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Characterizing peptides in individual mammalian cells using mass spectrometry

Nature Protocols volume 2, pages 1987–1997 (2007)Cite this article

Abstract

Cell-to-cell chemical signaling plays multiple roles in coordinating the activity of the functional elements of an organism, with these elements ranging from a three-neuron reflex circuit to the entire animal. In recent years, single-cell mass spectrometry (MS) has enabled the discovery of cell-to-cell signaling molecules from the nervous system of a number of invertebrates. We describe a protocol for analyzing individual cells from rat pituitary using matrix-assisted laser desorption/ionization MS. Each step in the sample preparation process, including cell stabilization, isolation, sample preparation, signal acquisition and data interpretation, is detailed here. Although we employ this method to investigate peptides in individual pituitary cells, it can be adapted to other cell types and even subcellular sections from a range of animals. This protocol allows one to obtain 20–30 individual cell samples and acquire mass spectra from them in a single day.

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Figure 1
Figure 2: Hardware setup used for single-cell preparation for MALDI MS investigation.
Figure 3: Preparation of single pituitary intermediate lobe cell for MALDI MS investigation.
Figure 4: Single-cell MALDI MS reveals expression of a number of POMC peptides in individual cells from the intermediate pituitary.
Figure 5: MS/MS allows confirmation of signal identity assignments made using the precise molecular mass of peptides and a variety of data such as the peptide's known distribution in tissue, its prohormone expression and the presence of other peptides originating from the same prohormone.

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References

  1. Garden, R.W., Shippy, S.A., Li, L., Moroz, T.P. & Sweedler, J.V. Proteolytic processing of the Aplysia egg-laying hormone prohormone. Proc. Natl. Acad. Sci. USA 95, 3972–3977 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Li, L., Garden, R.W. & Sweedler, J.V. Single-cell MALDI: a new tool for direct peptide profiling. Trends Biotechnol. 18, 151–160 (2000).

    Article  CAS  PubMed  Google Scholar 

  3. Rubakhin, S.S., Garden, R.W., Fuller, R.R. & Sweedler, J.V. Measuring the peptides in individual organelles with mass spectrometry. Nat. Biotechnol. 18, 172–175 (2000).

    Article  CAS  PubMed  Google Scholar 

  4. Rubakhin, S.S., Greenough, W.T. & Sweedler, J.V. Spatial profiling with MALDI MS: distribution of neuropeptides within single neurons. Anal. Chem. 75, 5374–5380 (2003).

    Article  CAS  PubMed  Google Scholar 

  5. Rubakhin, S.S., Li, L., Moroz, T.P. & Sweedler, J.V. Characterization of the Aplysia californica cerebral ganglion F cluster. J. Neurophysiol. 81, 1251–1260 (1999).

    Article  CAS  PubMed  Google Scholar 

  6. Sweedler, J.V., Rubakhin, S.S., Churchill, J.D. & Greenough, W.T. Assaying the neuropeptides in single mammalian neurons using mass spectrometry. Program no. 326.16. 2003 Abstract Viewer/Itinerary Planner (Society for Neuroscience, Washington, DC, 2003, http://sfn.scholarone.com/itin2003/).

  7. Rubakhin, S.S., Churchill, J.D., Greenough, W.T. & Sweedler, J.V. Profiling signaling peptides in single mammalian cells using mass spectrometry. Anal. Chem. 78, 7267–7272 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Research highlights. Biochemistry: cell detectives. Nature 443, 248–249 (2006).

  9. Pacak, K., Aguilera, G., Sabban, E. & Kvetnansky, R. (eds.) Stress: Current Neuroendocrine and Genetic Approaches Vol. 1018 (New York Academy of Sciences, New York, 2004).

    Google Scholar 

  10. Sandman, C.A. et al. (eds.). Neuropeptides: Structure and Function in Biology and Behavior Vol. 897 (New York Academy of Sciences, New York, 1999).

    Google Scholar 

  11. Strand, F.L. Neuropeptides: Regulators of Physiological Processes (MIT Press, Cambridge, MA, 1999).

    Google Scholar 

  12. Kastin, A.J. Handbook of Biologically Active Peptides (Academic, Amsterdam, Boston, 2006).

    Google Scholar 

  13. Walsh, J.H. & Dockray, G.J. Gut Peptides: Biochemistry and Physiology (Raven Press, New York, 1994).

    Book  Google Scholar 

  14. Sewald, N. & Jakubke, H.-D. Peptides: Chemistry and Biology (Wiley-VCH, Weinheim, 2002).

    Book  Google Scholar 

  15. Levin, E.R., Hu, R.M., Rossi, M. & Pickart, M. Arginine vasopressin stimulates atrial natriuretic peptide gene expression and secretion from rat diencephalic neurons. Endocrinology 131, 1417–1423 (1992).

    Article  CAS  PubMed  Google Scholar 

  16. Jensen, J. Regulatory peptides and control of food intake in non-mammalian vertebrates. Comp. Biochem. Physiol. A 128, 471–479 (2001).

    Article  CAS  Google Scholar 

  17. Gundlach, A.L., Burazin, T.C. & Larm, J.A. Distribution, regulation and role of hypothalamic galanin systems: renewed interest in a pleiotropic peptide family. Clin. Exp. Pharmacol. Physiol. 28, 100–105 (2001).

    Article  CAS  PubMed  Google Scholar 

  18. Okada, Y., Tsuda, Y., Bryant, S.D. & Lazarus, L.H. Endomorphins and related opioid peptides. Vitam. Horm. 65, 257–279 (2002).

    Article  CAS  PubMed  Google Scholar 

  19. Kieffer, B.L. & Gaveriaux-Ruff, C. Exploring the opioid system by gene knockout. Prog. Neurobiol. 66, 285–306 (2002).

    Article  CAS  PubMed  Google Scholar 

  20. Stefano, G.B., Fricchione, G., Goumon, Y. & Esch, T. Pain, immunity, opiate and opioid compounds and health. Med. Sci. Monit. 11, MS47–MS53 (2005).

    CAS  PubMed  Google Scholar 

  21. Hardy, J.A. & Higgins, G.A. Alzheimer's disease: the amyloid cascade hypothesis. Science 256, 184–185 (1992).

    Article  CAS  PubMed  Google Scholar 

  22. Taylor, A. & Jones, M.T. Chemical Communication Within the Nervous System and Its Disturbance in Disease (Pergamon, Oxford, New York, 1977).

    Google Scholar 

  23. Davison, A.N. & Thompson, R.H.S. The Molecular Basis of Neuropathology (Edward Arnold, London, 1981).

    Google Scholar 

  24. Laidlaw, J.P., Richens, A. & Oxley, J. A Textbook of Epilepsy (Churchill Livingstone, Edinburgh, New York, 1988).

    Google Scholar 

  25. Bradford, H.F. Chemical Neurobiology: An Introduction to Neurochemistry (W.H. Freeman, New York, 1986).

    Google Scholar 

  26. Ebner, K. & Singewald, N. The role of substance P in stress and anxiety responses. Amino Acids 31, 251–272 (2006).

    Article  CAS  PubMed  Google Scholar 

  27. Cummings, D.E. Ghrelin and the short- and long-term regulation of appetite and body weight. Physiol. Behav. 89, 71–84 (2006).

    Article  CAS  PubMed  Google Scholar 

  28. Crawley, J.N. & Corwin, R.L. Biological actions of cholecystokinin. Peptides 15, 731–755 (1994).

    Article  CAS  PubMed  Google Scholar 

  29. Charmandari, E., Tsigos, C. & Chrousos, G. Endocrinology of the stress response. Annu. Rev. Physiol. 67, 259–284 (2005).

    Article  CAS  PubMed  Google Scholar 

  30. Porte, D. Jr., Baskin, D.G. & Schwartz, M.W. Insulin signaling in the central nervous system: a critical role in metabolic homeostasis and disease from C. elegans to humans. Diabetes 54, 1264–1276 (2005).

    Article  CAS  PubMed  Google Scholar 

  31. Bach, P.H. & Baker, J.R.J. Histochemical and Immunohistochemical Techniques: Applications to Pharmacology and Toxicology (Chapman & Hall, London, 1991).

    Book  Google Scholar 

  32. Pool, C.W., Buijs, R.M., Swaab, D.G. & Boer, F.W. Immunohistochemistry. in IBRO Handbook Series Vol. 3 (ed. Cuello, A.C.) xvii 501 (Wiley, Chichester, West Sussex, New York, 1983).

    Google Scholar 

  33. Brownstein, M.J., Saavedra, J.M., Axelrod, J., Zeman, G.H. & Carpenter, D.O. Coexistence of several putative neurotransmitters in single identified neurons of Aplysia. Proc. Natl. Acad. Sci. USA 71, 4662–4665 (1974).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Irvine, G.B. & Williams, C.H. Neuropeptide Protocols (Humana Press, Totowa, NJ, 1997).

    Google Scholar 

  35. Silberring, J. & Ekman, R. Mass Spectrometry and Hyphenated Techniques in Neuropeptide Research (Wiley-Interscience, New York, 2002).

    Google Scholar 

  36. Michael, A.C. & Borland, L.M. Electrochemical Methods for Neuroscience (CRC Press/Taylor & Francis, Boca Raton, FL, 2007).

    Google Scholar 

  37. Xu, X.-H.N. (ed.) New Frontiers in Ultrasensitive Bioanalysis: Advanced Analytical Chemistry Applications in Nanobiotechnology, Single Molecule Detection, and Single Cell Analysis 308 (Wiley, Hoboken, NJ, 2007).

    Book  Google Scholar 

  38. Sweedler, J.V. & Arriaga, E.A. Single cell analysis. Anal. Bioanal. Chem. 387, 1–2 (2007).

    Article  CAS  Google Scholar 

  39. Baggerman, G., Cerstiaens, A., De Loof, A. & Schoofs, L. Peptidomics of the larval Drosophila melanogaster central nervous system. J. Biol. Chem. 277, 40368–40374 (2002).

    Article  CAS  PubMed  Google Scholar 

  40. Clynen, E. et al. Peptidomics of the pars intercerebralis–corpus cardiacum complex of the migratory locust, Locusta migratoria. Eur. J. Biochem. 268, 1929–1939 (2001).

    Article  CAS  PubMed  Google Scholar 

  41. Svensson, M., Skold, K., Svenningsson, P. & Andren, P.E. Peptidomics-based discovery of novel neuropeptides. J. Proteome Res. 2, 213–219 (2003).

    Article  CAS  PubMed  Google Scholar 

  42. Hummon, A.B. et al. From the genome to the proteome: uncovering peptides in the Apis brain. Science 314, 647–649 (2006).

    Article  CAS  PubMed  Google Scholar 

  43. Husson, S.J., Clynen, E., Baggerman, G., De Loof, A. & Schoofs, L. Discovering neuropeptides in Caenorhabditis elegans by two dimensional liquid chromatography and mass spectrometry. Biochem. Biophys. Res. Commun. 335, 76–86 (2005).

    Article  CAS  PubMed  Google Scholar 

  44. Wei, H. et al. Identification and quantification of neuropeptides in brain tissue by capillary liquid chromatography coupled off-line to MALDI-TOF and MALDI-TOF/TOF-MS. Anal. Chem. 78, 4342–4351 (2006).

    Article  CAS  PubMed  Google Scholar 

  45. Fricker, L.D., Lim, J., Pan, H. & Che, F.Y. Peptidomics: identification and quantification of endogenous peptides in neuroendocrine tissues. Mass Spectrom. Rev. 25, 327–344 (2006).

    Article  CAS  PubMed  Google Scholar 

  46. Hummon, A.B., Amare, A. & Sweedler, J.V. Discovering new invertebrate neuropeptides using mass spectrometry. Mass Spectrom. Rev. 25, 77–98 (2006).

    Article  CAS  PubMed  Google Scholar 

  47. Karas, M., Bahr, U. & Giessmann, U. Matrix-assisted laser desorption ionization mass-spectrometry. Mass Spectrom. Rev. 10, 335–357 (1991).

    Article  CAS  Google Scholar 

  48. Tanaka, K. et al. Protein and polymer analysis up to m/z 100,000 by laser ionisation time-of-flight mass spectrometry. Rapid Commun. Mass Spectrom. 2, 151–153 (1988).

    Article  CAS  Google Scholar 

  49. Hillenkamp, F., Karas, M., Beavis, R.C. & Chait, B.T. Matrix-assisted laser desorption ionization mass-spectrometry of biopolymers. Anal. Chem. 63, A1193–A1202 (1991).

    Article  Google Scholar 

  50. Ong, S.E. & Mann, M. Mass spectrometry-based proteomics turns quantitative. Nat. Chem. Biol. 1, 252–262 (2005).

    Article  CAS  PubMed  Google Scholar 

  51. Bienvenut, W.V. et al. Matrix-assisted laser desorption/ionization-tandem mass spectrometry with high resolution and sensitivity for identification and characterization of proteins. Proteomics 2, 868–876 (2002).

    Article  CAS  PubMed  Google Scholar 

  52. Medzihradszky, K.F. et al. The characteristics of peptide collision-induced dissociation using a high-performance MALDI-TOF/TOF tandem mass spectrometer. Anal. Chem. 72, 552–558 (2000).

    Article  CAS  PubMed  Google Scholar 

  53. Yergey, A.L. et al. De novo sequencing of peptides using MALDI/TOF-TOF. J. Am. Soc. Mass Spectrom. 13, 784–791 (2002).

    Article  CAS  PubMed  Google Scholar 

  54. Suckau, D. et al. A novel MALDI LIFT-TOF/TOF mass spectrometer for proteomics. Anal. Bioanal. Chem. 376, 952–965 (2003).

    Article  CAS  PubMed  Google Scholar 

  55. Ma, P.W.K. et al. Characterizing the Hez-PBAN gene products in neuronal clusters with immunocytochemistry and MALDI MS. J. Insect Physiol. 46, 221–230 (2000).

    Article  CAS  PubMed  Google Scholar 

  56. Jimenez, C.R. et al. Direct mass spectrometric peptide profiling and sequencing of single neurons reveals differential peptide patterns in a small neuronal network. Biochemistry 37, 2070–2076 (1998).

    Article  CAS  PubMed  Google Scholar 

  57. Jimenez, C.R. et al. Neuropeptide expression and processing as revealed by direct matrix-assisted laser desorption ionization mass spectrometry of single neurons. J. Neurochem. 62, 404–407 (1994).

    Article  CAS  PubMed  Google Scholar 

  58. Li, L. et al. Orcokinin peptides in developing and adult crustacean stomatogastric nervous systems and pericardial organs. J. Comp. Neurol. 444, 227–244 (2002).

    Article  CAS  PubMed  Google Scholar 

  59. Sweedler, J.V. et al. Identification and characterization of the feeding circuit-activating peptides, a novel neuropeptide family of Aplysia. J. Neurosci. 22, 7797–7808 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Standing, K.G. Peptide and protein de novo sequencing by mass spectrometry. Curr. Opin. Struct. Biol. 13, 595–601 (2003).

    Article  CAS  PubMed  Google Scholar 

  61. Nachman, R.J. et al. Occurrence of insect kinins in the flesh fly, stable fly and horn fly—mass spectrometric identification from single nerves and diuretic activity. Peptides 23, 1885–1894 (2002).

    Article  CAS  PubMed  Google Scholar 

  62. Jimenez, C.R. Batch introduction techniques. in Mass Spectrometry: Modified Proteins and Glycoconjugates Vol. 405 (ed. Burlingame, A.L.) 36–49 (Elsevier Academic Press, Boston, USA, 2005).

    Chapter  Google Scholar 

  63. Glass, D.J. Experimental Design for Biologists (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2007).

    Google Scholar 

  64. Townend, J. Practical Statistics for Environmental and Biological Scientists (Wiley, Chichester, New York, 2001).

    Google Scholar 

  65. Freshney, R.I. Culture of Animal Cells: A Manual of Basic Technique (Wiley-Liss, Hoboken, NJ, 2005).

    Book  Google Scholar 

  66. Hatoya, S. et al. Isolation and characterization of embryonic stem-like cells from canine blastocysts. Mol. Reprod. Dev. 73, 298–305 (2006).

    Article  CAS  PubMed  Google Scholar 

  67. Jiang, X.Y. et al. Methods for isolating highly-enriched embryonic spinal cord neurons: a comparison between enzymatic and mechanical dissociations. J. Neurosci. Methods 158, 13–18 (2006).

    Article  CAS  PubMed  Google Scholar 

  68. Weiler, I.J. et al. Fragile X mental retardation protein is translated near synapses in response to neurotransmitter activation. Proc. Natl. Acad. Sci. USA 94, 5395–5400 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Murray, G.I. & Curran, S. Laser Capture Microdissection: Methods and Protocols (Humana Press, Totowa, NJ, 2005).

    Book  Google Scholar 

  70. Nishino, S. The hypothalamic peptidergic system, hypocretin/orexin and vigilance control. Neuropeptides 41, 117–133 (2007).

    Article  CAS  PubMed  Google Scholar 

  71. Elias, C.F. et al. Chemically defined projections linking the mediobasal hypothalamus and the lateral hypothalamic area. J. Comp. Neurol. 402, 442–459 (1998).

    Article  CAS  PubMed  Google Scholar 

  72. Antoni, F.A. Vasopressinergic control of pituitary adrenocorticotropin secretion comes of age. Front. Neuroendocrinol. 14, 76–122 (1993).

    Article  CAS  PubMed  Google Scholar 

  73. Gerfen, C.R. & Young, W.S. III Distribution of striatonigral and striatopallidal peptidergic neurons in both patch and matrix compartments: an in situ hybridization histochemistry and fluorescent retrograde tracing study. Brain Res. 460, 161–167 (1988).

    Article  CAS  PubMed  Google Scholar 

  74. Bollheimer, L.C., Skelly, R.H., Chester, M.W., McGarry, J.D. & Rhodes, C.J. Chronic exposure to free fatty acid reduces pancreatic beta cell insulin content by increasing basal insulin secretion that is not compensated for by a corresponding increase in proinsulin biosynthesis translation. J. Clin. Invest. 101, 1094–1101 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  75. Monroe, E.B. et al. Massively parallel sample preparation for the MALDI MS analyses of tissues. Anal. Chem. 78, 6826–6832 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  76. Monroe, E.B., Koszczuk, B.A., Losh, J.L. & Sweedler, J.V. Measuring salty samples without adducts with MALDI MS. Int. J. Mass Spectrom. 260, 237–242 (2007).

    Article  CAS  Google Scholar 

  77. Wingerd, B.D. & Stein, G. Rat Dissection Manual (Carolina Biological Supply, Burlington, NC, 1988).

    Google Scholar 

  78. Walker, W.F. & Homberger, D.G. Anatomy and Dissection of the Rat (W.H. Freeman, New York, 1997).

    Google Scholar 

  79. Miao, H., Rubakhin, S.S. & Sweedler, J.V. Subcellular analysis of D-aspartate. Anal. Chem. 77, 7190–7194 (2005).

    Article  CAS  PubMed  Google Scholar 

  80. Cagney, G. & Emili, A. De novo peptide sequencing and quantitative profiling of complex protein mixtures using mass-coded abundance tagging. Nat. Biotechnol. 20, 163–170 (2002).

    Article  CAS  PubMed  Google Scholar 

  81. Deissler, H. et al. Rapid protein sequencing by tandem mass spectrometry and cDNA cloning of p20-CGGBP. A novel protein that binds to the unstable triplet repeat 5′-d(CGG)n-3′ in the human FMR1 gene. J. Biol. Chem. 272, 16761–16768 (1997).

    Article  CAS  PubMed  Google Scholar 

  82. Hunt, D.F., Yates, J.R. III, Shabanowitz, J., Winston, S. & Hauer, C.R. Protein sequencing by tandem mass spectrometry. Proc. Natl. Acad. Sci. USA 83, 6233–6237 (1986).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  83. Li, L., Garden, R.W., Romanova, E.V. & Sweedler, J.V. In situ sequencing of peptides from biological tissues and single cells using MALDI-PSD/CID analysis. Anal. Chem. 71, 5451–5458 (1999).

    Article  CAS  PubMed  Google Scholar 

  84. Mann, M. & Jensen, O.N. Proteomic analysis of post-translational modifications. Nat. Biotechnol. 21, 255–261 (2003).

    Article  CAS  PubMed  Google Scholar 

  85. Shevchenko, A. et al. Rapid 'de novo' peptide sequencing by a combination of nanoelectrospray, isotopic labeling and a quadrupole/time-of-flight mass spectrometer. Rapid Commun. Mass Spectrom. 11, 1015–1024 (1997).

    Article  CAS  PubMed  Google Scholar 

  86. Shevchenko, A., Wilm, M., Vorm, O. & Mann, M. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal. Chem. 68, 850–858 (1996).

    Article  CAS  PubMed  Google Scholar 

  87. Steen, H. & Mann, M. The ABC's (and XYZ's) of peptide sequencing. Nat. Rev. Mol. Cell Biol. 5, 699–711 (2004).

    Article  CAS  PubMed  Google Scholar 

  88. Li, L. et al. Cerebrin prohormone processing and distribution in Aplysia californica. J. Neurochem. 77, 1569–1580 (2001).

    Article  CAS  PubMed  Google Scholar 

  89. Li, L. et al. Peptide profiling of cells with multiple gene products: combining immunochemistry and MALDI mass spectrometry with on-plate microextraction. Anal. Chem. 72, 3867–3874 (2000).

    Article  CAS  PubMed  Google Scholar 

  90. Li, L.J. et al. Mass spectrometric survey of interganglionically transported peptides in Aplysia. Peptides 19, 1425–1433 (1998).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

In the mammalian single-cell study, coauthors James Churchill and William Greenough greatly contributed to the initial investigations. This work is supported by the National Institute on Drug Abuse through award nos. DA018310 and DA017940.

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  1. Department of Chemistry and the Beckman Institute, University of Illinois, Urbana, 61801, Illinois, USA

    Stanislav S Rubakhin & Jonathan V Sweedler

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Correspondence to Jonathan V Sweedler.

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Rubakhin, S., Sweedler, J. Characterizing peptides in individual mammalian cells using mass spectrometry. Nat Protoc 2, 1987–1997 (2007). https://doi.org/10.1038/nprot.2007.277

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