Waters nanoacquity uplc system

SRM-based targeted quantification using ¹⁸O-based reference²² was performed for 39 selected proteins. The peptides and SRM transitions was selected and screened as previously described²² , and were listed in Supplemental Table 7. At least 6 transitions of each peptide were monitored in initial screening to ensure the confident identification and detection of the targeted peptides. The best two transitions (without interference) for each peptide were selected for final quantification. The predicted collision energies from Skyline were used for all peptides. Prior to LC-SRM analyses, the ¹⁸O-labeled reference sample was spiked into each peptide sample in 1:1 mixing ratio. All peptide samples were analyzed on a Waters nanoACQUITY UPLC system (Waters Corporation, Milford MA) directly coupled to coupled on-line to a triple quadrupole mass spectrometer (TSQ Vantage; Thermo Fisher Scientific) using a 25-cm-long, 75-μm-inner diameter fused silica capillary column. 1 μl aliquots of each sample containing ~ 0.5 μg/μl peptides were injected onto the analytical column with a 40-min linear gradient of 10–50% acetonitrile and 0.1% formic acid. A fixed dwell time of 10 ms and a scan window of 0.002 m/z were employed. All datasets were analyzed by Skyline software. The peak area ratios were used for the evaluation of protein abundance changes.

Twenty-four hours after the end of the exposure, human bronchial epithelial cells were lysed in 1.5 ml mixture of 50 mM NH₄HCO₃, 8 M urea, 5 mM N-ethylmaleimide (ThermoFisher Scientific, Waltham, MA) and 0.5 mM Tris-(2-carboxyethyl)-phosphine hydrochloride (TCEP), which allowed protein denaturation, reduction, and alkylation. Approximately 400 μg protein per sample was subjected to trypsin digestion after diluting the samples eightfold with 50 mM NH₄HCO₃. Trypsin was added at the ratio of 1 to 50 (enzyme to protein) for digestion at 37 °C for 3 h. The digested peptides were further desalted using C18 solid phase extraction columns (Phenomenex, Torrance, CA). Finally, 5 μl of 0.1 μg/μl of peptides from each sample were analyzed by LC–MS/MS using a Waters nano ACQUITY UPLC system (Waters, Milford, MA) coupled with a Q-Exactive plus mass spectrometer (ThermoFisher Scientific, Waltham, MA).