The identification of metabolites from complex biological samples often involves matching experimental mass spectrometry data to signatures of compounds derived from massive chemical databases. However, misidentifications may result due to the complexity of potential chemical space that leads to databases containing compounds with nearly identical structures. Prior knowledge of compounds that may be enzymatically consumed or produced by an organism can help reduce misidentifications by restricting initial database searching to compounds that are likely to be present in a biological system. While databases such as UniProt allow for the identification of small molecules that may be consumed or generated by enzymes encoded in an organism's genome, currently no tool exists for identifying SMILES strings of metabolites associated with protein identifiers and expanding R-containing substructures to fully defined, biologically relevant chemical structures. Here we present Proteome2Metabolome (P2M), a tool that performs these tasks using external database querying behind a simple command line interface. Beyond mass spectrometry based applications, P2M can be generally used to identify biologically relevant chemical structures likely to be observed in a biological system.

While deprivation of dietary fiber has been associated with adverse health outcomes, investigations concerning the effect of dietary fiber on the gut microbiome have been largely limited to compositional sequence-based analyses or utilize a defined microbiota not native to the host. To extend understanding of the microbiome’s functional response to dietary fiber deprivation beyond correlative evidence from sequence-based analyses, approaches capable of measuring functional enzymatic activity are needed. In this study, we use an activity-based protein profiling (ABPP) approach to identify sugar metabolizing and transport proteins in native mouse gut microbiomes that respond with differential activity to the deprivation or supplementation of the soluble dietary fibers inulin and pectin. We found that the microbiome of mice subjected to a high fiber diet high in soluble fiber had increased functional activity of multiple proteins, including glycoside hydrolases, polysaccharide lyases, and sugar transport proteins from diverse taxa. The results point to an increase in activity of the Bifidobacterium shunt metabolic pathway in the microbiome of mice fed high fiber diets. In those subjected to a low fiber diet, we identified a shift from the degradation of dietary fibers to that of gut mucins, in particular by the recently isolated taxon “ Musculibacterium intestinale” , which experienced dramatic growth in response to fiber deprivation. When combined with metabolomics and shotgun metagenomics analyses, our findings provide a functional investigation of dietary fiber metabolism in the gut microbiome and demonstrates the power of a combined ABPP-multiomics approach for characterizing the response of the gut microbiome to perturbations.

The majority of methods for detecting differentially abundant proteins between samples in label-free LC-MS bottom-up proteomics experiments rely on statistically testing inferred protein abundances derived from peptide ionization intensities or averaging peptide level statistics. Here, we statistically test peptide ionization intensities directly and combine the resulting dependent P-values using the Empirical Brown's Method (EBM), avoiding error introduced through the estimation of protein abundances or summarizing test statistics. We show that on a spike-in proteomics dataset, a peptide level approach using EBM outperforms differential abundance detection using a protein level approach and several analysis workflows, including MSstats. Additionally, we demonstrate the effectiveness of this approach by detecting enriched proteins from an activity-based protein profiling dataset.

Microbial bile salt hydrolases (BSHs) found in the intestine catalyze the deconjugation of taurine‐ and glycine‐linked bile salts produced in the liver. The resulting bile salts are biological detergents and are critical in aiding lipophilic nutrient digestion. Therefore, the activity of BSHs in the gut microbiome is directly linked to human metabolism and overall health. Bile salt metabolism has also been associated with disease phenotypes such as liver and colorectal cancer. In order to reshape the gut microbiome to optimize bile salt metabolism, tools to characterize and quantify these processes must exist to enable a much‐improved understanding of how metabolism goes awry in the face of disease, and how it can be improved through an altered lifestyle and environment. Furthermore, it is necessary to attribute metabolic activity to specific members and BSHs within the microbiome. To this end, we have developed activity‐based probes with two different reactive groups to target bile salt hydrolases. These probes bind similarly to the authentic bile salt substrates, and we demonstrate enzyme labeling of active bile salt hydrolases by using purified protein, cell lysates, and in human stool.

This chapter provides the reader with an understanding of activity-based protein profiling (ABPP) and the ability to incorporate it into their own research. ABPP has been applied broadly across biological disciplines, ranging from plant science to clinical research. As the pool of available ABPs continues to expand and the sensitivity of chemoproteomics increases, this technique is becoming increasingly powerful. Techniques in mass-spectrometry (MS) can be used for the quantification of ABP target protein activity. The most common MS analysis of peptides derived from ABP-labeled proteins shares similarities with shotgun proteomics. As novel probes are developed and applied in unprecedented ways, the field of ABPP will continue to expand and reveal the functional mechanisms mediating biological phenomena. Drug discovery and assay development have benefited from the implementation of ABPP, particularly through defining the selectivity of small-molecule leads and the uncovering of pathways that contribute to off-target pharmacology.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants generated from combustion of carbon-based matter. Upon ingestion, these molecules can be bioactivated by cytochrome P450 monooxygenases to oxidized toxic metabolites. Some of these metabolites are potent carcinogens that can form irreversible adducts with DNA and other biological macromolecules. Conjugative enzymes, such as glutathione S-transferases or UDP-glucuronosyltransferases, are responsible for the detoxification and/or facilitate the elimination of these carcinogens. While responses to PAH exposures have been extensively studied for the bioactivating cytochrome P450 enzymes, much less is known regarding the response of glutathione S-transferases in mammalian systems. In this study, we investigated the expression and activity responses of murine hepatic glutathione S-transferases to benzo[ a]pyrene exposure using global proteomics and activity-based protein profiling for chemoproteomics, respectively. Using this approach, we identified several enzymes exhibiting increased activity including GSTA2, M1, M2, M4, M6, and P1. The activity of one GST enzyme, GSTA4, was found to be downregulated with increasing B[ a]P dose. Activity responses of several of these enzymes were identified as being expression-independent when comparing global and activity-based data sets, possibly alluding to as of yet unknown regulatory post-translational mechanisms.

Understanding the factors that regulate microbe function and microbial community assembly, function, and fitness is a grand challenge. A critical factor and an important enzyme cofactor and regulator of gene expression is cobalamin (vitamin B-12). Our knowledge of the roles of vitamin B-12 is limited, because technologies that enable in situ characterization of microbial metabolism and gene regulation with minimal impact on cell physiology are needed. To meet this need, we show that a synthetic probe mimic of B-12 supports the growth of B-12-auxotrophic bacteria and archaea. We demonstrate that a B-12 activity-based probe (B-12-ABP) is actively transported into Escherichia coli cells and converted to adenosyl-B-12-ABP akin to native B-12. Identification of the proteins that bind the B-12-ABP in vivo in E. coli, a Rhodobacteraceae sp. and Haloferax volcanii, demonstrate the specificity for known and novel B-12 protein targets. The B-12-ABP also regulates the B-12 dependent RNA riboswitch btuB and the transcription factor EutR. Our results demonstrate a new approach to gain knowledge about the role of B-12 in microbe functions. Our approach provides a powerful nondisruptive tool to analyze B-12 interactions in living cells and can be used to discover the role of B-12 in diverse microbial systems. IMPORTANCE We demonstrate that a cobalamin chemical probe can be used to investigate in vivo roles of vitamin B-12 in microbial growth and regulation by supporting the growth of B-12 auxotrophic bacteria and archaea, enabling biological activity with three different cell macromolecules (RNA, DNA, and proteins), and facilitating functional proteomics to characterize B-12-protein interactions. The B-12-ABP is both transcriptionally and translationally able to regulate gene expression analogous to natural vitamin B-12. The application of the B-12-ABP at biologically relevant concentrations facilitates a unique way to measure B-12 microbial dynamics and identify new B-12 protein targets in bacteria and archaea. We demonstrate that the B-12-ABP can be used to identify in vivo protein interactions across diverse microbes, from E. coli to microbes isolated from naturally occurring phototrophic biofilms to the salttolerant archaea Haloferax volcanii.

Glutathione S-transferases (GSTs) comprise a diverse family of phase II drug metabolizing enzymes whose shared function is the conjugation of reduced glutathione (GSH) to endo- and xenobiotics. Although the conglomerate activity of these enzymes can be measured, the isoform-specific contribution to the metabolism of xenobiotics in complex biological samples has not been possible. We have developed two activity-based probes (ABPs) that characterize active GSTs in mammalian tissues. The GST active site is composed of a GSH binding "G site" and a substrate binding "H site". Therefore, we developed (1) a GSH-based photoaffinity probe (GSTABP-G) to target the "G site", and (2) an ABP designed to mimic a substrate molecule and have "H site" activity (GSTABP-H). The GSTABP-G features a photoreactive moiety for UV-induced covalent binding to GSTs and GSH-binding enzymes. The GSTABP-H is a derivative of a known mechanism-based GST inhibitor that binds within the active site and inhibits GST activity. Validation of probe targets and "G" and "H" site specificity was carried out using a series of competition experiments in the liver. Herein, we present robust tools for the characterization of enzyme- and active site-specific GST activity in mammalian model systems.