Peptidomics pp 29-47 | Cite as

Approaches to Identify Endogenous Peptides in the Soil Nematode Caenorhabditis elegans

  • Steven J. Husson
  • Elke Clynen
  • Kurt Boonen
  • Tom Janssen
  • Marleen Lindemans
  • Geert Baggerman
  • Liliane Schoofs
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 615)

Abstract

The transparent soil nematode Caenorhabditis elegans can be considered an important model organism due to its ease of cultivation, suitability for high-throughput genetic screens, and extremely well-defined anatomy. C. elegans contains exactly 959 cells that are ordered in defined differentiated tissues. Although C. elegans only possesses 302 neurons, a large number of similarities among the neuropeptidergic signaling pathways can be observed with other metazoans. Neuropeptides are important messenger molecules that regulate a wide variety of physiological processes. These peptidergic signaling molecules can therefore be considered important drug targets or biomarkers. Neuropeptide signaling is in the nanomolar range, and biochemical elucidation of individual peptide sequences in the past without the genomic information was challenging. Since the rise of many genome-sequencing projects and the significant boost of mass spectrometry instrumentation, many hyphenated techniques can be used to explore the “peptidome” of individual species, organs, or even cell cultures. The peptidomic approach aims to identify endogenously present (neuro)peptides by using liquid chromatography and mass spectrometry in a high-throughput way. Here we outline the basic procedures for the maintenance of C. elegans nematodes and describe in detail the peptide extraction procedures. Two peptidomics strategies (off-line HPLC–MALDI-TOF MS and on-line 2D-nanoLC–Q-TOF MS/MS) and the necessary instrumentation are described.

Key words

Nematode Caenorhabditis elegans neuropeptide insulin FMRFamide-like peptide flp neuropeptide-like protein G-protein-coupled receptor mass spectrometry 

Notes

Acknowledgments

Research in the authors’ lab was sponsored by the Research Foundation Flanders (FWO-Vlaanderen grant G.0434.07 and 1.5.137.06). The authors strongly acknowledge the Interfacultary Centre for Proteomics and Metabolomics “Prometa”, K.U. Leuven, and wish to thank the Caenorhabditis Genetics Center for providing all the C. elegans strains. S.J. Husson, T. Janssen, M. Lindemans and E. Clynen are postdoctoral fellows of the Research Foundation Flanders (FWO-Vlaanderen).

References

  1. 1.
    The C. elegans Sequencing Consortium (1998) Genome sequence of the nematode C. elegans: a platform for investigating biology. Science 282, 2012–2018.Google Scholar
  2. 2.
    Kim, K. and Li, C. (2004) Expression and regulation of an FMRFamide-related neuropeptide gene family in Caenorhabditis elegans. J. Comp. Biol. 475, 540–550.Google Scholar
  3. 3.
    Li, C., Nelson, L.S., Kim, K., Nathoo, A., and Hart, A.C. (1999) Neuropeptide gene families in the nematode Caenorhabditis elegans. Ann. N.Y. Acad. Sci. 897, 239–252.PubMedCrossRefGoogle Scholar
  4. 4.
    Li, C., Kim, K., and Nelson, L.S. (1999) FMRFamide-related neuropeptide gene family in Caenorhabditis elegans. Brain Res. 848, 26–34.PubMedCrossRefGoogle Scholar
  5. 5.
    McVeigh, P., Leech, S., Mair, G.R., Marks, N.J., Geary, T.G., and Maule, A.G. (2005) Analysis of FMRFamide-like peptide (FLP) diversity in phylum Nematoda. Int. J. Parasitol. 35, 1043–1060.PubMedCrossRefGoogle Scholar
  6. 6.
    Nathoo, A.N., Moeller, R.A., Westlund, B.A., and Hart, A.C. (2001) Identification of neuropeptide-like protein gene families in Caenorhabditis elegans and other species. Proc. Natl. Acad. Sci. USA. 98, 14000–14005.PubMedCrossRefGoogle Scholar
  7. 7.
    Pierce, S.B., Costa, M., Wisotzkey, R., Devadhar, S., Homburger, S.A., Buchman, A.R., Ferguson, K.C., Heller, J., Platt, D.M., Pasquinelli, A.A., Liu, L.X., Doberstein, S.K., and Ruvkun, G. (2001) Regulation of DAF-2 receptor signaling by human insulin and ins-1, a member of the unusually large and diverse C. elegans insulin gene family. Genes Dev. 15, 672–686.PubMedCrossRefGoogle Scholar
  8. 8.
    Husson, S.J., Mertens, I., Janssen, T., Lindemans, M., and Schoofs, L. (2007) Neuropeptidergic signaling in the nematode Caenorhabditis elegans. Prog. Neurobiol. 82, 33–55.PubMedCrossRefGoogle Scholar
  9. 9.
    Marks, N.J., Shaw, C., Maule, A.G., Davis, J.P., Halton, D.W., Verhaert, P., Geary, T.G., and Thompson, D.P. (1995) Isolation of AF2 (KHEYLRFamide) from Caenorhabditis elegans: evidence for the presence of more than one FMRFamide-related peptide-encoding gene. Biochem. Biophys. Res. Commun. 217, 845–851.PubMedCrossRefGoogle Scholar
  10. 10.
    Marks, N.J., Maule, A.G., Geary, T.G., Thompson, D.P., Davis, J.P., Halton, D.W., Verhaert, P., and Shaw, C. (1997) APEASPFIRFamide, a novel FMRFamide-related decapeptide from Caenorhabditis elegans: structure and myoactivity. Biochem. Biophys. Res. Commun. 231, 591–595.PubMedCrossRefGoogle Scholar
  11. 11.
    Marks, N.J., Maule, A.G., Geary, T.G., Thompson, D.P., Li, C., Halton, D.W., and Shaw, C. (1998) KSAYMRFamide (PF3/AF8) is present in the free-living nematode, Caenorhabditis elegans. Biochem. Biophys. Res. Commun. 248, 422–425.PubMedCrossRefGoogle Scholar
  12. 12.
    Marks, N.J., Maule, A.G., Li, C., Nelson, L.S., Thompson, D.P., Alexander-Bowman, S., Geary, T.G., Halton, D.W., Verhaert, P., and Shaw, C. (1999) Isolation, pharmacology and gene organization of KPSFVRFamide: a neuropeptide from Caenorhabditis elegans. Biochem. Biophys. Res. Commun. 254, 222–230.PubMedCrossRefGoogle Scholar
  13. 13.
    Marks, N.J., Shaw, C., Halton, D.W., Thompson, D.P., Geary, T.G., Li, C., and Maule, A.G. (2001) Isolation and preliminary biological assessment of AADGAPLIRFamide and SVPGVLRFamide from Caenorhabditis elegans. Biochem. Biophys. Res. Commun. 286, 1170–1176.PubMedCrossRefGoogle Scholar
  14. 14.
    Rosoff, M.L., Doble, K.E., Price, D.A., and Li, C. (1993) The flp-1 propeptide is processed into multiple, highly similar FMRFamide-like peptides in Caenorhabditis elegans. Peptides 14, 331–338.PubMedCrossRefGoogle Scholar
  15. 15.
    Husson, S.J., Clynen, E., Baggerman, G., De Loof, A., and Schoofs, L. (2005) Discovering neuropeptides in Caenorhabditis elegans by two dimensional liquid chromatography and mass spectrometry. Biochem. Biophys. Res. Commun. 335, 76–86.PubMedCrossRefGoogle Scholar
  16. 16.
    Husson, S.J., Clynen, E., Baggerman, G., De Loof, A., and Schoofs, L. (2005) Peptidomics of Caenorhabditis elegans: in search of neuropeptides. Commun. Agric. Appl. Biol. Sci. 70, 153–156.PubMedGoogle Scholar
  17. 17.
    Husson, S.J., Landuyt, B., Thomas, N., Baggerman, G., Boonen, K., Clynen, E., Lindemans, M., Janssen, T., and Schoofs, L. (2008) Comparative peptidomics of Caenorhabditis elegans versus C. briggsae by LC–MALDI-TOF MS. Peptides, 30, 449–457.PubMedGoogle Scholar
  18. 18.
    Yew, J.Y., Dikler, S., and Stretton, A.O. (2003) De novo sequencing of novel neuropeptides directly from Ascaris suum tissue using matrix-assisted laser desorption/ionization time-of-flight/time-of-flight. Rapid Commun. Mass Spectrom. 17, 2693–2698.PubMedCrossRefGoogle Scholar
  19. 19.
    Yew, J.Y., Kutz, K.K., Dikler, S., Messinger, L., Li, L., and Stretton, A.O. (2005) Mass spectrometric map of neuropeptide expression in Ascaris suum. J. Comp. Neurol. 488, 396–413.PubMedCrossRefGoogle Scholar
  20. 20.
    Husson, S.J., Clynen, E., Baggerman, G., Janssen, T., and Schoofs, L. (2006) Defective processing of neuropeptide precursors in Caenorhabditis elegans lacking proprotein convertase 2 (KPC-2/EGL-3): mutant analysis by mass spectrometry. J. Neurochem. 98, 1999–2012.PubMedCrossRefGoogle Scholar
  21. 21.
    Husson, S.J. and Schoofs, L. (2006) Characterization of a key neuropeptide processing enzyme in C. elegans by mass spectrometry. Commun. Agric. Appl. Biol. Sci. 71, 171–174.PubMedGoogle Scholar
  22. 22.
    Husson, S.J., Janssen, T., Baggerman, G., Bogert, B., Kahn-Kirby, A.H., Ashrafi, K., and Schoofs, L. (2007) Impaired processing of FLP and NLP peptides in carboxypeptidase E (EGL-21)-deficient Caenorhabditis elegans as analysed by mass spectrometry. J. Neurochem. 102, 246–260.PubMedCrossRefGoogle Scholar
  23. 23.
    Husson, S.J. and Schoofs, L. (2007) Altered neuropeptide profile of Caenorhabditis elegans lacking the chaperone protein 7B2 as analyzed by mass spectrometry. FEBS Lett. 581, 4288–4292.PubMedCrossRefGoogle Scholar
  24. 24.
    Baggerman, G., Boonen, K., Verleyen, P., De Loof, A., and Schoofs, L. (2005) Peptidomic analysis of the larval Drosophila melanogaster central nervous system by two-dimensional capillary liquid chromatography quadrupole time-of-flight mass spectrometry. J. Mass Spectrom. 40, 250–260.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Steven J. Husson
    • 1
  • Elke Clynen
    • 1
  • Kurt Boonen
    • 2
  • Tom Janssen
    • 1
  • Marleen Lindemans
    • 1
  • Geert Baggerman
    • 3
  • Liliane Schoofs
    • 2
  1. 1.Functional Genomics and Proteomics, Department of BiologyK.U. LeuvenLeuvenBelgium
  2. 2.Functional Genomics and Proteomics Research Unit, Department of BiologyK.U. LeuvenLeuvenBelgium
  3. 3.ProMeta, Interfacultary Center for Proteomics and Metabolomics, K.U. LeuvenLeuvenBelgium

Personalised recommendations