Dataset Information

Global analysis of protein degradation rates in wildtype, Atg5-/- and Atg7-/- primary human fibroblasts

ABSTRACT: In this project, we employed a dynamic mass spectrometry-based proteomic approach to obtain global maps of basal autophagic flux in human primary fibroblasts (HCA2-hTert). The data provide a comparison of protein degradation rates between wildtype and autophagy deficient (Atg5-/- and Atg7-/-) cells. The media utilized for isotopic labeling was Eagle’s Minimum Essential Medium (ATCC) supplemented with 15% dialyzed fetal bovine serum (Thermo Scientific), 100U/mL penicillin, 100U/mL streptomycin. Prior to labeling, cells were gradually adapted to unlabeled media over the course of multiple passages. Cells were then plated at a density of 500,000 cells per 10 cm plate. 8 days after plating, the confluent quiescent 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). Cells were collected after 0d, 2d, 4d, 6d of labeling, washed with PBS and cell pellets were frozen prior to further analysis. In order to assess the precision of our measurements, biological replicate experiments were conducted for WT+vector and Atg5-/- experiments and cells were collected after 4d for analysis

INSTRUMENT(S): Q Exactive

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

SUBMITTER: Sina Ghaemmaghami