Infrared laser spectroscopy of mass-separated metabolites

描述（由申请人提供）：挑战：使能技术的发展支撑了生物分子研究的持续进步。例如，在过去的十年里，基于质谱（MS）的测序技术已经抛弃了蛋白质组学研究。目前，在代谢组学中还没有“金标准”技术，可以对这些样品中含有的数千种成分进行常规表征。由于灵敏度问题，核磁共振仅限于更丰富的分析物。另一方面，质谱通常能够检测到更多的特征，但通常无法对这些分子进行结构表征。原理：通过耦合可调谐红外（IR）激光器到质谱仪器，可以记录质量分离离子的红外光谱。红外激光离子光谱结合了高灵敏度和分析复杂混合质谱的能力与振动光谱增强的结构信息。因此，该技术允许基于诊断振动和红外光谱指纹的许多未知的化学解释。目的1：低温质谱和多路红外光谱的发展。为了使红外光谱技术成为一种有用的生物分子离子分析工具，红外光谱技术的应用至关重要

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.