Microson xl sonicator

CAL-120 cells were harvested and lysed in 20 mM Hepes at pH 8.0, containing 9 M urea, 1 mM sodium orthovanadate, 2.5 mM sodium pyrophosphate, and 1 mM β-glycerophosphate. Whole cell lysates from PBS-treated, FGF1-treated (20 min), and R1MAb2-treated (1 h) CAL-120 cells (3 bioreplicates per condition, total of nine samples) were digested with trypsin and prepared for proteomic analysis. Samples were sonicated using a Misonix Microson XL sonicator followed by centrifugation at 20,000g for 20 min at 15 °C. Protein concentration was determined using a Bradford assay (Bio-Rad). Samples were reduced in 5 mM DTT at 37 °C for 1 h followed by alkylation with 15 mM iodoacetamide at room temperature for 20 min in the dark. Proteins were subjected to a serial digestion using Lys-C (Wako) at an enzyme:substrate (E:S) ratio of 1:50 at 37 °C for 4 h followed by tryptic digestion (Promega) at an E:S ratio of 1:50 at 37 °C overnight in 2 M urea. The peptide mixture was acidified with 20% TFA and desalted using C₁₈ cartridge (500 mg absorbent) from Waters. Peptides were eluted with 3 × 2.0 ml of 60% acetonitrile (ACN)/0.1% TFA followed by peptide concentration measurement using a quantitative colorimetric peptide assay kit (Thermo Fisher Scientific). Equal amounts of peptides per condition (17 mg) were aliquoted and lyophilized overnight.