It is proposed to develop a leukocyte proteome map as a new research resource for scientists studying immune mediated events in mice. The proteome consists of all of the proteins that are expressed at any one time, in a cell or tissue. A proteomic assay is, therefore, by definition the most encompassing assay for changes at the protein level. It is the protein equivalent of DNA microarray assays. By evaluating shifts in 2D gel electrophoresis (2DGE) proteome patterns of blood leukocytes, immunologists can quickly find out if any of thousands of proteins are expressed at different levels in two leukocyte samples. Unfortunately, if interesting differences are found the process of identifying what protein is in the spot that changed expression requires resources and expertise not available to most scientists. High sensitivity mass spectrometry (MS) is used to characterize such proteins, via the molecular weight (mw) of the intact protein, and via peptide mapping (mw and amino acid sequence of e.g. tryptic peptides from the protein) It is proposed to make 2DGE proteome assay of mouse leukocytes available to most scientists by establishing a 2DGE proteome reference map of mouse blood leukocytes. This will allow instant identification of a large number of protein spots on 2D gels, eliminating the need for MS analysis to characterize spots on the gels. The initial three year proposal will be used to create a """"""""low detail"""""""" map and solve some important technical questions, that must be resolved before an application for creating a full scale detailed mouse leukocyte proteome map can be submitted. The proposal has the following Specific Aims:
Specific Aim 1. To establish a 2DGE proteome reference map of mouse blood leukocytes that will allow an instant identification of a number of protein spots on 2D gels, eliminating the need for MS analysis to characterize those protein spots.
Specific Aim 2. To evaluate the extent to which such maps are useful in mouse strains different from the one used to create the map. Polymorphism in immunologically important molecules may create pattern differences between mouse strains. The extent to which such differences will interfere with the use of a leukocyte proteome map created in another strain cannot presently be predicted.
Specific Aim 3. To evaluate the extent to which a general mouse leukocyte map can be used to study proteomes of individual leukocyte subsets. Important immunological events may occur only in specific subsets of leukocytes, and presently, it cannot be predicted to what extent the presence of proteins from all other leukocyte subsets may interfere with the ability to detect such events.
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