Picoelectrospray Ionization Mass Spectrometry for Top-down Proteomics

项目总结/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有力的工具 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，并已成为蛋白质组学的使能技术。然而，在这方面， 目前的蛋白质组学研究大多依赖于自下而上的方法。在这种情况下，蛋白质的混合物是 被蛋白酶之一消化（例如，胰蛋白酶），通过液相色谱（LC）分离，并通过HPLC分析。 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因素 在自下而上的蛋白质组学中：首先，很难鉴定所有的蛋白质同种型或蛋白质型，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丢失。目前 大力推动自上而下的蛋白质组学，全长蛋白质的鉴定和表征 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： 第一，蛋白质相对于肽的较低MS灵敏度;第二，对高分子量蛋白质的检测的限制。 蛋白质;第三，通过MS/MS片段化鉴定蛋白质效率低;第四， 多维蛋白质分离不适用于小体积的生物样品。该领域正在呼吁 变革性技术。在这个第二阶段项目中，Newomics Inc.建议进一步发展和 将一种新技术-纳米流LC-皮考电喷雾电离质谱（NanoLC- PicoESI-MS），用于小体积生物样品至单细胞的自上而下的蛋白质组学。技术 是建立在我们的微制造单片多喷嘴发射器（M3发射器）和多喷嘴发射器阵列（MEA） LC-nanoESI-MS芯片，共同为长期存在的问题提供了一种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，为大规模的 规模集成的微流控芯片的nanoLC-picoESI-MS。我们的新的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 小体积生物样品的自上而下的蛋白质组学，从而有助于精确医学。

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.