Measuring Lipid Flux By Ultra High Resolution Mass Spectrometry

通过超高分辨率质谱法测量脂质通量

• 批准号: 10027699
• 负责人: Matthew Alvin Mitsche
• 金额: $ 40.88万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-05 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10027699
• 关键词: Address; Analytical Biochemistry; Animal Model; Atherosclerosis; Biochemical Pathway; Biochemistry; Biomedical Research; Cardiovascular Diseases; Chromatography; Collaborations; Communities; Data; Data Collection; Deuterium; Development; Diabetes Mellitus; Diagnosis; Disease; Electrospray Ionization; Engineering; Environment; Fatty Liver; Fatty acid glycerol esters; Goals; Human; Infusion procedures; Intervention; Ions; Isotope Labeling; Isotopes; Label; Lead; Lipid Biochemistry; Lipids; Location; Malignant Neoplasms; Manufacturer Name; Maps; Mass Spectrum Analysis; Measures; Metabolic; Metabolic syndrome; Modernization; Nerve Degeneration; Noise; Patients; Proteomics; Protocols documentation; Radioisotopes; Research; Resolution; Signal Transduction; Software Tools; Source; Specificity; Stable Isotope Labeling; Techniques; Technology; Time; Tracer; Training; Translating; Water; base; effective therapy; effectiveness measure; experience; human disease; improved; in vivo; instrument; lipid biosynthesis; lipid metabolism; mass spectrometer; mouse genetics; mouse model; multidisciplinary; novel; programs; stable isotope; technology validation; tool; ultra high resolution

Project Summary: Many human diseases are caused by a dysregulation of lipid metabolism, including atherosclerosis, cancer, neurodegeneration, diabetes, and fatty liver. The development of effective treatments for lipid related disorders is hinder by a lack of modern in vivo biochemistry techniques for studying lipid metabolism. The overall goal of this proposal is to develop tools and protocol to measure the rates of lipid biosynthesis and remodeling by stable isotope labeling with sensitivity comparable to radio-isotope tracing with the specificity and broad coverage of modern mass spectrometry based lipidomics. This is enabled by an ultra-high resolution orbitrap mass spectrometer I developed in collaboration of Thermo Scientific, now commercially available as the Lumos 1M. This instrument has sufficient resolution to resolve the natural abundance 13C from a tracer isotope, for example 2H, in intact lipid ions. By resolving the dominant natural abundance ions from tracer isotopes will improve the signal to noise ratio by at least 2 orders of magnitude (1:1 vs >1:100) and increase the dynamic range. This advancement will allow in vivo analysis of lipid metabolism to study a variety of disease, and will ultimately lead to lipid fluxomics analysis that is translatable to human studies. By measuring lipid flux in patients we will be able to directly studying the progression of metabolic syndrome, potentially circumventing the need for animal models, and measure the effectiveness of therapies and interventions. To facilitate the development and widespread implementation of this technology, I will address the fundamental roadblocks to adapting this technology. Firstly, the commercial instrument is engineered for proteomics applications, in particular the electrospray ionization source. By working with the manufacturer and translating my lipidomics experience to this new platform I will overcome these issue. Secondly, I will develop novel data collection approaches for both chromatography and direct infusion based applications to accommodate the long transient time and coalescence issues associated with ultra-high resolution resonance based mass spectrometry. Thirdly, software tools will be developed to extract ultra-high resolution data in a time efficient manner, convert the data to physically interpretable parameters, and map data onto biochemical pathways. Lastly, I will develop protocols and platforms for stable isotope labeling by deuterium labeled water (D2O) and other isotope labeled metabolic tracers in mouse models of metabolic syndrome relevant to my lab’s research program studying the mechanism for fat accumulation. By accomplishing these aims this technology will be accessible to the biomedical research community. My multi-disciplinary training in engineering, physical, analytical and biochemistry, and mouse genetics makes me well-suited to develop this technology and the lipid centric research environment at UT Southwestern is the ideal location for the initial application.

项目摘要： 许多人类疾病是由脂质代谢的失调引起的，包括动脉粥样硬化， 癌症，神经变性，糖尿病和脂肪肝。开发有关脂质相关的有效治疗 由于缺乏研究脂质代谢的现代体内生物化学技术而造成的疾病阻碍了疾病。这 该提案的总体目标是开发工具和协议，以衡量脂质生物合成和 通过稳定的同位素标记重塑，灵敏度可与放射性异位座相媲美，并具有特异性 以及基于现代质谱脂质组学的广泛覆盖范围。这是由超高启用的 分辨率Orbitrap质谱仪我开发了Thermo Scientific的合作，现在是商业上的 作为Lumos 1m可用。该仪器具有足够的分辨率来解决自然丰度13C 从完整脂质离子中的示踪剂同位素（例如2H）。通过解决主要的自然丰度离子 从示踪剂同位素可以将信号与噪声比至少提高2个数量级（1：1 vs> 1：100），并且 增加动态范围。这种进步将允许对脂质代谢的体内分析来研究 疾病的种类，最终将导致可翻译成人类研究的脂质通量分析。经过 测量患者的脂质通量，我们将能够直接研究代谢综合征的进展， 有可能规避对动物模型的需求，并衡量疗法的有效性和 干预措施。 为了促进该技术的开发和广泛实施，我将解决 基本障碍，以适应这一技术。首先，商业仪器是为 蛋白质组学应用，特别是电喷雾电离源。通过与制造商合作 将我的脂肪组学经验转换为这个新平台，我将克服这些问题。其次，我会发展 针对色谱和直接输注应用的新型数据收集方法 适应与超高分辨率分辨率相关的较长瞬态时间和合并问题 基于质谱。第三，将开发软件工具以提取超高分辨率数据 时间效率的方式，将数据转换为可解释的参数，然后将数据映射到生化 途径。最后，我将开发通过氘标记的水的稳定同位素标记的协议和平台 （D2O）和其他同位素在与我的实验室相关的代谢综合征模型中标记为代谢示踪剂 研究计划研究脂肪积累机制。通过完成这些技术 生物医学研究界将可以使用。我的工程学多学科培训， 物理，分析和生物化学以及鼠标遗传学使我非常适合开发这项技术 UT西南地区的以脂质为中心的研究环境是最初应用的理想场所。