Biology subjects

Dau, T.

Publications and source records attributed to Dau, T..

2 recordsLinked to original sources

bioRxiv2018

Sequential digestion with Trypsin and Elastase in cross-linking/mass spectrometry

Cross-linking/mass spectrometry has become an important approach for studying protein structures and protein-protein interactions. The amino acid composition of some protein regions impedes the detection of cross-linked residues, although it would yield invaluable information for protein modelling. 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.

biochemistry↗

bioRxiv2018

An integrated workflow for cross-linking/mass spectrometry

We present a concise workflow to enhance the mass spectrometric detection of cross-linked peptides by introducing sequential digestion and the cross-link identification software Xi. Sequential digestion enhances peptide detection by shortening long tryptic peptides while avoiding over-digestion. We demonstrate our simple 12-fraction protocol for cross-linked multi-protein complexes and cell lysates, quantitative analysis, and high-density cross-linking, without requiring specific cross-linker features. This overall approach reveals dynamic protein-protein interaction sites, which are accessible, have fundamental functional relevance and are therefore ideally suited for the development of small molecule inhibitors.

biochemistry↗