Proteomics in neurotoxicology

National Institute Of Mental Health

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Abstract

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, are being devised and tested. A combination of immunoaffinity strategies is used to separate proteins from patient sera. Mass spectral patterns of proteins are being compared to determine whether there are statistically significant characteristics of patient state and the disease trait. We prepared several sub-proteome fractions and have applied chemometric analyses to test whether MALDI/TOF measurements of the intact proteins produce disease characteristic profiles. A second strategy is to isolate proteins implicated from genomic studies as being associated with schizophrenia. Dysbindin antibody immunoaffinity is being tested with available genetically dysbindin-gene modified mouse strains. 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?s Disease is being tested. Elucidation of the complexity of a protein?s function and its role in biological pathways involves first identifying potential protein-protein interactions. Comparison of these interactions for wild-type vs. modified protein forms with a biologically relevant complex protein mixture can yield quantitative differences in the extent of these interactions relative to its stage in the cellular pathway. The standard methods of pull down assays combined with solution digestion, LC-MS/MS and data base searching provide for a high-throughput strategy for the identification differences in protein-protein interactions. Equally useful are the quantitative differences/similarities imbedded in the measured ion currents for specific peptides. Much attention has been given to the use of stable isotope labeling for relative quantization of protein interactions. The stability of high-throughput LC-MS/MS ion currents are such that integration of ion current alone can be used for rapid screening of up/down regulated proteins occurring between multiple samples. 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 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.