Research:

Introduction

An important aspect of chemical biology is the elucidation of the molecular mechanisms that underlie important biological and pathological processes. Global metabolite profiling promises accelerate our understanding of these mechanisms by providing access to a portion of biomolecular space currently inaccessible by other methods. Our lab will focus on the development and application of liquid chromatography-mass spectrometry (LC-MS) based global metabolite profiling methods through research at the interface of chemistry and biology.

Endogenous biochemical characterization of enzymes

Enzymes play important roles in the regulation (production and degradation) of biologically active metabolites in vivo. Understanding the biochemical role of an enzyme in vivo requires a detailed understanding of the metabolic substrates of that enzyme. Traditionally, the substrate scope of an enzyme is defined through in vitro assays. However, these assays fail to account for important aspects of in vivo biology, such as post-translational modifications, competing metabolic pathways, and the existence of uncharacterized metabolites. This difficulty can be overcome through the use of a comparative metabolite profiling strategy to identify the endogenous substrates of an enzyme by quantitatively measuring changes in metabolite levels that result from the inhibition (i.e. chemical inhibition or genetic knockout) of the enzyme in vivo (Figure 1). Application of this approach to the study of biomedically important family enzymes will uncover the relevant endogenous substrates of these enzymes and will lay the foundation for future experiments designed to understand the impact of these enzymatic nodes in cellular and physiological processes.

Figure 1. Connecting the proteome and metabolome through global metabolite profiling. (A) Assigning enzyme function using comparative metabolite profiling begins by disruption of the enzyme activity of interest through either chemical or genetic means. Changes in the metabolite profile between disrupted (knockout) and undisrupted (wildtype) systems are then identified, using liquid chromatography-mass spectrometry (LC-MS) based comparative global metabolite profiling, structurally characterized through further MS analysis, FTMS and MSn approaches, and chemical synthesis. These compounds are then directly examined as substrates for the enzyme of interest. (B) Comparative global metabolite profiling of mouse tissue, where mass ion intensity ratios (knockout/wildtype) of metabolites are presented on three-dimensional surface plots. Global view of the relative levels of metabolites in brains, plotted over a mass range of 200-1200 Da and liquid chromatography retention times of 0-105 minutes. Knockout animals possess highly elevated levels of two classes of metabolites while other metabolites were unaltered in these samples. (Saghatelian, A.; Trauger, S. A.; Want, E. J.; Hawkins, E. G.; Siuzdak, G.; Cravatt, B. F., Assignment of endogenous substrates to enzymes by global metabolite profiling. Biochemistry, 2004, 43, 14332-14339.)

Protein-Metabolite Interactions (PMIs)

The assignment of enzyme-substrate interactions in vivo underscores the capacity of global metabolite profiling to establish important connections between the proteome and metabolome in complex biological settings. Of course, biology provides us with a multitude of other important protein-metabolite interactions (PMIs), such as the binding of metabolites to receptors and transport proteins. Thus, extending our metabolomics platform to include these other important, yet hard to identify, PMIs will be of tremendous value. However, unlike the enzymatic conversion of a substrate to a product the binding of a ligand to a receptor lacks a detectable metabolic signature. As a result, binding information between ligands and receptors is overlooked by our current metabolite profiling protocols and the challenge remains to refine our methodology to identify these important interactions. The key to identifying endogenous PMIs is the isolation of endogenous protein metabolite complexes from cells and tissues. The lab will approach this problem using two different and complementary strategies for the identification of endogenous protein metabolite interactions (Scheme 1).

Scheme 1. Metabolite profiling of endogenous protein-metabolite interactions (PMIs). (A) The enzyme catalyzed conversion of a substrate (gray) results in a measurable changes in the concentrations of the substrate as well as the product (red). These differences are revealed through the comparison of WT and KO samples by comparative global metabolite profiling. In contrast, the lack of a biochemical conversion in non-enzymatic protein-metabolite interactions (PMIs) requires the development of alternative strategies for their identification. (B) Two such strategies, affinity purification-metabolite profiling (AP-MP) and size exclusion chromatography-MP (SEC-MP), focus on the isolation of intact protein-metabolite complexes (PMCs) (center, brown protein-blue ligand structure) from complicated biological milieu and subsequent metabolite profiling of bound ligands. SEC-MP (left side) separates the proteome, including protein bound metabolites, from the metabolome by SEC. Comparison of the resulting metabolite profiles from WT samples against KO or antagonist treated samples (bottom center) will reveal specific endogenous PMIs. Alternatively, AP-MP (right side), uses a straightforward affinity purification (AP) step, such as an immunoprecipitation, to directly isolate a single protein-metabolite complex of interest.

Integrated molecular profiling

A complete molecular understanding of human physiology and pathology requires new technologies that enable the large-scale analysis of genes, proteins, and metabolites in complex biological systems. The application of integrated genomic, proteomic, and metabolomic methods will facilitate the coordinated profiling and functional characterization of biomolecules in complex biological systems (Figure 2). Such an integrated molecular profiling platform would retain all the advantages of more classical genomics/proteomics approaches, while at the same time gaining the capacity to elucidate endogenous biochemical activities for genes/proteins through the inclusion of metabolite profiling. To test this method we plan to study the process of adipocyte differentiation, which is of interest due to the health problems associated with obesity, such as coronary heart disease and diabetes. A complete inventory of the biomolecules that show altered expression and activity during adipogenesis would provide key insights into the metabolic and signaling pathways that support this cellular process. We anticipate that this study will yield an unprecedented boon of molecular information that, when viewed as a whole, will illuminate new biochemical networks that support the preadipocyte to adipocyte transition.

Figure 2. Integration of global molecular profiling technologies. The application of genomics, proteomics, and metabolomics reveals biochemical networks (from gene to metabolite) that support physiological and pathological processes.