Multibatch TMT Reveals False Positives, Batch Effects and Missing Values
Multibatch TMT Reveals False Positives, Batch Effects and Missing Values
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DOI：
10.1074/mcp.ra119.001472
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发表时间：
2019-10-01
影响因子：
7
通讯作者：
Lamond, Angus, I
中科院分区：
文献类型：
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作者：
Brenes, Alejandro;Hukelmann, Ens;Lamond, Angus, I
大规模蛋白质组分析的多重分析策略已变得越来越普遍，特别是串联质量标签 (TMT)。在这里，我们使用了具有 24 个 10 重 TMT 批次的大型 iPSC 蛋白质组实验来评估在单次分析中整合多个 TMT 批次的效果。我们发现，随着多个批次的整合，蛋白质缺失值出现显着的膨胀率，并表明这种模式在肽水平上更加严重。我们还表明，如果没有标准化策略来解决批次效应，则当集成多个 TMT 批次的数据时，无法再现单个多重 TMT 批次内的高精度定量。此外，使用Y染色体肽作为内部对照来研究假阳性的发生率。该数据集中量化的 iPSC 系均来自男性和女性供体，因此女性系中应该不存在映射到 Y 染色体的肽。尽管如此，这些 Y 染色体特异性肽在所有 TMT 批次的女性通道中均被检测到。然后，我们使用相同的 Y 染色体特异性肽来量化离子共隔离水平以及初级和次级报告离子干扰的影响。这些结果用于提出解决方案，以减轻多批次 TMT 分析的局限性。我们确认，在每批中包含公共参考线可以通过促进批次之间的标准化来提高精度，并且我们提出了最大限度地减少跨群体报告离子干扰影响的实验设计。
Multiplexing strategies for large-scale proteomic analyses have become increasingly prevalent, tandem mass tags (TMT) in particular. Here we used a large iPSC proteomic experiment with twenty-four 10-plex TMT batches to evaluate the effect of integrating multiple TMT batches within a single analysis. We identified a significant inflation rate of protein missing values as multiple batches are integrated and show that this pattern is aggravated at the peptide level. We also show that without normalization strategies to address the batch effects, the high precision of quantitation within a single multiplexed TMT batch is not reproduced when data from multiple TMT batches are integrated. Further, the incidence of false positives was studied by using Y chromosome peptides as an internal control. The iPSC lines quantified in this data set were derived from both male and female donors, hence the peptides mapped to the Y chromosome should be absent from female lines. Nonetheless, these Y chromosome-specific peptides were consistently detected in the female channels of all TMT batches. We then used the same Y chromosome specific peptides to quantify the level of ion coisolation as well as the effect of primary and secondary reporter ion interference. These results were used to propose solutions to mitigate the limitations of multi-batch TMT analyses. We confirm that including a common reference line in every batch increases precision by facilitating normalization across the batches and we propose experimental designs that minimize the effect of cross population reporter ion interference.