Time-resolved analysis of proteome dynamics by TMT-SILAC hyperplexing

In this project, a more efficient methodology for obtaining multi-timepoint kinetic data for proteome-wide analyses of protein turnover is developed and used to globally measure the kinetics of protein turnover in primary human fibroblasts (HCA2-hTert). This approach takes advantage of the multiplexing capabilities of isobaric tandem mass tags (TMT) to measure fractional SILAC labeling of multiple time-points in a single LC-MS/MS run. Human dermal fibroblasts (HCA2-hTert) (19, 20) were maintained in Eagle’s Minimum Essential Medium (ATCC) supplemented with 15% fetal bovine serum (Invitrogen), 100 U/mL penicillin, 100 U/mL streptomycin at 37℃ with 5% CO2. The media utilized for isotopic labeling was Eagle’s minimum essential medium (ATCC) supplemented with 15% dialyzed fetal bovine serum (Thermo Scientific), 100 U/ml penicillin, and 100 U/ml streptomycin. Cells were gradually adapted to the labeling media and were then plated at a density of 500,000 cells per 10 cm plate. For dividing cells, one day after plating, the cultures were switched to MEM labeling media for SILAC (Thermo Scientific) supplemented with L-arginine:HCl (13C6, 99%) and L-lysine:2HCl (13C6, 99%; Cambridge Isotope Laboratories) at concentrations of 0.1264 g/l and 0.087 g/l and 15% dialyzed fetal bovine serum (Thermo Scientific) and were subsequently collected after 0, 24, 48, 72 hours of labeling. For quiescent cells, the cultures were grown for 8 days to achieve a state of quiescence by contact inhibition. The confluent quiescent cultures were switched to the labeling media and cells were collected after 0, 6, 12, 24, 36, 48, 72, 96, 144, 192, 336 hours of labeling. All cells were washed with PBS and frozen as cell pellets prior to further analysis.