Xevo acquity uplc

The mixed peptide sample fractioning was performed on a Chromatographic column (BEH C18, 300A, 1.7 µm, 1 mm × 150 mm) coupled to a Waters XevoTM AC-QUITY UPLC (Waters, Milford, MA, USA) with an 80 min liquid phase gradient. We collected the first 4 min of liquid as the first fraction, the liquid of the 64–68 min as the last fraction, and discarded the liquid of the last 12 min. We collected the liquid sample every minute during the gradient of 4–64 min. The first fraction was mixed with the last one and the rest were mixed in pairs every 30 fractions. Finally, 31 fractions were obtained and vacuum-centrifuge dried. All 31 fractions were reconstituted in 10 µL Milli-Q water with 0.1 vol% formic acids (FA). IRT peptides were spiked before the data-dependent analysis (DDA).

Approximately 88 µg mixed peptides were fractioned on a Chromatographic column (BEH C18, 300 Å, 1.7 µm, 1 mm × 150 mm) coupled to a Waters Xevo^TM ACQUITY UPLC (Waters, USA) within 80 min gradient and concatenated into 62 fractions. The first fraction is mixed with the last fraction, and the rest is mixed with two fractions every 30 fractions sequentially. Finally, 31 fractions were obtained. All fractions were vacuum-centrifuged to dryness and reconstituted in 10 μl Milli-Q water with 0.1 vol% FA. iRT peptides were spiked before data-dependent acquisition (DDA) analysis.