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. Current 2-D protein electrophoresis methodology has a major inadequacy 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. One of the techniques that we are investigating uses confocal laser technology. To facilitate this effort we have established a collaboration with the researchers in the NIST Optical and Analytical Chemistry Division. With help from these collaborators we are developing a micro gel chamber 10 microns thick. This is approximately 10x thinner than existing micro gels. The first prototypes are in the testing phase. The separation chamber is thin to prevent quenching of the laser dyes. Unlike CE (capillary electrophoresis) techniques our detection dye is added after separation, avoiding mobility changes by bound fluorescent dye molecules. The trailing edge of the extended stack zone compresses all the protein in a microscopically thin and highly ordered (anisotropic) zone. Our immediate goal with the micro gel chamber is to resolve numerous proteins with unique dye characteristics with a detection sensitivity approaching single molecules. In addition to efforts aimed at enhancing protein detection we also engaged in fundamental studies to facilitate protein separations. SDS-PAGE gels are run everyday but the underlying mechanisms of SDS-PAGE are not clearly understood. Up to 40% of proteins in the first dimension of a 2-D gel do not enter the second dimension. Proteins >200 kD and <10 kD are not well resolved. We hope to dramatically improve the SDS protein gel dimension separation and detection. These studies are ongoing with the collaboration of Andreas Chrambach, NICHD and Lori Goldner's group at NIST. This effort has already provided two important observations: (1) The stacking mechanism is being studied using mass spectroscopy to measure the sodium ion and sulfur ion content in critical zones on the gel. One preliminary finding is that sodium ion content is greatly diminished in the protein-separating zone. Counter-ion type is critical to micelle formation, protein solubility and electophoretic mobility. Recognition that trailing ion buffers and hydrogen ions have largely replaced the sodium in an """"""""SDS"""""""" gel will be crucial to explaining the """"""""stack"""""""" and protein mobility in SDS-PAGE. The stack has also been explored with a series of pH and detergent-binding dyes. We have for the first time been able to identify the true leading/trailing boundary in a discontinuous gel and determine an approximate pH value of the boundary stack by pre-coating the gel with dilute pH dyes and driving the L/T boundary into the pre-positioned dye. (2) The structure of the """"""""extended stack"""""""" ES is being studied in collaboration with the Optical Division at NIST. We are examining the structure of the detergent complex in the stack. Proteins and anionic dyes are excluded from the ES zone, but cationic dyes are not. Preliminary data using a confocal polarized emission pulse laser and wavelength-specific C-12 cationic dyes indicate anisotropy (orderedness) to the ES zone. We have a proposed model for this zone: interdigitated polarizable dodecyl sulfate (-) monomers and hydrogen ion (+) counter ions forming compacted laminar arrays due to the alignment in an electric field. A systematic approach to studying the ES zone using single molecule measurements with confocal laser microscopy, reinterpretation of existing free solution surfactant models and electrophoresis techniques has not been previously reported. All proteins """"""""stacked"""""""" in an """"""""SDS-gel"""""""" - approach - but never reach the ES zone. The existing Ogston model for protein separation in SDS-PAGE does not adequately explain current findings. Our data indicates a combination of the low pH (enriched hydrogen counter ion) and previously undescribed laminar array of detergent monomers will provide an important insight into why proteins stack in gels and how to control band sharpness with modified buffer conditions. In addition we are exploring methods to extend the number of proteins that can be characterized by 2-Dimensional electrophoresis. These efforts include exploration of prefractionation by micro solution isoelectrofocusing. This approach allows for the removal of major proteins, such as albumin in serum or actin in cell lysates, so that trace proteins can be detected. 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 could 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 |
