Spectral Counting Label-Free Proteomics
Spectral Counting Label-Free Proteomics
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DOI：
10.1007/978-1-4939-0685-7_14
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发表时间：
2014-01-01
期刊：
影响因子：
--
通讯作者：
Peil, Lauri
中科院分区：
文献类型：
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作者：
Arike, Liisa;Peil, Lauri
基于自下而上质谱的蛋白质组学中使用的无标记蛋白质组定量方法越来越受欢迎，因为它们易于应用，并且可以集成到不同的工作流程中，而无需任何额外的工作或成本。在无标记蛋白质组定量方法中，单独制备和分析感兴趣的样品。质谱法通常不被认为是一种定量方法，因为肽的电离效率取决于肽的组成。无标记定量方法必须通过额外的计算来克服这一限制。有多种算法可以考虑肽的序列和长度并计算样品中蛋白质的预测丰度。无标记方法可分为两类：基于肽峰强度的定量和依赖于从给定蛋白质中识别的肽数量的光谱计数定量。该协议将集中于光谱计数定量-指数修改的蛋白质丰度指数（emPAI）。标准化 emPAI 最常见于 Mascot 搜索结果，可用于整个蛋白质组的广泛比较。将演示基于 emPAI 值（添加或不添加标准品）的蛋白质绝对定量。将给出如何轻松将 emPAI 集成到现有数据中的指南；例如，从 iTRAQ 数据计算基于 emPAI 的绝对蛋白质丰度，无需添加标准。
Label-free proteome quantification methods used in bottom-up mass-spectrometry based proteomics are gaining more popularity as they are easy to apply and can be integrated into different workflows without any extra effort or cost. In the label-free proteome quantification approach, samples of interest are prepared and analyzed separately. Mass-spectrometry is generally not recognized as a quantitative method as the ionization efficiency of peptides is dependent on composition of peptides. Label-free quantification methods have to overcome this limitation by additional computational calculations. There are several algorithms available that take into account the sequence and length of the peptides and compute the predicted abundance of proteins in the sample. Label-free methods can be divided into two categories: peptide peak intensity based quantification and spectral counting quantification that relies on the number of peptides identified from a given protein.This protocol will concentrate on spectral counting quantification-exponentially modified protein abundance index (emPAI). Normalized emPAI, most commonly derived from Mascot search results, can be used for broad comparison of entire proteomes. Absolute quantification of proteins based on emPAI values with or without added standards will be demonstrated. Guidelines will be given on how to easily integrate emPAI into existing data; for example, calculating emPAI based absolute protein abundances from iTRAQ data without added standards.