Biochemical Genetics And Metabolic Disease

National Institute Of Mental Health

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Abstract

Traditionally neuroscience has relied on protein studies performed on samples from brain regions. However, even when regions of the central nervous system are microscopic they generally contain numerous cell types, each with different functions and morphology. Current technology does not permit detection of proteins from single cells or even small numbers of cells. The commonly used organic stains such as Coomassie Blue provides for the detection of proteins in the microgram range. The introduction of fluorescent stains, such as SYPRO Ruby Red fluorescent stain and the silver stains, advanced the limits of detection into the nanogram range. While the most sensitive silver stains that we have developed, under ideal conditions, have been demonstrated to be able to detect as little as 0.01 ng of protein, this level of sensitivity for a 30-kDa protein represents approximately 200 million molecules. If we are to study protein alterations in single cells in heterogeneous organs such as the brain, methods of detection levels sufficient to detect as little as 10 to 100 molecules are needed. We are exploring methods to extend the number of proteins that can be characterized. To facilitate our efforts we have been investigating, in collaboration with researchers in the NIST Optical and Analytical Chemistry Division, a number of optical methods to enhance the sensitivity of protein detection. One class of agents that our NIST collaborators encouraged us to look at are the fluorescent semiconductor nanocrystals or quantum dots (qdots). These qdots can be attached to proteins and nucleic acids and they have a photostability several orders of magnitude greater than conventional dyes. They also have an additional advantage in their long fluorescence lifetime which permits the use of time-gated detection, a characteristic that provides for lower backgrounds as the background autofluorescence generally has a shorter half life. Initial experiments used Qdot?605 Streptavidin Conjugate (Quantum Dot Corporation to detect ImmunoPure? Biotinylated Bovine Serum (BSA) Albumin (Pierce Biotechnology). In tests with serial dilutions of biotinylated BSA dot-blotted onto PVDF membranes the best results were achieved by using a carrier protein blocking agent (3%BSA) with Qdot? Streptavidin Conjugate. The blots imaged in collaboration with Dr. Jon Marsh on his FluorChem 8800? , using a 630 nm filter with a 30 nm band pass (BioRad,) achieved sensitivity levels representing 1 to 10 molecules. We are currently developing protocols for the direct detection of proteins in electrophoretic gels. The high sensitivity of the qdots also offers the possibility of their use in a cooperative two component systems for the detection and quantization of proteins in crude cell lysates. In this system, proteins of interest are first bound with an antibody attached to an immobilizing agent (a magnetic bead, a microscope slide or a multiwell plate). The immobilized proteins are extensively washed and the bound proteins are then exposed to a second antibody attached to a qdot (which emits at a specific wave length). Proteins of interest would have to interact with both antibodies to be recorded. Such a system permits high sensitivity and selectivity. We are also developing this approach for nucleic acids. Given the sensitivity of detection permitted by the qdots it may be able to replace PCR for some applications. The section has also been working with other groups in the NIMH on protein separation and detection problems. Our collaborative study with Howard Nash, using 2D electrophoretic and mass spectroscopy techniques to study mutant strains of Drosophila that display specific phenotypic responses to anesthetic agents has progressed well. In these studies proteins spots of interest were dissected from the gels and analyzed by mass spectrometric methods by Drs. Sanford P. Markey and Jeffrey A. Kowalak, (ABC, IRP, NIMH). The confounding presence of ?contaminating proteins? found in the analysis of single spots identified by the mass spectrometric analysis became an issue that needed to be addressed. It has been suggested that by a number of researchers that such ?contamination? is primarily associated with the reagents and working conditions of electrophoretic separations. However, an experiment was performed that indicates that the problem is more general than that. In this experiment sealed empty glass ampoules that should not contain any proteins or peptides, were supplied for mass spectrometric analysis. These ampoules were prepared by heating them with a Bunsen burner, until the glass of the ampoule had a red glow. The heating was initiated at the bottom of the ampoule and progressed upward toward the top opening. When the opening was reached, they were sealed by melting the glass at the tip with the heat of the gas flame. This procedure should have assured that any proteins present in the vials were destroyed by the heat before the ampoules were sealed. Two such vials were provided to the mass spectrometry laboratory. Mass spectrometric analysis reported for the contents of these vials yielded 20 to 99 strong matches (75-100%) with various types of keratin from many species, and 1 to 46 strong matches with non-keratin proteins. Many of these matches were with human peptides. These results indicate that for unambiguous protein profiles the work (both electrophoretic separation and mass spectrometric analysis) should be preformed with the same "clean room" technology currently employed by the computer chip industry. At this time, without the luxury of such ?clean room? technology most researchers use various schemes to determine the most probable protein associated with spots dissected from gels and analyzed by mass spectrometric. Using this approach we have identified 70 proteins in an effort to develop a protein map of Drosophila head proteins. Currently a manuscript entitled "A High Resolution Protein Map of Drosophila head Proteins" is in preparation. We established a collaborative effort with Drs Giulian and Charlie Xiang and Michael Brownstein to examine the effects of lithium on a human cortical neuron-derived cell line (HCN-1A obtained from the ATTC, originally derived from a non-malignant tumor of the CNS in a patient with epilepsy at Johns Hopkins). The goal of this study was to look at alterations of mRNA (using the microarray technologies developed by the LOG) and protein variations with 2D electrophoresis. In preliminary studies a number of mRNAs were found to be significantly changed following Lithium in both the HCN-1A cells and cells derived from human eye tissue. Manuscripts ?Does the Lithium-induced down regulation of the human aquaporin mRNAs account for many of the known Lithium human side-effects?? (Giulian and Merril) and ?mRNA array analysis of human neuronal and glial cell culture after therapeutic treatment levels of Li salts for 1 and 17 days.? (Giulian, Merril, Brownstein, von Kollmar, Xiang) are in preparation from these preliminary experimental results.