Ultimate 3000 rslc nanoflow system

SDS-PAGE was performed with 4%–20% precast gels in Tris-based buffer system from SERVA Electrophoresis (#43289.01) according to the manufacturer’s instructions. PageBlue Protein Staining Solution (Fermentas, #R0571) was used for coomassie-staining the gels, images were taken with iPhone 6s (Apple) and the contrast was adjusted using ImageJ (version 1.52d). Global identification and quantification of MSU- and zymosan-binding proteins in the presence of different donor sera was done as previously described (25 (link)). In brief, eluted proteins were reduced with DTT, alkylated with acrylamide, and separated using SDS-PAGE (4%–20%, Sigma-Aldrich). Whole lanes were cut into three individual slices and proteins therein were in-gel digested with trypsin. Generated peptides were analyzed using an LC-MS system consisting of an Orbitrap Velos mass spectrometer coupled to an Ultimate 3000 RSLC nanoflow system (Thermo Fisher Scientific). Raw data were analyzed with the Andromeda search engine implemented in MaxQuant software (version 1.5.3.30; www.maxquant.org). Proteins were identified based on a false discovery rate (FDR) of less than 0.01 on protein and peptide level.

Following phosphopeptide enrichment, dried phosphopeptides were reconstituted in 2% ACN/0.1% TFA and analyzed by an Obritrap Velos mass spectrometer connected to an Ultimate 3000 RSLC nanoflow system (Thermo Fisher). Samples were loaded on a trap column (2 cm length, 75 µm ID, 3 µm C18 particles) at a flow rate of 6 µL/min of 0.1% TFA for 5 min. The trap column was switched online with the analytical column (Acclaim PepMap, Thermo Fisher, 50 cm length, 75 µm ID, 2 µm C18 particles), and peptides were eluted at a flow rate of 250 nL/min and at 45 °C column temperature by an increasing gradient from 4 to 50% in 105 min. The column outlet was directly connected to the nano electrospray source of the mass spectrometer and peptides were ionized with a spray voltage of 1.35 kV using metal-coated fused silica emitter.
The Orbitrap Velos mass spectrometer was operated in data-dependent acquisition mode, recording survey scans in the Orbitrap mass analyzer with a mass range from 300–1600 at a resolution of 60,000 at m/z 400. The five most intense precursors with a charge state of +2 or higher were selected for CID fragmentation with a normalized collision energy of 38, using multi-stage activation for the neutral loss masses of phosphoric acid and MS/MS spectra were acquired in the linear ion trap mass analyzer. Dynamic exclusion duration was set to 30 s.

Samples were dissolved in 0.1% TFA/2% acetonitrile (ACN) and analyzed in an Orbitrap Fusion Lumos mass spectrometer (Thermo Scientific, Waltham, MA, USA) equipped with a nanoelectrospray source and connected to an Ultimate 3000 RSLC nanoflow system (Thermo Scientific, Waltham, MA, USA). Peptides were loaded on an Acclaim PepMap C18 trap (Thermo Scientific, Waltham, MA, USA) and separated by a 50 cm µPAC™ (PharmaFluidics, Gent, Belgium) analytical column at 35 °C column temperature, utilizing 0.1% formic acid solvent as solvent A and 100% ACN with 0.1% formic acid as solvent B. We used a 120 min gradient at a flow rate of 500 nL/min, increasing it from 3.4% B to 21% B in 65 min, to 42% B in 32 min and to 75.6% B in 2 min; maintaining it for 3 min; then decreasing it to 3.6% B in 2 min; and maintaining it for 16 min. The spray voltage was set to 2 kV. A data-dependent acquisition method was used with a cycle time of 3 s and top N setting. Dynamic exclusion was set to 60 s, an AGC target of 4 × 10⁵, a maximum injection time of 50 msec and an Orbitrap resolution of 120,000 for MS1 scan. For all runs, an MS2 method was used with HCD fragmentation at 38%, first mass at 100 m/z, an MS² maximum injection time of 110 msec, an MS² isolation width of 0.8 m/z and an Orbitrap resolution of 60,000.