GNPS - Pride Project PXD011163 Bulk Hela Cells

This data was originally uploaded to pride project PXD011163. More details can be found there.

Cells were lysed, reduced, and alkylated in lysis buffer (1% SDC, 10 mM TCEP, 40 mM CAA, and 100 mM TRIS, pH 8.0) supplemented with complete EDTA-free protease inhibitor mixture and phosSTOP phosphatase inhibitor mixture. Cells were heated for 5 min at 95 C, sonicated with a Bioruptor Plus, and diluted 1:10 with 50 mM ammonium bicarbonate, pH 8.0. Proteins were digested overnight at 37 C with trypsin and Lys-C (enzyme:substrate ratio of 1:50 and 1:75). SDC was precipitated by acidification to 5% of formic acid. Samples were desalted using Sep-Pak C18 cartridges and directly subjected to phosphopeptide enrichment. Samples for proteome analysis were instead dried down and stored at -80 C until nLC-MS analysis. Phosphopeptides enrichment was performed using Fe(III)-NTA in an automated fashion using the AssayMAP Bravo Platform. Reversed phase nLC-MS/MS analysis was performed with an Agilent 1290 Infinity UHPLC system coupled to an Orbitrap Q Exactive Plus mass spectrometer, or Orbitrap Fusion mass spectrometer for the phosphoproteome analysis. The UHPLC was equipped with a double frit trapping column (Reprosil C18, 3 um, 2 cm x 100 um) and a single frit analytical column (Poroshell EC-C18, 2.7 um, 50 cm x 75 um). Trapping was performed in solvent A (0.1% FA in water) at 5 uL/min, while for the elution the flow rate was passively split to 300 nL/min. The linear gradient was as follows: 13-40% solvent B (0.1% FA in 80% ACN) in 220 min, or 8-32% in 95 min for phosphopeptide analysis. Total analysis time was 235 min for the proteome samples and 110 min for the phosphoproteome samples. The mass spectrometers were operated in data-dependent mode. The Orbitrap Q Exactive Plus full-scan MS spectra from m/z 375-1600 were acquired at a resolution of 35000 (FWHM) after accumulation to a target value of 3e6. Up to 10 most intense precursor ions were selected for fragmentation, with the isolation window set to 1.5 m/z. HCD fragmentation was performed at normalized collision energy of 25% after the accumulation to a target value of 5e4. MS/MS was acquired at a resolution of 17500 (FWHM). The Orbitrap Fusion full-scan MS spectra from m/z 375-1500 were acquired at a resolution of 120000 (FWHM) after accumulation to a target value of 4e5. The most intense peptide ions fitting within a 3 s cycle were selected for HCD fragmentation, with the isolation window set to 1.6 m/z, and a normalized collision energy of 30%, after the accumulation to a target value of 5e4. MS/MS was acquired at a resolution of 30000 (FWHM).