The next Step: Exploring the Proteome:
Translation and Beyond

Proteome Stargazing

Jonathan Minden, Department of Biological Sciences and The NSF Science and Technology Center for Light Microscope Imaging and Biotechnology,
Carnegie Mellon University


Abstract:

Proteomics is a new and burgeoning field that has emerged as a major focus since the sequencing of the human genome and the genomes of many other model organisms. The proteome is defined as the collection of proteins found in cells, tissues and organisms. Individual cells may express 5,000-10,000 different proteins, while a whole organism can express >1,000,000 protein isoforms. One of the main goals of proteomics is to identify protein changes that occur normally during development and tissue differentiation and as a result of disease, drug treatment and environmental change. There are two main components to proteomics: separation of cellular proteins into individual components and identification of the genes that encode individual proteins. Protein separation is usually conducted by two-dimensional gel electrophoresis, which separates proteins based on size and charge. Identification of the gene encoding isolated proteins is done by mass spectrometry where one compares the mass of specifically-generated protein fragments to a database of predicted protein fragments.

The scale of the genome sequencing projects pales in comparison to the goal of proteome projects. There are plans to search for protein changes in dozens of different tissues at dozens of different stages of development and dozens of different diseases from hundreds of samples and in the presence of thousands of different drugs and conditions. The number of possible experiments is truly staggering. There is great need to increase the rate of proteome analysis 10-100 fold, or more. We have developed a method, called difference gel electrophoresis (DIGE), to rapidly identify protein changes between two or three samples on the same two-dimensional electrophoresis gel. This method relies on fluorescently tagging all proteins in each sample with one of a set of matched fluorescent dyes that do not affect the relative mobility of proteins during electrophoresis. This method greatly reduces the complexity of the search for protein differences from several thousand proteins observed in the protein extract to several dozen candidate proteins that are significantly different between the samples. DIGE is more sensitive than silver staining and can detect changes as little as 0.01% of total protein. The imaging system can detect a 1000-fold range of protein concentrations; silver staining can only detect a 30-fold concentration range. This level of sensitivity and discrimination challenges the performance of mass spectrometers to identify the genes that encode the proteins difference that we can detect using DIGE. This presentation will describe the uses of DIGE and future challenges for proteome analysis.

Biographical sketch:

Jonathan Minden, Ph.D.Associate Professor

EDUCATION/TRAINING
University of Toronto, Toronto, Canada: B.Sc., 1980, Chemistry/Biochemistry
Albert Einstein College of Medicine, Bronx, NY: M.A., 1982, Molecular Biology
Albert Einstein College of Medicine, Bronx, NY: Ph.D., 1985, Biochemistry & Molecular Biology

Professional Positions:
1980-1981. Graduate student, Department of Molecular Biology at Albert Einstein college of Medicine, Bronx, NY. Advisor Dr. Margaret Clarke.
1982-1985. Graduate student, Department of Developmental Biology and Cancer at Albert Einstein College of Medicine, Bronx, NY. Advisor Dr. Kenneth Marians.
1986-1990. Postdoctoral fellow, Department of Biochemistry and Biophysics, University of California at San Francisco, San Francisco, CA. Advisor Dr. Bruce M. Alberts.
1991-1997. Assistant professor, Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA.
1997-present. Associate professor, Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA.

Awards and Other Professional Activities:
1980 - 1984 NIH Predoctoral Training Grant1986 - 1989 Helen Hay Whitney Foundation Postdoctoral Fellowship Award
1989 - 1996 Lucille P. Markey Charitable Trust Scholarship Award

Research Projects Ongoing or Completed During the Last 3 Years:

Drosophila Pattern Repair: Cell Fate and Death Mapping
Principle Investigator: Jonathan Minden
Agency: National Institute of Child Health and Human Development
Type: 5R01HD31642-06 Period: 1-DEC-97 to 30-NOV-02
The long term objective of this grant is to understand how fields of embryonic cells monitor and correct patterning defects. Using Drosophila embryogenesis as a model system, we have studied how embryos contend with defects such as perturbation of the anterior-posterior axis and cell density. Embryos deal with these defects by killing excess cells by apoptosis, but cannot induce extra divisions to increase diminished regions. We have developed a wide range of time-lapse microscopy methods to monitor cell division, shape, death and gene expression. We have also mapped cell death patterns in the embryonic epithelium. Recently, we have narrowed our studies to understanding how small groups of cell behave under normal and perturbed conditions. We have developed a method to selectively turn on cloned transgenes by light using a caged-GAL4 and UAS-transgene expression system. We are currently single-cell fate mapping the pro-cephalic mitotic domains.

Rapid detection of Cellular Protein Differences
Principle Investigator: Jonathan Minden
Agency: National Institute for Human Genome Research
Type: 5R01HG01724-03 Period: 30-SEP-97 to 31-AUG-00
The goal of this grant was to further develop difference gel elctrophoresis, DIGE. There were three goals: to develop improved DIGE dyes, to construct an improved fluorescence imager robotic gel cutting tool, and to develop software for automated protein difference detection. At the writing of this proposal, we are in the final year of this grant and have completed the first two goals. The final goal is well under way. We have improved the sensitivity of DIGE several hundred fold and the imager/gel cutter is fully operational. This grant will end in August, 2000. A competing renewal of this grant will not be submitted.

Relevant Publications (Partial List)
1. Minden, J.S. and Marians, K.J. (1985) Replication of pBR322 DNA in Vitro with Purified Proteins: Requirement for Topoisomerase I in the Maintenance of Template Specificity. J. Biol. Chem., 260, 9316-9325.
2. Minden, J.S., Agard, D.A., Sedat, J.W., and Alberts, B.M. (1989) Direct Cell Lineage Analysis in Drosophila melanogaster by Time-Lapse, Three-Dimensional Optical Microscopy in Living Embryos, J. Cell Biol., 109, 505-516. ,BR>3. Hiraoka, Y., Minden, J.S., Swedlow, J.R., Sedat, J.W., and Agard, D.A. (1989) Focal Points for Chromosome Condensation and Decondensation Revealed by Three-dimensional in vivo Time-lapse Microscopy. Nature, 342, 293-296.
4. Minden, J.S., Kam, Z., Agard, D.A., Sedat, J.W., and Alberts, B.M. (1990) Embryonic Lineage Analysis using Three-dimensional Time-lapse in vivo Fluorescence Microscopy. In Bioimaging and Two-dimensional Spectroscopy. (l. Smith. Ed.), SPIE vol 1205, pp 29-42.
5. Kam, Z., Minden, J.S., Agard, D.A., Sedat, J.W., and Leptin, M. (1991) Drosophila Gastrulation: Analysis of Cell Shape Changes in Living Embryos by Three-dimensional Fluorescence Microscopy. Development, 112, 365-370.
6. Valdes-Perez, R.E. and Minden, J.S. (1995) Drosophila melanogaster Syncytial Nuclear Divisions are Patterned: Time-lapse Images, Hypothesis, and Comuptational Evidence. J. Theor. Biol., 175, 525-532.
7. Minden, J.S. (1996) Synthesis of a New Substrate for Detection of lacZ Gene Expression in Live Drosophila embryos. BioTechniques, 20, 122-129.
8. Namba, R., Pazdera, T. M., Cerrone, R., and Minden, J. S. (1997) Drosophila embryonic pattern repair: How embryos respond to bicoid mRNA dosage alteration. Development. 124, 1393-1403.
9. Ünlü, M., Morgan, M. E., and Minden, J. S. (1997). Difference gel electrophoresis: a single gel method for detecting changes in cell extracts. Electrophoresis, 18, 2071-2077.
10. Cambridge, S. B., Davis, R. L. and Minden, J. S. (1997). Drosophila mitotic domain boundaries represent cell fate boundaries. Science, 277, 825-828.
11. Francis-Lang, H., Minden, J. S., Sullivan, W. and Oegema, K. (1999). Live confocal analysis with fluorescently labeled proteins. Methods Mol. Biol. 122, 223-39.
12. Pazdera, T. M., Janardhan, P., and Minden, J. S. (1998). Patterned cell death in the epidermis of wild-type and segment-polarity mutant Drosophila embryos, Development 125, 3427-3436.
13.Minden, J. S. (1999). Three-dimensional, time-lapse microscopy of Drosophila embryogenesis. In A Comparative Approach to the Study of Oocytes and Embryos. Ed. Richter. Oxford University Press, Inc.. New York, NY.
14. Namba, R. and Minden, J. S. (1999). Fate mapping Drosophila embryonic mitotic domain 20 reveals that the larval visual system is derived from a subdomain of a few cells, Developmental Biology, 212, 465-476.
15. Li, Q-J., Pazdera, T, M., and Minden, J. S. (1999). Drosophila embryonic pattern repair: How embryos responded to cyclin E-induced ectopic division, Development, 126, 2299-2307.
16. Minden, J. S., Namba, R. and Cambridge, S. B. (1999). Single-cell fate mapping. In Drosophila: A laboratory manual. Eds. Sullivan, Hawley, Ashburner. Cold Spring Harbor Press. Cold Spring Harbor, NY.

Patents:
1. Minden, J. S. and Waggoner, A. (1999). Difference Gel Electrophoresis using Matched Multiple Dyes. S.N.:08/425,480

Contact Information:

Jonathan S. Minden, Department of Biological Sciences, Carnegie Mellon University, 4400 5th Avenue, Pittsburgh, PA 15213, Tel: (412) 268-2669, Lab: (412) 268-3448, Fax: (412) 268-7129, Email: minden@cmu.edu.