Metabolomics: Small Molecules, Single Cells

TECHNOLOGY FEATURE METABOLOMICS: SMALL MOLECULES, SINGLE CELLS Sensitive mass spectrometry and innovative cell-sampling techniques allow researchers to profile metabolites in single cells, but the field is still in its infancy. TORSTEN WITTMANN/SPL TORSTEN

Researchers are developing innovative methods to understand the inner workings of individual cells.

BY MARISSA FESSENDEN a clonal population can develop antibacterial between universities and companies in Japan resistance. It also gives rise to the different cell launched the Society for Single-Cell Surveyor, itting in his first-floor office, in an indus- subpopulations in the brain. And it explains which awards grants and holds symposia on trial lab space that opens onto a field of tumour relapses. The tools to detect those single-cell analysis and technologies. And in grazing cattle, analytical chemist Renato differences are only just becoming available. October, experts discussed launching the Inter- SZenobi explains one of the fundamental prob- “Recent technological advancements — national Human Cell Atlas Initiative, which lems facing today’s cell biologists. He traces out especially those made just in the past two aims to chart every type of human cell and its a curve representing the average concentra- years — have revealed that individual cells properties — an ambitious task that relies heav- tion of a molecule in a theoretical cell popula- within the same population may differ dramati- ily on single-cell analysis. tion — a simple bell-shaped distribution. That cally,” says Ananda Roy, programme leader of Much of this work focuses on revealing distribution, he explains, can obscure complex- the National Institutes of Health (NIH) Com- cell-to-cell differences at the DNA level. Yet ity. To prove the point, he sketches two curves mon Fund working group for single-cell analysis profiles of genes and epigenetic modifications overlapping either side of the single peak, each in Bethesda, Maryland. “And these differences merely outline a cell’s potential. The rapid, representing a distinct phenotype in the popula- can have important consequences for health dynamic responses a living cell has to its envi- tion — and also consistent with that bell-shaped and disease.” Funders worldwide have queued ronment are better reflected in the metabolic curve. “To really figure out if the distribution is up to support single-cell research. The NIH has transformations — and the resulting patterns of multimodal or bimodal, you need to go down invested in special initiatives to support single- small molecules — that keep the cell powered, to the single-cell level,” says Zenobi, at the Swiss cell profiling, starting with US$2 million in cycling and communicating with others. Federal Institute of Technology (ETH) in Zurich. 2014, and almost 60 groups have now received “The metabolome is most directly related to Cell heterogeneity is why some bacteria in awards under the programme. A collaboration the phenotype,” says Caroline Johnson, an

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analytical chemist at Yale School of Public doesn’t have immediate biomedical applica- micrometres in diameter. When the research- Health in New Haven, Connecticut. It reveals tions, Zenobi admits, but it does illuminate a ers add a dilute solution of cells to the slide, the the products of the genome and its protein out- fundamental way in which cells work. coating’s repellent properties ensure that some put, as well as metabolites from diet, drugs and To draw such insights, Zenobi’s team uses liquid, and one or two cells, end up in each toxic compounds. sophisticated techniques to isolate cells and to reservoir. The researchers then direct the spec- Yet its complexity means that metabolomics boost the sensitivity of its analytical approaches. trometer’s laser to target each well in turn. One has lagged behind other ‘omics’. Unlike DNA Researchers generally use either mass spectrom- configuration of these slides, called microarrays and RNA, metabolites cannot be ampli- etry or nuclear magnetic resonance (NMR) for mass spectrometry (MAMS), is available fied. Although some metabolites can reach to drive metabolomics studies. But because from MilliporeSigma in St. Louis, Missouri. millimolar concentrations, a single cell offers a NMR is less sensitive, mass spectrometry has Across the hall from Zenobi’s office, graduate limited volume for analysis. Exquisitely sensi- emerged as the method of choice, and there are student Robert Steinhoff demonstrates how the tive methods are needed to detect rarer com- many variations to improve its detection abili- slide fits into a mass spectrometer. The research- pounds — and the abundances can change in ties, throughput or simply make it easier to pull ers use matrix-assisted laser desorption/ioni- seconds in a living cell. Metabolites also have a the contents out of a cell. “The whole arsenal zation (MALDI) coupled to a time-of-flight bewildering variety of structures. The Human of mass spectrometry has been thrown at this analyser, which requires them to spot their Metabolome Database contains records for problem,” Zenobi says. slides with a chemical matrix to drive ionization. more than 42,000 metabolites, from sugars to Mass spectrometry involves ionizing a sample By using a matrix that minimizes interference peptides to cofactors. But the total may be sig- to lend a charge to its constituent molecules. with the signal given off by small molecules, nificantly higher, and single analytical methods The charge then means that magnetic plates the team can detect often struggle to capture the chemical diversity. can nudge the molecules as they fly through a metabolites in the Still, the field is advancing, thanks to leaps in vacuum. Each molecule deflects by a different “Individual cells low attomole (10–18 detection capabilities, increasingly sophisticated amount, depending on its mass-to-charge ratio, behave very moles) range — and ways of isolating and handling single cells and such that by the time the molecules reach the interestingly and do so for about 1,000 developments in bioinformatics. “We are get- detector, they have resolved into their compo- unexpectedly.” cells per chip, which ting close to making single-cell metabolomics nent parts. The data appear as a complicated is relatively high robust,” says Jonathan Sweedler, an analytical plot of unidentified peaks, each corresponding throughput in the single-cell world. chemist at the University of Illinois at Urbana– to a different molecular entity. Steinhoff is also working to alter the substrate Champaign. “I can’t see exactly how it is going The method is straightforward — except used in his reservoirs. “Making nanostruc- to work, but I look at the increase in detectability when applied to single cells. Attempting to tures — pillars — within the spot helps us see and throughput of mass spectrometry and I say detect just a few molecules in a vanishingly things more reliably,” he explains. The mecha- it will happen.” small volume pushes the limits of modern nisms behind this effect aren’t yet clear, but instrumentation. Steinhoff says that the cells end up clinging to PUSHING SENSITIVITY Zenobi uses a specialized silicon slide to the tops of the silicon columns. Profiling individual yeast cells allowed Zenobi’s individually deliver hundreds of single cells Sweedler has developed a high-throughput group to spot two phenotypes lurking in a into the mass spectrometer. To the naked approach that uses a computer to guide the genetically identical sample — one character- eye, the slides seem to be covered with a fine laser to individual cells spread across a slide. ized by low levels of a metabolite called fructose black mesh. The mesh is a coating of a poly- His team can process about 10,000 cells per 1,6-bisphosphate, and another with high levels1. mer called polysilazane, which has been laser slide in this way. But for a more comprehensive The difference probably comes down to differ- micro-machined to create hundreds to thou- look at the metabolome, Sweedler takes it one ent glucose-utilization strategies. The insight sands of reservoirs, each a couple of hundred cell at a time. He uses a modified patch-clamp tool, which typically records cellular electrical signals, to withdraw roughly three picolitres of SELECTED METABOLOMICS SOFTWARE cytoplasm (about 10–40% of the total volume) Name Description from individual brain cells and deliver it into a mass spectrometer. Human Metabolome Free database of metabolites in the human body, including details relevant to Database chemical profiles, molecular biology and clinical chemistry. Links to pathway and The limited throughput restricts analysis to a structure-viewing tools and to other databases. few dozen cells per experiment. Still, Sweedler’s KEGG Pathway Visualization tool linked to a collection of manually drawn pathway maps (KEGG group has used it to detect about 60 metabolites database) with ability to locate and colour specific metabolites and pathways. from 30 neurons and astrocytes in slices of rat 2 LipidBlast A tandem-mass-spectral database for lipid identification. brain . The team focused on neurochemicals such as glutamate, but also detected amino acids MapMan Tool for converting large data sets, such as those from gene-expression arrays, into metabolic pathway diagrams. Focuses on plant metabolomics. and derivatives of ATP, among others. From that, they compiled unique profiles for the dif- MetaboAnalyst Complete workflow for metabolomics data processing and analysis for both mass spectrometry and nuclear magnetic resonance data with common statistical ferent cell types, providing a window into the analyses and heat-map visualizations. cell-to-cell heterogeneity that makes the brain so complex, Sweedler says. Metabolites Biological A server that integrates chemical and biological annotations from a handful of Role (MBRole) databases to identify potentially important metabolites and other molecules. DIFFERENT STROKES FOR DIFFERENT SIZES MS-Dial Downloadable program that spots and identifies peaks in mass-spectrometry data, developed for small molecules. When dealing with single cells, size matters. Plant cells can be anywhere from 10 to OpenMS Open-source library for C++-based data analysis and management that includes infrastructure for software development. Mainly for processing raw data. 100 micrometres across. Mammalian cells tend to be smaller, on the scale of 10–20 micrometres. XCMS Online Cloud-based platform for metabolite profiling from raw liquid-chromatography and mass-spectrometry data and for data sharing. Microbial cells are smaller still, reaching down to the submicrometre range. As cell size varies, Yeast Metabolome Manually curated collection of yeast metabolites and their chemical properties, Database with links to spectral and chemical databases. so too does the volume and thus the absolute number of metabolites. “It is obvious from an

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analytical perspective there will be no single to profile tens or maybe a hundred different method that can deal with all these volumes,” molecules. But a single yeast cell can have some says Akos Vertes, a chemist at George Washing- 600 metabolites7. As a result, even the most sen- ton University in Washington DC. His lab uses sitive analytical techniques are picking up only

ET AL./ANAL. CHEM. ET different methods for different cell sizes. the easiest-to-detect, most prevalent molecules For the largest cells, the team uses a sharp- in a cell; less-common ones fall below the radar. ened optical fibre to deliver infrared light A possible solution to this problem, says J. T. AERTS AERTS J. T. directly into the cell. The light excites the computational biologist Jianguo Xia of McGill oxygen–hydrogen bonds in the water of a cell, University in Montreal, Canada, may lie in causing the cell to explode and eject its contents. population-based tools and in various ’omics. The escaping material then meets an aero- “The pipelines and tools that have been devel- solized, ionized liquid called an electrospray, oped for metabolomics data sets generated from which charges the molecules for mass spec- bulk cell populations can be reused,” he says. All trometry. The advantage of this technique is that that may be needed are slight modifications to single cells can be profiled while still embedded the data-processing and normalization methods in tissue. But it also can be very slow, because that researchers use. “Single-cell transcriptom- each cell generally needs to be poked with the ics is already established, and we can learn from fibre by “a very patient graduate student”, Vertes it to accelerate bioinformatics development for says. He recently automated the process, using a single-cell metabolomics,” he adds. computer to manoeuvre the sample stage. Other researchers are pursuing single-cell The smallest cells are deposited on a The cytoplasm of a cell can now be extracted and strategies that are based on methods other than nanopillar-covered surface, also made of its contents analysed. mass spectrometry — in particular, using living silicon although fabricated differently from cells. Matthias Heinemann, for instance, who Steinhoff’s. Imaging the entire surface reveals inject the contents into the mass spectrometer. collaborated with Zenobi during his postdoc, where the instrument’s ion beam needs to aim Adding the visual component allows his group now heads a molecular systems-biology group at to target single cells. With this method, the to complete delicate manoeuvres, such as cap- the University of Groningen in the Netherlands. team can routinely detect metabolites at femto- turing and analysing the amino acid and lipid There, he uses his background in biochemi- mole (10–15 moles) levels3. But the investigators contents of single white blood cells and tumour cal engineering to “find other ways to zoom estimate their lower limit of detection at about cells circulating in the blood5. into single cells”. In one approach, his group 800 zeptomoles (one zeptomole is 10–21 of a But it also allows his team to estimate molec- uses fluorescent molecular sensors to quantify mole), or about 482,000 molecules. ular abundance. Often, such a seemingly trivial metabolites, such as ATP. In a forthcoming paper It is possible to go even smaller. Bioanalyti- calculation is complicated, because researchers in Molecular Cell, his group has used time-lapse cal chemist Andrew Ewing at the University aren’t sure exactly how much volume they are microscopy to watch levels of ATP and another of Gothenburg in Sweden examines the small- analysing. So Masujima and his colleagues use metabolite, NADH (which is autofluorescent), molecule content of neural vesicles. These 3D microscopy to observe the cell before and oscillate as the cells go through the cell cycle8. vesicles are key to delivering and releasing after removing a small amount of cytoplasm. Key technical challenges have already been chemicals into the space between cells to facili- By measuring the resulting deformation, they solved when it comes to single-cell metabo- tate cell-to-cell communication. “It’s tricky can deduce the volume they removed as well as lomics, Heinemann says. “What is needed now because you get very little signal with so few the location from which it was taken6. In one is to do tedious development and validation molecules in there,” he says. case, they sampled a cytosolic metabolite called work.” The process might not be glamorous, he Ewing uses an approach called nanoscale methionine sulfoxide, determining that they admits, but it is essential if single-cell metabo- secondary ion mass spectrometry (NanoSIMS), had captured 5.9 zeptomoles of it. lomics is ever to make the leap from proof of which bombards a sample surface with a high- concept to answering fundamental biological energy beam of caesium ions. This assault ejects MAKING SENSE OF THE DATA questions. charged particles from the surface, and they Fortunately, says Gary Siuzdak, a chemist who “We are often so happy to detect unique can then be analysed in a mass spectrometer to heads the Scripps Center for Metabolomics and molecules, but my point is: why?” Masujima determine the material’s composition. Ewing’s Mass Spectrometry in La Jolla, California, bio- says. “What is behind this finding, why does group uses the method to assess distribution of informatics tools are available to make sense of this molecule come up?” Without that insight, the neurotransmitter dopamine. And by cor- such findings. techniques run the risk of being mere gim- relating the NanoSIMS data with transmission Siuzdak’s centre runs a cloud-based micks that fail to address questions of biological electron microscopy to observe a single vesicle metabololomic analysis platform called XCMS significance. “I don’t want to be a scientist who as it loads and unloads dopamine, the research- Online (see ‘Selected metabolomics soft- does that kind of science,” he says. ■ ers determined that a vesicle’s inner shape might ware’), whose 12,000-plus users have collec- regulate how quickly this process happens4. tively shared more than 120,000 jobs. Few of Marissa Fessenden is a freelance writer in those jobs have involved single cells, Siuzdak Bozeman, Montana. SEEING IS BELIEVING acknowledges, but that doesn’t mean that they 1. Ibáñez, A. J. et al. Proc. Natl Acad. Sci. USA 110, At the RIKEN Quantitative Biology Center in are inherently incompatible with the software. 8790–8794 (2013). 2. Aerts, J. T. et al. Anal. Chem. 86, 3203–3208 (2014). Osaka, Japan, chemist Tsutomu Masujima uses “On the bioinformatics side, I don’t see a major 3. Walker, B. N., Antonakos, C., Retterer, S. T. & Vertes, a video feed to target single cells for mass spec- issue with doing these experiments,” he says. A. Angew. Chem. Int. Ed. Engl. 52, 3650–3653 trometry. Rather, the main challenge with single- (2013). 4. Lovrić, J. et al. ACS Nano (in the press). “Individual cells behave very interestingly cell metabolomics is one of instrumentation: 5. Hiyama, E. et al. Anal. Sci. 31, 1215–1217 (2015). and unexpectedly, so I like to see [them] as devices that can analyse enough single cells, and 6. Ali, A. et al. Anal. Sci. 32, 125–127 (2016). much as possible,” Masujima says. His approach enough metabolites per cell, for the results to 7. Förster, J., Famili, I., Fu, P., Palsson, B. Ø. & Nielsen, J. involves inserting a nanospray needle directly be statistically meaningful. “The primary issue Genome Res. 13, 244–253 (2003). 8. Papagiannakis, A., Niebel, B., Wit, E. & into a cell under video observation, sucking out with single cells is that the hardware still needs Heinemann, M. Mol. Cell http://dx/doi. the contents and then using the same needle to to be improved,” Siuzdak says. Studies tend org/10.1016/j.molcel.2016.11.018 (in the press).

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CORRECTION The Technology Feature ‘Metabolomics: small molecules, single cells’ (Nature 540, 153–155; 2016) erroneously stated that Matthias Heinemann was a former postdoc in Renato Zenobi’s lab. Although he worked with Zenobi, Heinemann was a postdoc in another lab at the time. Also, Heinemann’s background was in biochemical engineering, not analytical chemistry.