Aeris 3 mm c18 analytical column

PC9-BrM and PC9-Tr-BrM cells were cultured at 80% confluency. Cells were washed three times with cold PBS and collected in TBS buffer (25 mmol/L Tris-HCl, 150 mmol/L NaCl, pH 7.2) supplemented with protease inhibitor (Roche) and phosphatase inhibitor (Thermo Scientific) on ice. After ultrasound sonication, cell lysates were centrifuged at 18,000 × g for 10 minutes, and supernatants were collected for proteomic analysis, which was performed at the Whitehead Proteomics Core Facility (WPCF). At the WPCF, the fractionated samples were further purified by TCA precipitation, resuspended in a Tris/urea buffer, reduced, alkylated, and digested with trypsin at 37°C overnight. This solution was subjected to solid-phase extraction to concentrate the peptides and remove unwanted reagents, followed by injection onto a Waters NanoAcquity HPLC equipped with a self-packed Aeris 3-mm C18 analytical column, 0.075 mm by 20 cm (Phenomenex). Peptides were eluted using standard reverse-phase gradients. The effluent from the column was analyzed using a Thermo Orbitrap Elite mass spectrometer (nanospray configuration) that was operated in a data-dependent manner for 120 minutes. The resulting fragmentation spectra were correlated against custom databases using PEAKS Studio X (Bioinformatics Solutions).