Developing High-Resolution Ion Mobility Spectrometry-Charge Detection-Mass Spectrometry for Rapid Analysis in the Megadalton to Gigadalton Regime

项目摘要 本项目将推进一种新的质谱技术--电荷检测质谱 （CDMS）用于分析10至100 nm尺寸范围内的大生物分子组装体（即，具有分子 重量为约IMDa至IGDa）。在这个大小范围内的物种，如病毒和脂蛋白，在人类免疫系统中起着关键作用。 人体健康然而，它们很难检测和表征。传统的MS仪器可以 确定许多类型的生物分子的质量和碎片模式，包括组件;但是， 测量仅限于低于约1 MDa的物质。在CDMS中，单个离子的质量直接 通过同时测量每个离子的质荷比和电荷来确定。原理证明 使用我们的CDMS原型进行的研究表明，可以将精确的质量确定为MDa到GDa 政权如本提案所示，这是一个有利的进展。然而，我们的原型设计具有有限的 质量分辨本领（我们测得的最大值为m/Δm ~ 330）。测量所需的时间 完整的光谱使得该仪器对于常规分析不切实际。该项目描述了进步 这将提高CDMS的分辨能力和频谱获取速度，每一个都至少提高一个数量级 数量级。这将使大物种的高分辨率质谱首次被常规记录下来。 时间我们还将开发一个离子迁移谱（IMS）接口的CDMS。IMS分离将 通过CDMS分析提高总峰容量并简化非均相混合物的分析。在 此外，离子的迁移率取决于其结构，因此提供了补充 电荷和质量信息。组合的高分辨率IMS-CDMS仪器将提供 分析大生物分子的强大新方法。经过校准和测试后， 仪器将被用来研究许多对人类健康很重要的问题。包括：病毒 组装和拆卸，表征基因治疗载体，药物和抗体与病毒的结合， 以及脂蛋白亚类的鉴定。这种测量具有变革的潜力。病毒基因 例如，治疗载体难以表征，因为它们的尺寸大，并且因为遗传学上的缺陷， 物质包含在病毒衣壳内。关键问题，例如衣壳是否包含完整的基因组， 必须解决部分基因组或无基因组的问题。高分辨率IMS-CDMS测量将提供 关于小的结构缺陷的信息，这些缺陷很难（如果不是不可能的话）通过其他手段检测。 同样，IMS-CDMS分析将影响其他领域，如心血管疾病-导致 死在美国。血浆脂蛋白在动脉粥样硬化的主要潜在原因中起关键作用。然而，在这方面， 主要类别HDL、LDL和VLDL都以尺寸和组成的广泛分布存在。划定 将使发展更可靠的诊断测试和更好的治疗。初步 结果表明高分辨率IMS-CDMS将能够分辨关键的脂蛋白亚类。

Project Summary This project will advance a new mass spectrometry (MS) technology, charge detection mass spectrometry (CDMS) for analyzing large biomolecular assemblies in the 10 to 100 nm size range (i.e., having molecular weights of ~1 MDa to 1 GDa). Species in this size range, such as viruses and lipoproteins, play critical roles in human health. However, they are difficult to detect and characterize. Conventional MS instruments can determine masses and fragmentation patterns of many types of biomolecules, including assemblies; but, such measurements are limited to species below ~1 MDa. In CDMS, the masses of individual ions are directly determined from simultaneous measurement of each ion’s mass-to-charge ratio and charge. Proof-of-principle studies using our CDMS prototype, show that accurate masses can be determined into the MDa to GDa regime. As shown in this proposal, this is an enabling advance. However, our prototype design has a limited mass resolving power (the maximum we have measured is m/Δm ~ 330). And, the time required for measuring a complete spectrum makes this instrument impractical for routine analyses. This project describes advances that will improve both the CDMS resolving power and spectrum acquisition speed – each by at least an order of magnitude. This will allow high-resolution mass spectra for large species to be routinely recorded for the first time. We will also develop an ion mobility spectrometry (IMS) interface for CDMS. The IMS separation will improve the overall peak capacity and simplify the analysis of heterogeneous mixtures by CDMS analysis. In addition, the mobility of an ion depends on its structure and thus provides information that complements the charge and mass information from CDMS. The combined high-resolution IMS-CDMS instrument will provide a powerful new approach for the analysis of large biomolecules. Once calibrated and tested, the IMS-CDMS instrument will be used to investigate numerous problems important to human health. These include: virus assembly and disassembly, the characterization gene therapy vectors, drug and antibody binding to viruses, and the identification of lipoprotein subclasses. Such measurements have transformative potential. Viral gene therapy vectors, for example, are difficult to characterize because of their large sizes and because the genetic material is contained within virus capsids. Critical issues, such as whether capsids contain the full genome, partial genomes, or no genome, must be addressed. High-resolution IMS-CDMS measurements will provide information about small structural defects, which are difficult (if not impossible) to detect by other means. Similarly, IMS-CDMS analyses will impact other areas, such as cardiovascular diseases - the leading cause of death in the US. Plasma lipoproteins play a key role in the main underlying cause, atherosclerosis. However, the major classes, HDL, LDL, and VLDL, all exist as broad distributions of sizes and compositions. Delineation of subclasses will enable development of more reliable diagnostic tests and better therapeutics. Preliminary results indicate that high-resolution IMS-CDMS will be able to resolve key lipoprotein subclasses.