Measurement of nitric-oxide-modified proteins by cavity ring-down spectroscopy

通过腔衰荡光谱法测量一氧化氮修饰的蛋白质

• 批准号: 8576343
• 负责人: Kevin K. Lehmann
• 金额: $ 17.46万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-01 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8576343
• 关键词: Area; Biological; Biological Assay; Biology; Blood; Blood Vessels; Cells; Cellular biology; Chemical Engineering; Chemistry; Clinical; Coupled; Cyclic GMP; Cysteine; Detection; Devices; Disease; Endothelial Cells; Epithelial Cells; Eukaryotic Cell; Guanylate Cyclase; Health; Heme; Human; Label; Light; Location; Lung; Mass Spectrum Analysis; Measurement; Measures; Medicine; Membrane; Metabolism; Metalloproteins; Methods; Modification; Movement; Muscle relaxation phase; Neurons; Nitric Oxide; Nitric Oxide Synthase; Nitrogen Oxides; Noise; Optics; Phase; Post-Translational Protein Processing; Process; Proteins; Proteome; Proteomics; Reaction; Reproducibility; S-Nitrosothiols; SKIL gene; Sampling; Scheme; Sensitivity and Specificity; Serum; Signal Pathway; Signal Transduction; Specimen; Spectrum Analysis; Sulfhydryl Compounds; System; Techniques; Technology; Testing; Time; Vesicle; absorption; biological research; body system; cytotoxic; design; improved; insight; instrument; metabolic abnormality assessment; novel; photolysis; public health relevance; uptake

DESCRIPTION (provided by applicant) Nitric oxide synthase products signal through interactions with heme centers in metalloproteins, such as guanylate cyclase and through covalent reactions with protein cysteine thiols. These nitrosylation reactions have broad relevance to eukaryotic cell biology in health and disease. However, nitrosylated proteins have often proven difficult to detect with the sensitivity and specificity needed for clinical and reseach samples. We are developing cavity ring-down spectroscopy to measure these protein modifications. We believe that this technology will dramatically improve both sensitivity and specificity it has the potential to will revolutionize the field. Here, we propose to design and fabricate an improved cavity ring-down instrument with a sample photolysis interface. This instrument will use advanced optical spectroscopy techniques to measure S- nitrosothiols in biological specimens. It will be used to studying a broad range of diseases and organ systems. It will be more powerful and more precise than previous technologies, measuring NO-modified proteins accurately in pM concentrations with minimal sample disruption. In the second Aim, it will also be applied to proteomic microarrays for rapid, high-throughput discovery.

说明（由申请人提供）一氧化氮合酶产物通过与金属蛋白（如鸟苷酸环化酶）中的血红素中心的相互作用以及通过与蛋白质半胱氨酸硫醇的共价反应来发出信号。这些亚硝基化反应与健康和疾病中的真核细胞生物学具有广泛的相关性。然而，亚硝基化蛋白质通常被证明难以以临床和研究样品所需的灵敏度和特异性进行检测。我们正在开发腔衰荡光谱来测量这些蛋白质修饰。我们相信这项技术将大大提高灵敏度和特异性，并有可能彻底改变该领域。在这里，我们提出了一种改进的光腔衰荡仪器的设计和制作与样品光解接口。该仪器将使用先进的光谱技术来测量生物样品中的S-亚硝基硫醇。它将用于研究广泛的疾病和器官系统。它将比以前的技术更强大，更精确，以pM浓度准确测量NO修饰的蛋白质，并将样品破坏降至最低。在第二个目标中，它也将被应用于蛋白质组微阵列，以实现快速，高通量的发现。