博文
Nat Methods:从研究PPIs的酵母双杂交谈到不同的方法学验证
||||
今天看到Nature Methods一篇关于protein-protein interactions(PPIs)的Y2H的Correspondence和Author Reply，个人认为特别对现今众多研究方法的选择和比较有借鉴意义，所以作以记录分享。
2009年Braun,P在Nature Methods发表一项方法学比较研究(Nat Methods. An experimentally derived confidence score for binary protein-pro.pdf)，比较了5种检测PPIs的方法的tool kit —— 1. The Y2H system ；2. MAPPIT；3. The YFP reconstitution of the PCA assay；4. The LUMIER pull-down assay；5. The completely in vitro performed wNAPPA。
5种kit单独检测92种金标准protein pairs的检出率都不超过36%，每个方法平均检出率只有31.3%，联合检测则达到67.4%。由此，Braun希望能对每个方法检测出的每种protein-protein interactions 作出可信度评价，以重新评估相互作用网络中PPIs的真实情况。
2010年Yu-chi Chen在Nature Methods发表了一个相似结论的Correspondence (Nat Methods. Exhaustive benchmarking of the yeast two-hybrid system.pdf)，方法是采用5种不同的Yeast Two-Hybrid System(Bait-Prey Vectors)——pGBGT7g-pGADCg；pGBGT7g-pGADT7g；pDEST32-pDEST22； pGBKCg-pGADT7g；pGBKCg-pGADCg，目的是展示出相似蛋白的不同亚型，最后得到与Braun相似的数据是92种金标准protein pairs检出率也只有40%，每个方法的平均检出率为25.3%，联合检测也只达到79.3%。
Chen的目的在于想阐明：其实只要采用不同的Y2H载体，即便是单独Y2H系统就能得到和联合其他4种方法相似的数据；也就是因为这些PPIs检测方法原理上显著性差异，所以未来必须结合multiple protocols——可以是Braun描述的different methods，也可以是Chen描述的same method的不同变化。即是他赞成系统间验证，也认为系统内验证也是合理可行的！
Braun在同期的Reply里首先对Chen的工作表示欢迎，和部分肯定。也重新表明了自己希望建立“可信度”的观念，即一个特定方法检测筛选到的任何protein interaction都必须再经过multiple orthogonal validation assays（多重正交确认试验），筛选和验证实验必须相互独立才能尽可能消除系统假阳性的危险.筛选和验证采用同一试验，即使应用不同的构象，也可能引入这样的系统偏差。
Braun reply 原文
Braun et al. reply: We applaud the thorough and revealing study by Chen et al. in this issue of Nature Methods. The work expands our previous findings in thoroughly characterizing different yeast two-hybrid (Y2H) implementations, with respect to overall assay sensitivity, by testing each implementation against a panel of reference protein-protein interactions. The standardized reference sets make the data easily comparable to our previous analyses and clearly demonstrate that different Y2H assays detect different sub-sets of interacting pairs of proteins. Given the proven utility of using several assay configurations, the next question is where and how to deploy them. The high-throughput capabilities of Y2H make it an ideal primary screening assay. Having multiple versions of Y2H that detect different subsets of interactions will be of a great value to generate more comprehensive data sets, which would then need to be validated using a scheme such as the “confidence scoring” scheme that we proposed. A key concept of our confidence scoring method is that any interaction detected by a given screening assay is subsequently confirmed by multiple orthogonal validation assays. The screening and validation assays must be as independent from each other as possible to eliminate the danger of systematic assay dependent artifacts, which could make protein pairs appear as robustly interacting when they may not be. Use of a single type of assay for both screening and validation, even if implemented in different configurations, may introduce such systematic biases. It is therefore critical to obtain orthogonal validation ideally of all interacting pairs identified in an initial screen.
https://m.sciencenet.cn/blog-396054-411913.html
上一篇：Phase Lock Gel —— 酚氯仿抽提核酸的最佳伴侣
