Ultimate 3000 rlsc nano system

The enriched peptides were desalted using a SepPak SPE C-18 column and dried using a SpeedVac centrifuge. Cross-linked peptides were analyzed by nano-LC-MS/MS (Dionex Ultimate 3000 RLSC nano system interfaced with Q Exactive HF (Thermo Fisher Scientific, Waltham, MA, USA)). The samples were loaded into a self-packed 100-μm by 2-cm trap (Magic C18AQ; 5 μm and 200 Å; Michrom Bioresources, Inc., Auburn, CA, USA) and washed with buffer A (0.1% trifluoroacetic acid) for 5 min at a flow rate of 10 μL/min. The trap was brought in line with the analytical column (self-packed Magic C18AQ; 3 μm and 200 Å; 75 μm by 50 cm), and peptides were eluted at 300 nl/min using a segmented linear gradient of 4%–15% solution A (0.2% formic acid) for 30 min, followed by a 15%–25% gradient of solution B (0.16% formic acid and 80% acetonitrile) for 40 min and continued with a 25%–50% solution B for 44 min and 50%–90% of solution B for 11 min. Mass spectrometric data were acquired using a data-dependent acquisition procedure with a cyclic series of a full scan with a resolution of 120,000, followed by MS/MS (higher-energy C-trap dissociation; relative collision energy: 27%) of the 20 most intense ions and a dynamic exclusion duration of 20 s.

The immunoprecipitated samples were run ∼1 cm into SDS-PAGE (Invitrogen NuPAGE Bis-Tris 1.5 mm 10% gel) and stained with Coomassie R250. Gel plugs were subjected to in-gel reduction, alkylation, tryptic digestion, and peptide extraction as described in Shevchenko et al. (1996) (link) and Sleat et al. (2008) (link). Resulting peptides were analyzed by nano LC-MS/MS (Dionex Ultimate 3000 RLSCnano System interfaced with a Velos-LTQ-Orbitrap (Thermo Fisher Scientific, San Jose, CA, United States) as described in (Sleat et al., 2013 (link)).