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Anal Chem. 2019 Apr 2;91(7):4472-4478. doi: 10.1021/acs.analchem.8b05222. Epub 2019 Mar 13.

Sequential Digestion with Trypsin and Elastase in Cross-Linking Mass Spectrometry.

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Wellcome Centre for Cell Biology, Institute of Cell Biology, School of Biological Sciences , University of Edinburgh , Edinburgh EH9 3BF , United Kingdom.
BrisSynBio Centre, The School of Biochemistry, Faculty of Biomedical Sciences , University of Bristol , Bristol BS8 1TD , United Kingdom.
Bioanalytics, Institute of Biotechnology , Technische Universität Berlin , 13355 Berlin , Germany.


Cross-linking mass spectrometry has become an important approach for studying protein structures and protein-protein interactions. The amino acid compositions of some protein regions impede the detection of cross-linked residues, although it would yield invaluable information for protein modeling. Here, we report on a sequential-digestion strategy with trypsin and elastase to penetrate regions with a low density of trypsin-cleavage sites. We exploited intrinsic substrate-recognition properties of elastase to specifically target larger tryptic peptides. Our application of this protocol to the TAF4-12 complex allowed us to identify cross-links in previously inaccessible regions.

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