A Method for Label-Free, Differential Top-Down Proteomics.
A Method for Label-Free, Differential Top-Down Proteomics.
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DOI：
10.1007/978-1-4939-3524-6_8
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发表时间：
2016
期刊：
影响因子：
--
通讯作者：
Kelleher NL
中科院分区：
文献类型：
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作者：
Ntai I;Toby TK;LeDuc RD;Kelleher NL
生物标志物的发现在翻译研究中严重依赖于标记和无标记的定量自下而上的蛋白质组学。在这里，我们描述了一种生物标志物研究的新方法，该方法利用高通量自上而下的蛋白质组学，并且是第一个在同一实验中提供全蛋白表征和相对定量的方法。使用酵母作为模型，我们报告了一种无标记方法的程序，以量化在两种不同状态下从0到30 kDa的完整蛋白的相对丰度。在本章中，我们描述了通过液相色谱-质谱（LC-MS）对完整蛋白质组进行大规模分析和定量的综合方法，而无需进行代谢或化学标记。定量自顶向下蛋白质组学的最新进展最好是在高分辨率质谱仪上获取数据之前，通过强大且高度控制的样品制备工作流程来实现，并应用分层线性统计模型来解释基础和临床研究中样品定量蛋白质组学比较中包含的多个水平的方差。
Biomarker discovery in the translational research has heavily relied on labeled and label-free quantitative bottom-up proteomics. Here, we describe a new approach to biomarker studies that utilizes high-throughput top-down proteomics and is the first to offer whole protein characterization and relative quantitation within the same experiment. Using yeast as a model, we report procedures for a label-free approach to quantify the relative abundance of intact proteins ranging from 0 to 30 kDa in two different states. In this chapter, we describe the integrated methodology for the large-scale profiling and quantitation of the intact proteome by liquid chromatography-mass spectrometry (LC-MS) without the need for metabolic or chemical labeling. This recent advance for quantitative top-down proteomics is best implemented with a robust and highly controlled sample preparation workflow before data acquisition on a high-resolution mass spectrometer, and the application of a hierarchical linear statistical model to account for the multiple levels of variance contained in quantitative proteomic comparisons of samples for basic and clinical research.