Senescence-specific translation dysregulation desensitizes cells to stress by inhibiting activation of the integrated stress response

ABSTRACT: IMR90 human fibroblasts were used to generate etoposide-induced senescent cells (Etop), contact-inhibited quiescent cells (Qui), and cycling cells (Cyc). Briefly, Etop cells were prepared with three treatments of 50 uM etoposide over 10 days, Qui cells were incubated for four days after 100% confluency, and Cyc cells were harvested at confluency between 40-60%. Whole cell lysates were prepared in lysis buffer(100 mM Tris, 150 mM NaCL, 4% SDS, 1% Triton X-114, 0.1M DTT, and 1X Halt Protease Cocktail (Sigma)) and then subjected to water sonication and heat inactivation. Protein concentrations of cell lysates were then measured using BCA method and an equal amount of total protein (50 ug) for each sample was processed for MS analysis. In brief, samples were subjected to trypsin digestion followed by TMT-multiplex labeling using a TMT16plex kit (ThermoFisher). 15 unique TMT tags were used to label trypsin-digested peptides from 15 samples and one TMT tag was used to label a master mix of equal amount tryptic peptides from the 15 samples. After TMT labeling, peptides were mixed and then fractionated using basic reversed phase UHPLC. 24 fractions from the TMT16plex (raw files F1-F24) were generated and analyzed sequentially by 24 100-min LC/MS/MS runs. The 24 raw MS data files acquired from analysis of the 24 peptide fractions were analyzed by using Proteome Discoverer 2.4 for protein ID and TMT-tag based quantification. 7995 proteins were quantitatively identified in this study.

INSTRUMENT(S): Q Exactive Plus

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Matthew Payea  

PROVIDER: MSV000091077 | MassIVE | Tue Jan 17 12:54:00 GMT 2023

REPOSITORIES: MassIVE