Acquity m class beh130 c18 analytical column

Samples were analyzed using a Maxis II QTOF instrument equipped with a CaptiveSpray nanoBooster ion source coupled to a Dionex UltiMate 3000 RSLCnano system (Bruker Daltonics GmbH, Bremen, Germany). Peptides were separated on an Acquity M-Class BEH130 C₁₈ analytical column (1.7 μm, 75 μm × 250 mm, Waters, Budapest, Hungary) using gradient elution (isocratic hold at 4% for 11 min, then elevating B solvent content to 25% in 75 min, and to 40% in 15 min) following trapping on an Acclaim PepMap100 C₁₈ (5 μm, 100 μm × 20 mm, Thermo Fisher Scientific, Waltham, MA, USA) trap column. Solvent A consisted of 0.1% FA in water, Solvent B was 0.1% FA in ACN, and the sample loading buffer was 0.1% TFA + 0.01% HFBA in water.
For MS analysis, DDA measurements were performed. The cycle time was set at 2.5 s, with a dynamic MS/MS exclusion of the same precursor ion for 2 min, or if its intensity was at least 3 times larger than before. Preferred charge states were set between +2 and +5. MS spectra were acquired at 3 Hz in the 150–2200 m/z range, while MS/MS spectra were at 4 or 16 Hz, depending on the intensity of the precursor. Internal calibration was performed by infusing sodium formate and raw data was recalibrated using the Compass DataAnalysis software 4.3 (Bruker Daltonics, Bremen, Germany).

Samples were dissolved in injection solvent (98% H₂O, 2% ACN, and 0.1% FA) and were subjected to nanoLC-MS/MS analysis using a Dionex Ultimate 3000 RSLC nanoHPLC (Dionex, Sunnyvale, CA, USA). Trapping was performed on an Acclaim PepMap100 C18 (5 µm, 100 µm × 20 mm, Thermo Fisher Scientific, Waltham, MA, USA) trap column with 0.1% TFA + 0.01% HFBA (H₂O) as the transport liquid. Peptides were separated on an Acquity M-Class BEH130 C₁₈ analytical column (1.7 µm, 75 µm × 250 mm Waters, Milford, MA, USA) using the gradients listed in Table 6. Solvent A was 0.1% FA in H₂O, Solvent B was 0.1% FA in ACN, the flow rate was 300 nL min−1, and the column temperature was 48 °C. The method 4-25-40 was used for the separation during the SPE optimization phase with 600 ng injected amounts.