Proximity labelling and LCMSMS analysis of spliceosome complexes using SUGP1-miniTurbo

The purpose of the project is to identify proteins binding differentially to the human specific spliceosomal protein SUGP1 in the context of point mutations to its G-patch domain. SUGP1 wt and mutant (G603N or L570E) C-terminally tagged with mini-Turbo-NLS was overexpressed for 48 h in HEK293T cells prior to a 2 min labelling pulse with 200 µM biotin. A non-transfection control was included and all samples were prepared in four independent biological replicates. Biotinylated proteins were captured by incubating total cell lysates for 30 min at 4 °C with streptavidin beads. The enrichment was then performed exactly as described in Cho et al. Nat Protoc 15, 3971–3999 (2020) pmid: 33139955