Autophagy is a catabolic process during which cellular components including
protein aggregates and organelles are degraded via a lysosome-dependent process to sustain metabolic homeostasis during nutrient or energy deprivation. Measuring the rate of proteolysis of long-lived
proteins is a classical assay for measurement of autophagic flux. However, traditional methods, such as a
radioisotope labeling assay, are technically tedious and have low sensitivity. Here, we report a novel method for quantification of long-lived protein degradation based on L-
azidohomoalanine (AHA) labeling in mouse embryonic fibroblasts (MEFs) and in human
cancer cells. AHA is a surrogate for
L-methionine, containing a bio-orthogonalazide moiety. When added to cultured cells, AHA is incorporated into
proteins during active
protein synthesis. After a click reaction between an
azide and an
alkyne, the
azide-containing
proteins can be detected with an
alkyne-tagged
fluorescent dye, coupled with flow cytometry. Induction of autophagy by
starvation or mechanistic target of
rapamycin (
MTOR) inhibitors was able to induce a significant reduction of the fluorescence intensity, consistent with other autophagic markers. Coincidently, inhibition of autophagy by pharmacological agents or by Atg gene deletion abolished the reduction of the fluorescence intensity. Compared with the classical
radioisotope pulse-labeling method, we think that our method is sensitive, quantitative, nonradioactive, and easy to perform, and can be applied to both human and animal cell culture systems.