We are applying proteomic methodology to unresolved problems in neuropathologic diseases. Methods to identify protein biomarkers of neuropsychiatric disorders, such as the obsessive compulsive syndrome that follows streptococcal infections (PANDAS) in pediatric patients, were tested. A combination of immunoaffinity strategies was used to separate proteins from patient sera. Mass spectral patterns of proteins were compared to determine whether there were statistically significant characteristics of patient state and the disease trait. We prepared several sub-proteome fractions and applied chemometric analyses to test whether MALDI/TOF measurements of the intact proteins produce disease characteristic profiles. There was no significant difference among the spectra related to disease state by principle components analysis. Using an alternative sample preparation (Conconavin A affinity), plasma and serum glycoproteomes associated with PANDAS, Sydenham chorea patients and controls were screened for differentially expressed proteins using SELDI-TOF-MS, 1D and 2D-gels. The absence of demonstrable differential proteomes from IVIgG, IgG or glycoproteomes precluded further analyses. A second strategy is to isolate proteins implicated from genomic studies as being associated with schizophrenia. We tested dysbindin antibody immunoaffinity, but were unsuccessful in capturing peptides related to dysbindin. Consequently, we are exploring the expression of affinity tag labeled dysbindin in an in vitro translation system. We will then explore transient or stable expression transfection of mammalian cells. In collaborative studies with NHGRI, the mitochondrial electron transport complex-I proteomics are being characterized. The hypothesis that deficiency in this complex results in Parkinson Disease is being tested. Preliminary work suggested phosphorylation dependent differences in proteins interacting with the C-terminus of alpha-synuclein. Of particular interest is the interaction of only the non-phosphorylated aS form with mitochondrial electron transport chain proteins. This data suggests that the function and localization of alpha-synuclein might be regulated by kinases in the synapse and that phosphorylation may be an important factor in protein aggregation and Lewy body formation. Although overexpression of alpha-synuclein induces signs of neurodegeneration, alpha-synuclein knockout mice show little to no obvious phenotype. Therefore, our proposal to identify proteins interacting with the alpha-synuclein tail under a variety of conditions will be of importance to understanding the function and localization of alpha-synuclein, possible physiological pathways of regulation and insight into its role in Parkinson Disease. Collaborative studies with NINDS have focused on defining the composition of the post-synaptic density complex. PSD-95, a specialized scaffold protein with multiple protein interaction domains, forms the backbone of an extensive postsynaptic protein complex that organizes receptors and signal transduction molecules at the synaptic contact zone. Large, detergent-insoluble PSD-95-based postsynaptic complexes can be affinity-purified from conventional postsynaptic density (PSD) fractions using magnetic beads coated with a PSD-95 antibody. Purified PSD-95 complexes were analyzed by mass spectrometry. In order to assess enrichment/depletion of proteins upon affinity purification, the relative abundances of proteins in the parent PSD fraction and the isolated PSD-95 complexes were evaluated, based on cumulative ion current intensities of corresponding peptides. The affinity-purified preparation was largely depleted of presynaptic proteins, spectrin, intermediate filaments and other contaminants prominent in the parent PSD fraction. A group of 26 proteins were identified as major components in the PSD-95 complex on the basis of abundance ranking and enrichment upon affinity purification. This group of constituent proteins includes, in addition to the specialized scaffolds and NMDA receptors, an abundance of AMPA receptors, as well as certain proteins that were not previously acknowledged as major elements of the postsynaptic complex. Two of these, BRAG1 (O60275) and BRAG2b (Q6DN90), are of the same family of brefeldin A-resistant regulators of the small G-protein Arf; another is a hypothetical protein (Q8BZM2) with two SAM domains. The properties of these new major components suggest that they may serve pivotal functions in spine structure and plasticity, including reorganization of the actin cytoskeleton, and insertion and retrieval of proteins to and from the plasma membrane. In collaborative studies with NYU, we are performing proteome analyses of several affinity purified protein extracts from HeLa cells expressing the amino terminus of the Huntington?s Disease protein with different numbers of glutamines (the wild type and disease states). The objective of this project is to test the hypothesis that there is transcriptional dysregulation associated with Huntington?s Disease. The first objective is to identify and compare proteins that co-purify with Huntintin (Htt) and Huntintin mutant (HttMut) from nuclear and cytoplasmic fractions. The retroviral vector pOZ-N was used to establish several HeLa cell lines that express FLAG/HA epitope-tagged Htt N-terminus in order to purify Htt-containing protein complexes. Htt amino-terminal constructs that express the 171 or 590 amino acids with 25, 46, or 97 Qs have been made. The 25Q constructs are representative of the wild- type protein. The 46 Q represents an expansion near the pathogenic threshold, and the 97 Q represents the fully expanded form. The Htt-FLAG/HA gene is under the control of the MMLV LTR promoter and the tagged proteins are expressed at levels only slightly higher than that of endogenous Htt. Immunopurification conditions were optimized to maximize positive identifications while keeping nonspecific identifications at a minimum, using small scale coimmuno-precipitations of endogenous proteins to investigate previously reported Htt interactions. A collaborative study with NIDCR is to identify relevant proteins involved in TRPC3 function and regulation in the rat brain. Mammalian transient receptor potential canonical (TRPC) family of cation channels consists of seven members (TRPC1-TRPC7) that are activated in response to agonist-stimulated PIP2 hydrolysis in a variety of tissues. Mass spectrometric analyses have been completed, and significant identified proteins confirmed by Western blot.