The quantitative multiplexing capacity of isobaric tandem mass tags (TMT) has increased the throughput of affinity purification mass spectrometry (AP-MS) to characterize protein interaction networks of immunoprecipitated bait proteins. However, variable bait levels between replicates can convolute interactor identification. We compared the Student’s t-test and Pearson’s R correlation as methods to generate t-statistics and assessed the significance of interactors following TMT-AP-MS. Using a simple linear model of protein recovery in immunoprecipitates to simulate reporter ion ratio distributions, we found that correlation-derived t-statistics protect against bait variance while robustly controlling type I errors (false positives). We experimentally determined the performance of these two approaches for determining t-statistics under two experimental conditions: irreversible prey association to the Hsp40 mutant DNAJB8^H31Q followed by stringent washing, and reversible association to 14-3-3ζ with gentle washing. Correlation-derived t-statistics performed at least as well as Student’s t-statistics for each sample and with substantial improvement in performance for experiments with high bait-level variance. Deliberately varying bait levels over a large range fails to improve selectivity but does increase the robustness between runs. The use of correlation-derived t-statistics should improve identification of interactors using TMT-AP-MS. Data are available via ProteomeXchange with identifier PXD016613.

中文翻译：

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诱饵相关性通过串联质谱标签亲和纯化-质谱技术改善了相互作用物的鉴定。

等压串联质谱（TMT）的定量多路复用能力提高了亲和纯化质谱（AP-MS）的通量，以表征免疫沉淀的诱饵蛋白的蛋白相互作用网络。但是，重复试验之间诱饵水平的变化可能会使交互作用者的识别复杂化。我们比较了学生的t检验和Pearson的R相关作为生成t统计的方法，并评估了TMT-AP-MS后相互作用子的重要性。使用免疫沉淀物中蛋白质回收的简单线性模型来模拟报告子离子比率分布，我们发现相关性衍生的t-统计数据可以防止诱饵变化，同时可以有效控制I型错误（误报）。我们通过实验确定了这两种方法在两种实验条件下确定t统计量的性能：与Hsp40突变体DNAJB8 ^H31Q不可逆地结合，随后进行严格的洗涤；与^14-3-3ζ进行可逆结合，并进行温和洗涤。对于每个样本，相关派生的t统计量至少与学生的t统计量一样好，并且对于高诱饵水平方差的实验，其性能有显着提高。在较大范围内故意改变诱饵水平无法提高选择性，但确实会增加两次运行之间的鲁棒性。相关性的使用t统计量应使用TMT-AP-MS改善对相互作用者的识别。数据可通过ProteomeXchange获得，其标识符为PXD016613。