Vogel Christine, Marcotte Edward M
Department of Biology, Center for Genomics and Systems Biology, New York University, New York, NY, USA.
Methods Mol Biol. 2012;893:321-41. doi: 10.1007/978-1-61779-885-6_20.
Mass spectrometry (MS)-based shotgun proteomics allows protein identifications even in complex biological samples. Protein abundances can then be estimated from the counts of MS/MS spectra attributable to each protein, provided that one corrects for differential MS-detectability of the contributing peptides. We describe the use of a method, APEX, which calculates Absolute Protein EXpression levels based on learned correction factors, MS/MS spectral counts, and each protein's probability of correct identification.The APEX-based calculations consist of three parts: (1) Using training data, peptide sequences and their sequence properties, a model is built that can be used to estimate MS-detectability (O (i)) for any given protein. (2) Absolute abundances of proteins measured in an MS/MS experiment are calculated with information from spectral counts, identification probabilities and the learned O (i)-values. (3) Simple statistics allow for significance analysis of differential expression in two distinct biological samples, i.e., measuring relative protein abundances. APEX-based protein abundances span more than four orders of magnitude and are applicable to mixtures of hundreds to thousands of proteins from any type of organism.
基于质谱（MS）的鸟枪法蛋白质组学即使在复杂的生物样品中也能实现蛋白质鉴定。只要对构成肽段的质谱检测差异进行校正，就可以根据归因于每种蛋白质的二级质谱（MS/MS）谱图计数来估计蛋白质丰度。我们描述了一种名为APEX的方法的应用，该方法基于学习到的校正因子、MS/MS谱图计数以及每种蛋白质正确鉴定的概率来计算绝对蛋白质表达水平。基于APEX的计算包括三个部分：（1）利用训练数据、肽段序列及其序列特性，构建一个模型，该模型可用于估计任何给定蛋白质的质谱检测能力（O(i)）。（2）利用谱图计数、鉴定概率和学习到的O(i)值信息，计算在MS/MS实验中测得的蛋白质绝对丰度。（3）简单的统计方法可对两个不同生物样品中的差异表达进行显著性分析，即测量相对蛋白质丰度。基于APEX的蛋白质丰度跨越四个以上数量级，适用于来自任何类型生物体的数百到数千种蛋白质的混合物。
