A Novel Alignment Method and Multiple Filters for Exclusion of Unqualified Peptides To Enhance Label-Free Quantification Using Peptide Intensity in LC-MS/MS
A Novel Alignment Method and Multiple Filters for Exclusion of Unqualified Peptides To Enhance Label-Free Quantification Using Peptide Intensity in LC-MS/MS
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DOI：
10.1021/pr2005633
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发表时间：
2011-10-01
影响因子：
4.4
通讯作者：
Witzmann, Frank A.
中科院分区：
文献类型：
--
作者：
Lai, Xianyin;Wang, Lianshui;Witzmann, Frank A.
尽管已经开发了许多软件包来利用LC-MS/MS产生的多肽强度对复杂生物样品中的蛋白质进行无标记定量，但在这一领域中通常忽略了两个关键问题：(I)多肽在实验中具有多种洗脱模式，以及(Ii)许多多肽不能用于蛋白质定量。为了解决这两个关键问题，我们开发了一种新的比对方法，以实现准确的多肽峰保留时间测定和多个过滤器来排除不合格的多肽用于蛋白质定量。使用六种不同的样品，即标准多肽、肾组织裂解产物、HT29-MTX细胞裂解产物、耗尽的人血清、人血清白蛋白结合蛋白和肾组织裂解产物中添加的标准蛋白质，对重复性和线性进行了测试。在7种浓度下，至少90.8%的蛋白质(最多1,390)的变异系数为0.9500。在两组的八次注射中，在复杂生物样品中添加等量的标准蛋白质，获得了8.6%的变异系数。通过将质谱学结果与免疫检测结果进行比较，进行了进一步的评估，得到了一致的结果。这种新的方法具有新颖而具体的特征，可以实现准确的无标记定量。
Though many software packages have been developed to perform label-free quantification of proteins in complex biological samples using peptide intensities generated by LC-MS/MS, two critical issues are generally ignored in this field: (i) peptides have multiple elution patterns across runs in an experiment, and (ii) many peptides cannot be used for protein quantification. To address these two key issues, we have developed a novel alignment method to enable accurate peptide peak retention time determination and multiple filters to eliminate unqualified peptides for protein quantification. Repeatability and linearity have been tested using six very different samples, i.e., standard peptides, kidney tissue lysates, HT29-MTX cell lysates, depleted human serum, human serum albumin-bound proteins, and standard proteins spiked in kidney tissue lysates. At least 90.8% of the proteins (up to 1,390) had CVs = 0.9500 across 7 concentrations. Identical amounts of standard protein spiked in complex biological samples achieved a CV of 8.6% across eight injections of two groups. Further assessment was made by comparing mass spectrometric results to immunodetection, and consistent results were obtained. The new approach has novel and specific features enabling accurate label-free quantification.