On-beads trypsin digest and Mass Spectrometry

Following the immunoprecipitation procedure described above, beads were incubated with 100 µl of 10 ng µl⁻¹ trypsin solution in 1 M Urea and 50 mM NH₄HCO₃ for 30 min at 25°C for trypsin digestion. The supernatant was collected, beads washed twice with 50 mM NH₄HCO₃, and all three supernatants collected together and incubated overnight at 25°C at 800 rpm after addition of dithiothreitol to 1 mM. Iodoacetamide was added at a final concentration of 27 mM and the samples were incubated at 25°C for 30 min in the dark. 1 µl of 1 M dithiothreitol was added to the samples and incubated for 10 min to quench the iodoacetamide. Finally, 2.5 µl of trifluoroacetic acid was added and the samples were subsequently desalted using C18 Stage tips. Samples were evaporated to dryness, resuspended in 15 µl of 0.1% formic acid solution, and injected into an Ultimate 3000 RSLCnano system (Thermo), separated by a 15-cm analytical column (75 μm ID home-packed with ReproSil-Pur C18-AQ 2.4 μm from Dr. Maisch) with a 50 min gradient from 5% to 60% acetonitrile in 0.1% formic acid. The effluent from the HPLC was directly electrosprayed into a Qexactive HF (Thermo) operated in data-dependent mode to automatically switch between full-scan MS and MS/MS acquisition. Survey full-scan MS spectra (from m/z 375–1600) were acquired with resolution R = 60,000 at m/z 400 (AGC target of 3 × 10⁶). The 10 most intense peptide ions with charge states between 2 and 5 were sequentially isolated to a target value of 1 × 10⁵ and fragmented at 27% normalized collision energy. Typical mass spectrometric conditions were: spray voltage, 1.5 kV; no sheath and auxiliary gas flow; heated capillary temperature, 250°C; ion selection threshold, 33,000 counts. MaxQuant 1.5.2.8 was used to identify proteins and quantify by intensity-based absolute quantification (iBAQ) with the following parameters: Database, uniprot_proteomes_Bacteria_151113.fasta; MS tol, 10 ppm; MS/MS tol, 10 ppm; Peptide FDR, 0.1; Protein FDR, 0.01 Min. peptide Length, 5; Variable modifications, Oxidation (M); Fixed modifications, Carbamidomethyl (C); Peptides for protein quantitation, razor and unique; Min. peptides, 1; Min. ratio count, 2. Identified proteins were considered as interaction partners if their MaxQuant iBAQ values were greater than log2 twofold enrichment and p-value 0.05 (ANOVA) when compared to the control.