Ultimate 3000 rslcnano pro flow system

Platelet digests of 3 μg in 15 μL 0.1% TFA were separated with an Ultimate 3000 RSLCnano Pro Flow system (Thermo Scientific, Waltham, MA, USA), concentrated on an Acclaim PepMapTM 100 C18 trapping column (2 cm length, 100 μm i.d., 5 μm particle size, pore size 100 Å, Thermo Scientific), and separated on an Acclaim PepMap^® RSLC C18 column (50 cm length, 75 μm i.d., 2 μm particle size, pore size 100 Å, Thermo Scientific) heated to 60 °C. Sample preconcentration was performed for 5 min with a flow of 20 µL/Min and 0.1% TFA. A 120 min gradient from 3% to 45% solvent B (84% ACN/0.1% FA, 250 nL/Min) was applied for peptide separation. The nanoHPLC was coupled to a Q Exactive Hybrid Quadrupole-Orbitrap Plus mass spectrometer (Thermo Scientific) and eluting peptides were ionized directly via a silica emitter (FS360-20-10-D-20, New Objective, Ringoes, NJ, USA) of the nanoESI source. Peptides of interest were analyzed using a scheduled parallel reaction monitoring method, which was performed with a resolution of 17,500, a maximum injection time of 50 ms, and an automatic gain control (AGC) value of 3 × 10⁶. The isolation window was 0.4 m/z for the respective masses of endogenous and SIL peptides.

Proteins were digested on the beads with 1 µg of trypsin (Promega, Leiden, Netherlands) at 37 °C for 4 h while spinning at 161× g. Beads were removed, an additional 1 µg of trypsin was added, and proteins were further digested at 37 °C, overnight. The resulting peptide mixture was purified using OMIX C18 pipette tips (Agilent, Santa Clara, California, USA). The purified peptides were dried completely and re-suspended in 20 µl loading solvent (0.1% TFA in water/ acetonitrile, 2/98 (v/v)) of which 5 µl were injected for LC-MS/MS analysis on an Ultimate 3000 RSLCnano ProFLow system connected online to a Q Exactive HF mass spectrometer (Thermo, Waltham, MA, USA). Peptide trapping and elution and mass spectrometer operation details are described in Appendix A (Sample injection and mass spectrometer operation).