This proposal will develop and apply new methods for differential proteomics. Our hypothesis is that 15N stable isotope labeling of rats will allow differential proteomics using mammals. The long term objective is to develop advanced proteomic capabilities that will: identify proteins in complex mixtures by shotgun proteomics, measure differences in abundance of protein species by quantitative differential proteomics, identify and quantify global differences in protein post-translational modifications, and identify sequence variations in complex protein mixtures. A centerpiece of these objectives is a method to metabolically label the proteins of rats with 15N for quantitative analysis of proteins and protein modifications. New methods will be used to prepare tissues from brain for analysis by shotgun proteomics. Cell membranes will be labeled and then digested to preserve protein topology information. Multidimensional separations will be advanced to increase the numbers of proteins that can be identified from stable isotope labeled peptide mixtures. These methods will be used to create differential proteomic profiles of cell membranes from the cerebellum, cerebrum, and striatum/hypothalamus of a fetal and an adolescent rat. Information from these studies will lead to the development of specific hypotheses of receptor and protein populations during brain development. These methods will enable the use of powerful genetic models of development and disease for whole organism proteomics. ? ?
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