Differential Proteome Analysis of Germ-Line Maturaturation-Dependent Proteins of C. elegans

Prof. Dr. Hans-Juergen Thiesen
Proteome Center Rostock
Medical Faculty
University of Rostock,
Joachim-Jungius Str. 9
18059 Rostock
telephone: +49 381 4059687
fax: +49 381 4059686
email: hans-juergen.thiesen@med.uni-rostock.de
prestype: Platform
presenter: Hans-Juergen Thiesen

H.-J. Thiesen1, M. Bantscheff1, B. Ringel1, R. Schnabel2 and M.O. Glocker1
1Proteome Center Rostock, Medical Faculty, University of Rostock, Joachim-Jungius Str. 9, 18059 Rostock, Germany
2
TU Braunschweig, Biozentrum / Genetik A, Spielmannstr. 7, 38106 Braunschweig, Germany

Caenorhabditis elegans ( C. elegans) is a small (about 1 mm long) soil nematode found in temperate regions and may be handled as a microorganism. Although C. elegans is a primitive organism it nonetheless shares many of the essential biological characteristics that are central problems of human biology (1). The development and function of this diploid organism that has been fully sequenced (2-4), is encoded by an estimated 17,800 distinct genes. C. elegans is regarded as a model system to facilitate the purification of low abundant proteins involved in regulatory protein networks. Our initial investigations are focussed on studying germ line formation during maturation of C. elegans as a model system for regulatory and developmental processes in multicellular organisms. In this project we are studying protein expression patterns of the temperature-sensitive C. elegans mutant glp-1(e2144) (5). This mutant presents a normal phenotype when grown at 15 °C. However, when grown at 25 °C formation of germ line cells is lacking.

Differential proteome analyses (6-8) of the C. elegans mutant grown at different temperatures were performed and characteristic alterations in protein expression were observed by 2D-gel electrophoresis (2-DE). The protein extracts (1.5 ml) contained a total of 4.1 mg protein, each. 2D-gel electophoresis (30 x 23 cm2) was performed with 15 µl aliquots, applying high resolution conditions (9). Isoelectric focussing was carried out using a pH gradient ranging from 4 to 10. After staining with silver approx. 3000 spots were detected by computer assisted image analysis. The protein spots were excised using a picking robot. From the approx. 100 protein spots that were analyzed and identified by MALDI-TOF MS up to now, about 15 spots / spot families were found reproducibly different in abundance. While for many of them the function is still unknown, others are associated with germ cell function. The major sperm protein, for instance, was found in high abundance only in the 2-DE gels from the culture grown at 15 °C. This finding correlates well with normal germ cell development at this temperature. By contrast, in the culture grown at 25 °C vitellogenins were present in much higher abundance in the corresponding 2-DE gels than in the gels from the 15 °C culture. This can be explained by the lack of germ cells at 25 °C, as these proteins after production in the intestine are taken up by the healthy gonads (10).

Hence, mass spectrometric analyses of in-gel digested proteins and subsequent data base searches are applicable for the identification of protein candidates associated with the observed phenotypes. The obtained informations lay the grounds for detailed biochemical and cell biological investigations for studying regulatory and developmental processes in multicellular organisms.

References

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  10. J. Spieth, K. Denison, S. Kirtland, J. Cane and T. Blumenthal (1985). Nucleic Acid Res. 13, 5283-5295.



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