Figure 1.

Workflow for AHA-labeling of de novo protein synthesis in autophagy. (A) Structure of L-azidohomoalanine (AHA), an analog of methionine with an azide tag. (B) AHA, which can be tagged with a fluorophore- or biotin alkyne, is used for labeling, detection and identification of newly synthesized proteins. (C) General workflow for the combined BONCAT-iTRAQ approach to detect de novo proteins synthesized during autophagy. HeLa cells were labeled with AHA (50 µM) or DMSO in amino acid-free medium for 2 h. The newly synthesized proteins were enriched by affinity isolation, labeled with isobaric tags and analyzed by LC-MS/MS.