Universität zu Köln

Arlt, Christian, Goetze, Michael, Ihling, Christian H., Hage, Christoph, Schaefer, Mathias ORCID: 0000-0002-5943-4335 and Sinz, Andrea (2016). Integrated Workflow for Structural Proteomics Studies Based on Cross-Linking/Mass Spectrometry with an MS/MS Cleavable Cross-Linker. Anal. Chem., 88 (16). S. 7930 - 7938. WASHINGTON: AMER CHEMICAL SOC. ISSN 1520-6882

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Abstract

Cross-linking combined with mass spectrometry (MS) has evolved as an alternative strategy in structural biology for characterizing three-dimensional structures of protein assemblies and for mapping protein protein interactions. Here, we describe an integrated workflow for an automated identification of cross-linked products that is based on the use of a tandem mass spectrometry (MS/MS) cleavable cross-linker (containing a 1,3-bis-(4oxo-butyl)-urea group, BuUrBu) generating characteristic doublet patterns upon fragmentation. We evaluate different fragmentation methods available on an Orbitrap Fusion mass spectrometer for three proteins and an E. coli cell lysate. An updated version of the dedicated software tool MeroX was employed for a fully automated identification of cross-links. The strength of our cleavable cross-linker is that characteristic patterns of the cross-linker as well as backbone fragments of the connected peptides are already observed at the MS/MS level, 'eliminating the need for conducting MS3 or sequential CID (collision-induced dissociation) and ETD (electron transfer dissociation)-MS/MS experiments. This makes our strategy applicable to a broad range of mass spectrometers with MS/MS capabilities. For purified proteins and protein complexes, our workflow using CID-MS/MS acquisition performs with high confidence, scoring cross-links at 0.5% false discovery rate (FDR). The cross-links provide structural insights into the intrinsically disordered tetrameric tumor suppressor protein p53. As a timeconsuming manual inspection of cross-linking data is not required, our workflow will pave the way for making the cross-linking/ MS approach a routine technique for structural proteoinics studies.

Item Type:	Journal Article
Creators:	Creators Email ORCID ORCID Put Code Arlt, Christian UNSPECIFIED UNSPECIFIED UNSPECIFIED Goetze, Michael UNSPECIFIED UNSPECIFIED UNSPECIFIED Ihling, Christian H. UNSPECIFIED UNSPECIFIED UNSPECIFIED Hage, Christoph UNSPECIFIED UNSPECIFIED UNSPECIFIED Schaefer, Mathias UNSPECIFIED orcid.org/0000-0002-5943-4335 UNSPECIFIED Sinz, Andrea UNSPECIFIED UNSPECIFIED UNSPECIFIED
URN:	urn:nbn:de:hbz:38-266491
DOI:	10.1021/acs.analchem.5b04853
Journal or Publication Title:	Anal. Chem.
Volume:	88
Number:	16
Page Range:	S. 7930 - 7938
Date:	2016
Publisher:	AMER CHEMICAL SOC
Place of Publication:	WASHINGTON
ISSN:	1520-6882
Language:	English
Faculty:	Faculty of Mathematics and Natural Sciences
Divisions:	Faculty of Mathematics and Natural Sciences > Department of Chemistry > Institute of Organic Chemistry
Subjects:	no entry
Uncontrolled Keywords:	Keywords Language MASS-SPECTROMETRY; GAS-PHASE; P53; COMPLEX; IDENTIFICATION; CHAPERONIN; TRIC/CCT Multiple languages Chemistry, Analytical Multiple languages
Refereed:	Yes
URI:	http://kups.ub.uni-koeln.de/id/eprint/26649