Workflow of a typical click chemistry-based nascent proteomics study. Briefly, cells are incubated with AHA for a desired period of time (eg, 30 min). Proteins are extracted from cells. The extracted proteins can be directly subjected to analysis either by MS for proteomics or WB for individual proteins of interest, or to the click reaction to biotinylate AHA-labeled, newly synthesized proteins. The insert in the dashed box explains the principle of the click reaction. Biotinylated, AHA-labeled proteins are then isolated from the rest of the protein extracts by incubation with streptavidin beads, and subjected to MS or WB analysis. The same workflow can be applied to whole animal labeling for in vivo studies with adjustments of AHA concentration and duration of labeling. AHA, azidohomoalanine; MS, mass spectrometry; WB, western blotting.
Proteomics in stroke research: potentials of the nascent proteomics.
Journal of Investigative Medicine,64(8),
Accessed March 25, 2017.https://doi.org/10.1136/jim-2016-000186.