Proteomics

Dataset Information

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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 (human)

TISSUE(S): Fibroblast

SUBMITTER: Tian Zhang  

LAB HEAD: Sina Ghaemmaghami

PROVIDER: PXD003562 | Pride | 2016-06-14

REPOSITORIES: Pride

Dataset's files

Source:
Action DRS
Atg5_KO_0d.raw Raw
Atg5_KO_2d.raw Raw
Atg5_KO_2d.zip Other
Atg5_KO_4d.raw Raw
Atg5_KO_4d.zip Other
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Publications

Global Analysis of Cellular Protein Flux Quantifies the Selectivity of Basal Autophagy.

Zhang Tian T   Shen Shichen S   Qu Jun J   Ghaemmaghami Sina S  

Cell reports 20160303 10


In eukaryotic cells, macroautophagy is a catabolic pathway implicated in the degradation of long-lived proteins and damaged organelles. Although it has been demonstrated that macroautophagy can selectively degrade specific targets, its contribution to the basal turnover of cellular proteins has not been quantified on proteome-wide scales. In this study, we created autophagy-deficient primary human fibroblasts and quantified the resulting changes in basal degradative flux by dynamic proteomics. O  ...[more]

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