Picoelectrospray Ionization Mass Spectrometry for Top-down Proteomics

用于自上而下蛋白质组学的皮电喷雾电离质谱法

• 批准号: 10546686
• 负责人: DAOJING WANG
• 金额: $ 86.54万
• 依托单位: NEWOMICS, INC
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-05-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10546686
• 关键词: Academia; Address; Aging; Antibodies; Apoptosis; Biological; Biological Assay; Biological Models; Biomedical Research; Blood capillaries; Caliber; Cell Separation; Cells; Charge; Coupling; Detection; Development; Digestion; Disease; Early Diagnosis; Ensure; Foundations; Future; Housing; Human; Individual; Industry; Length; Liquid Chromatography; Liquid substance; Marketing; Mass Spectrum Analysis; Measures; Mesenchymal Stem Cells; Methods; Microfluidic Microchips; Modification; Molecular Weight; Monitor; Pathology; Patients; Peptides; Performance; Phase; Physiology; Plasma; Positioning Attribute; Protein Chemistry; Protein Isoforms; Proteins; Proteome; Proteomics; Publishing; RNA Splicing; Resolution; Sales; Sampling; Signal Transduction; Silicon; Small Business Innovation Research Grant; Solid; Source; Spectrometry, Mass, Electrospray Ionization; Tail; Technology; Trypsin; Validation; Vendor; commercialization; disease diagnostic; experience; imaging system; improved; industry partner; innovation; ionization; lipidomics; liquid chromatography mass spectrometry; mass spectrometer; new technology; novel; precision medicine; protease E; protein biomarkers; protein complex; prototype; research and development; senescence; single cell analysis; success; targeted treatment; tool; treatment response

Project Summary/Abstract The proteome reflects the physiology and pathology states of a patient therefore proteomics is a powerful tool for early diagnostics of diseases and monitoring of therapeutic responses. Mass spectrometry (MS) measures the mass-to-charge ratio of charged species and has become the enabling technology for proteomics. However, majority of the current proteomics studies rely on bottom-up approaches. In this case, mixtures of proteins are digested by one of the proteases (e.g., trypsin), separated by liquid chromatography (LC), and analyzed by electrospray mass spectrometry (ESI-MS). Despite tremendous successes, there remain two major limitations in bottom-up proteomics: first, it is difficult to identify all protein isoforms or proteoforms, including splicing, modifications, cleavages, etc.; second, the native state of proteins is always lost after digestion. There is currently a great push to implement top-down proteomics, i.e., identification and characterization of full-length proteins by LC-MS. Unfortunately, top-down proteomics proves to be much more challenging. There are several bottlenecks: first, lower MS sensitivity of proteins relative to peptides; second, limitation on detection of high molecular weight proteins; third, inefficient identification of proteins by MS/MS fragmentation; and fourth, laborious multidimensional protein separation not suitable for small volumes of biological samples. The field is calling for transformative technologies. In this Phase II project, Newomics Inc. proposes to further develop and commercialize a new technology, nanoflow LC-picoelectrospray ionization mass spectrometry (NanoLC- PicoESI-MS), for top-down proteomics of small-volume biological samples down to single cells. The technology is built on our microfabricated monolithic multinozzle emitters (M3 emitters) and multinozzle emitter array (MEA) chips for LC-nanoESI-MS, which collectively offer a straightforward yet novel solution to the longstanding problem of the efficient coupling between silicon microfluidic chips and ESI-MS, and pave the way for the large- scale integration on the proposed microfluidic chips for nanoLC-picoESI-MS. Our new PicoESI-MS platform will directly address the aforementioned bottlenecks, and thus enable high-sensitivity, high-throughput, and multiplex top-down proteomics of small volumes of biological samples, thereby contributing to precision medicine.

项目摘要/摘要 蛋白质组反映了患者的生理和病理状态，因此蛋白质组学是一种强大的工具 用于早期诊断疾病和监测治疗反应。质谱（MS）测度 带电物种的质量与电荷比，已成为蛋白质组学的促成技术。然而， 当前大多数蛋白质组学研究都依赖于自下而上的方法。在这种情况下，蛋白质的混合物是 由一种蛋白酶（例如胰蛋白酶）消化，被液相色谱分离（LC），并通过 电喷雾质谱法（ESI-MS）。尽管取得了巨大的成功，但仍有两个主要限制 在自下而上的蛋白质组学中：首先，很难识别所有蛋白质同工型或蛋白质成型，包括剪接， 修改，分裂等；其次，消化后蛋白质的本地状态总是丢失。目前有 伟大的推动以实施自上而下的蛋白质组学，即通过 LC-MS。不幸的是，自上而下的蛋白质组学被证明更具挑战性。有几个瓶颈： 首先，蛋白质相对于肽的MS敏感性较低。其次，检测高分子量的限制 蛋白质；第三，通过MS/MS碎片对蛋白质的效率低下；第四，费力 多维蛋白分离不适合少量生物样品。该领域正在要求 变革性技术。在此第二阶段项目中，Newomics Inc.建议进一步发展和 商业化一项新技术，纳米流LC-Picoelectrospray电离质谱法（nanolc- Picoesi-ms），用于向单个细胞的小体积生物样品的自上而下的蛋白质组学。技术 建在我们的微型整体多弹力发射器（M3发射器）和多蛋白发射器阵列（MEA）上建立 LC-Nanoesi-MS的芯片，该芯片共同为长期存在的简单而新颖 硅微流体芯片和ESI-MS之间有效耦合的问题，并为大型的道路铺平了道路 在纳米果皮MS的拟议的微流体芯片上进行比例整合。我们的新Picoesi-MS平台将 直接解决上述瓶颈，从而实现高敏感，高通量和多路复用 自上而下的蛋白质组学的少量生物样品，从而有助于精密医学。