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

职业：Nanoflow RPLC-CZE-MS/MS：一种深度、高灵敏度自下而上蛋白质组学的新方法

• 批准号: 1846913
• 负责人: Liangliang Sun
• 金额: $ 67.04万
• 依托单位: Michigan State University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-01 至 2025-07-31
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1846913&HistoricalAwards=false
• 关键词: CAREER; Nanoflow; RPLC; CZE; MS

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.

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，改变基于质谱学的分析平台，使其能够从目前无法检测到的生物样本中识别和量化蛋白质分子。新的平台将提供更高的灵敏度和特异度，部分是通过开发比通常使用的基于质谱学的方法更高的分离能力。这些进展的影响将是广泛的，因为生物学中的许多基础研究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，它将为跨学科研究培养新一代研究生和本科生。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，激发他们积极的学习体验，并鼓励他们追求科学事业。自下而上的蛋白质组学是大规模和高通量表征细胞中蛋白质的强大工具。在给定数百微克蛋白质材料的情况下，使用先进的自下而上的蛋白质组学方法可以从人类细胞中识别超过8000种蛋白质。然而，为了在使用自下而上的蛋白质组学分析质量有限的蛋白质组样本方面取得进展，这些样本只包含低微克到纳克的蛋白质，将需要具有显著更高灵敏度的新型分析工具。该项目将开发一种创新的、自动化的纳米流反相液相色谱-毛细管区带电泳-串联质谱仪(NanRPLC-CZE-MS/MS)平台，用于高灵敏和自下而上的蛋白质组学。这种变革性的方法将使自下而上的蛋白质组学的灵敏度提高两个数量级以上，能够从仅几毫微克的人类细胞蛋白质材料中识别约8000个蛋白质作为概念证明。这种新的方法将使各种科学界受益，以寻求涉及质量限制的蛋白质组分析的重要基本问题的答案。这项研究还将应用纳米RPLC-CZE-MS/MS技术来发现斑马鱼胚胎中染色质相关蛋白在母体到受精卵转变(MZT)期间的丰度水平的变化。MZT是发育的关键，它协调早期胚胎发生的重要事件，例如合子基因组的激活。定量蛋白质组学数据集将提供更好的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。该项目的结果可以在https://sungroupchemmsu.wixsite.com/sungroup/publications.This上找到，该奖项反映了国家科学基金会的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

项目成果

Capillary Zone Electrophoresis-Tandem Mass Spectrometry As an Alternative to Liquid Chromatography-Tandem Mass Spectrometry for Top-down Proteomics of Histones.

毛细管区电泳串联质谱法作为液态色谱串联质谱法，用于组蛋白自上而下的蛋白质组学。

• DOI: 10.1021/acs.analchem.0c04237
• 发表时间: 2021-03-16
• 期刊: Analytical chemistry
• 影响因子: 7.4
• 作者: Chen D;Yang Z;Shen X;Sun L
• 通讯作者: Sun L

Comprehensive Detection of Single Amino Acid Variants and Evaluation of Their Deleterious Potential in a PANC-1 Cell Line.

• DOI: 10.1021/acs.jproteome.9b00840
• 发表时间: 2020-02
• 期刊: Journal of proteome research
• 影响因子: 4.4
• 作者: Zhijing Tan;Jianhui Zhu;P. Stemmer;Liangliang Sun;Zhichang Yang;Kendall Schultz;Matthew J. Gaffrey;Anthony J. Cesnik;Xinpei Yi;Xiaohu Hao;M. Shortreed;Tujin Shi;D. Lubman

Automated Capillary Isoelectric Focusing-Tandem Mass Spectrometry for Qualitative and Quantitative Top-Down Proteomics.

自动毛细管等电聚焦量质谱法，用于定性和定量自上而下的蛋白质组学。

• DOI: 10.1021/acs.analchem.0c03266
• 发表时间: 2020-12-15
• 期刊: Analytical chemistry
• 影响因子: 7.4
• 作者: Xu T;Shen X;Yang Z;Chen D;Lubeckyj RA;McCool EN;Sun L
• 通讯作者: Sun L

A Mini Review on Capillary Isoelectric Focusing-Mass Spectrometry for Top-Down Proteomics.

• DOI: 10.3389/fchem.2021.651757
• 发表时间: 2021
• 期刊: Frontiers in chemistry
• 影响因子: 5.5
• 作者: Xu T;Sun L
• 通讯作者: Sun L

Large-scale top-down proteomics of the Arabidopsis thaliana leaf and chloroplast proteomes.

• DOI: 10.1002/pmic.202100377
• 发表时间: 2023-02
• 期刊: Proteomics
• 影响因子: 3.4