Stowers Institute for Medical Research, 1000 East 50th Street, Kansas City, Missouri 64110, USA.
Anal Chem. 2010 Mar 15;82(6):2272-81. doi: 10.1021/ac9023999.
Quantitative shotgun proteomics is dependent on the detection, identification, and quantitative analysis of peptides. An issue arises with peptides that are shared between multiple proteins. What protein did they originate from and how should these shared peptides be used in a quantitative proteomics workflow? To systematically evaluate shared peptides in label-free quantitative proteomics, we devised a well-defined protein sample consisting of known concentrations of six albumins from different species, which we added to a highly complex yeast lysate. We used the spectral counts based normalized spectral abundance factor (NSAF) as the starting point for our analysis and compared an exhaustive list of possible combinations of parameters to determine what was the optimal approach for dealing with shared peptides and shared spectral counts. We showed that distributing shared spectral counts based on the number of unique spectral counts led to the most accurate and reproducible results.
定量鸟枪法蛋白质组学依赖于肽的检测、鉴定和定量分析。对于存在于多个蛋白质之间的肽，会出现这样一个问题：它们源自哪种蛋白质，以及应该如何在定量蛋白质组学工作流程中使用这些共享肽？为了系统地评估无标记定量蛋白质组学中的共享肽，我们设计了一个由来自不同物种的六种已知浓度的白蛋白组成的明确定义的蛋白质样品，并将其添加到高度复杂的酵母裂解物中。我们使用基于谱计数的归一化谱丰度因子 (NSAF) 作为分析的起点，并比较了一整套可能的参数组合，以确定处理共享肽和共享谱计数的最佳方法。我们表明，根据独特谱计数的数量分配共享谱计数可得到最准确和可重复的结果。
