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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

基本信息

批准号：
10693274
负责人：
DAOJING WANG
金额：
$ 86.54万
依托单位：
NEWOMICS, INC
依托单位国家：
美国
项目类别：
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/10693274
关键词：
Academia Acceleration Address Aging Antibodies Apoptosis Back Biological Biological Assay Biological Models Biomedical Research Blood capillaries Cell Separation Cells Charge Collaborations Coupling Detection Development Diameter Digestion Dimensions Disease Early Diagnosis Ensure Foundations Fractionation Future Housing Human Individual Industry Length Liquid Chromatography Marketing Mass Spectrum Analysis Measures Mesenchymal Stem Cells Methods Microfluidic Microchips Modification Molecular Weight Monitor Pathology Patients Peptide Hydrolases 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 Tail Technology Trypsin Tube Validation Vendor commercialization disease diagnostic experience imaging system improved industry partner innovation ionization lipidomics liquid chromatography mass spectrometry mass spectrometer nano new technology novel precision medicine pressure protein biomarkers protein complex prototype research and development senescence single cell analysis success targeted treatment tool treatment response

项目摘要

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.

项目概要/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个强大的工具用于疾病的早期诊断和治疗反应的监测。质谱 (MS) 测量带电物质的质荷比，已成为蛋白质组学的支持技术。然而，目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，蛋白质的混合物是通过其中一种蛋白酶（例如胰蛋白酶）消化，通过液相色谱 (LC) 分离，并通过电喷雾质谱（ESI-MS）。尽管取得了巨大的成功，但仍然存在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异构体或蛋白质异构体，包括剪接，修饰、裂解等；其次，蛋白质的天然状态在消化后总是会丢失。目前有大力推动自上而下的蛋白质组学的实施，即通过以下方法对全长蛋白质进行鉴定和表征 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 灵敏度较低；二、高分子量检测的局限性蛋白质；第三，通过 MS/MS 碎片鉴定蛋白质的效率低下；第四，辛苦多维蛋白质分离不适合小体积的生物样品。领域正在呼唤变革性技术。在这个二期项目中，Newomics Inc.建议进一步开发和将新技术纳流LC-皮电喷雾电离质谱（NanoLC- PicoESI-MS），用于小体积生物样品直至单细胞的自上而下的蛋白质组学。技术基于我们的微加工单片多喷嘴发射器（M3 发射器）和多喷嘴发射器阵列 (MEA) 用于 LC-nanoESI-MS 的芯片，这些芯片共同为长期存在的问题提供了简单而新颖的解决方案解决了硅微流控芯片与ESI-MS之间的高效耦合问题，为大规模应用铺平了道路。在所提出的 nanoLC-picoESI-MS 微流控芯片上进行规模集成。我们的新 PicoESI-MS 平台将直接解决上述瓶颈，从而实现高灵敏度、高通量和多路复用少量生物样本的自上而下的蛋白质组学，从而有助于精准医学。