植物标记和非标记定量蛋白质组学技术

Labeling and label-free quantitative proteomics in plant biology

蛋白质是细胞中重要的有机大分子,是生命活动的主要执行者.植物在受到外界信号干扰时其主要反应是通过调节细胞内蛋白质的合成和降解来恢复稳态.因而,蛋白质的定量分析有助于人们更深入地揭示植物细胞中蛋白质的动态变化及其分子功能.随着高分辨质谱技术的发展,使用质谱方法对蛋白质进行高通量定量和定性分析的蛋白质组学技术已经成为研究蛋白质功能的重要分析手段,同时也是植物生物学研究领域的热点之一.结合本课题组取得的研究成果,本文总结和概述了植物蛋白质组学定量方法的最新进展,包括非标记定量和稳定同位素标记分析方法,比较不同方法在蛋白定量的精确度、蛋白组覆盖率和稳定性等方面的优缺点,分析其在植物生物学研究中的应用范围,并对这方面的研究热点进行展望.

Proteins not only construct cellular structures but also play regulatory functions in plant development and growth. Accurate quantification of protein dynamics, turnover rates, and post-translational modifications provide crucial insights into the molecular mechanisms of biological systems. While early studies only consisted of listing proteins identified in given organs or under specific conditions. There is growing need to apply quantitative proteomic techniques to be biased or unbiased studies of plant protein dynamic changes. With rapid improvements in mass spectrometry(MS) technologies,various proteomic quantification methods have been developed and widely used in biological research. In the meantime,MS-based quantitative proteomics has emerged as a powerful approach for gaining insights into functional aspects of proteins in biological systems. MS is not inherently quantitative;thus, several strategies have been developed to assist MS for protein quantification. These strategies can be distinguished not only by the method of quantification, such as labeled or label-free method, but also by the quantitative goal, such as relative or absolute quantification. They can be also distinguished by the scopes of the experiments performed, such as unbiased or targeted studies.Even within these different research goals, numerous options exist, since each strategy has advantages and disadvantages. For example, labeling methods can tag proteins/peptides with chemical, metabolic, or enzymatic reagents,whereas label-free quantitation methods use spectra ion counting, or peak intensity-based analysis. In comparison with labeling strategies, label-free methods are widely used because of the dynamic quantitative range, costing less, and without labeling steps. However, label-free methods are limited in the sample multiplex capacity and quantitative accuracy. For labeling-based quantitation, materials or samples are labeled by different isotope reagents, the paired MS spectra ions are extracted for relative or absolute quantification. In combination with high accuracy MS instruments, various labeling strategies can be used in quantitative proteome analysis, these methods can be distinguished by quantification ions, such as precursor ion-based, reporter ion-based, peptide-coupled reporter ion-based, and fragment ion-based quantitation. For stable isotope labeled methods, proteins or peptides labeled with different stable isotopes are combined into one vial for LC-MS analysis. These strategies have gained popularity due to the diversity and flexibility of stable isotope reactions, the power to analyze multiple samples simultaneously, and the high quantification accuracy. In this review, we discuss the most widely used label-free and stable isotope-labeling-based proteomic strategies for relative and absolute quantitative proteomics in plants. We compare their strengths and weaknesses in terms of quantitation accuracy, proteome coverage and robustness. We summarize the most promising recent developments and their applications for solving plant biological complexities.