Fractionation and Desalting for Mass Spectrometry

Pooled and dried samples were resuspended in 20 μl 2% (vol/vol) acetonitrile for fractionation. Basic reversed phase fractionation was performed on a quaternary Agilent 1290 Infinity II UPLC system equipped with a Kinetex Evo-C₁₈ column (150 by 2.1 mm, 2.6 μm, 100 Å, Phenomenex) that was operated at 40°C. Solvent A consisted of high-performance liquid chromatography (HPLC)-grade H₂O, solvent B consisted of 100% acetonitrile, and solvent C consisted of 25 mM ammonium bicarbonate. Fractionation was carried out at a constant flow rate of 100 μl/minute using a linear gradient from 2 to 25% (vol/vol) acetonitrile within 50 min, followed by column washing and equilibration. Over the whole gradient, solvent C was kept constant at 10% (vol/vol). In total, 32 fractions with 200 μl per fraction were collected in conical 96-well plates (Greiner). The organic solvent was removed in a vacuum concentrator (default settings for organic solvents) for 1 h and fractions were concatenated into 8 final samples as described before (64 (link)). Peptides were acidified with formic acid (Thermo Fisher Scientific) to a final concentration of 1% (vol/vol), desalted using OASIS HLB 96-well cartridges (Waters), dried down at room temperature, and resuspended in 30 μl 2% (vol/vol) acetonitrile, 0.1% (vol/vol) trifluoroacetic acid (TFA) prior to mass spectrometry (MS) analysis.