Anal. Chem. 2003, 75, 5374-5380
Spatial Profiling with MALDI MS: Distribution of Neuropeptides within Single Neurons Stanislav S. Rubakhin, William T. Greenough, and Jonathan V. Sweedler*
Department of Chemistry, Psychology and the Beckman Institute, University of Illinois, Urbana, Illinois 61801
MALDI MS imaging and single-cell profiling are important new capabilities for mass spectrometry. The distribution of neuropeptides within a cell plays an important role in the functioning of the cells in a neuronal network. Protocols for subcellular MALDI MS are described that allow comparative peptide profiling of cell bodies and the neuronal processes (neurites) using single isolated neurons from the neuronal model Aplysia californica. The seawater surrounding the neurons is problematic for mass spectrometry and so must be removed in a manner that does not cause morphological changes or a redistribution of the neuropeptides. Several protocols have been investigated for subcellular spatial profiling, including the use of air-drying, replacement of the seawater with deionized water, and substitution of the cell matrix with fluorinert, mineral oil and glycerol, as well as paraformaldehyde fixation. Glycerol stabilization offers the best combination of preservation of cell morphology and prevention of neuropeptide redistribution. The profiles of the peptides in specific neuronal processes and the cell bodies demonstrate a variety of differences that appear to be cell-specific. These methods are suitable for smaller cells and subcellular MS imaging. The distal regions of neurons, the axons and dendrites, play an important and specific role in the functional organization of the nervous system. Many Aplysia neurons have multiple processes (termed neurites) terminating at synaptic contacts, where the release of a number of neurotransmitters/neuromodulators occurs. The chemical composition of these releasates at a specific site depends on synthesis, transport, and reuptake of signaling molecules. Profiling the biochemical content of a neurite is complicated by its small diameter and complex morphology. For example, a neurite that is a few micrometers in diameter may be intertwined throughout a distance of up to 1 m in length. Immunohistochemical methods are used for detection of neurotransmitters and neuromodulators in individual neurites and produce excellent spatial information.1,2 A limitation is potential cross-reactivity of the antibody with other compounds, especially for neuropeptides in which particular peptides can be part of * Corresponding author address: 600 S. Mathews Ave. 63-5, University of Illinois, Urbana, IL 61801. Phone: (217) 244-7359. Fax: (217) 244-8068. E-mail:
[email protected]. (1) Hockfield, S. Selected methods for antibody and nucleic acid probes; Cold Spring Harbor Laboratory Press: Plainview, NY, 1993. (2) Boulton, A. A.; Baker, G. B.; Campagnoni, A. T. Molecular neurobiological techniques; Humana Press: Clifton, NJ, 1990.
5374 Analytical Chemistry, Vol. 75, No. 20, October 15, 2003
structurally similar peptide families. In addition, immunocytochemistry can reveal distribution only of predetermined molecules. It is difficult to apply many analytical approaches, such as HPLC and Edman degradation, for peptide detection and identification in a neuronal process because of the minute amounts of analytes present and the difficulty in performing such manipulations. We investigate the extension of single-cell MALDI MS profiling to subcellular domains. MALDI MS appears to be well-suited for the study of the biochemical composition of neurites because of its high sensitivity, good spatial resolution, and selectivity. Previously, MALDI MS was used to profile the neuropeptides in single molluscan and insect neurons,3-12 connective nerves,13 and single micrometersized peptidergic vesicles.14 In addition, imaging MS, in which a laser is rastered across a biological tissue, maps peptides and proteins from a variety of tissue samples.15-20 However, neither approach has been applied to examining the neuropeptide distribution within the processes of single isolated or cultured (3) Van Veelen, P. A.; Jimenez, C. R.; Li, K. W.; Wildering, W. C.; Geraerts, W. P. M.; Tjaden, U. R.; Vandergreef, J. Org. Mass Spectrom. 1993, 28, 15421546. (4) Jimenez, C. R.; Li, K. W.; Dreisewerd, K.; Spijker, S.; Kingston, R.; Bateman, R. H.; Burlingame, A. L.; Smit, A. B.; van Minnen, J.; Geraerts, W. P. Biochemistry 1998, 37, 2070-2076. (5) Floyd, P. D.; Li, L. J.; Rubakhin, S. S.; Sweedler, J. V.; Horn, C. C.; Kupfermann, I.; Alexeeva, V. Y.; Ellis, T. A.; Dembrow, N. C.; Weiss, K. R.; Vilim, F. S. J. Neurosci. 1999, 19, 7732-7741. (6) Garden, R. W.; Shippy, S. A.; Li, L.; Moroz, T. P.; Sweedler, J. V. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 3972-3977. (7) Li, L. J.; Romanova, E. V.; Rubakhin, S. S.; Alexeeva, V.; Weiss, K. R.; Vilim, F. S.; Sweedler, J. V. Anal. Chem. 2000, 72, 3867-3874. (8) Li, L.; Garden, R. W.; Sweedler, J. V. Trends Biotechnol. 2000, 18, 151160. (9) Li, L. J.; Garden, R. W.; Romanova, E. V.; Sweedler, J. V. Anal. Chem. 1999, 71, 5451-5458. (10) Rubakhin, S. S.; Li, L.; Moroz, T. P.; Sweedler, J. V. J. Neurophysiol. 1999, 81, 1251-1260. (11) Predel, R.; Eckert, M.; Holman, G. M. Ann. N.Y. Acad. Sci. 1999, 897, 282-290. (12) Li, L. J.; Pulver, S. R.; Kelley, W. P.; Thirumalai, V.; Sweedler, J. V.; Marder, E. J. Compar. Neurol. 2002, 444, 227-244. (13) Li, L. J.; Moroz, T. P.; Garden, R. W.; Floyd, P. D.; Weiss, K. R.; Sweedler, J. V. Peptides 1998, 19, 1425-1433. (14) Rubakhin, S. S.; Garden, R. W.; Fuller, R. R.; Sweedler, J. V. Nat. Biotechnol. 2000, 18, 172-175. (15) Caprioli, R. M.; Farmer, T. B.; Gile, J. Anal. Chem. 1997, 69, 4751-4760. (16) Chaurand, P.; Stoeckli, M.; Caprioli, R. M. Anal. Chem. 1999, 71, 52635270. (17) Stoeckli, M.; Chaurand, P.; Hallahan, D. E.; Caprioli, R. M. Nat. Med. 2001, 7, 493-496. (18) Stoeckli, M.; Staab, D.; Staufenbiel, M.; Wiederhold, K. H.; Signor, L. Anal. Biochem. 2002, 311, 33-39. (19) Spengler, B.; Hubert, M. J. Am. Soc. Mass Spectrom. 2002, 13, 735-748. (20) Bergquist, J. Chromatographia 1999, 49, S41-S48. 10.1021/ac034498+ CCC: $25.00
© 2003 American Chemical Society Published on Web 09/12/2003
neurons. A number of challenges arise during MALDI MS application to neurite profiling, including preservation of the molecule’s spatial location; neuropeptides, unlike many proteins, are small, highly soluble molecules so that the addition of MALDI matrix can easily distort the original spatial distribution of the molecules. Methods to replace the physiological solution with MALDI matrix,21 paraformaldehyde fixation,22 slow analyte entrapping during matrix crystal formation,17,18 and spatially restricted MALDI matrix application14 have been explored. In this work, we demonstrate protocols for profiling of neuronal processes with MALDI MS, including methods to isolate and culture neurons with long processes, to remove the physiological solution without damaging these neuronal processes, and spatially to restrict the mixing of MALDI matrix with analyte. These methods allow peptide profiling in different regions of single isolated neurons with
