ELECTROSPRAY TANDEM MASS SPECTROMETER

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The board objective of this grant application is to make available state- of-the-art electrospray mass spectrometry to investigators at the National Jewish Center/University of Colorado Health Sciences Center. The electrospray ion source permits molecular weight determination of intact proteins and sequence determination of peptides. Instrumentation will be applied in studies of antigen receptors on lymphocytes which play an important role in immunologic defenses against potential pathogens. Electrospray/MS/MS will be used to determine the structure of antigenic peptide fragments bound to the grooves in the histocompatibility complex (MHC) from class 1 as well as class 2 proteins. The distribution of peptides is not only important in resistance to infection, but also may play a role in multiple sclerosis. Phosphorylation plays a prominent role in regulation of the B cell antigen receptor and direct mass spectrometry will be used to assess receptor phosphorylation under a variety of stimulatory conditions. Protein phosphorylation is also thought to play an important role in the cascade of enzyme activation steps involved in signal transduction events. Specific studies of the site of phosphorylation are planned with one of these, MEK kinase (MAP kinase/ERK kinase). The suicide inactivation of the enzyme 5-lipoxygenase, the central enzyme in the synthesis of leukotrienes, will be studied using electrospray mass spectrometry. The stoichiometry of the covalent attachment of LTA4 to 5-lipoxygenase will be directly assessed. The inactivated enzyme will be enzymatically digested into fragments that can be analyzed to determine sites of LTA4 attachment (specific immunoassay residues) as well as the metabolic fate of the LTA4 structure. Electrospray mass spectrometer will also be used for the analysis of small molecular weight molecules, in particular metabolites of arachidonic acid. The instrument will facilitate structure elucidation of metabolites of leukotriene B4 isolated from monkey as well as human cell incubations. The instrument will also be used for quantitative analysis of the major human urinary metabolites of the cysteinyl leukotrienes, 16-carboxy-LTE4 and 14-carboxy-LTE3. Another small lipid mediator that will be analyzed is platelet activating factor. The instrument will provide sensitive quantitative assays for this polar, nonvolatile lipid molecule as well as various CoA esters in experiments designed to test the hypothesis that the synthesis of PAF is regulated by the concentration of substrates lyso-PAF and fatty acyl CoA esters.