Reprosil pur 120 c18 aq 3 μm reversed phase material

LC/MS/MS analyses were performed with an Orbitrap Fusion Lumos (Thermo Fisher Scientific) connected to an Ultimate 3000 pump (Thermo Fisher Scientific) and an HTC-PAL autosampler (CTC analytics). Peptides were separated by a self-pulled needle column (150 mm length, 100 μm ID, 6 μm needle opening) packed with Reprosil-Pur 120 C18-AQ 3 μm reversed-phase material (Dr. Maisch GmbH), using a 20 min or 65 min gradient of 5–40% B (mobile phase A: 0.5% acetic acid, mobile phase B: 0.5% acetic acid / 80% acetonitrile) at a flow rate of 500 nL/min. The applied ESI voltage was 2.4 kV. For TMT-labeled samples, the following parameters were applied: MS scan range of 375–1500, MS1 orbitrap resolution of 120,000, quadrupole isolation window of 0.7, HCD (higher-energy collision dissociation) collision energy of 38%, MS2 orbitrap resolution of 50,000, AGC target value of 50000. For non-labeled samples, the following parameters were applied: MS scan range of 300–1500, MS1 orbitrap resolution of 120,000, quadrupole isolation window of 1.6, HCD collision energy of 30%, MS2 orbitrap resolution of 15,000, MS2 AGC target value of 50,000.

NanoLC/TIMS/Q/TOF analyses were performed on a timsTOF Pro (Bruker, Bremen, Germany) connected to an Ultimate 3,000 pump (Thermo Fisher Scientific) and an HTC-PAL autosampler (CTC Analytics, Zwingen, Switzerland). Peptides were separated on self-pulled needle columns (150 mm length, 100 μm ID, 6 μm needle opening) packed with Reprosil-Pur 120 C18-AQ 3 μm reversed-phase material (Dr. Maisch, Ammerbuch, Germany). The injection volume was 5 μL, and the flow rate was 500 nL/min. Separation was achieved by applying a linear gradient of 4−32% ACN in 50 min with 0.5% acetic acid as an ion-pair reagent. The TIMS section was operated with a 200 ms ramp time and a scan range of 0.6–1.5 Vs cm⁻². One cycle was composed of 1 MS scan followed by 10 PASEF MS/MS scans. MS and MS/MS spectra were recorded from m/z 100 to 1,700. Singly charged ions were excluded from the precursor ions based on their m/z and 1/K₀ values. The quadrupole isolation width was set to 2 Da. The TIMS elution voltage was calibrated linearly to obtain the reciprocal of reduced ion mobility (1/K₀) using three selected ions (m/z 622, 922, and 1,222) of the ESI-L Tuning Mix (Agilent, Santa Clara, CA, USA).