New Mass Spectrometry Measurement Tools for Characterizing Individual Cells

New developments in measurement science are providing chemical information on single cells. Single-cell chemical assays provide unique information on cell heterogeneity and allow detailed studies of the metabolome and proteome for both snapshot and time-resolved studies (1,2). Recent papers describe multimodal chemical imaging (3), single-cell MALDI-MS supervised by immunocytochemical classification (4), integrating mass spectrometry with microphysiological systems for improved neurochemical studies (5), high throughput approaches (6), and others. This interview with Jonathan Sweedler explores the latest developments in mass spectrometry and separations for single cell analysis.

You have published on the use of multimodal chemical imaging, microfluidic devices, and spheroid cultures, hydrogels, scaffolds, and fibers combined with mass spectrometric technologies for improvement of gathering chemical information on cells. What prompted you to investigate this problem and methodology? What is unique or novel about your approach?

One of my group’s research major areas is to improve the chemical information we can obtain from individual cells. We have focused on enhancements to sampling, sample cleanup, separations, and detection for single-cell measurements. Rather than summarize the analytical enhancements we have created over the past >25 years, it is worth noting that using our approaches, we have discovered hundreds of neuropeptides and reported novel neurochemical pathways and unusual neuromodulators in animal models ranging from comb jellies, sea slugs, flatworms, and sea urchins to song birds, rodents, camels, and others. I think it is the intertwined nature of our method development and the application of the methods to cellular analyses that has enabled us to excel in both areas.

Would you explain for our readers the best current technology for studying the chemistry of single cells using MS?

This is a difficult question to answer as the “best” depends on the experimental goals. For example, if one needs information on the shape of a cell, microscopy is the “best” option. My research involves measuring the chemical contents of cells, with a special interest in measuring and understanding the molecules that are involved in cell-to-cell communication. These are the neurotransmitters, neuromodulators, and hormones in a cell. Perhaps one of the largest issues is that if a cell has a volume of a picoliter, then a chemical of interest at a concentration of 10 µM is at 10 amol. Said differently, a single-cell metabolomics measurement only measures the more abundant analytes because our measurements are always fighting detectability. Thus, many of the approaches incorporated into analytical workflows with larger samples, such as desalting and multistep sample cleanup approaches, do not work as well at the single-cell scale because of sample losses.

In terms of specific MS approaches, we have been measuring the small-molecule content of individual cells with capillary electrophoresis (CE) for more than two decades. What has changed is the information we can now gain from an optimized interface between a CE system and a mass spectrometer, allowing us to characterize a greater range of metabolites (7). Another option for some molecular classes is direct matrix-assisted laser desorption/ionization (MALDI)-MS. For this approach, we deposit cells onto a surface, which keeps the analytes at the relatively high concentration found within the cell. In fact, we performed single-cell neuropeptide measurements via MALDI-MS several decades ago, and have used this approach to characterize more than a thousand novel brain peptides across the animal kingdom.

From your perspective what are the most exciting developments in MS over the past five years-in terms of both applications and instrumentation development?

From a general perspective, the number of approaches that are being published on single-cell MS is exciting-according to a recent PubMed search, more than 1000 articles (and more than 100 in the last year alone) involve single-cell mass spectrometry! The fields of single-cell metabolomics and proteomics are exploding with innovative sampling approaches, improved small-volume separations, better interfaces to mass spectrometers, enhanced data analysis, and amazing applications. It is an exciting time.

Regarding my group’s research efforts, I am encouraged about a high throughput approach that allows us to deposit thousands of cells (or even cellular organelles) onto a surface, visualize their locations using optical microscopy, filter them based on size, shape, and even distance between closest neighbors, and then feed the selected locations into the MS instrument to acquire spectra only from the cells or objects of interest (8). Because MALDI-MS does not have to be destructive, we can measure the cell contents using MALDI time-of-flight (TOF)-MS, and perform another measurement on the cells using MALDI-TOF, secondary ion MS, or even MALDI ion cyclotron resonance MS (8,9). A recent enhancement allows us to use a liquid microjunction probe and select a few cells from among the thousands and perform CE–MS (10). In other words, we are now able to gain additional information by combining MS measurements from the same cells in a fairly high throughput analysis. For example, we recently demonstrated the ability to characterize lipids from 30,000 rodent brain cells (6).

What are some major gaps in knowledge for single-cell analysis and discovery that you would like to see more research and development time devoted to?

This is an interesting question. As the individual approaches improve both in the information content obtained from each cell and the throughput to measure many cells, addressing a range of exciting biological questions now becomes more feasible. When is single-cell data needed? The obvious answer is for samples of cell populations that are heterogeneous. My group (and others) study the brain, which contains intermingled cells types that can change state and function depending on animal state, behavior, and even memory (1,2). Some researchers look at how an oocyte becomes a complete organism, and others examine cell heterogeneity in terms of health and disease progression as in cancer. All are exciting areas. As far as technology development is concerned, we need better sampling approaches.

Another area that is lacking involves the currently available informatics tools and databases. Many data repositories have strict policies, including file size limitations, that must be met in order to deposit the mass spectra. As an example, this spring my group published a series of single-cell lipid profiles in Angewandte Chemie International Edition and Analytical Chemistry (4,6). The smaller dataset was 2 TB, and the larger was about 8 TB, and we could not find a repository that would accept the MS data. We were able to deposit the smaller dataset in our university databank (https://databank.illinois.edu/), but even they would not accept the larger one (from 30,000 individual cells). Single-cell data is somewhat different from what common repositories want; for example, for technical reasons we do not acquire tandem MS data for all cells, and so cannot deposit our individual cell spectra because they do not fit existing repository guidelines, but the cell heterogeneity data is unique and advances our understanding of lipid distributions within the brain.

What do you anticipate is your next major area of research?

It is hard to know where our research will take us and what will be our next area of attention. I can say that we will always work to create the newest technologies for small-volume measurements.

Besides technology development, I am excited about our recent focus towards understanding neuropeptides and hormones that have an enigmatic and unusual modification, the switch of a single amino acid from the L-form to the D-form, thereby creating a D-amino acid-containing (neuro)peptide (DAACP). While this modification had been known for several toxins across the animal kingdom (from cone snails to spiders, frog skin secretions, and platypus venom), it is a difficult modification to find because there is no associated mass shift. We have created several approaches to look for this modification (11), and have found a surprising number of DAACPs. Recently we characterized receptors and even the impact on animal function. In one case, the DAACP modification is required for the peptide to bind to the receptor and in another, both the all L-form and the DAACP bind, but the DAACP has a much longer half-life in the blood (12,13). We are finding more and more evidence of this modification in other animals. Nature continues to surprise us.

I also run a neurometabolomics and neuroproteomics center (http://neuroproteomics.scs.illinois.edu/) for the National Institutes of Drug Abuse, and we are becoming more involved in characterizing the brain’s response to pain, especially in terms of hormone and neuropeptide changes. Pain and the opioid epidemic continues to be a large issue in our society. I hope our research will help in charting a path forward.

What is one of the largest surprises of your career?

Without a doubt, becoming Editor-in-Chief of Analytical Chemistry. I really enjoyed being an associate editor for the journal, and did not foresee moving up to become Editor as part of my career path. As Analytical Chemistry continues to grow and thrive, its success raises some challenges. For example, in the seven years since I became Editor, the number of submissions has doubled (and my day remains 24 hours long). Obviously, with one part of my job increasing in this way, something else has to give-finding the right balance has presented some challenges. Regardless, I enjoy reading the manuscripts, interacting with authors, and helping move the journal forward while making sure all of the subfields of analytical chemistry thrive, including, of course, separations and MS.

References

1. E.K. Neumann T.D. Do, T.J. Comi, J.V. Sweedler, Exploring the Fundamental Structures of Life: Non‐targeted, Chemical Analysis of Single Cells and Subcellular Structures. Ange Chemie Int Ed.58, 9348–9364 (2018).
2. T.J. Comi, T.D. Do, S.S. Rubakhin J.V. Sweedler, Categorizing Cells on the Basis of their Chemical Profiles: Progress in Single-Cell Mass Spectrometry. J Am Chem Soc. 139, 3920–3929 (2017).
3. T. Cao, N. Morales-Soto, J. Jia, N.F. Baig, S.J. Dunham, J. Ellis, J.V. Sweedler, J.D. Shrout, P.W. Bohn, Spatiotemporal Dynamics of Molecular Messaging in Bacterial Co-Cultures Studied by Multimodal Chemical Imaging. In Photonic Diagnosis and Treatment of Infections and Inflammatory Diseases II, International Society for Optics and Photonics. 10863, 108630A (2019).
4. E.K. Neumann, T.J. Comi, S.S. Rubakhin, J.V. Sweedler, Lipid Heterogeneity Between Astrocytes and Neurons Revealed With Single Cell MALDI MS Supervised by Immunocytochemical Classification. Ange Chemie Int Ed.58, 5910–5914 (2019).
5. E.G. Tillmaand, and J.V. Sweedler, Integrating Mass Spectrometry With Microphysiological Systems for Improved Neurochemical Studies. Microphysiol Syst.2(4), (2018). doi: 10.21037/mps.2018.05.01.
6. E.K. Neumann, J.F. Ellis, A.E. Triplett, S.S. Rubakhin, J.V. Sweedler, Lipid Analysis of 30 000 Individual Rodent Cerebellar Cells Using High-Resolution Mass Spectrometry. Anal Chem.91, 7871–7878 (2019).
7. J.T. Aerts, K.R. Louis, S.R. Crandall, G. Govindaiah, C.L. Cox, J.V. Sweedler, Patch Clamp Electrophysiology and Capillary Electrophoresis-Mass Spectrometry Metabolomics for Single Cell Characterization. Anal Chem.86, 3203–3208 (2014).
8. T.J. Comi, E.K. Neumann, T.D. Do, J.V. Sweedler, microMS: A Python Platform for Image-Guided Mass Spectrometry Profiling. J Am Soc Mass Spectrom.28,1919–1928 (2017).
9. T.D. Do, J.F. Ellis, E.K. Neumann, T.J. Comi, E.G. Tillmaand, A.E. Lenhart, S.S. Rubakhin, J.V. Sweedler, Optically Guided Single Cell Mass Spectrometry of Rat Dorsal Root Ganglia to Profile Lipids, Peptides and Proteins. Chemphyschem.19, 1180–1191 (2018).
10. T.J. Comi, M.A. Makurath, M.C. Philip, S.S. Rubakhin, J.V. Sweedler, MALDI MS Guided Liquid Microjunction Extraction for Capillary Electrophoresis-Electrospray Ionization MS Analysis of Single Pancreatic Islet Cells. Anal Chem.89, 7765–7772 (2017).
11. I. Livnat, H.C. Tai, E.T. Jansson, L. Bai, E.V. Romanova, T.T. Chen, K. Yu, S.A. Chen, Y. Zhang, Z.Y. Wang, D.D. Liu, K.R. Weiss, J. Jing, J.V. Sweedler, A d-Amino Acid-Containing Neuropeptide Discovery Funnel. Anal Chem.88, 11868–11876 (2016).
12. J.W. Checco, G. Zhang, W.D. Yuan, Z.W. Le, J. Jing, J.V. Sweedler, Aplysia Allatotropin-Related Peptide and its Newly Identified d-Amino Acid-Containing Epimer Both Activate a Receptor and a Neuronal Target. J Biol Chem.293, 16862–16873 (2018).
13. J.W. Checco , G. Zhang, W.D. Yuan, K. Yu, S.Y. Yin, R.H. Roberts-Galbraith, P.M. Yau, E.V. Romanova, J. Jing, J.V. Sweedler, Molecular and Physiological Characterization of a Receptor for d-Amino Acid-Containing Neuropeptides. ACS Chem Biol.13, 1343–1352 (2018).

Prof. Jonathan Sweedler is the James R. Eiszner Family Endowed Chair in Chemistry and the Director of the School of Chemical Sciences at the University of Illinois at Urbana-Champaign, and has appointments in Neuroscience, Molecular and Integrative Physiology, Bioengineering, and Medicine. His research interests focus on assaying individual cells and other small-volume samples for their small molecules and peptides using a range of separations and mass spectrometry approaches. He and his group have uncovered novel neurotransmitters and neuromodulators, including unusual D-amino acids, neuropeptides, and other cell–cell signaling molecules. He is receiving the 2019 CASSS Award for Outstanding Achievements in Separation Science at HPLC 2019 Kyoto.  He is currently the Editor-in-Chief of Analytical Chemistry.