Stowers Institute for Medical Research, Kansas City, MO 64110, USA.
Mol Cell Proteomics. 2011 Feb;10(2):M110.000687. doi: 10.1074/mcp.M110.000687. Epub 2010 Nov 3.
The use of quantitative proteomics methods to study protein complexes has the potential to provide in-depth information on the abundance of different protein components as well as their modification state in various cellular conditions. To interrogate protein complex quantitation using shotgun proteomic methods, we have focused on the analysis of protein complexes using label-free multidimensional protein identification technology and studied the reproducibility of biological replicates. For these studies, we focused on three highly related and essential multi-protein enzymes, RNA polymerase I, II, and III from Saccharomyces cerevisiae. We found that label-free quantitation using spectral counting is highly reproducible at the protein and peptide level when analyzing RNA polymerase I, II, and III. In addition, we show that peptide sampling does not follow a random sampling model, and we show the need for advanced computational models to predict peptide detection probabilities. In order to address these issues, we used the APEX protocol to model the expected peptide detectability based on whole cell lysate acquired using the same multidimensional protein identification technology analysis used for the protein complexes. Neither method was able to predict the peptide sampling levels that we observed using replicate multidimensional protein identification technology analyses. In addition to the analysis of the RNA polymerase complexes, our analysis provides quantitative information about several RNAP associated proteins including the RNAPII elongation factor complexes DSIF and TFIIF. Our data shows that DSIF and TFIIF are the most highly enriched RNAP accessory factors in Rpb3-TAP purifications and demonstrate our ability to measure low level associated protein abundance across biological replicates. In addition, our quantitative data supports a model in which DSIF and TFIIF interact with RNAPII in a dynamic fashion in agreement with previously published reports.
使用定量蛋白质组学方法研究蛋白质复合物有可能深入了解不同蛋白质成分的丰度及其在各种细胞条件下的修饰状态。为了使用鸟枪法蛋白质组学方法研究蛋白质复合物的定量，我们专注于使用无标记多维蛋白质鉴定技术分析蛋白质复合物，并研究了生物学重复的重现性。对于这些研究，我们专注于三种高度相关且必不可少的多蛋白酶，来自酿酒酵母的 RNA 聚合酶 I、II 和 III。我们发现，当分析 RNA 聚合酶 I、II 和 III 时，使用光谱计数进行无标记定量在蛋白质和肽水平上具有高度重现性。此外，我们表明肽采样不遵循随机采样模型，并表明需要先进的计算模型来预测肽检测概率。为了解决这些问题，我们使用 APEX 方案根据使用相同多维蛋白质鉴定技术分析获得的全细胞裂解物来模拟预期的肽可检测性，该分析用于蛋白质复合物。这两种方法都无法预测我们使用重复多维蛋白质鉴定技术分析观察到的肽采样水平。除了 RNA 聚合酶复合物的分析外，我们的分析还提供了关于几种与 RNAP 相关的蛋白质的定量信息，包括 RNAPII 延伸因子复合物 DSIF 和 TFIIF。我们的数据表明，DSIF 和 TFIIF 是 Rpb3-TAP 纯化中最丰富的 RNAP 辅助因子，并证明了我们能够在生物学重复中测量低水平相关蛋白质丰度的能力。此外，我们的定量数据支持一种模型，即 DSIF 和 TFIIF 以与先前发表的报告一致的动态方式与 RNAPII 相互作用。
