Ultimate 3000 binary rslcnano system

An aliquot of the tryptic digest (in 2% acetonitrile/0.1% formic acid in water) was analyzed by LC/MS/MS on an Orbitrap Fusion™ Tribrid™ mass spectrometer (Thermo Scientific™) interfaced with a Dionex UltiMate 3000 Binary RSLCnano System. Peptides were separated onto a Acclaim™ PepMap ™ C₁₈ column (75 μm ID × 15 cm, 2 μm) at a flow rate of 300 nl/min. Gradient conditions were: 3–22% B for 120 min; 22–35% B for 10 min; 35–90% B for 10 min; and 90% B held for 10 min (solvent A, 0.1% formic acid in water; solvent B, 0.1% formic acid in acetonitrile). The peptides were analyzed using data-dependent acquisition method, Orbitrap Fusion was operated with measurement of FTMS1 at resolutions 120,000 FWHM, scan range 350–1,500 m/z, AGC target 2E5, and maximum injection time of 50 ms; during a maximum 3 s cycle time, the ITMS2 spectra were collected at rapid scan rate mode, with CID NCE 35, 1.6 m/z isolation window, AGC target 1E4, maximum injection time of 35 ms, and dynamic exclusion was employed for 60 s.

An aliquot of the tryptic digest was analysed by LC–MS/MS on an Orbitrap Fusion Tribrid mass spectrometer (Thermo Scientific) interfaced with an UltiMate 3000 Binary RSLCnano System (Dionex), as previously described^{74 (link)}. In our experiments, dynamic exclusion was employed for 40 s.

Purified complexes were denatured by boiling in Laemmli’s buffer, electrophoresed a distance of 1 cm into an SDS-(12%) polyacrylamide gel, and stained with Coomassie blue. After thorough destaining, the stained area of each lane was excised, dehydrated in acetonitrile, washed extensively and subjected to thiol reduction by Tris (2-carboxyethyl) phosphine hydrochloride (TCEP) and alkylation with iodoacetamide. Samples were digested overnight with trypsin endoproteinase. An aliquot of the tryptic digest (in 2 % acetonitrile/0.1% formic acid in water) was analyzed by LC/MS/MS on an Orbitrap FusionTM TribridTM mass spectrometer (Thermo ScientificTM) interfaced with a Dionex UltiMate 3000 Binary RSLCnano System. The raw data files were processed using Thermo ScientificTM Proteome DiscovererTM software version 1.4, spectra were searched against the NCBI bacterial database using the Mascot search engine v2.3.02(Matrix Science).