Poroshell ec120 c18

All samples were analyzed on a Q-Exactive Plus (Thermo Scientific) mass spectrometer that was coupled to an EASY nLC 1000 UPLC (Thermo Scientific). Peptides were loaded with solvent A (0.1% formic acid in water) onto an in-house packed analytical column (50 cm × 75 µm I.D., filled with 2.7 µm Poroshell EC120 C18, Agilent). Peptides were chromatographically separated at a constant flow rate of 250 nL/min using the following gradient: 5–30% solvent B (0. 1% formic acid in 80% acetonitrile) within 119 min, 30–50% solvent B within 19 min, followed by washing and column equilibration. The mass spectrometer was operated in data-dependent acquisition mode. The MS1 survey scan was acquired from 300–1750 m/z at a resolution of 70,000. The top ten most abundant peptides were isolated within a 2 Da window and subjected to HCD fragmentation at a normalized collision energy of 27%. The AGC target was set to 5 × 10E5 charges, allowing a maximum injection time of 55 ms. Product ions were detected in the Orbitrap at a resolution of 17,500. Precursors were dynamically excluded for 20 s.

Peptides were eluted from SDB-RPS tips with 30 μl of 5 % (w/v) ammonium hydroxide in 80% acetonitrile (ACN) and dried a speed vac.
All samples were analyzed on a Q-Exactive Plus (Thermo Scientific) mass spectrometer that was coupled to an EASY nLC 1000 UPLC (Thermo Scientific). After resuspension in 10 μl 5% formic acid, 2% ACN the peptides were loaded with 8 μl solvent A (0.1% formic acid in water) onto an in-house packed analytical column (50 cm × 75 μm I.D., filled with 2.7 μm Poroshell EC120 C18, Agilent). Peptides were separated at a constant flow rate of 250 nL/min using the following gradient: 3 - 5% solvent B (0.1% formic acid in 80 % ACN) within 1 min, 5-30% solvent B within 40 min, 30-50% solvent B within 8 min, followed by washing with 95 % solvent B for 10 min. The mass spectrometer was operated in data-dependent acquisition mode.
The MS1 survey scan was acquired from 300-1750 m/z at a resolution of 70.000. The top 10 most abundant peptides were isolated within a 1.8 Th window and subjected to HCD fragmentation at normalized collision energy of 27%. The AGC target was set to 5e5 charges, allowing a maximum injection time of 110 ms. Product ions were detected in the Orbitrap at a resolution of 35.000. Precursors were dynamically excluded for 20 s.

The chromatographic separation was performed on a reversed-phase analytical column Poroshell EC-120 C18 (50 mm × 2.1 mm, 2.7 µm), supplied by Agilent Technologies (Santa Clara, CA, USA) preceded by a similar precolumn (30 mm × 2.1 mm, 2.7 µm). The chromatographic separation was performed at 25°C. The mobile phase was composed of (A) H₂O : acetonitrile : formic acid (95 : 5 : 0.1%, v/v/v) and (B) H₂O : acetonitrile : formic acid (5 : 95 : 0.1%, v/v/v), and the isocratic elution mode was used with 70% (A) and 30% (B). The flow rate was 0.2 mL·min⁻¹ with a run time of 4 min and injection volume of 5 µL.
The following ionization conditions were established for the ESI-QTOF/MS system: positive ionization mode, capillary voltage: 2.5 kV, detector voltage: 1.850 kV, sample cone voltage: 20.0 V, extraction cone voltage: 2.0 V, source temperature: 100°C, desolvation gas temperature: 300°C, nitrogen gas flow in the cone: 50 L·h⁻¹, and desolvation flow: 400 L·h⁻¹. The molecules of interest were quantified by monitoring the signal related to the protonated molecular ion m/z (M + H⁺). The sulfonamide and trimethoprim identity was confirmed by obtaining the accurate mass of the protonated molecular ion, as well as by the consideration of fragment ions in order to obtain the identification points (IPs) according to Commission Decision 2002/657/EC [22 ] (Table 1).