Proxeon easy nlc 1200

All samples were analyzed on a Q Exactive Plus mass spectrometer (Thermo Fischer Scientific) coupled with a Proxeon EASY-nLC 1200 liquid chromatography (LC) pump (Thermo Fisher Scientific). Peptides were separated on a 75 µm inner diameter microcapillary column packed with 35 cm of Kinetex C18 resin (1.7 µm, 100 Å, Phenomenex). For each sample, a 2 μg aliquot was loaded in buffer A (0.1% formic acid, 2% acetonitrile) and eluted with a linear 60 min gradient of 2–20% of buffer B (0.1% formic acid, 80% acetonitrile), followed by an increase to 40% buffer B within 10 min and afterwards a 10 min wash at 98% buffer A. The flow rate was kept at 250 nL/min. MS data were acquired with the Thermo Xcalibur software version 4.27.19, a topN method where N could be up to 15. Target values for the full scan MS spectra were 1 × 10⁶ charges in the 300–1500 m/z range with a maximum injection time of 25 ms. Transient times corresponding to a resolution of 70,000 at m/z 200 were chosen. A 1.6 m/z isolation window and fragmentation of precursor ions was performed by higher-energy C-trap dissociation (HCD) with a normalized collision energy of 30 eV. MS/MS scans were performed at a resolution of 17,500 at m/z 200 with an ion target value of 1 × 10⁶ and a maximum injection time of 50 ms. Dynamic exclusion was set to 45 s to avoid repeated sequencing of peptides.

Samples were analyzed on an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific, San Jose, CA) coupled to a Proxeon EASY-nLC 1200 liquid chromatography (LC) pump (Thermo Fisher Scientific). Peptides were separated on a 100 μm inner diameter microcapillary column packed with 35 cm of Accucore C18 resin (2.6 μm, 150 Å, ThermoFisher). For each analysis, approximately 2 μg of peptides were separated using a 150 min gradient of 8–28% acetonitrile in 0.125% formic acid at a flow rate of 450–500 nL/min. Each analysis used an MS3-based TMT method,^{170 (link),171 (link)} which has been shown to reduce ion interference compared to MS2 quantification.^{172 (link)} The data were collected using the SPS-MS3 method.^{173 (link)}