN: 1520-6882
ISSN: 0003-2700
TU Affiliation(s): Fak. 3 Prozesswissenschaften » Inst. Biotechnologie » FG Bioanalytik
Appears in Collections:Technische Universit├Ąt Berlin » Publications

Files in This Item:


Format: Adobe PDF | Size: 1.87 MB
DownloadShow Preview

Supporting Information

Format: Adobe PDF | Size: 20.82 MB
DownloadShow Preview

Item Export Bar

This item is licensed under a Creative Commons License Creative Commons