Advanced click chemistry strategies for in vivo enzyme activity profiling.

摘要

Methods for profiling the activity of enzymes in vivo are needed to understand the role that these proteins and their endogenous regulators play in physiological and pathological processes. This thesis describes the introduction and optimization of a tag-free strategy for activity-based protein profiling (ABPP) that utilizes the copper(I)-catalyzed azide-alkyne cycloaddition reaction ("click" chemistry) to analyze the functional state of enzymes in living cells and organisms. We show that enzymes can be labeled in an activity-based manner both in vitro and in vivo by an azido and alkynyl phenyl sulfonate ester probe and that these labeling events can be detected in whole proteomes by copper-catalyzed ligation with the complementary rhodamine-alkyne or -azide tag. We report a detailed characterization of the reaction parameters that affect click chemistry-based ABPP and identify conditions that maximize the speed, sensitivity, and bioorthogonality of this approach. Using these optimized conditions, we compare the enzyme activity profiles of living and homogenized breast cancer cells, resulting in the identification of several enzymes that are labeled by activity-based probes in situ but not in vitro.; One of the most critical aspects of activity-based protein profiling methodology is determining whether or not labeling events are truly activity-based. Controls such as the comparative profiling of native and heat-denatured proteomes can provide great insight into the nature of the labeling event; however, to formally establish that probe labeling occurs in an activity-dependent manner, it is necessary to identify the site of modification. This is particularly important for in situ and in vivo labeling experiments, which are not amenable to the use of protein denaturation as biochemical controls. We report a tandem orthogonal on-bead proteolysis (TOP) strategy for the parallel identification of enzyme activities and sites of modification. Click chemistry is used to introduce a biotin tag with an orthogonal protease cleavage domain, which for the selective elution of active site peptides following on-bead trypsin digestion. Preliminary results using this TOP-ABPP to identify sites of alkyne-phenyl sulfonate labeling are presented.