Infrared laser spectroscopy of mass-separated metabolites

描述（由申请人提供）：挑战：使能技术的发展支持生物分子研究的持续进步。例如，在过去的十年中，基于质谱（MS）的测序技术已经抛弃了蛋白质组学研究。目前，在代谢组学中没有“金标准”技术，可以对这些样品中包含的数千种成分进行常规表征。由于灵敏度问题，NMR仅限于更丰富的分析物。另一方面，MS通常能够检测更多的特征，但通常不能从结构上表征这些分子。原理：通过将可调谐红外（IR）激光器耦合到质谱仪，可以记录质量分离离子的IR光谱。离子的IR激光光谱结合了高灵敏度和分析MS的复杂混合物的能力以及来自振动光谱的增强的结构信息。因此，该技术允许基于诊断振动和IR光谱指纹对许多未知物质进行化学解析。目标1：低温质谱和多路红外光谱的发展。为了使红外光谱成为生物分子离子的有用的生物分析工具， 分析物的光谱是很好分辨的，因此是可区分的，并且混合物中的多种分析物可以以多路复用的方式同时探测。我们建议开发一种定制的低温线性离子阱，其中离子被标记有弱结合的分子（例如N2），这些分子在共振IR吸收后选择性地分离。目的2：质量分离代谢物的红外光谱。我们的生物分子红外光谱的应用集中在代谢物，我们希望该技术具有最大的潜力。标准代谢物的对照实验将确定在多重方法中可以成功探测多少分析物。然后将该方法应用于来自结肠癌研究的选定代谢物样品，这些样品先前已通过高通量液相色谱法和高分辨率质谱法进行了分析。目的3：通过与计算红外光谱和生物信息学方法的比较，对未知代谢物进行结构解析。该提案的最终目标是对目前MS方法无法识别的未知生物标志物进行化学表征。这需要将每种分析物的实验数据（即其质量和IR光谱）与代谢物数据库中的推定匹配进行比较。已知标准品的IR光谱（来自目标2）将作为训练集和实施该鉴别方法的基准。创新与影响：预计这里开发的技术将对全球代谢组学产生最大的影响，目前的串联质谱方法限制了可以在这些混合物中识别的成分数量。我们期望从振动光谱增强的结构信息产生许多新的见解，在生物标志物的发现。

DESCRIPTION (provided by applicant): Challenge: Developments of enabling technologies underpin continuing advances in biomolecular research. For instance, mass spectrometry (MS)-based sequencing techniques have spurned proteomics research in the past decade. Currently, there is no "gold standard" technique in metabolomics that allows a routine characterization of the thousands of constituents contained in these samples. NMR is limited to the more abundant analytes due to sensitivity issues. On the other hand, MS is typically capable of detecting many more features, but is often not able to structurally characterize these molecules. Rationale: By coupling tunable infrared (IR) lasers to mass spectrometry instrumentation, the IR spectra of mass- separated ions can be recorded. IR laser spectroscopy of ions combines the high sensitivity and ability to analyze complex mixtures of MS with the enhanced structural information from vibrational spectroscopy. The technique hence allows a chemical elucidation of many unknowns based on diagnostic vibrations and IR spectral fingerprints. Aim 1: Development of cryogenic mass spectrometry and multiplexed IR spectroscopy. In order to make IR spectroscopy a useful bioanalytical tool for biomolecular ions, it is essential that the IR spectra of analytes are well- resolved, and thus distinguishable, and that multiple analytes in mixtures can be probed simultaneously in a multiplexed fashion. We propose to develop a custom-built, cryogenic linear ion trap, where the ions are tagged with weakly-bound molecules (e.g. N2), which are selectively detached upon resonant IR absorption. Aim 2: IR spectroscopy of mass-separated metabolites. Our application of IR spectroscopy of biomolecules focuses on metabolites, where we expect the technique to have most potential. Control experiments on standard metabolites will establish how many analytes can be successfully probed in a multiplexed approach. The methodology will then be applied to selected metabolite samples from colon cancer studies, which have previously been analyzed by high- throughput liquid chromatography and high-resolution mass spectrometry. Aim 3: Structural elucidation of unknown metabolites by comparison to computed IR spectra and bioinformatics approaches. The ultimate goal of this proposal is to chemically characterize unknown biomarkers that cannot be identified by current MS approaches. This requires a comparison of the experimental data for each analyte, namely its mass and its IR spectrum, to putative matches from metabolite databases. The IR spectra of known standards (from aim 2) will serve as a training set and as a benchmark for implementing this identification methodology. Innovation and Impact: The techniques developed here are expected to have the largest impact in global metabolomics, where current tandem mass spectrometry methodologies limit the number of constituents that can be identified in these mixtures. We expect the enhanced structural information from vibrational spectroscopy to yield many new insights in biomarker discovery.