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，目前大多数蛋白质组学研究依赖于自下而上/鸟枪法。在这种情况下， 蛋白质混合物被蛋白酶之一消化（例如，胰蛋白酶），通过液相色谱法分离 (LC)通过电喷雾质谱法（ESI-MS）分析。尽管取得了巨大的成功， 在自下而上的蛋白质组学中仍然存在两个主要限制：首先，很难鉴定所有的蛋白质同种型， 蛋白形式，包括剪接、修饰、裂解等;第二，蛋白质的天然状态总是 消化后消失。目前有一个很大的推动，以实现自上而下的蛋白质组学，即，识别和 通过LC-MS表征全长蛋白质。不幸的是，自上而下的蛋白质组学被证明是更多 挑战性存在几个瓶颈：第一，蛋白质相对于肽的MS灵敏度较低;第二， 限制了高分子量蛋白质的检测;第三，通过MS/MS鉴定蛋白质的效率低 片段化;第四，费力的多维蛋白质分离，不适合小体积的蛋白质分离。 生物样本该领域正在呼唤变革性技术。根据PA-11-215，Newomics Inc.建议开发一种新技术，皮电喷雾电离质谱（picoESI-MS）， 基于我们突破性的多喷嘴发射器阵列，用于小体积样品的自上而下的蛋白质组学。的 技术将建立在我们的微型单片多喷嘴发射器（M3发射器）和多喷嘴 LC-nanoESI-MS发射极阵列（MEA）芯片，共同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之间的有效耦合这一长期存在的问题，并为 为LC-picoESI-MS微流控芯片的大规模集成提供了一种新的途径。 平台将直接解决上述瓶颈，从而实现高灵敏度，高通量， 和小体积生物样品的多重自上而下的蛋白质组学。

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, the majority of the current proteomics studies rely on bottom-up/shotgun 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 protein 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 response to PA-11-215, Newomics Inc. proposes to develop a new technology, picoelectrospray ionization mass spectrometry (picoESI-MS), based on our breakthrough multinozzle emitter array, for top-down proteomics of small-volume samples. The technology will be 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 LC-picoESI-MS. Our 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.