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Mini-metabolomics Glucose 6.7 Recently, metabolomics, a biotransfer metabolites such as ATP logical field devoted to studying and NADH. First, metabolite Glucose-6-phosphate/fructose-6-phosphate 1.8 ± 0.4 cellular processes by measuring standards of known identity and Fructose-1,6-bisphosphate 0.6 ± 0.0 chemical signatures, has expanded concentration were run through dramatically. The characterization the LC/MS system. Then, the Glyceraldehyde-3-phosphate 0.5 ± 0.1 of metabolites by analytical techresearchers matched retention Glycolysis niques such as MS and NMR can times and MS spectra from these 0.9 ± 0.2 1,3-Bisphosphoglycerate yield information about signaling standards to the data from an islet pathways, gene function, pharmaextract. To further solidify an as2/3-Phosphoglycerate 1.2 ± 0.3 ceutical effects, and diseases. In signment, they used a technique a paper published in AC (2008, called induced dissociation, which Phosphoenolpyruvate 4.6 ± 0.7 80, 3539–3546), Robert Kennedy Kennedy describes as “breaking Pyruvate 13.7 ± 2.5 and co-workers at the University molecules up to see if they break of Michigan Ann Arbor describe up the same way.” The researchers Acetyl-CoA 7.7 ± 1.1 2.4 ± 0.7 Oxaloacetate a miniaturized metabolomics apcould identify 22 of the 200 preproach for analyzing single islets of sumed metabolites observed in an Langerhans. These clusters of cells, 4.5 ± 0.8 Malate islet sample. located in the pancreas, secrete inWith these important meCitrate/isocitrate 4.4 ± 1.0 sulin; consequently, their malfunc- 3.2 ± 1.1 Fumarate TCA cycle tabolites identified, it was time tion can lead to diabetes. to begin asking biologically relSo far, there hasn’t been a large evant questions and to determine 8.3 ± 1.9 Succinate push in the field of metabolomics whether the micromethod was α-Ketoglutarate 1.0 ± 0.2 to go to smaller scales. Most studies sensitive enough to detect differ2.6 ± 0.4 Succinyl-CoA have focused on detecting metaboences between islets exposed to lites from abundant sources: cell Glycolysis and TCA cycle intermediates identified by capil- varying environments. Kennedy cultures, plants, microbes, or bodily lary LC/MS. The ratio of metabolite concentration in single and co-workers compared the fluids. But Kennedy saw a need to islets cultured in 20 mM vs 3 mM glucose is given. The metabolite concentrations from develop metabolomics methods arrow indicates the direction of change. islets cultured in 3 mM versus 20 for smaller, more precious samples. mM glucose (a concentration that “Working on a small scale makes sense “The column we make ourselves,” elicits insulin secretion). Their results because of the difficulty of obtaining Kennedy says. “We like to make them square with previous studies but pro[islets],” he says. “It takes a steady hand.” a little smaller than what is available vide additional metabolic insights. For After isolation from a mouse pancommercially.” example, says Kennedy, “one thing I’m creas, each individual islet, which conEven though each islet’s metabolites still kind of surprised at was how big tains ~3000 cells, was transferred to an are reconstituted in 20 μL of formic of a change we saw in pyruvate.” The Eppendorf tube. Cellular metabolism acid, only 15 nL is injected at a time into meaning of this and other observations was then arrested by a dip in liquid the 75 μm i.d. column, making it posis still being sorted out. nitrogen, followed by the extraction of sible to perform multiple assays on the Kennedy says that this study was intracellular metabolites with methasame islet. As the sample exits the colmore about demonstrating high sennol–water, ultrasonication, and centrifu- umn, it is continuously fed into an ESI sitivity in their miniaturized LC/MS gation. The final product is a powder of mass spectrometer that is connected disystem than about biology, but in the metabolites that can be stored for up to rectly to the column, and several spectra long term, his group is definitely inter11 days at –80 °C and then reconstituted are collected per second. The detection ested in applying its system to biological in a formic acid solution for analysis. limit for the capillary LC/MS system in questions. Currently, his research group This dense broth of metabolites this study was calculated to be 0.1–0.33 is analyzing islets from mouse models of would overwhelm a mass spectrometer fmol, which translates into metabolomic diabetes, but the method isn’t limited if injected directly, so before running analysis of as few as two cells at a time. to islets. “It should be applicable for the sample through the instrument, the The next task was to identify signals anything with a really small sample,” researchers separated the metabolites from metabolites involved in insulin says Kennedy—which is good. It’s a by reversed-phase LC. But for such tiny secretion, such as glycolysis and TCA small world, after all. a samples, they needed a microcolumn. cycle intermediates, as well as energy —Erika Gebel J u n e 1 , 2 0 0 8 / A n a ly t i c a l C h e m i s t r y
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