Computational protocol: Constitutive and activation-dependent phosphorylation of lymphocyte phosphatase-associated phosphoprotein (LPAP)

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[…] LPAP immune-purified from lysate of 5 × 108 CEM cells was subjected to 10% SDS-PAGE. The position of the band containing unlabeled LPAP on the gel was identified by a parallel running of Cy3-labeled LPAP. Bands were manually excised and fixed in acetic acid. To remove SDS gel pieces were washed twice in 40% methanol with 10% glacial acetic acid and rinsed three times in deionized water. For trypsin digestion gel fragments were equilibrated with 50 mM triethyammonium bicarbonate buffer (pH 8.0). Trypsin (Promega) was added in a ratio of 1/50 (w/w) and samples were incubated at 42°C for 5 h. In case of pepsin digestion gel fragments were dried under vacuum, then rehydrated in 3% trifluoracetic acid with pH 1.0 or alternatively in 30 mM hydrochloric acid (pH 2.0–2.5). Pepsin (Sigma) was added in a ratio 1/30 and in a ratio 1/50 in 4 h after beginning of reaction. Digestion with pepsin was carried out at 37°C overnight. Peptides were extracted in three consequent change of 1% trifluoracetic acid and dried under vacuum. The pellet was reconstituted in 10–15 μL of 0.5% formic acid and transferred in glass inserts for LC-MS.Liquid chromatography (LC) separation was performed on an Ultimate 300 RSLCnano system (Thermo Scientific) using an analytical Acclaim® RSLC PepMap column (75 μm x 150 mm, 1.8 μm particle size, 100A pore size; Thermo Scientific) coupled with enrichment μ-precolumn C18 PepMap (300 μm x 5 mm, 5 μm particle size, 100A pore size; Thermo Scientific). Separation was carried out in eluting gradient of mobile phase A (water with 0.08% formic acid, 0.02% acetic acid; pH 2.7) and mobile phase B (acetonitrile with 0.08% formic acid and 0.02% acetic acid). The initial starting condition was 2% of B for 3.5 min following linear increasing to 35% of B for 37.5 min and hold in isocratic mode for the following 4 min.The MS analysis was performed on Orbitrap Fusion (Thermo Scientific) mass spectrometer equipped with NSI ion source. The instrument was operated in the positive ionization mode. The capillary voltage was set at -2.1 kV, and the ion transfer tube temperature was 260°C. The electrodynamic ion funnel voltage (s-lens RF level) was adjusted to 75%. In the data-dependent analysis (ddMS2) the precursor ions were surveyed in a range of 420–1200 m/z at a resolution of R = 60K using quadrupole mass analyzer and Orbitrap detector type. The acquisition gain control (AGC) target was set to 4e5 ion, or maximum integration time of 85 ms. Dynamic exclusion after 3 consequent scans within 20 s (independent on single charge state per precursor) for 45 s and active apex triggered detection were applied during analysis. The precursor ions were selected for MS/MS using top-speed mode. The high-energy collision dissociation (HCD) was used as an activation type. Fragmentation was performed at 27% normalized HCD collision energy with stepped energy ramping within ±20%. Detection of the fragment ion was accomplished in Orbitrap detector type at a resolution of R = 15K at a normal scan speed using fixed first mass as 110 m/z. The AGC target was set to 5e4 ions, or maximum integration time of 60 ms. Full duty cycle time was 2.5 s.After preliminary data-dependent survey and data processing, the further analysis was performed in targeted MS2 mode (t-MS2) at a resolution of R = 30K and AGC of 1e5–2.5e5 ions, or varied integration time of 125–200 ms. The normalized collision energy was set on 22–26% depending on the target precursor ion fragmentation efficiency inspected from the data-dependent analysis. Ions were isolated using quadrupole mass analyzer and the isolation window for all target ions was 3 m/z with offset of 0.5 m/z.Spectra were processed and analyzed using MASCOT search engine version 2.2 for conventional proteins and peptides identification. Phosphorylated sites searching following further identification and annotation were accomplished in support of Xcalibur version 3.0.63 (Thermo Scientific) and Peptide Shaker version 1.16.0 (Compomics) supporting MS-GF and OMMSA search algorithms. Theoretical calculation of peptides decomposition was performed and validated using Spectrum Mill Workbench version B.04.01.141 (Agilent Technology). […]

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