mitoPROT:

Novel assays to quantify disease-associated proteins

State of the art

Different approaches to identify deficiencies of mitochondrial complexes on the protein level are currently used that offer both specific advantages and limitations. Direct enzymatic analysis is optimal for functional and inhibitor-sensitivity studies if sufficient sample is available. Isolation and quantification of mitochondrial complexes followed by in-gel activity stain, western blotting or densitometric analysis are rather complicated and time-consuming. Potentially useful novel approaches rely on the high sensitivity of fluorescence detection similar to classical "Fluorescence Difference Gel Electrophoresis" (DIGE) approaches. No attempts have been made so far to optimize the labelling conditions to quantify mitochondrial complexes in 1-D and 2-D gels.

Aims of the project

Major aims of the project are to develop a simple and highly sensitive assay to quantify the five mitochondrial oxidative phosphorylation complexes from patient samples, and to screen the cohort of patients of the consortium by the novel protocol. This will guide the collaboration partners with their search for the underlying genetic alterations. We expect that clinical institutions and biochemical research in general will benefit from the novel protocols. Modifications of the basic protocol should be useful to elucidate the protein interfaces in supramolecular complexes.

Project management and contact addresses

Ilka Wittig Dr. rer. nat. Ilka Wittig
Molekulare Bioenergetik
Zentrum der Biologischen Chemie
Goethe-Universität Frankfurt
Theodor-Stern-Kai 7, Haus 26, 60590 Frankfurt, Deutschland

Tel: +49-69-6301-4180
Fax: +49-69-6301-6970
E-mail: wittig@zbc.kgu.de


Hermann Schägger Prof. Dr. rer. nat. Hermann Schägger
Molekulare Bioenergetik
Zentrum der Biologischen Chemie
Goethe-Universität Frankfurt
Theodor-Stern-Kai 7, Haus 26, 60590 Frankfurt, Deutschland

Tel: +49-69-6301-4180
Fax: +49-69-6301-6970
E-mail: schagger@zbc.kgu.de

Publications:

  1. Wittig, I., Meyer, B., Heide, H., Steger, M., Bleier, L., Wumaier, Z., Karas, M. and Schägger, H. (2010) Assembly and oligomerization of human ATP synthase lacking mitochondrial subunits a and A6L. Biochim. Biophys. Acta - Bioenergetics 1797:1004-1011.
  2. Haack, T.B., Danhauser, K., Haberberger, B., Hoser, J., Strecker, V., Boehm, D., Uziel, G., Lamantea, E., Invernizzi, F., Poulton, J., Rolinski, B., Iuso, A., Biskup, S., Schmidt, T., Mewes, H.-W., Wittig, I., Meitinger, T., Zeviani, M. and Prokisch, H. (2010) Exome sequencing identifies ACAD9 mutations as a cause of complex I deficiency. Nature Genetics 42:1131-1134.
  3. Strecker, V., Wumaier, Z., Wittig, I. and Schägger, H. (2010) Large pore gels to separate mega protein complexes larger than 10 MDa by blue native electrophoresis: Isolation of putative respiratory strings or patches. Proteomics 18:3379-3387.
  4. Wittig, I., Beckhaus, T., Wumaier, Z., Karas, M. and Schägger, H. (2010) Mass estimation of native proteins by blue-native electrophoresis: principles and practical hints. Mol. Cell. Proteom. 9:2149-2161.
  5. Sukhorukov, V.M., Dikov, D., Busch, K., Strecker, V., Wittig, I. and Bereiter-Hahn, J. (2010) Determination of protein mobility in mitochondrial membranes of living cells. Biochim. Biophys. Acta - Biomembranes 1798:2022-2032.
  6. Muster, B., Kohl, W., Wittig, I., Strecker, V., Joos, F., Haase, W., Bereiter-Hahn, J. and Busch, K. (2010) Respiratory chain complexes in dynamic mitochondria display a patchy distribution in life cells. PLoS ONE 5:e11910