Acquity uplc beh300 c18 column

The peptides were analysed by HPLC and high-resolution ESI-MS mass spectrometry. HPLC analyses were carried out on an Agilent system equipped with a Chromolith^® High Resolution RP-18e column (150 Å, 100 × 4.6 mm) at a flow rate of 3 mL/min, with a XSelect Peptide CSH C18 column (130 Å, 2.5 μm, 150 × 4.6 mm) or with an Agilent AdvanceBio Peptide column (2.7 μm, 100 × 2.1 mm or 250 × 2.1 mm) at a flow rate of 1 mL/min and 214 nm. Solvents A and B were 0.1% TFA in H₂O and 0.1% TFA in MeCN. The acquisition of the LC-ESI-MS data was carried out on a Waters Q-TOF Xevo G2S mass spectrometer with an Acquity UHPLC system and Lockspray source equipped with a Waters Acquity UPLC BEH300 C18 column (1.7 μm, 2.1 × 150 mm). Peptide elution was performed at a flow rate of 0.4 mL/min with a 10–70% gradient of buffer B over 10 min. Solvents A and B were 0.1% formic acid (FA) in H₂O and 0.1% FA in MeCN. All calculated and found masses were monoisotopic. Purification of the peptides was performed by HPLC on a preparative Agilent 1260 system equipped with a Phenomenex Jupiter column (4 µm Proteo 90 Å, C12, 250 × 4.6mm) at a flow rate of 20 mL/min or a semi-preparative Agilent 1260 system equipped with an Agilent Poroshell EC-C18 column (120 Å, 4 µm, 250 × 9.4 mm) at a flow rate of 4.5 mL/min.

Samples were separated by a reverse-phase Acquity UPLC BEH 300 C18 column (1.7 μm, 2.1 × 150 mm) equipped with an Acquity UPLC BEH C18 VanGuard Pre-column (300 Å, 1.7 μm, 2.1 × 5 mm; Waters, Milford, MA, USA) in an UPLC system coupled with ESI and MS (UPLC Acquity with a single quadrupole detector (SQD); Waters, Milford, MA, USA).
Gradient elution was set as follows with eluent A (H₂O with 0.2% CH₃CN and 0.1% HCOOH) and eluent B (CH₃CN with 0.1% HCOOH)): 0 to 7 min, isocratic 100% A; 7 to 47 min, linear gradient from 100% A to 53.5% A; 47 to 48 min, from 53.5% A to 0% A; 48 to 53 min, isocratic 0% A; and reconditioning. The flow rate was 0.2 mL/min.
The analysis parameters were as follows: flow rate, 0.2 mL/min; analysis time, 67 min; column temperature, 35 °C; sample temperature, 18 °C; injection volume, 5 μL; acquisition time, 0–67 min; ionization type, positive ion mode; capillary voltage, 3.2 kV; cone voltage, 30 V; source temperature, 150 °C; desolvation temperature, 300 °C; cone gas flow, 100 L/h; and desolvation gas flow, 650 L/h. Samples were analyzed in full-scan mode, with a scan range of 100–2000 m/z.

For nrPEM, a 100‐µg sample was denatured and alkylated for 1 hour in 8‐M urea with 6‐mM NEM at 30°C. The alkylated sample was digested for 2 hours with 2‐µg endoproteinase LysC at 30°C, followed by a 1‐hour digestion with 2‐µg trypsin in 50‐mM Tris–HCl, 0.6‐mM CaCl₂ buffer at 30°C and finalized by the addition of 2‐µL TFA. A 10‐µl sample was injected onto an Ultimate 3000 UPLC system (Dionex, Thermo Scientific) equipped with an Acquity UPLC BEH300 C18 column (2.1 cm × 150 mm, 1.7‐µm particle size, Waters), a DAD detector and connected to an Orbitrap Fusion Tribrid mass spectrometer (Thermo Scientific). Peptides were eluted using a gradient increasing 0.1% formic acid (FA) in 80% ACN (MPB) from 0% to 80% in 0.1% FA (MPA) in 66 min at 60°C with a flowrate of 0.25 mL/min. Their UV absorbance was detected at 214, 280, and 330 nm, followed by ionization with an ESI source. MS data acquisition was done using Orbitrap detection with quadrupole isolation mode. MS was performed at positive polarity, Orbitrap resolution at 120 000 over a scan range m/z 400–1600. MS/MS was performed using electron‐transfer dissociation activation and an isolation window of 1.6 m/z over a normal scan range and Orbitrap resolution at 60 000. Data were analyzed by Xcalibur and PeptideFinder software (Thermo Scientific Corp.).