ELECTROSPRAY TANDEM MASS SPECTROMETER

这项拨款申请的董事会目标是使国家- 最先进的电喷雾质谱仪给研究人员 国家犹太人中心/科罗拉多大学健康科学中心。这个 电喷雾离子源可用于完整分子量的测定 蛋白质和多肽的序列测定。仪器设备将是 在淋巴细胞表面抗原受体研究中的应用 在对潜在病原体的免疫防御中发挥重要作用。 电喷雾/MS/MS将用于确定抗原的结构 与组织相容性复合体中的凹槽结合的多肽片段 (MHC)来自1类和2类蛋白。分布情况： 多肽不仅在抵抗感染方面很重要，而且还可能 在多发性硬化症中起一定作用。磷酸化起着突出的作用。 B细胞抗原受体的调节与直接质谱学 将被用来评估在各种不同的 刺激条件。蛋白质的磷酸化也被认为起了作用 在一系列酶激活步骤中扮演着重要的角色 信号转导事件。对古城遗址的具体研究 计划与其中之一，MEK激酶(MAP激酶/ERK)一起进行磷酸化 激酶)。5-脂氧合酶的自杀性失活， 白三烯合成中的中心酶，将使用 电喷雾质谱仪。共价键的化学计量 将直接评估LTA4与5-脂氧合酶的结合。这个 失活的酶将被酶消化成片段，可以 被分析以确定LTA4的附着位置(特异性免疫分析 残基)以及LTA4结构的代谢命运。 电喷雾质谱仪也将用于分析 小分子，特别是其代谢物 花生四烯酸。该仪器将有助于结构的阐明 猴和人白三烯B4代谢物的研究 细胞孵化。该仪器还将用于定量 人体尿中主要半胱氨酸代谢物的分析 白三烯，16-羧基-LTE4和14-羧基-LTE3。另一小块脂类 需要分析的介质是血小板活化因子。这个 仪器将为这一极地提供灵敏的定量分析， 实验中的非挥发性脂类分子以及各种辅酶A酯 旨在检验PAF的合成受到调控这一假设 通过底物Lyso-PAF和脂肪酰基CoA酯的浓度。

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.