Cross-linking mass spectrometry sample preparation

One hundred fifty micrograms of purified holo-TFIIH at a concentration of 1 mg/ml in buffer 300 [20 mM Hepes (pH 7.6), 300 mM potassium acetate, 5% glycerol, and 2 mM DTT] was mixed with 6 mM DSBU (Thermo Fisher Scientific) and incubated on ice for 2 hours. The reaction was quenched by adding 50 mM ammonium bicarbonate, and the reaction was further stopped by trichloroacetic acid (TCA) precipitation. Cross-linked proteins were precipitated with 20% (w/v) TCA (Sigma-Aldrich) on ice for 90 min. Proteins were pelleted by centrifugation at 21,000g for 15 min and washed with 10% TCA in 0.1 M tris-HCl and then with acetone (Thermo Fisher Scientific). The solvent was discarded, the pellet was air-dried and then stored at −80°C for analysis by MS.

Cross-linked proteins were resuspended in 50 μl of resuspension buffer (2.5% SDS and 50 mM triethylammonium bicarbonate final concentrations) and reduced with final 10 mM DTT (US Biological) for 30 min at 30°C, followed by alkylation with final 50 mM iodoacetamide (Sigma-Aldrich) for 30 min at 30°C. The proteins were processed using an S-Trap column according to the protocol recommended by the supplier (Protifi, C02-mini) and digested with trypsin (Thermo Fisher Scientific) in 1:10 (w/w) enzyme/protein ratio for 1 hour at 47°C. Peptides eluted from this column were vacuum-dried and resuspended with the peptide fractionation-elution buffer [70% (v/v) liquid chromatography–MS (LC-MS) grade water (Thermo Fisher Scientific), 30% (v/v) acetonitrile (Thermo Fisher Scientific), and 0.1% (v/v) trifluoroacetic acid (TFA; Thermo Fisher Scientific)]. Peptides were first fractionated using AKTA Pure 25 with Superdex 30 Increase 3.2/300 (GE Life Sciences) at a flow rate of 30 μl min⁻¹ of the elution buffer, and 100-μl fractions were collected. On the basis of the elution profile, fractions containing enriched cross-linked peptides of higher molecular masses were vacuum-dried and resuspended with LC-MS grade water containing 0.1% (v/v) TFA for MS analysis. One-half of each fraction was analyzed by a Q-Exactive HF mass spectrometer (Thermo Fisher Scientific) coupled to a Dionex Ultimate 3000 UHPLC system (Thermo Fisher Scientific) equipped with an in-house–made 15-cm-long fused silica capillary column (75 μm inner diameter), packed with reversed-phase ReproSil-Pur C18-AQ 2.4-μm resin (Dr. Maisch GmbH, Ammerbuch, Germany) column. Elution was performed using a gradient from 5 to 45% B (90 min), followed by 90% B (5 min), and reequilibration from 90 to 5% B (5 min) with a flow rate of 400 nl/min (mobile phase A: water with 0.1% formic acid; mobile phase B: 80% acetonitrile with 0.1% formic acid). Data were acquired in data-dependent tandem MS (MS/MS) mode. Full-scan MS settings were as follows: mass range, 300 to 1800 (mass/charge ratio); resolution, 120,000; MS1 AGC target 1E6; MS1 Maximum IT, 200 ms. MS/MS settings were as follows: resolution, 30,000; AGC target 2E5; MS2 Maximum IT, 300 ms; fragmentation was enforced by higher-energy collisional dissociation with stepped collision energy of 25, 27, 30; loop count, top 12; isolation window, 1.5 m/z; fixed first mass, 130; MS2 Minimum AGC target, 800; charge exclusion: unassigned, 1, 2, 3, 8 and > 8; peptide match, off; exclude isotope, on; dynamic exclusion, 45 s. Raw files were converted to mgf format with TurboRawToMGF 2.0.8 (54).