Nanoacquity uplc device

NanoLC-MS/MS analysis was performed on a nanoAcquity UPLC device (Waters, Milford, USA) coupled to a TripleTOF 5600 mass spectrometer (Sciex, Framingham, MA, USA). Peptide separation was performed on an ACQUITY UPLC BEH130 C18 column (250 mm × 75 μm with 1.7 μm diameter particles) and a Symmetry C18 precolumn (20 mm × 180 μm with 5 μm diameter particles, Waters). The solvent system consisted of 0.1% FA in water (solvent A) and 0.1% FA in ACN (solvent B). The samples (1 μL) were loaded into the enrichment column over 3 min at 5 μL/min with 99% of solvent A and 1% of solvent B. The peptides were eluted at 300 μL/min with the following gradient of solvent B: from 3 to 35% over 110 min, and 35 to 85% over 5 min.
The Ion Spray Voltage Floating was set to 2.6 kV and the interface heater at 100 °C. The system was operated in data-dependent-acquisition mode with automatic switching between MS (mass range 400–1250 m/z) and MS/MS (mass range 100–1800 m/z in high sensitivity mode) modes. The fifty most abundant peptides (intensity threshold of 150 counts), were selected on each MS spectrum for further isolation and collision induced dissociation fragmentation, preferably from 2+ to 5+ charged ions. The dynamic exclusion time was set to 6 s. Complete datasets are available via ProteomeXchange with identifier PXD008656.

Before BCR stimulation (T0) and at eight time points after stimulation (1 h, 2 h, 4 h, 7 h, 12 h, 24 h, 48 h, 96 h), 8.10⁶ cells were resuspended in lysis buffer. Proteins were acetone precipitated and 10 µg of each sample were concentrated in a stacking gel band, in-gel reduced, alkylated, and trypsin digested. NanoLC-MS/MS analyses of extracted trypsic peptides were performed on a nanoAcquity UPLC device (Waters Corporation) coupled to a Q-Exactive Plus mass spectrometer (Thermo Scientific) operated in data dependent acquisition mode. Label-free extracted ion chromatogram-based quantification was performed using MaxQuant software (version 1.5.5.1) [19 ]. The proteomics dataset was deposited to the ProteomeXchange Consortium via the PRIDE partner repository (PXD013573).