Class assignment of sequence-unrelated members of enzyme superfamilies by activity-based protein profiling.
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Amino Acid Sequence↗
Biomedical subjects
Matthew P Patricelli
Publications and source records attributed to Matthew P Patricelli.
High-resolution functional proteomics by active-site peptide profiling.
Characterization and functional annotation of the large number of proteins predicted from genome sequencing projects poses a major scientific challenge. Whereas several proteomics techniques have been developed to quantify the abundance of proteins, these methods provide little information regarding protein function. Here, we present a gel-free platform that permits ultrasensitive, quantitative, and high-resolution analyses of protein activities in proteomes, including highly problematic samples such as undiluted plasma. We demonstrate the value of this platform for the discovery of both disease-related enzyme activities and specific inhibitors that target these proteins.
Animals↗
Mapping enzyme active sites in complex proteomes.
Genome sequencing projects have uncovered many novel enzymes and enzyme classes for which knowledge of active site structure and mechanism is limited. To facilitate mechanistic investigations of the numerous enzymes encoded by prokaryotic and eukaryotic genomes, new methods are needed to analyze enzyme function in samples of high biocomplexity. Here, we describe a general strategy for profiling enzyme active sites in whole proteomes that utilizes activity-based chemical probes coupled with a gel-free analysis platform. We apply this gel-free strategy to identify the sites of labeling on enzymes targeted by sulfonate ester probes. For each enzyme examined, probe labeling was found to occur on a conserved active site residue, including catalytic nucleophiles (e.g., C32 in glutathione S-transferase omega) and bases/acids (e.g., E269 in aldehyde dehydrogenase-1; D204 in enoyl CoA hydratase-1), as well as residues of unknown function (e.g., D127 in 3 beta-hydroxysteroid dehydrogenase/isomerase-1). These results reveal that sulfonate ester probes are remarkably versatile activity-based profiling reagents capable of labeling a diversity of catalytic residues in a range of mechanistically distinct enzymes. More generally, the gel-free strategy described herein, by consolidating into a single step the identification of both protein targets of activity-based probes and the specific residues labeled by these reagents, provides a novel platform in which the proteomic comparison of enzymes can be accomplished in unison with a mechanistic analysis of their active sites.
Aldehyde Dehydrogenase↗
Activity-based probes for functional proteomics.
Achieving an understanding of the functional role of all proteins expressed by a complex organism will require the development of methods that enable the rapid monitoring of protein function on a global scale. Current genomics and proteomics technologies fall short of this goal since they measure only the relative abundance of transcripts and proteins, respectively. Recent efforts in several laboratories have led to the development of tagged chemical probes that selectively react with families of active enzymes based on shared mechanistic features. These activity-based probes (ABPs) permit the quantitation and comparison of multiple protein activities simultaneously in complex proteomes. In this paper, the general properties and design features of ABPs will be discussed with an emphasis on the use of ABPs for activity-based proteome analysis.
Affinity Labels↗