LC-MSMS of cell secretome for MAP1LC3B interactors

ABSTRACT: Description Growing evidence implicates autophagy in cell secretion. Identifying the repertoire of proteins involved with autophagy dependent secretions is key for understanding the underlying mechanism. We initially use a proximity-dependent biotinylation proteomics strategy to label protein that engage the autophagy regulator MAP1LC3B (LC3/ATG8) in cells; the labeled proteins are then secreted, captured with neutravidin, tryptically digested, and identified by LC-MS/MS. Cells stably expressing BirA* alone serves as control for non-specific cytosolic labeling. SILAC is employed to quantify the degree of LC3B interaction over the background. We follow up with proteomic comparisons of purified exosomes from HEK293T, and autophagy related, ATG7-/- and ATG12-/-, knockout HEK293T cell lines. We quantified differentially secreted proteins in the exosomes by isobaric TMT labels.

INSTRUMENT(S): Q Exactive Plus

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture, Fibroblast

DISEASE(S): Disease Free

SUBMITTER: Hector Huang  

LAB HEAD: Arun Paul Wiita

PROVIDER: PXD015479 | Pride | 2020-01-09

REPOSITORIES: Pride