Non-radioactive targeting of multiple classes of protein post-translational modifications (PTMs) with click chemistry

摘要

Characterizing changes in multiple classes of protein post-translational modifications (PTMs) is key to deciphering the complex signaling networks involved in cellular regulation. PTMs directly affect protein activities and orchestrate signal transduction processes by regulating both spatial and temporal protein expression parameters. Disruption of these regulatory processes often leads to disease states. In the past, radioactive metabolic precursors have been used to label, track, and detect changes in protein PTMs, however the use of radioactivity has many shortcomings. It is labor-intensive, hazardous, and expensive to dispose of. Although the method is quite sensitive, it is not compatible with in-gel protein detection or cellular microscopy, and X-ray film exposure times can be painstakingly long. Here we provide an alternative non-radioactive approach to the labeling and detection of multiple types of post-translationally modified proteins using a twostep labeling(1-4) and detection(5,6) platform that employs a "toolbox" of metabolic PTM protein labeling compounds. We demonstrate dynamic changes in protein farnesylation, geranylgeranylation, palmitoylation, myristoylation, O-GlcNAc modification using cellular models for apoptosis in HeLa cells and insulin stimulation in 3T3-L1 adipocytes.