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. Recently, we introduced 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. Here, 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.
Activity-based protein profiling (ABPP) utilizes active site-directed chemical probes to monitor the functional state of enzymes directly in native biological systems. Identification of the specific sites of probe labeling on enzymes remains a major challenge in ABPP experiments. In this protocol, we describe an advanced ABPP platform that utilizes a tandem orthogonal proteolysis (TOP) strategy coupled with mass spectrometric analysis to simultaneously identify probe-labeled proteins together with their exact sites of probe modification. Elucidation of probe modification sites reveals fundamental insights into the molecular basis of specific probe-protein interactions. The TOP-ABPP method can be applied to any type of proteomic sample, including those derived from in vitro or in vivo labeling experiments, and is compatible with a variety of chemical probe structures. Completion of the entire protocol, including chemical synthesis of key reagents, requires approximately 8-10 days.
Proteomics aims to assign molecular and cellular functions to the numerous proteins encoded by eukaryotic and prokaryotic genomes. 1 The daunting size and diversity of the proteome has inspired efforts to enrich specific classes of proteins based on shared functional properties. 2 Within this realm of "targeted proteomics", chemical strategies have proven particularly valuable. For example, chemical probes have been created that label proteins based on their catalytic properties [e.g., activity-based protein profiling (ABPP) 3 ] and post-translational modifications (PTMs). 4 These chemical methods offer key insights into protein function, distinguishing, for example, active from inactive enzymes in cells and tissues. 3 The theoretical information content in chemical proteomic experiments greatly exceeds the actual data procured, due in large part to limitations in existing analytical technologies. Ideally, the identities of all probe-labeled proteins and their sites of modification could be determined in a single experiment. However, the low abundance of probe-modified peptides, coupled with their conjugation to large affinity tags, complicates mass spectrometry (MS) analysis, especially in the background of whole proteome proteolytic digestions. 5 Strategies have been introduced for the enrichment of probe-labeled peptides, 4,6a but these methods discard the rest of the proteome digest, which makes it difficult to differentiate proteins of high similarity, renders protein assignments less statistically significant, and prohibits molecular analysis of entire protein sequences. We sought to address these issues by designing a tandem orthogonal proteolysis (TOP) strategy for the parallel characterization of probe-labeled proteins and sites of probe modification.The TOP method was combined with ABPP by exploiting click chemistry (CC) techniques, 7 as outlined in Scheme 1. Following proteome labeling with an alkynyl ABPP probe, CC is used to introduce a biotin tag with a tobacco etch virus protease (TEV) 8 cleavage site. Tagged proteins are then subject to streptavidin enrichment and on-bead trypsin digestion. The supernatant is isolated by filtration, and the probe-labeled peptides are eluted from the beads by incubation with TEV. The trypsin and TEV digests are then analyzed in sequential MudPIT 9 experiments to characterize probe-labeled proteins and site(s) of probe modification, respectively.For initial experiments, a small library of biotinylated TEV-N 3 tags 1-4 with variable spacer regions ( Figure 1A) was synthesized and reacted under CC conditions with a mouse heart proteome pretreated with an alkynyl phenyl-sulfonate ester ABPP probe (PSt; Scheme 1). 7b Enoyl-CoA hydratase-1 (ECH1), a target of PS probes, is abundant in this proteome, and its labeling site has been characterized as D204. 6 Avidin blotting confirmed that all tags were conjugated to ECH1 ( Figure 1A). After proteome enrichment with streptavidin beads and sequential trypsin and TEV digestions, liquid chromatography (LC)-MS/MS revealed s...
The development of small-molecule inhibitors for perturbing enzyme function requires assays to confirm that the inhibitors interact with their enzymatic targets in vivo. Determining target engagement in vivo can be particularly challenging for poorly characterized enzymes that lack known biomarkers (e.g., endogenous substrates and products) to report on their inhibition. Here, we describe a competitive activity-based protein profiling (ABPP) method for measuring the binding of reversible inhibitors to enzymes in animal models. Key to the success of this approach is the use of activity-based probes that show tempered rates of reactivity with enzymes, such that competition for target engagement with reversible inhibitors can be measured in vivo. We apply the competitive ABPP strategy to evaluate a newly described class of piperazine amide reversible inhibitors for the serine hydrolases LYPAL1 and LYPLA2, two enzymes for which selective, in vivo-active inhibitors are lacking. Competitive ABPP identified individual piperazine amides that selectively inhibit LYPLA1 or LYPLA2 in mice. In summary, competitive ABPP adapted to operate with moderately reactive probes can assess the target engagement of reversible inhibitors in animal models to facilitate the discovery of small-molecule probes for characterizing enzyme function in vivo.
The assignment of molecular and cellular functions to the numerous protein products encoded by prokaryotic and eukaryotic genomes presents a major challenge for the field of proteomics. To address this problem, chemical approaches have been introduced that utilize small-molecule probes to profile dynamics in enzyme activity in complex proteomes. These strategies for activity-based protein profiling enable both the discovery and functional analysis of enzymes associated with human disease.
ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.