LC-MSMS of cell secretome for MAP1LC3B interactors

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 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.