Abstract
Background: Cross-linking mass spectrometry (XL-MS) is a powerful technology capable of yielding structural insights across the complex cellular protein interaction network. However, up to date most of the studies utilising XL-MS to characterise individual protein complexes' topology have been carried out on over-expressed or recombinant proteins, which might not accurately represent native cellular conditions. Methods: We performed XL-MS using MS-cleavable crosslinker disuccinimidyl sulfoxide (DSSO) after immunoprecipitation of endogenous BRG/Brahma-associated factors (BAF) complex and co-purifying proteins. Data are available via ProteomeXchange with identifier PXD027611. Results: Although we did not detect the expected enrichment of crosslinks within the BAF complex, we identified numerous crosslinks between three co-purifying proteins, namely Thrap3, Bclaf1 and Erh. Thrap3 and Bclaf1 are mostly disordered proteins for which no 3D structure is available. The XL data allowed us to map interaction surfaces on these proteins, which overlap with the non-disordered portions of both proteins. The identified XLs are in agreement with homology-modelled structures suggesting that the interaction surfaces are globular. Conclusions: Our data shows that MS-cleavable crosslinker DSSO can be used to characterise in detail the topology and interaction surfaces of endogenous protein complexes without the need for overexpression. We demonstrate that Bclaf1, Erh and Thrap3 interact closely with each other, suggesting they might form a novel complex, hereby referred to as BET complex. This data can be exploited for modelling protein-protein docking to characterise the three-dimensional structure of the complex. Endogenous XL-MS might be challenging due to crosslinker accessibility, protein complex abundance or isolation efficiency, and require further optimisation for some complexes like the BAF complex to detect a substantial number of crosslinks.
Highlights
Cross-linking mass spectrometry (XL-MS) is a powerful technique that enables the identification of proximal amino acid residues within a single protein as well as residues in close proximity in interacting proteins
Bclaf1, Erh and Thrap3 interact directly with each other To investigate interaction surfaces of Arid1a and the Brahma-associated factors (BAF) complex, we carried out five experiments where immunoprecipitation of endogenous Arid1a from mouse embryonic stem cells was coupled to crosslinking using MS cleavable crosslinker disuccinimidyl sulfoxide (DSSO) and MS3 mass spectrometry
Interactions between Bclaf1 and Thrap3 with Erh have been identified through immunoprecipitation of Erh (Kavanaugh et al, 2015), but these interactions were not further validated
Summary
Cross-linking mass spectrometry (XL-MS) is a powerful technique that enables the identification of proximal amino acid residues within a single protein as well as residues in close proximity in interacting proteins. Cross-linking mass spectrometry (XL-MS) is a powerful technology capable of yielding structural insights across the complex cellular protein interaction network. Methods: We performed XL-MS using MS-cleavable crosslinker disuccinimidyl sulfoxide (DSSO) after immunoprecipitation of endogenous BRG/Brahma-associated factors (BAF) complex and copurifying proteins. Conclusions: Our data shows that MS-cleavable crosslinker DSSO can be used to characterise in detail the topology and interaction surfaces of endogenous protein complexes without the need for overexpression. We demonstrate that Bclaf, Erh and Thrap interact closely with each other, suggesting they might form a novel complex, hereby referred to as BET complex This data can be exploited for modelling protein-protein docking to characterise the threedimensional structure of the complex. Endogenous XL-MS might be challenging due to crosslinker accessibility, protein complex abundance or isolation efficiency, and require further optimisation for
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
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
