Omix c18 pipet tips

The whole-cell lysates prepared from MCF-7 or MDA-MB-231 breast cancer cells and their radioresistant counterparts were combined at 1:1 ratio, incubated with 8 M urea for protein denaturation, and then treated with dithiothreitol and iodoacetamide for cysteine reduction and alkylation, respectively. The proteins were subsequently digested with modified MS-grade trypsin (Pierce) at an enzyme/substrate ratio of 1:100 in 50 mM NH₄HCO₃ (pH 8.5) at 37 °C overnight. The peptide mixture was then dried in a Speed-vac, desalted with OMIX C18 pipet tips (Agilent Technologies), and analyzed by LC-MS and MS/MS in the PRM mode.

The protein lysates prepared from the aforementioned light and heavy SILAC-labeled PC3 cells were combined respectively with the heavy and light SILAC-labeled PC3MLN4 cells at a 1:1 ratio (in mass based on quantification from Bradford assay). Tryptic peptides for LC-PRM analysis were generated following the filter-aided sample preparation (FASP) protocol.^{21 (link)} Approximately 50 μg of cell lysates was washed with 8 M urea for protein denaturation using a Microcon centrifugal filter with a molecular weight cutoff of 30 kDa, and the urea buffer was then removed by centrifugation at 10000 rpm. The ensuing denatured proteins were treated with dithiothreitol and iodoacetamide for cysteine reduction and alkylation, respectively. The proteins were subsequently digested with modified MS-grade trypsin (Pierce) at an enzyme/substrate ratio of 1/100 in 50 mM NH₄HCO₃ (pH 8.5) at 37 °C overnight. The resultant peptide mixture was dried in a Speed-vac, desalted with OMIX C18 pipet tips (Agilent Technologies), and subjected to LC-MS/MS analysis in the PRM mode. Samples from three replicates (two forward and one reverse SILAC labeling experiment) of lysates from the PC3/PC3MLN4 pair of prostate cancer cells were prepared for LC-PRM analyses.