Picoelectrospray Ionization Mass Spectrometry for Top-down Proteomics

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 用于疾病的早期诊断和治疗反应的监测。质谱仪(MS)测量 带电物种的质荷比，已成为蛋白质组学的使能技术。然而， 目前的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 用其中一种酶(例如胰酶)消化，用液相色谱(LC)分离，并通过 电喷雾质谱(ESI-MS)。尽管取得了巨大的成功，但仍然存在两大局限性 在自下而上的蛋白质组学中：首先，很难识别所有的蛋白质异构体或蛋白质异构体，包括剪接， 第二，蛋白质的天然状态总是在消化后丢失。目前有 大力推动实现自上而下的蛋白质组学，即通过以下方式鉴定和鉴定全长蛋白质 LC-MS不幸的是，事实证明，自上而下的蛋白质组学更具挑战性。有几个瓶颈： 第一，蛋白质相对于多肽的较低的MS灵敏度；第二，对高分子量检测的限制 蛋白质；第三，通过MS/MS裂解鉴定蛋白质的效率低下；第四，费力 多维蛋白质分离不适合小量生物样品。这一领域正在呼唤 变革性的技术。在这个第二阶段的项目中，Neomeics Inc.计划进一步开发和 将一项新技术--纳米流LC-微电喷雾电离质谱仪(NanoLC-MS)商业化 PicoESI-MS)，用于从上到下的小体积生物样本到单个细胞的蛋白质组学。技术 是建立在我们的微型单片式多喷嘴发射器(M3发射器)和多喷嘴滴头阵列(MEA)的基础上的 用于LC-NanESI-MS的芯片，它们共同为长期存在的 解决了硅微流控芯片与电喷雾串联质谱仪之间的有效耦合问题，为大规模的微流控芯片与电喷雾串联质谱仪的实现铺平了道路。 用于纳米LC-PicoESI-MS的微流控芯片的规模集成我们的新PicoESI-MS平台将 直接解决上述瓶颈，从而实现高灵敏度、高吞吐量和多路复用 自上而下的少量生物样本的蛋白质组学，从而有助于精准医学。

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.