Abstract
Knowledge of protein structures and protein-protein interactions is essential for understanding biological processes. Chemical cross-linking 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 cross-links (because of inefficient cross-linking reactions) and by the difficulty of confidently identifying the sequences of cross-linked peptide pairs from their fragmentation spectra. Here we present an approach based on a new MS labile cross-linking reagent, BDRG (biotin-aspartate-Rink-glycine), which addresses these issues. BDRG incorporates a biotin handle (for enrichment of cross-linked peptides prior to MS analysis), two pentafluorophenyl ester groups that react with peptide amines, and a labile Rink-based bond between the pentafluorophenyl groups that allows cross-linked 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 cross-linked peptides derived from purified or partially purified protein complexes, including software to aid in the identification of different classes of cross-linker-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. The results show that the method is effective at identifying cross-linked 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.
Highlights
Most cellular processes are carried out by macromolecular complexes, and knowledge of the structure of these complexes is an essential step toward understanding how they function to control diverse cellular functions [1]
The BDRG Cross-linking Approach—To localize sites of protein-protein interactions in protein complexes and to map the topology of complexes, we have developed an MS-based approach to identify cross-linked peptides derived from protein complexes
It is based on a new, homo-bifunctional, MS labile cross-linking reagent called BDRG (Fig. 1A) that we designed with the following features: 1) BDRG has two PFP ester groups that react with primary amines, and to a lesser extent with secondary amines, at pH 8 [36]
Summary
Pol II, RNA polymerase II; BDRG, biotin-aspartate-Rink-glycine; BLG, -lactoglobulin; CID, collisioninduced dissociation; DIC, N,NЈ-diisopropylcarbodiimide; PFP, pentafluorophenyl; Fmoc, N-(9-fluorenyl)methoxycarbonyl; SCX, strong cation exchange. Except for the D–P-based cross-linker, all of these reagents can produce multiple fragments (four or more) during CID of interpeptide cross-links because of the presence of two labile bonds This can negatively impact peptide identification caused by reduced product ion intensity and duty cycle limitations. CID of interpeptide cross-links containing any of these reagents, except for the Rink-based cross-linker, produces modified peptides with different modification masses This issue must be addressed by considering multiple amine modification masses during database searching. This approach involves a protocol for the application of BDRG to identify cross-linked peptides derived from purified and partially purified protein complexes and software for identification of different classes of cross-linkermodified peptides. It provides an attractive method to study the topology of protein complexes and to infer sites of protein-protein interactions within complexes
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
