Reprosil pur 120 ods 3

Mass spectrometric analysis was basically carried out as described in Schwenk et al. (2014) (link). Briefly, peptides obtained from tryptic in-gel digests were dissolved in 0.5% trifluoroacetic acid and aliquots were loaded onto a C18 PepMap100 precolumn (particle size 5 mm; Dionex/Thermo Scientific, Germany) with 0.5% (v/v) acetic acid using a split-based UltiMate 3000 HPLC (Dionex/Thermo Scientific, Germany). Bound peptides were eluted and separated with an aqueous-organic gradient from 0.5% (v/v) acetic acid to 0.5% (v/v) acetic acid in 80% (v/v) acetonitrile (1 h 20′ total) in a PicoTip emitter (i.d. 75 mm; tip 8 mm; New Objective, United States) manually packed with ReproSil-Pur 120 ODS-3 (C18; particle size 3 mm; Dr. Maisch HPLC, Germany) and electrosprayed (2.3 kV; transfer capillary temperature 250°C) into an LTQ Orbitrap XL tandem mass spectrometer with the described settings.

The trypsin-digested peptides were dissolved in 20 µl sample buffer (0.5% (v/v) trifluoroacetic acid in H₂O) and 1 µl aliquots (or less) were taken for LC–MS/MS analysis. Loading onto the precolumn (PepMap 100, C18 stationary phase) was achieved through an autosampler of a split-free UltiMate 3000 RSLCnano HPLC (Dionex/Thermo Fisher Scientific). Subsequent elution and separation on the SilicaTip column emitter (inner diameter, 75 µm; tip, 8 µm; New Objective,; packed 23 cm with ReproSil-Pur 120 ODS-3 (C18 stationary phase; Dr. Maisch HPLC)) occurred during a three-step linear gradient generated from eluent A (0.5% (v/v) acetic acid) and eluent B (0.5% (v/v) acetic acid in 80% (v/v) acetonitrile): after 5 min equilibration in 3% B, 90 min from 3% B to 30% B; 20 min from 30% B to 50% B; and 10 min from 50% B to 99% B. Subsequent column washing/regeneration comprised 5 min 99% B; 5 min from 99% B to 3% B; 10 min 3% B. The flow rate was set to 300 nl min⁻¹. Electrospray parameters were positive ion mode, spray voltage 2.3 kV, transfer capillary temperature 300 °C. Data were acquired on the QExactive HF-X mass spectrometer (Thermo Fisher Scientific) with the following settings: maximum MS/MS injection time = 200 ms; dynamic exclusion time = 45 s; minimum signal intensity threshold = 40,000 (counts), fragmentation = 15 top precursors; mass isolation width = 1.0 m/z.

Analysis was performed on an Orbitrap XL (Thermo scientific) mass spectrometer that was coupled to an Ultimate 3000 micro pump (Thermo scientific). Buffer A was 0.5 % acetic acid, buffer B 0.5 % acetic acid in 80 % acetonitrile (HPLC grade). Liquid phases were applied at a flow rate of 300 nl/min with an increasing gradient of organic solvent for peptide separation. Reprosil-Pur 120 ODS-3 (Dr. Maisch, Ammerbuch, Germany) was used to pack column tips of 75 µm inner diameter and 11 cm length. The MS was operated in data dependent mode and each MS scan was followed by a maximum of five MS/MS scans.