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.

项目概要： 许多人类疾病都是由脂质代谢失调引起的，包括动脉粥样硬化、 癌症、神经退行性疾病、糖尿病和脂肪肝。脂质相关有效治疗方法的开发 由于缺乏研究脂质代谢的现代体内生物化学技术而阻碍了疾病的发展。这 该提案的总体目标是开发工具和协议来测量脂质生物合成和 通过稳定同位素标记进行重建，其灵敏度与放射性同位素示踪的特异性相当 以及基于现代质谱的脂质组学的广泛覆盖。这是通过超高 我与 Thermo Scientific 合作开发的分辨率轨道阱质谱仪，现已商业化 以 Lumos 1M 形式提供。该仪器具有足够的分辨率来解析自然丰度 13C 来自完整脂质离子中的示踪同位素，例如 2H。通过解析占主导地位的自然丰度离子 来自示踪同位素的信号将提高信噪比至少 2 个数量级（1:1 vs >1:100） 增加动态范围。这一进展将允许对脂质代谢进行体内分析来研究 多种疾病，并最终将导致可转化为人类研究的脂质通量组学分析。经过 通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， 可能规避对动物模型的需求，并衡量治疗的有效性和 干预措施。 为了促进这项技术的发展和广泛实施，我将解决 采用该技术的基本障碍。首先，商用仪器的设计目的是 蛋白质组学应用，特别是电喷雾电离源。通过与制造商合作并 将我的脂质组学经验转化为这个新平台，我将克服这些问题。其次，我会发展 适用于色谱法和直接输注应用的新颖数据收集方法 适应与超高分辨率共振相关的长瞬态时间和聚结问题 基于质谱分析。第三，将开发软件工具来提取超高分辨率数据 时间高效的方式，将数据转换为物理上可解释的参数，并将数据映射到生化 途径。最后，我将开发氘标记水稳定同位素标记的协议和平台 (D2O) 和其他同位素标记的代谢示踪剂在与我实验室相关的代谢综合征小鼠模型中 研究脂肪积累机制的研究计划。通过实现这些目标，这项技术 生物医学研究界将可以使用。我在工程学方面接受过多学科训练， 物理、分析和生物化学以及小鼠遗传学使我非常适合开发这项技术 德克萨斯大学西南分校以脂质为中心的研究环境是初始应用的理想场所。