To understand normal and pathophysiological processes at the molecular level in the central nervous system it is essential to develop methods for studying proteins expressed in individual cells. The cellular heterogeneity of the brain, in which glial cells generally surround and outnumber neurons at a ratio of 10 to 1 and the existence of different types of neurons in close proximity to one another requires a technology that permits the study of proteins from single neurons and single glial cells. High- resolution 2-D protein electrophoresis should help facilitate such studies. Current 2-D protein electrophoresis methodology has two major inadequacies. The first, is caused by the limits of sensitivity for protein detection. While silver staining can detect as little as 0.01 nanogram of protein, for an average protein of molecular weight 30kD this represents approximately 200 million molecules. At this level of detection it is not possible to detect many proteins from a single cell, such as those in the brain, since the average number of molecules of any specific cellular protein is considerably below this range. The second problem is that it has proven to be difficult, and in some instances impossible, to characterize proteins that appear to be biologically interesting but present as silver stained spots at the 0.1 to 0.01ng level. While the use of mass spectrometric analysis of the silver stained spots is beginning to alleviate this problem the analysis of trace proteins spots is still a problem. Given these problems, both of these inadequacies could be overcome by using either bacterial viruses (phage) or bacteria as protein detection reagents. These organisms have the potential to serve as detection reagents with high sensitivity because of their ability to replicate exponentially and their capacity to display specific ligands on their surface. It is currently possible to incorporate ligand or antibody genes into phage or bacterial genomes in such a manner that these ligands or antibodies are expressed as fusion products with normal surface proteins on these viruses or bacteria. The display of such ligands or antibodies on the surface of these organisms can facilitate their binding to appropriate protein-binding motifs. The use of such fusion product displays can be employed both for general and for specific protein detection by using organisms displaying a specific ligand or a collection of organisms displaying a ligand library respectively. As both phage and bacteria can grow exponentially they can provide a detection system that has the theoretically possibility of detecting as few as 1 to 10 molecules of a specific protein. We have tested this approach by using the phage M13 to detect serial dilution of proteins on membranes and blots of serial dilutions of proteins separated on electrophoretic gels. Preliminary experiments have demonstrated sensitivities close to the theoretical limits of 1 to 10 molecules of protein. A major biotech company has recently confirmed our preliminary results. We have also used E.coli displaying ligand on flagella to detect proteins directly on electrophoretic gels. When a ligand library is used to provide an amplification detection system for protein separated from cells on two-dimensional electrophoretic gels, each phage plaque or bacterial colony will contain specific phage or bacteria that bind to a specific protein spot. These agents could then be replicated to provide for assay systems for identification of these specific proteins. They may also be used as reagents to aid in the purification of quantities of the specific proteins necessary for structural characterization. Based on these preliminary results a patent for the use of organisms capable of exponential growth and ligand display for high sensitivity detection of proteins has been filed. Over the past year I have been invited to give numerous lectures including a plenary lecture at a symposium on """"""""Current 2-D separation technologies applied to the study of complex protein mixtures"""""""" in Frederick, MD, as well as at """"""""The Third Annual Phage Display Technologies Symposium"""""""" in Cambridge, MA. In addition, there has been some interest on the part of biotechnology companies to establish CRADA's with the LBG to help facilitate the development of more sensitive protein detection methods. Currently the LBG is conducting a study on childhood onset schizophrenia (COS), defined as onset of psychotic symptoms before the 13th birthday, This disease is clinically and neurobiologically continuous with adult onset schizophrenia, however is more severe and progressive. The cause of COS, as well as schizophrenia in general, has yet to be determined. Evidence points to a strong genetic component, but environmental factors, such as pre- or postnatal infections, are also likely, and may play a significant role in COS. The results of a large number of studies searching to identify specific pathogen in schizophrenia (i.e. influenza, rubella, Borna virus, etc) are controversial. The difficulty lies in the enormous number of possible pathogens, which making screening for specific organisms or antibodies an extremely laborious task. We developed a scheme using phage display that would allow us to screen for large numbers of antibodies simultaneously. We are using serum samples of 15 COS subjects to screen for common immunological epitopes. If COS patients share common antibodies to a specific agent, panning and selection of a phage library against multiple patients, along with subtractive panning against normal controls, should yield sub-libraries that display peptide epitopes common to the patients. Identifying a causative agent from such epitopes may provide a platform from which to search for such causative agents as well as for development of potentially useful diagnostic tools. Besides infectious agents, we may also identify other possible antigens for an immune response such as toxins or autoimmune phenomena. We are also continuing our work on protein expression in neuronal cultures. Protein patterns from primary neuronal cultures, derived from cortical, striatal and hippocampal brain regions dissected from 19 day Sprague-Dawley rat embryos, are being compared with each other and with tissue from similar regions from an adult brain. Currently a manuscript is in preparation for submission to Electrophoresis. The goal of this effort is to develop a rat neuronal protein database on the WWW and to use this type of neuronal culture system as a model for studying the effects of stress such as hypoxia, growth factors, toxins, and as a guide to interpret protein patterns with the protein detection system being developed which may permit detection of proteins from single neurons. This database will be linked with the SWISSPROT database. One of the major problems with 2D electrophoresis has been the identification of the protein spots. Until recently it was difficult to characterize proteins that appeared to be of interest due to two major problems: direct N-terminal sequencing required relatively large amounts protein, difficult to achieve for trace proteins, and many of our proteins were N-terminal blocked (estimates indicate that ~ 45% of proteins resolved by 2DE are N-terminally blocked). Initial attempts to characterize proteins isolated from gels with mass spec were disappointing. Our collaborators recently demonstrated that they can successfully characterize proteins from our gels by using data generated by mass MALDI-TOF and tandem spectrometry in concert with data from both protein and genomic databases. This technology, along with the LBG's high-resolution two-dimensional protein electrophoresis methodologies, should permit us to pursue clinical and basic studies of the proteome more effectively than we were able to in the past.
| Zullo, S J; Parks, W T; Chloupkova, M et al. (2005) Stable transformation of CHO Cells and human NARP cybrids confers oligomycin resistance (oli(r)) following transfer of a mitochondrial DNA-encoded oli(r) ATPase6 gene to the nuclear genome: a model system for mtDNA gene therapy. Rejuvenation Res 8:18-28 |
