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Activity-based proteomic and metabolomic approaches for understanding metabolism - PubMed

Review

Activity-based proteomic and metabolomic approaches for understanding metabolism

Devon Hunerdosse et al. Curr Opin Biotechnol. 2014 Aug.

Abstract

There are an increasing number of human pathologies that have been associated with altered metabolism, including obesity, diabetes, atherosclerosis, cancer, and neurodegenerative diseases. Most attention on metabolism has been focused on well-understood metabolic pathways and has largely ignored most of the biochemical pathways that operate in (patho)physiological settings, in part because of the vast landscape of uncharacterized and undiscovered metabolic pathways. One technology that has arisen to meet this challenge is activity-based protein profiling (ABPP) that uses activity-based chemical probes to broadly assess the functional states of both characterized and uncharacterized enzymes. This review will focus on how ABPP, coupled with inhibitor discovery platforms and functional metabolomic technologies, have led to discoveries that have expanded our knowledge of metabolism in health and disease.

Copyright © 2014 Elsevier Ltd. All rights reserved.

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Figures

Figure 1
Figure 1. Activity-based protein profiling (ABPP)

ABPP uses active site-directed chemical probes to broadly assess the functional state of enzymes across enzyme families. These probes consist of a reactive group and a detection handle, most commonly rhodamine (Rh) or biotin (B). In gel-based ABPP, native proteomes are reacted with the probe and proteins are separated by SDS-PAGE and visualized by fluorescent scanning. MS-based ABPP facilitates the identification and quantification of enzyme activities following avidin enrichment, on-bead tryptic digest, and resolution by Multidimensional Protein Identification Technology (MudPIT).

Figure 2
Figure 2. Competitive ABPP and fluopol ABPP for the discovery of enzyme inhibitors

(A) Competitive ABPP assesses the potency and selectivity of small molecule inhibitors in native proteomes by competing with the ability of the probe to bind. Enzyme inhibition is indicated by a loss of fluorescent intensity by gel or by a loss of spectral counts by MS. (B) Fluopol ABPP is a HTS version of competitive ABPP conducted with pure or recombinant protein. Fluorescence polarization is high if enzyme activity is high (inactive inhibitor) and low if enzyme activity is low (active inhibitor).

Figure 3
Figure 3. Discovery Metabolite Profiling

In untargeted metabolomics, the mass spectrometer scans a large mass range (m/z 100-1,200) for known and unknown metabolites. Datasets are then analyzed by bioinformatics platforms which align, quantify, and identify metabolites that are significantly altered between treatment groups.

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