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

Author:
Dau, Therese, Gupta, Kapil, Berger, Imre, Rappsilber, Juri
Source:
Analytical chemistry 2019 v.91 no.7 pp. 4472-4478
ISSN:
1520-6882
Subject:
crosslinking, digestion, elastase, mass spectrometry, models, peptides, protein structure, protein-protein interactions, protocols, trypsin
Abstract:
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.
Agid:
6355693