Proxeon easy nlc 1000 liquid

Mass spectrometry data were collected using an Orbitrap Fusion mass spectrometer (Thermo Fischer Scientific) equipped with a Proxeon EASY-nLC 1000 liquid chromatography (LC) system (Thermo Fisher Scientific). Peptides were separated on a 100 μm inner diameter microcapillary column packed with ~35 cm of Accucore C18 resin (2.6 μm, 150 Å, Thermo Fisher Scientific). We loaded ~2 μg sample onto the column. Peptides were separated using a 3 hour gradient of acidic acetonitrile. We used the multinotch MS3-based TMT method (McAlister et al., 2014 (link)). The scan sequence began with a MS1 spectrum (Orbitrap analysis; resolution 120,000; mass range 400–1400 Th). MS2 analysis followed collision-induced dissociation (CID, CE = 35) with a maximum ion injection time of 150 ms and an isolation window of 0.7 Da. The 10 most abundant MS1 ions of charge states 2–6 were selected for MS2/MS3 analysis. To obtain quantitative information, MS3 precursors were fragmented by high-energy collision-induced dissociation (HCD, CE = 65) and analyzed in the Orbitrap (resolution was 60,000 at 200 Th) with a maximum ion injection time of 2 ms and a charge state-dependent variable isolation window of 0.7 to 1.2 Da(Paulo et al., 2016 (link)).

GR, PR, or FLAG peptides were biotinylated using the EZ-Link TFPA-PEG3-Biotin kit (Thermo Fisher). Splicing reaction mixtures containing these peptides (10 µM) were separated on Sephacryl-S500 gel filtration columns in a buffer containing 20 mM Tris, pH 7.8, 60 mM KCl, 0.1% Triton X100, and 0.2 mM PMSF. Proteins in the gel filtration fractions were analyzed on NuPAGE Novex 4–12% Bis-Tris Gels (Thermo Fisher) followed by western blotting. Total RNAs from the fractions were analyzed on an 8% denaturing polyacrylamide gel stained with ethidium bromide. The data for rows FLAG, SNRPC, SNRPB2, SF3A1, SF3A2/3, FUS and TARDBP shown in Figure 2A and the RNA gel shown in Figure 2B were from the gel filtration column containing the GR peptide. Similar results were obtained with gel filtration fractions containing PR and FLAG peptides. Pulldowns were carried out from fractions 33–69 in the gel filtration buffer using streptavidin magnetic particles (Roche) at 4°C for 2 hours and washed 5 times with 1X PBS/0.1% Triton X100. Proteins associated with biotinylated peptides were labeled using tandem mass tag (TMT) (McAlister et al., 2012 (link)) and analyzed with an Orbitrap Fusion mass spectrometer coupled to a Proxeon EASY-nLC 1000 liquid chromatography (LC) pump (Thermo Fisher Scientific).