With a new strategy for global metabolite profiling that identifies all substrates for a given enzyme comes a link between proteome and metabolome.
Let's imagine the following scenario: a research group starts working on a particular enzyme with the question, "what are the substrates my enzyme is acting on?" As fundamental as this question is, no method for obtaining a comprehensive answer has yet been described. Alan Saghatelian and his colleagues in Benjamin Cravatt's lab at the Scripps Research Institute sought to change that. They developed a strategy that uses untargeted liquid chromatography−mass spectrometry (LC-MS) to analyze all metabolites generated by a particular enzyme. Their approach, which they call discovery metabolite profiling (DMP), is described in a recent paper in Biochemistry (Saghatelian et al., 2004).
DMP is based on the comparison of changes in metabolite levels in the presence or the absence of an enzyme. The lipase fatty acid amide hydrolase (FAAH) was the enzyme of choice for Cravatt's group. They extracted lipophilic compounds from brains and spinal cords of wild-type or FAAH knockout mice and compared the LC-MS spectra over a wide mass range. Peaks seen in the knockout but not in the wild-type sample represented FAAH substrates.
The originality and strength of DMP lies in the fact that it is untargeted and uses no standard for quantification of compounds, thus allowing a comprehensive analysis of the whole metabolome. In targeted LC-MS methods, researchers start with a known metabolite of interest and use a standard that is similar in mass. This allows absolute quantitative measurements, but eliminates the possibility of detecting new substrates.
Saghatelian was first able to identify known substrates with DMP, thus validating the method. What came as a surprise was that he also discovered a new class of metabolites, taurine-conjugated fatty acids, which have not been associated with FAAH previously. This finding is intriguing for several reasons. First, it demonstrates the ability of DMP to identify a set of completely unexpected substrates. Second, it underscores the importance of a method that starts with an in vivo sample to characterize the enzymatic regulation of metabolites. The taurine conjugates proved to be extremely poor substrates for FAAH in vitro and their connection to FAAH would never have been suspected based on in vitro data.
To show the potential of DMP even more clearly, Saghatelian plans to expand it in several ways. As the method requires an enzyme-depleted sample, he wants to use RNA interference or chemical inhibitors, rather than the more cumbersome gene knockout method, to reduce enzyme levels. To show that DMP is reproducible and not restricted to discovering FAAH metabolites, he intends to test it with other lipases. Finally, by modifying the way samples are fractionated and prepared for LC-MS, Saghatelian will no longer be restricted to lipophilic compounds but will also be able to include water-soluble metabolites in the analysis, thereby expanding the list of enzymes DMP can be used for.
With all its potential, this method will most likely not be confined to one laboratory for long, and Cravatt has high hopes for the future of DMP: "We anticipate that these findings will inspire every scientist to apply DMP to his or her enzyme of interest to elucidate its endogenous function and facilitate its integration into larger metabolic networks in the cell."
Saghatelian, A. et al. Assignment of endogenous substrates to enzymes by global metabolite profiling. Biochemistry43, 14332−14339 (2004). | Article | PubMed | ChemPort |
