Catherine Shaffer Shaffer is a freelance writer based in Mich. USA
Dated: 2/1/2006
 Chemical reactions in a biological system result in a number of intermediate molecules known as metabolites. Studying the nature of these metabolites can shed light on the functioning of the entire cellular system. The pursuit of this information has been variously described as metabolite profiling, metabolomics, and metabolomics. At this time, the use of these neologisms is still flexible, allowing for a great deal of overlap in their meaning, but in general, metabolite profiling is a study more likely to be found in the context of pharmaceutical research, whereas metabolomics is the domain
of systems biologists and metabonomics more of an environmental or ecological pursuit.
Challenges in metabolomics include the presence of a wide range of molecular weights and large variations in concentration, and the presence of polar and nonpolar, as well as organic and inorganic molecules. Mass spectrometry (MS) has emerged as the analytical method he study of metabolites, in large part because these differences are not as important in MS as in other techniques. Advances in technology are also making MS more efficient, faster, versatile, accessible, and affordable for researchers.
There are innumerable choices for MS and hyphenated MS techniques, each with its own contribution to the alphabet soup of mass spec acronyms. GC-MS and LC-MS, which combine chromatographic separation with a mass analysis, are some of the most popular hyphenated forms of MS.
At the Samuel Noble Roberts Foundation, Ardmore, Okla., an integrated functional genomics project focused on the stress response of the legume Medicago truncatulawas launched. The foundation is a private, nonprofit organization, founded in 1950 by Lloyd Noble, which strives for the improvement of agriculture. There is substantial interest at the foundation in legumes such as soybeans, alfalfa, beans, and peas, which fix atmospheric nitrogen and provide a nitrogen source for the plant, resulting in a high protein content. However, the study of legumes is complicated by the fact that they have large, complex polyploid genomes, and thus the genetics are difficult to understand and dissect.
The stress study, funded by National Science Foundation: Plant Genome Research Program, resulted in 640 samples taken from M. truncatula cell cultures at 21 time points when exposed to UV radiation (a sunburn), methyl jasmonate (to simulate a wound), and yeast elicitor (to simulate a fungal infection). Lloyd Sumner, PhD, an associate scientist at the foundation, presented findings related to the metabolomics portion of the project at the recent American Society for Mass Spectrometry Conference held in San Antonio, Texas. Sumner’s group took a multipronged approach, creating 3,000 metabolic profiles from the 640 samples using many different technologies, including GC-MS and LC-MS.
Using custom software, Sumner’s group extracted and processed the metabolic profiling data into an organized matrix for further statistical analysis. Cluster and correlation analyses indicated key metabolic changes in response to stress. Results included perturbation of the glycine, serine, and threonine biosynthetic pathways, identification of a threonine aldolase previously unknown in plants, and a suggestion of altered metabolism of coenzyme A, all of which Sumner and his colleagues describe as “a fundamental metabolic repartitioning of carbon resources following elicitation from primary toward secondary metabolism.” Sumner’s data illustrated metabolomics as a maturing tool useful in gene validation, gene discovery, mechanistic insight, and hypothesis generation.
The M. truncatula project has broad applications in its design and technology, and could be used with many other organisms and systems. “That’s the thing about the technology, we’re using it for plants, but it’s applicable to any system you want to study: yeast, microbes, humans, mice. . . . You have some differences in metabolites you’re looking at, but primary metabolites are conservedacross the system. Glucose is glucose, whether it’s from a plant or a rat,” says Sumner. The studies may yield useful information about natural products too. “Plants can’t get up and run away from their enemies,” says Sumner. “Over the eons they’ve evolved secondary metabolites—natural products. They are not necessary for survival, but provide competitive advantage and are associated with natural defense.”
Moving from plants to mammals, it becomes more difficult to design and execute metabolite profiling experiments. The traditional method of tracking metabolites in animals is by radioisotope tracer study, wherein the animal (or human) consumes a metabolite synthesized with an isotope such as carbon- 13. However, the costs, inconvenience, and hazards of the method have made it largely impractical, especially for the large-scale metabolomics projects stimulated by new advances in genomics.
Scientists at Target Discovery Inc., Palo Alto, Calif., are pioneering a metabolic profile method called MetaSIRMS (SIRMS, for stable isotope ratio mass spectrometry).
In MetaSIRMS, an animal or human subject consumes a bolus of metabolite synthesized with a stable isotope atom such as carbon-13, mixed with unlabeled metabolite. The ratio of labeled to unlabeled metabolite in the initial dose is known at the start of the experiment. Fluids are then monitored for the decrease in the ratio of labeled/unlabeled metabolite over time using mass spectrometry. The result is a metabolic profiling experiment with dramatically improved accuracy and reproducibility.
Luke Schneider, PhD, chief scientific officer at Target Discovery, is a passionate advocate for MetaSIRMS, systems biology, and hypothesis-based science in general. “The origins of the company were in proteomics and metabolomics. We knew at that time that the current global techniques being practiced were not going to address the questions at either the proteomics or metabolomics end of things. First, there was the complexity issue. With so many metabolites . . . looking at how the patterns change was going to be so cost-prohibitive that nobody could afford to do it, so we developed hypothesisdriven approaches. The second issue was with reproducibility of results. . . . MetaSIRMS gets you away from doing radioisotope work. It reduces the cost of doing an ADME study substantially.”
Target Discovery has just begun a collaborative study with PrecisionMed Inc., Solana Beach, Calif., working on wheat and milk protein metabolism in autistic children. It will use MetaSIRMS to pin down a relationship between the symptoms of autism and wheat and dairy in the diet that until now has been purely anecdotal. Autistic children will be given a meal containing a mixture of unlabeled metabolite and a metabolite made with a safe, stable isotope. Mass spectrometric analysis should, eventually, reveal the truth about whether there is an abnormality in the digestion or absorption of these proteins in autistic children.
The ultimate in precision and limit of detection for mass measurement in metabolomics is the Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer. These instruments retail for around $1 million. A collaboration between scientists from Bruker Daltonics and the University of North Carolina (UNC), Chapel Hill, N.C., used a 12-tesla FT-ICR to analyze metabolites in human blood samples, in order to demonstrate the application of FT-ICR to metabolomics. Twelve teslas is the highest field strength magnet commercially available in an FT-ICR. Results from this pilot experiment demonstrated that it was possibleto separate several hundred metabolites with better than 0.5 ppm resolution (with an internal calibration standard).
Bruker’s FT spectrometer is a hybrid which consists of a normal ionization source such as electrospray followed by a Q interface and then the FT-MS analyzer. The detection occurs when ions are injected into the magnetic field. The ions move in a orbit around the center of the field and the frequency of that motion (in rotations per unit time) is characteristic of the ion’s mass. This provides a very large advantage over a time-of-flight (TOF) instrument. The ions are not destroyed as they rotate, and thus a very long path length can be measured, which improves the accuracy and precision compared to the relatively short path length of the TOF detector. Performance also increases with increasing field strength.
The extremely high mass accuracy of the instrument allows researchers to calculate the elemental composition of each molecule. The high resolution makes it possible to detect hundreds of different metabolites by direct infusion, completely bypassing other separation steps such as liquid or gas chromatography. Furthermore, the total run time for each sample is less than a minute, making it a suitable instrument for highthroughput screening. Christoff Borschers, PhD, of UNC-Chapel Hill, cites intense interest in FT-MS for metabolomics. “I haven’t seen many papers, only one or two where they’re using FT-ICR for metabolomics. . . . We started this last year, and there’s so muchinterest. This is unbelievable.” Borschers has recently been awarded a $1 million grant from the NIH to purchase the instrument for a proteomics and metabolomics study.
FT-ICR MS will emerge as a dominant analytical method for metabolomics studies, especially as the daunting price tag of the instrument is mitigated by the high speed of analysis. For high-throughput laboratories, the cost of a FT instrument may well be lower calculated on a per sample basis. At the same time, different needs and experimental designs will keep other forms of mass spectrometry in use for a long time. Mass spectrometry is a tool that has the potential to untangle the highly complex human (or mammalian) metabolome, which is estimated at approximately 3,000 compounds.
Choosing the Right Mass Spectrometer The MetaSIRMS method is applicable to many studies, and can be used with different types of mass spectrometers. For a study focusing on the kinetics of a process, for example cholesterol catabolism, a simple, low-cost instrument is sufficient. Target Discovery Inc., Palo Alto, Calif., runs these types of assays on an ABI Mariner electrospray mass spectrometer.
For more exacting work, such as the identification of a drug product, a higher-end instrument is required. TOF (time-of-flight) mass spectroscopy provides the accuracy for most such studies. “People use all kinds of mass specs for this,” says Luke Schneider, chief scientific officer at Target Discovery. “We stick with the TOF, or rarely the Fourier-transform ion cyclotron resonance(FT-ICR). We do the up-front steps by liquid chromatography or capillary electrophoresis. Other people are using ion trap mass specs, and using the mass spec itself as a separation tool. We can get away with TOF-MS because we’re trying to drive to specific questions. People doing cfglobal searches use ion trap mass specs in real time.”
For this and other related articles visit www.genpromag.com. |
|
