Universität zu Köln

Iacobucci, Claudio ORCID: 0000-0001-9592-3606, Goetze, Michael, Ihling, Christian H., Piotrowski, Christine, Arlt, Christian, Schaefer, Mathias ORCID: 0000-0002-5943-4335, Hage, Christoph, Schmidt, Rico and Sinz, Andrea ORCID: 0000-0003-1521-4899 (2018). A cross-linking/mass spectrometry workflow based on MS-cleavable cross-linkers and the MeroX software for studying protein structures and protein-protein interactions. Nat. Protoc., 13 (12). S. 2864 - 2890. LONDON: NATURE PUBLISHING GROUP. ISSN 1750-2799

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Abstract

Chemical cross-linking in combination with mass spectrometric analysis of the created cross-linked products is an emerging technology aimed at deriving valuable structural information from proteins and protein complexes. The goal of our protocol is to obtain distance constraints for structure determination of proteins and to investigate protein-protein interactions. We present an integrated workflow for cross-linking/mass spectrometry (MS) based on protein cross-linking with MS-cleavable reagents, followed by enzymatic digestion, enrichment of cross-linked peptides by strong cation-exchange chromatography (SCX), and LC/MS/MS analysis. To exploit the full potential of MS-cleavable cross-linkers, we developed an updated version of the freely available MeroX software for automated data analysis. The commercially available, MS-cleavable cross-linkers (DSBU and CDI) used herein possess different lengths and react with amine as well as hydroxy groups. Owing to the formation of two characteristic 26-u doublets in their MS/MS spectra, many fewer false positives are found than when using classic, non-cleavable cross-linkers. The protocol, exemplified herein for BSA and the whole Escherichia coli ribosome, is robust and widely applicable, and it allows facile identification of cross-links for deriving spatial constraints from purified proteins and protein complexes. The cross-linking/MS procedure takes 2-3 days to complete.

Item Type:	Journal Article
Creators:	Creators Email ORCID ORCID Put Code Iacobucci, Claudio UNSPECIFIED orcid.org/0000-0001-9592-3606 UNSPECIFIED Goetze, Michael UNSPECIFIED UNSPECIFIED UNSPECIFIED Ihling, Christian H. UNSPECIFIED UNSPECIFIED UNSPECIFIED Piotrowski, Christine UNSPECIFIED UNSPECIFIED UNSPECIFIED Arlt, Christian UNSPECIFIED UNSPECIFIED UNSPECIFIED Schaefer, Mathias UNSPECIFIED orcid.org/0000-0002-5943-4335 UNSPECIFIED Hage, Christoph UNSPECIFIED UNSPECIFIED UNSPECIFIED Schmidt, Rico UNSPECIFIED UNSPECIFIED UNSPECIFIED Sinz, Andrea UNSPECIFIED orcid.org/0000-0003-1521-4899 UNSPECIFIED
URN:	urn:nbn:de:hbz:38-164588
DOI:	10.1038/s41596-018-0068-8
Journal or Publication Title:	Nat. Protoc.
Volume:	13
Number:	12
Page Range:	S. 2864 - 2890
Date:	2018
Publisher:	NATURE PUBLISHING GROUP
Place of Publication:	LONDON
ISSN:	1750-2799
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 ASSISTED-LASER-DESORPTION/IONIZATION; MASS-SPECTROMETRY; AUTOMATED ASSIGNMENT; RNA-POLYMERASE; IDENTIFICATION; PRODUCTS; P53; TOPOLOGIES; PROTEOMICS; REVEALS Multiple languages Biochemical Research Methods Multiple languages
Refereed:	Yes
URI:	http://kups.ub.uni-koeln.de/id/eprint/16458