Unlocking Mysteries of Neurochemistry

The study of neurochemistry may lead to a better understanding of a wide range of biological phenomena, from circadian rhythms to the processes involved in pain sensation. Jonathan V. Sweedler, a professor in the Department of Chemistry at the University of Illinois, and the 2015 ANACHEM Award winner, has focused his group’s major research efforts on analytical neurochemistry, developing new measurement tools to characterize small-volume samples for their cell–cell signaling molecules, and applying these technologies to the study of the distribution and dynamic release of neuropeptides, classical transmitters, and other cell–cell signaling molecules from the brain. Sweedler’s analytical developments have centered around making measurements work on smaller samples such as individual cells. He spoke to LCGC about some of this work.

In a recent paper (1), you discussed using a combination of two mass spectrometry imaging (MSI) approaches to study well-defined regions of the mammalian peripheral sensory-motor system, including the dorsal root ganglia (DRG) and adjacent nerves. Why did you combine the two MSI techniques? And what did your study, including the use of gas chromatography–mass spectrometry (GC–MS) to corroborate your findings, reveal about the utility of this analytical approach?

Sweedler: In this JASMS manuscript, our goal was to obtain a more complete metabolome of the DRG and several associated nerves. We used two MSI approaches, secondary ion mass spectrometry (SIMS) and matrix-assisted laser desorption–ionization (MALDI) MSI, as each offers different characteristics: SIMS has higher spatial resolution and MALDI MS provides an extended mass range. Our SIMS instrument is unusual as it, just as many MALDI time-of-flight (TOF) MS instruments do, incorporates tandem MS capabilities. Lastly, we went retro and used GC–MS. It is compatible with small samples and is a well validated measurement approach capable of generating information on the metabolome of specific regions of the neuronal networks we were studying.

What are your research goals for the work described above? How will this research potentially impact the field in the future?

Sweedler: The DRG is a complex heterogeneous structure responsible for pain sensation, among other functions. We are interested in understanding its cellular scale heterogeneity and are continuing these studies to understand the chemical changes that occur with both acute and inflammatory pain.

In another recent paper (2), you discuss peptidomic analyses that were examined to characterize variation in peptides that maintain circadian rhythms using three different label-free quantitation approaches: spectral count, spectra index, and SIEVE. What did your study show about the strengths and weakness of these three approaches? That paper (2) also stated that this research could lead to “pathways to target for pharmaceutical intervention to corrected altered circadian rhythms in various disorders and […] improved understanding of the link between circadian rhythm, mood disorders and drug addiction.” How far along is the research on those relationships? What challenges still need to be overcome?

Sweedler: A small structure in the mammalian brain, the suprachiasmatic nucleus, acts as the body’s master circadian clock and synchronizes the other biological clocks to time of day. In collaboration with Martha Gillette, we have been characterizing the neuropeptides and hormones found in this structure and have uncovered more than 200 putative neuropeptides. It can take our collaborator a year to test the biological activity of a single one of these peptides. The obvious question: Which peptides from among the hundreds of detected peptides are biologically active? We have created a method to follow peptide release from this structure. Another way to gain information on the actions of these peptides is to determine which peptides change levels as a function of time of day; in this manuscript, we compared the effectiveness of three different label-free quantitative MS approaches for characterizing neuropeptides. Several worked well and allowed us to determine subsets of peptides that change levels as a function of time of day. Using these data, our collaborators are testing the biological activity of only a few  peptides. This collaboration is a great example of new sampling and peptide characterization efforts that, when used in conjunction with the deep biological expertise of our collaborator, enable the discovery of new biologically active molecules.

You have also published work on capillary electrophoresis (CE) coupled to MS and a patch clamp method for single-cell characterization (3). How does this method work, and what type of information do you obtain?

Sweedler: CE–MS enables us to take small-volume samples and determine many of the small molecules present in the sample. In other words, we can determine the single-cell metabolome. The data we obtain from a single cell does not have the depth of metabolome coverage one would achieve when using larger samples, but it does provide a unique glimpse into cellular heterogeneity. The most difficult aspect of single-cell CE–MS is cell sampling. In the Analytical Chemistry manuscript, we used the well-established patch clamp method to patch onto a cell and record its electrophysiological parameters, which enabled us to characterize the cell type and its activity. Then about 3 pL of cytoplasm was drawn up into the patch pipette and introduced into our CE–MS system. Thus, we were able to correlate the major compounds within a specific cell to its type and activity. This is a low-throughput but high-information approach that we are using to examine the differences between functional categories of the cells in the brain.

What are the next steps in your research?

Sweedler: As described here, our research involves new sampling and measurement approaches applied to measure novel brain chemistry. This is a research area limited by our ability to probe the brain with the required spatial, temporal, and chemical detail. Our current work involves increasing the throughput of these measurement approaches and improving the spatial and chemical information we gain from our measurements. The other aspect is to determine the functional aspects of the new compounds we uncover. Both areas are major thrusts within my group.

References

(1)S.S. Rubakhin, A. Ulanov, and J.V. Sweedler, J. Am. Soc. Mass Spectrom. DOI: 10.1007/s13361-015-1128-8 (2015).

(2)B.R. Southey, J.E. Lee, L. Zamdborg, N. Atkins, Jr, J.W. Mitchell, M. Li, M.U. Gillette, N.L. Kelleher, and J.V. Sweedler, Anal. Chem.86(1), 443–452 (2014).

(3)J.T. Aerts, K.R. Louis, S.R. Crandall, G. Govindaiah, C.L. Cox, and J.V. Sweedler, Anal. Chem. 86(6), 3203–3208 (2014).