An integrated chemical crosslinking and mass spectrometry approach to study protein complex architecture and function.

Publication Type:

Journal Article

Source:

Molecular & cellular proteomics : MCP, Volume 119, Issue 2, p.M111.008318 (2012)

Keywords:

2012, Basic Sciences Division, Center-Authored Paper, November 2011

Abstract:

Knowledge of protein structures and protein-protein interactions is essential for understanding biological processes. Chemical crosslinking combined with mass spectrometry is an attractive approach for studying protein-protein interactions and protein structure, but to date its use has been limited largely by low yields of informative crosslinks (due to inefficient crosslinking reactions) and by the difficulty of confidently identifying the sequences of crosslinked peptide pairs from their fragmentation spectra. Here we present an approach based on a new MS labile crosslinking reagent, Biotin-Aspartate(D)-Rink-Glycine (BRDG), which addresses these issues. BRDG incorporates a biotin handle (for enrichment of crosslinked peptides prior to MS analysis), two pentafluorophenyl (PFP) ester groups that react with peptide amines, and a labile Rink-based bond between the PFP groups that allows crosslinked peptides to be separated during MS and confidently identified by database searching of their fragmentation spectra. We developed a protocol for the identification of BDRG crosslinked peptides derived from purified or partially purified protein complexes, including software to aid in the identification of different classes of crosslinker-modified peptides. Importantly, our approach permits the use of high-accuracy precursor mass measurements to verify the database search results. We demonstrate the utility of the approach by applying it to purified yeast TFIIE, a heterodimeric transcription factor complex, and to a single step affinity purified preparation of the 12-subunit RNA polymerase II complex (Pol II). The results show that the method is effective at identifying crosslinked peptides derived from purified and partially purified protein complexes and provides complementary information to that from other structural approaches. As such, it is an attractive approach to study the topology of protein complexes.