3c18 cl 120 separation column

Analysis was performed on a TripleTOF 5600+ mass-spectrometer (Sciex, Canada) coupled with a NanoLC Ultra 2D+ nano-HPLC system (Eksigent, USA). The HPLC system was configured in a trap-elute mode. Samples were eluted through a 3C18-CL-120 separation column (3 µm, 120 Å, 75 μm × 150 mm; Eksigent, USA) at a flow rate of 300 nl/min during 120 min. Information-dependent mass-spectrometer experiment included one survey MS1 scan followed by 50-dependent MS2 scans. Detailed methodology is included in the Supplemental Information.

Analysis was performed on a TripleTOF 5600 + mass spectrometer with a NanoSpray III ion source (ABSciex, US) coupled with a NanoLC Ultra 2D + nano-HPLC system (Eksigent, US). The HPLC system was configured in a trap-elute mode. For the sample loading buffer and buffer A, the mixture of 98.9% water, 1% methanol, and 0.1% formic acid (by volume) was used. Buffer B was 99.9% acetonitrile and 0.1% formic acid (by volume). Samples were loaded on a Chrom XP C18 trap column (3 µm, 120 A, and 350 µm × 0.5 mm; Eksigent) at a flow rate of 3.5 µL/min for 10 minutes and eluted through a 3C18-CL-120 separation column (3 µm, 120 A, and 75 µm × 150 mm; Eksigent) at a flow rate of 300 nL/min at 35 °C. The gradient was from 5% to 40% of buffer B for 90 minutes followed by 10 minutes at 95% of buffer B and a 20-minute equilibration at 5% of buffer B. The blank 90-minute run of a wave form (5%-95%-5% of buffer B) was applied between samples to wash the system and to prevent carryover. β-Galactosidase tryptic solution (20 fmol) was run after every 2 samples using a 15-minute gradient (5–25% of buffer B) to calibrate the mass spectrometer and to control the system’s overall performance, stability, and reproducibility.