TECHNOLOGY FEATURE Time to apply 119 Building pathways 120 Box 1: Metabolomics in situ 121 Metabolomics: from small molecules to big ideas Monya Baker The focus of metabolomic studies is shifting from cataloging chemical structures to finding biological stories. In 2004, when Alan Saghatelian wanted to know what reactions a particular enzyme catalyzed in vivo, he had to plow through a stack of paper two feet high. Standard in vitro techniques to uncover enzymes’ functions would have been much more straightfor- ward, but such studies work only when the right substrates are screened and even then might represent reactions that only occur in a test tube. To get a read on enzymatic activity in actual cells, Saghatelian, then a postdoc in Benjamin Cravatt’s laboratory at The Scripps Research Institute, developed an ‘untargeted metabolomics’ strategy to find previously unidentified substrates. Saghatelian lysed cells with and without the enzyme, ran the Diego San California, of University laboratory, Palsson Lewis, Nathan lysates through a mass spectrometer and Genomic, proteomic and metabolomic data can all be integrated using genome-scale metabolic compared the small molecule profiles found network reconstructions. in each sample. Nature America, Inc. All rights reserved. All rights Inc. America, Nature 1 This approach, which they termed dis- organism. The assumption was that a list Unlike genes, transcripts and proteins, covery metabolite profiling, yielded sub- of human metabolites could be as useful metabolites are not encoded in the genome. © 201 strates unlikely to have been found in vitro as a list of human genes. Such a list could They are also chemically diverse, consist- and led to the discovery of a new class of help researchers probe multiple pathways ing of carbohydrates, amino acids, lipids, mammalian lipids as substrates for an simultaneously, and collections of metabo- nucleotides and more. enzyme considered well characterized1. lites consistently associated with biological Extraction, separation and analytic Identifying the substrates, though, was traits could become biomarkers for identi- techniques that work for one class of slow going. “I had to manually look at each fying diseases and optimal treatments. metabolites are often useless for others. mass range [of chromatograms from the Researchers have only recently been With nucleic acids and proteins one detec- spectrometer], looking for ions that had able to integrate all the necessary steps to tion technique will usually suffice, but the changed between the two samples,” says produce profiles consistently. “It’s been a metabolomic community must rely on Saghatelian, now at Harvard University. decade-long progression to measure more a suite of detection techniques, includ- Things are different now, he says. Not only metabolites more reliably,” says Joshua ing capillary electrophoresis–mass spec- are mass spectrometers better at resolving Rabinowitz at Princeton University. trometry, liquid chromatography–mass and measuring compounds, instrument Profiling the complete metabolome spectrometry, gas chromatography–mass vendors all now sell software for compar- remains an impossible task for now, but spectrometry and nuclear magnetic reso- ing samples automatically. with dependable methods in hand, he nance, all paired with various enrichment The word ‘metabolomics’ was coined in says, the field is shifting from cataloging and separation technologies. 1998. It refers to biochemistry writ large, metabolites to asking broader biological “Over the last 10 years, we have learned the study of the entire set of small mole- questions about how metabolites reflect that there is no one platform; there is no cules in a biological sample. In 2001, when and affect cell function. one method,” says Rima Kaddurah-Daouk a handful of biochemists organized them- at Duke University, one of the scientists selves into the Metabolomics Society, their The known unknowns who founded the Metabolomics Society. In chief goal was to produce a catalogue of the Small molecules are less tractable to cata- fact, she says, it is hard even to pin down metabolites that existed in any particular loging than are the objects of other ‘omics’. how many compounds a sample contains. NATURE METHODS | VOL.8 NO.2 | FEBRUARY 2011 | 117 TECHNOLOGY FEATURE a few thousand they make themselves as researchers. Members of Oliver Fiehn’s lab well as nutrients and pollutants from their at the University of California, Davis, for environment and substances produced by example, have ten different kinds of mass microbes in the gut. spectrometers and five protocols geared to “We use three different detection various compound classes. methods, and we still miss stuff,” says And detection is just part of the chal- Michael Snyder of Stanford University, lenge. Many compounds can be detected describing his work to detect metabo- lites that interact with proteins. Though researchers in Snyder’s laboratory have now developed techniques to look at hydrophilic compounds, their initial studies looked at hydrophobic ones not because these were more likely to be interesting but because these were technically easier to analyze2. Even so, he says, developing these assays required years of optimization and quality Denise Applewhite, Princeton Office of Communications of Office Princeton Applewhite, Denise control. Graduate student Xiyan Li often Josh Rabinowitz wants to remake the classic ran assays six times to make sure everything biochemistry diagrams to include quantitative was working and highly reproducible. information. Scientists in a few highly dedicated laboratories can look at all sorts of com- Most bacteria are believed to produce a few pounds, from volatile compounds to Oliver Fiehn is creating software to show how hundred metabolites; a plant may produce complex lipids, but that capacity requires different mass spectrometry setups will portray a few thousand. Animals contain perhaps extensive equipment and well-trained the same molecule. Nature America, Inc. All rights reserved. All rights Inc. America, Nature 1 © 201 118 | VOL.8 NO.2 | FEBRUARY 2011 | NATURE METHODS TECHNOLOGY FEATURE researchers identify genetic variation that predicts how a patient will respond to certain antidepressants, says Fiehn, one of several authors of a study showing how metabolomics can inform genomic studies3. Metabolomic profiling found that levels of the amino acid glycine dif- fered between individuals who stopped responding to antidepressants and those who continued to benefit from them. This led researchers to analyze genetic varia- tion in a range of metabolic genes in these individuals, focusing on genes associated with glycine synthesis and degradation. The team ultimately identified genetic variations that could help predict how an Alan Saghatelian says metabolomic profiling can uncover enzymes and pathways with therapeutic relevance. individual will respond to certain antide- pressants. Without metabolomic profiling, but not identified. Researchers can make experiments,” says Saghatelian. “If you Fiehn says, his colleagues would have been informed guesses and usually deduce com- find something that hasn’t been identified drowning in data. “It lets you know what pounds’ molecular formulas, but typically yet, you don’t know what to do with that. to look for,” he says. only about 25% of observed compounds When you find a new metabolite, you can’t In a different approach, Jeremy Nicholson can be tentatively identified. “Compound just knock it out like a gene or a protein; of Imperial College London is applying ana- identification is improving the fastest as it you have to figure out the pathways that lytical tools developed for genetic research had the furthest to come,” says Steven M. make it and break it down, and what it to metabolomics. Metabolite-wide asso- Fischer, senior applications specialist at binds to.” ciation studies, he says, could be equally Agilent Technologies. Recently, software But profiling studies can have big pay- or more useful than genome-wide associa- from Gary Siuzdak and Colin Smith at The offs. The metabolites present in a cell offer tion studies to classify disease and predict Scripps Research Institute has automated a better snapshot of what is happening at a responses to treatment. data analysis, making metabolomic analy- given moment than genes or even proteins, Metabolites themselves may have a sis much more accessible. Still, confirm- says Saghatelian. “What’s nice about these more important biological role than ing an unknown metabolite’s identity often metabolic profiling experiments is that you many scientists have assumed. Whereas requires not just puzzling over spectra can measure some changes you can’t see in the goal of hundreds of studies has been and running samples against known stan- genome sequencing, but it’s still clear that a to understand regulatory networks by Nature America, Inc. All rights reserved. All rights Inc. America, Nature 1 dards but also synthesizing compounds. pathway is important.” hunting for protein-protein and protein- Such unknowns can discourage research- DNA interactions, few studies have been © 201 ers from looking broadly at metabolites. Time to apply designed to look at protein-small molecule “If you’re not a synthetic chemist, you’re Combining metabolomic profiling interactions. In the first high-throughput not likely to want to do these kinds of with other omics techniques has helped study to do so, researchers led by Stanford Table 1 | Bioinformatic