Nanorslc q exactive plus rslc ultimate 3000

Purified SFB fractions were labeled at RT with 2 mM sulfosuccinimidyl biotin NHS-LC-LC-Biotin (ThermoFisher, 21343) in PBS for 45 min, quenched with 500 mM glycine, washed twice in PBS and frozen at -80°C. Thawed samples were lysed in PBS with protease inhibitors (Roche, Complete Mini) by sonication (5x30 sec rounds, 30 Khz sonicator at 80%, Hielscher UP50H), and ultracentrifuged at 100,000xg for one hour at 4°C. Pellets were resuspended in CHAPS(4%)/ASB-14(4%) (Sigma), dissociated by 3x20 sec rounds with the FastPrep dissociator (MPBio, 0.1 mm beads), and incubated for 20 min on ice. The streptavidin bead purification was performed as per vendor’s manual (Dynabeads MyOne Streptavidin C1, ThermoFisher). Proteins were eluted at 95°C for 5 min in 1x Laemmli and digested in S-Trap™ micro spin columns (Protifi) as per vendor’s protocol. After elution, peptides were injected in a nanoRSLC-Q Exactive PLUS (RSLC Ultimate 3000) (ThermoScientific) as previously described (PMID:31042281). MS files were processed with Proteome Discoverer software (v1.4) and searched with Mascot search engine against SFB proteins.

Each sample was dried with a vacuum and resuspended in $50\mathrm{\mu }\mathrm{L}$ of 0.1% trifluoroacetic acid, 10% acetonitrile for liquid chromatography–tandem mass spectrometry (LC-MS/MS). For each run, $1\mathrm{\mu }\mathrm{L}$ was injected into a nanoRSLC-Q Exactive PLUS (RSLC Ultimate 3000; Thermo Scientific). Separation of the peptides were obtained with a $50\mathrm{cm}$ reversed-phase LC column (Pepmap C18, Thermo Scientific). The solvents were a) 0.1% formic acid in water, and b) 0.08% formic acid, 80% acetonitrile. Elution of the peptides was performed with the following gradient: 5% to 40% B (120 min), 40% to 80% B (10 min). At 131 min, the gradient was returned to 5% B to re-equilibrate the column for 30 min (before performing the next injection). Two blank conditions were run between each triplicate to prevent sample carryover. The analysis of the eluted peptides was performed by data-dependent MS/MS (the top-10 acquisition method was applied). The resolution was set to 70,000 [mass spectrometric (MS) scans] and 17,500 (data-dependent MS/MS scans). The MS AGC target was set to 3.106 counts (whereas MS/MS AGC target was set to 1.105 counts). We used an MS scan range from 400 to $\mathrm{2,000}\mathrm{m}/\mathrm{z}$ . Records of both MS and MS/MS scans were performed in the profile mode. Dynamic exclusion was set to 30 s. duration. For each sample, three replicates were analyzed by the nanoLC-MS/MS.