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Photoaffinity labeling (PAL) methodologies have proven to be instrumental for the unbiased deconvolution of protein-ligand binding events in physiologically relevant systems. However, like other chemical proteomic workflows, they are limited in many ways by time-intensive sample manipulations and data acquisition techniques. Here, we describe an approach to address this challenge through the innovation of a carboxylate bead-based protein cleanup procedure to remove excess small-molecule contaminants and couple it to plate-based, proteomic sample processing as a semiautomated solution. The analysis of samples via label-free, data-independent acquisition (DIA) techniques led to significant improvements on a workflow time per sample basis over current standard practices. Experiments utilizing three established PAL ligands with known targets, (+)-JQ-1, lenalidomide, and dasatinib, demonstrated the utility of having the flexibility to design experiments with a myriad of variables. Data revealed that this workflow can enable the confident identification and rank ordering of known and putative targets with outstanding protein signal-to-background enrichment sensitivity. This unified end-to-end throughput strategy for processing and analyzing these complex samples could greatly facilitate efficient drug discovery efforts and open up new opportunities in the chemical proteomics field.

The gut microbiome is a key contributor to xenobiotic metabolism. Polycyclic aromatic hydrocarbons (PAHs) are an abundant class of environmental contaminants that have varying levels of carcinogenicity depending on their individual structures. Little is known about how the gut microbiome affects the rates of PAH metabolism. This study sought to determine the role that the gut microbiome has in determining the various aspects of metabolism in the liver, before and after exposure to two structurally different PAHs, benzo[ a ]pyrene and 1-nitropyrene. Following exposures, the metabolic rates of PAH metabolism were measured, and activity-based protein profiling was performed. We observed differences in PAH metabolism rates between germ-free and conventional mice under both unexposed and exposed conditions. Our activity-based protein profiling (ABPP) analysis showed that, under unexposed conditions, there were only minor differences in total P450 activity in germ-free mice relative to conventional mice. However, we observed distinct activity profiles in response to corn oil vehicle and PAH treatment, primarily in the case of 1-NP treatment. This study revealed that the repertoire of active P450s in the liver is impacted by the presence of the gut microbiome, which modifies PAH metabolism in a substrate-specific fashion.

Bile is a digestive fluid produced in the liver and stored in the gallbladder. It participates in absorption of fatty nutrients and vitamins, and aids in elimination of metabolic waste and toxins. The major chemical components of bile are bile salts that, apart from their function in digestion, are also known to participate in cell signaling by binding host farnesoid X (FXR), vitamin D (VDR), and G-protein coupled bile acid (TGR5) receptors. Microbial bile salt hydrolases (BSHs) catalyze bile salt deconjugation, a gatekeeper reaction that is a prerequisite for all subsequent microbial transformations of bile acids. As a result, BSH determines the composition of the bile salt and acid pools, which in turn affects its nutrient absorption and signaling capabilities. BSH profiling remains a challenge due to a paucity of tools that enable scientists to study its function. In this chapter, we discuss current BSH profiling approaches and demonstrate a novel fluorogenic probe-based assay that circumvents laborious and resource intensive BSH quantification methods. Alongside our assay protocol, we provide the reader with a detailed method for microbial cell extraction from fecal matter. We also cover probe validation protocols that can be adapted for Michaelis-Menten analysis with any BSH expressing strain., (Copyright © 2022 Elsevier Inc. All rights reserved.)