CAREER: Nanoflow RPLC-CZE-MS/MS: a novel approach for deep and highly sensitive bottom-up proteomics

在这个项目中，密歇根州立大学的研究人员将开发新的方法，改造基于质谱的分析平台，使其能够从目前无法检测到的生物样品中识别和量化蛋白质分子。新平台将提供更高的灵敏度和特异性，部分原因是通过开发比常用的基于质谱的方法更高的分离能力。这些进步的影响将是广泛的，因为生物学基础研究的许多领域都涉及质量有限的蛋白质分析，如发育生物学、癌症生物学和神经科学。该项目需要在化学和生物学方面取得进展，通过参与研究，它将培养新一代的研究生和本科生从事跨学科研究。项目人员将接待来自代表性不足群体的当地高中生作为项目的参与者，培养他们对科学的兴趣，促进积极的学习经验，并鼓励他们追求科学事业。自底向上的蛋白质组学是细胞中大规模和高通量蛋白质表征的有力工具。使用先进的自下而上的蛋白质组学方法，可以完成来自人类细胞的8000多种蛋白质鉴定，给定数百微克的蛋白质材料。然而，使用自下而上的蛋白质组学来分析质量有限的蛋白质组样本（仅含有低微克到纳克的蛋白质），将需要具有更高灵敏度的新型分析工具。本项目将开发一个创新的自动化纳米流反相液相色谱-毛细管带电泳-串联质谱（nanorplc - cce -MS/MS）平台，用于高灵敏度和深度自下而上的蛋白质组学。这种变革性的方法将把自下而上的蛋白质组学的灵敏度提高两个数量级以上，使仅从纳克的人类细胞蛋白质材料中就能识别出大约8000种蛋白质，作为概念的证明。这种新颖的方法将使各种科学团体受益，以寻求涉及质量有限的蛋白质组分析的重要基本问题的答案。该研究还将应用nanoRPLC-CZE-MS/MS技术来发现斑马鱼胚胎中染色质相关蛋白的丰度水平在母体到合子过渡（MZT）期间的变化。MZT是发育的关键，它协调早期胚胎发生中的重要事件，例如合子基因组激活。定量蛋白质组学数据集将更好地了解染色质相关蛋白在MZT期间调节合子基因组激活中所起的作用。该项目的结果可在https://sungroupchemmsu.wixsite.com/sungroup/publications.This上找到，该奖项反映了美国国家科学基金会的法定使命，并通过基金会的智力价值和更广泛的影响审查标准进行评估，认为值得支持。

In this project, researchers at the Michigan State University will develop novel methods that transform a mass spectrometry-based analytical platform so that it can identify and quantify protein molecules from biological samples where they currently cannot be detected. The novel platform will provide much greater sensitivity and specificity, in part by developing higher separation capacities than the commonly used mass spectrometry-based methodologies. The impact of these advances will be wide, as many areas of fundamental research in biology involve mass-limited protein analyses, such as developmental biology, cancer biology, and neuroscience. The project requires advances in both chemistry and biology, and through involvement in the research it will train a new generation of graduate and undergraduate students for careers in interdisciplinary research. Project personnel will host local high school students from under-represented groups as participants in the project, nurturing their interest in science, prompting active learning experiences, and encouraging them to pursue science careers.Bottom-up proteomics is a powerful tool for large-scale and high-throughput characterization of proteins in the cell. Over 8000 protein identifications from human cells can be accomplished using advanced bottom-up proteomic methodologies, given hundreds of micrograms of protein material. However, to progress in using bottom-up proteomics for the analysis of mass-limited proteome samples, containing only low micrograms to nanograms of proteins, will require novel analytical tools with drastically better sensitivity. This project will develop an innovative and automated nanoflow reversed-phase liquid chromatography-capillary zone electrophoresis-tandem mass spectrometry (nanoRPLC-CZE-MS/MS) platform for highly sensitive and deep bottom-up proteomics. This transformative approach will boost the sensitivity of bottom-up proteomics by over two orders of magnitude, enabling the identification of about 8000 proteins from only nanograms of human cell protein material as proof of concept. The novel methodology will benefit a variety of scientific communities for pursuing answers to important fundamental questions that involve mass-limited proteome analyses. The research will also apply the nanoRPLC-CZE-MS/MS technology in discovering the changes in the level of abundance of chromatin-associated proteins in zebrafish embryos during the maternal-to-zygotic transition (MZT). The MZT is key for development, and it coordinates important events during early embryogenesis, e.g., zygotic genome activation. The quantitative proteomic dataset will provide a better understanding of the roles played by chromatin-associated proteins in modulating the zygotic genome activation during the MZT. Results of the project can be found at https://sungroupchemmsu.wixsite.com/sungroup/publications.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.