Stowers Institute for Medical Research, 1000 East 50th Street, Kansas City, Missouri 64110, USA.
Anal Chem. 2009 Aug 1;81(15):6317-26. doi: 10.1021/ac9004887.
To increase proteome coverage, dynamic exclusion (DE) is a widely used tool. When DE is enabled, more proteins can be identified, although the total spectral counts will decrease. To investigate the effects of DE duration on spectral-counting based quantitative proteomics, we analyzed the same sample via multidimensional protein identification technology while enabling different DE durations (15, 60, 90, 300, 600 s) or turning DE off. Normalized spectral abundance factors (NSAFs) measured for abundant proteins varied little with or without DE, while enabling DE lead to higher peptide counts, higher NSAFs, and better reproducibility of detection for proteins of relatively lower abundance. The optimal DE duration, which generated the maximum number of peptides, proteins, and peptides per protein, was observed to be 90 s in our settings. We developed a mathematical model for analyzing the effects of DE duration on peptide spectral counts. We found that the optimal DE duration depends on the average chromatographic peak width at the base of eluting peptides and mass spectrometry parameters, leading us to calculate an optimized DE duration of 97.9 s, in excellent agreement with our observations. In this study, we provide a systematic approach for the optimization of spectral counts for improved quantitative proteomics analysis.
为了增加蛋白质组覆盖度，动态排除（DE）是一种广泛使用的工具。启用 DE 时，可以鉴定更多的蛋白质，尽管总光谱计数会减少。为了研究 DE 持续时间对基于光谱计数的定量蛋白质组学的影响，我们通过多维蛋白质鉴定技术分析了同一样本，同时启用了不同的 DE 持续时间（15、60、90、300、600 秒）或关闭 DE。对于丰度蛋白，测量的归一化光谱丰度因子（NSAF）无论是否启用 DE，变化都很小，而启用 DE 则会导致更高的肽计数、更高的 NSAF 和相对较低丰度蛋白的检测重现性更好。在我们的设置中，观察到产生最大肽、蛋白质和每个蛋白质的肽数的最佳 DE 持续时间为 90 秒。我们开发了一个分析 DE 持续时间对肽光谱计数影响的数学模型。我们发现，最佳 DE 持续时间取决于洗脱肽的基峰处的平均色谱峰宽和质谱参数，这使我们计算出最佳 DE 持续时间为 97.9 秒，与我们的观察结果非常吻合。在这项研究中，我们提供了一种系统的方法来优化光谱计数，以改进定量蛋白质组学分析。
