Wps 3000rs

An Ultimate 3000 system equipped with a pump (LPG-3400RS), an autosampler (WPS-3000RS), a column oven (TCC-3000RC), and a VWD-3400RS UV/Vis detector (all from Thermo Scientific, Waltham, MA, USA) was used for the chromatographic separation, and the UV-Vis detector was operated at 214 nm. Data were collected and processed by Chromeleon software 7.2 SR5 (Thermo Scientific, Waltham, MA, USA). Separation was carried out on a Hypersil GOLD C18 column (100 mm × 2.1 mm, 5 μm) (Thermo Scientific, Waltham, MA, USA) at 45 °C, with an injection volume of 10 μL. Mobile phase A was 20 mM formic acid containing 5% of methanol (v/v), and mobile phase B was methanol containing 5% of 20 mM formic acid (v/v). Mobile phase B was increased from 20% to 80% within 3.5 min at a flow rate of 0.8 mL min⁻¹; afterward, it was decreased to 20% within 0.1 min, and this condition was kept for 2.5 min for equilibration.

85 µL was injected onto a HPLC system containing an anti-peptide antibody column to enrich the tryptic peptides of interest prior to reverse phase nanoflow chromatography comprised of Ultimate 3000 (ThermoFisher Scientific) with the following modules: WPS-3000RS Temperature Controlled autosampler fitted with a 250 µL sample loop; HPG-3400RS Rapid Separation Binary Pump (Micro Pump); NCS-3500RS Nano LC system (Loading pump and Nano Pump); TCC-3200RS (antibody column) column oven; NCS 3500 RSC column oven that encloses valve 2, 3 and the trap column (Thermo µ-Precolumn Cartridge P/N 164649) fitted with an Acclaim PepMap100 C18, 5 µm, 100 Å 300 µm i.d. × 5 mm (60 °C). The LC was connected to a Thermo Quantiva Triple Quadrupole with a Thermo Fisher Easy Spray Ionization Source. The analytical column is Thermo Easy Spray PepMap C18, 75 µm × 15 cm (p/n ES800) (60 °C). MRM transitions monitored in the LC–MS/MS assay for both proteins can be found in Table S5.

The Thermo Scientific UHPLC Ultimate 3000 apparatus (Thermo Fisher Scientific, Waltham, MA, USA) consisted of an LPG-3400RS quaternary pump with a vacuum degasser, a WPS-3000RS autosampler and a TCC-3000SD column oven. The ESI-qTOF Compact (Bruker Daltonics, Bremen, Germany) was connected as the MS detector. The separation was achieved on a Kinetex C-18 column (150 × 2.1 mm) of 2.6 μm particle size, core-shell type (Phenomenex, Torrance, CA, USA). The gradient elution system consisted of 0.1% HCOOH in water (mobile phase A) and 0.1% HCOOH in acetonitrile (mobile phase B). At the flow rate of 0.3 mL/min, the following elution program was used: 0→1 min (2%→30% B), 1→31 min (30%→60% B), 31→31.5 min (60%→100% B), 31.5→35.5 min (100% B). The column was equilibrated for 7 min before the next analysis. Blanks were added after each run to avoid any sample carryover. All analyses were carried out isothermally at 30 °C. The injection volume for samples and standard solutions was 5 μl. Each analysis was calibrated in the first segment of analysis and performed in duplicate. The method was as in Reference [32 (link)].