用于大规模相互作用研究的基于 TMT 的优化定量交联质谱策略,Analytical Chemistry

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用于大规模相互作用研究的基于 TMT 的优化定量交联质谱策略
Analytical Chemistry ( IF 6.7 ) Pub Date : 2022-03-15 , DOI: 10.1021/acs.analchem.1c04812
Max Ruwolt ₁ , Lennart Schnirch ₁ , Diogo Borges Lima ₁ , Michal Nadler-Holly ₁ , Rosa Viner ₂ , Fan Liu ₁

Affiliation

交联质谱 (XL-MS) 是研究高度复杂样品中蛋白质-蛋白质相互作用 (PPI) 的有力方法。XL-MS 与串联质量标签 (TMT) 标记相结合，有望实现大规模 PPI 量化。然而，由于缺乏基准数据集和对各种 MS 参数的全面评估，尚未建立稳健且有效的基于 TMT 的 XL-MS 量化方法。为了解决这些限制，我们通过在 TMT 标记的交联大肠杆菌中加标生成两个交互组数据集使用定义的混合方案将裂解物转化为 TMT 标记的交联 HEK293T 裂解物。使用此基准数据集，我们使用不同的 MS 采集策略评估交叉链接识别的有效性和交叉链接量化的准确性。为了识别，我们比较了各种基于 MS2 和 MS3 的 XL-MS 方法，并优化了 TMT 标记交联的阶梯式高能碰撞解离 (HCD) 能量。我们观察到与未标记的交联相比，需要显着更高的碎片能量。对于量化，我们评估了基于 MS2、MS3 和同步前体选择-MS3 的方法的量化准确性和分散性。我们表明，具有阶梯式碰撞能量 36-42-48 的阶梯式 HCD-MS2 方法提供了大量具有高量化精度的可量化交联。

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Optimized TMT-Based Quantitative Cross-Linking Mass Spectrometry Strategy for Large-Scale Interactomic Studies

Cross-linking mass spectrometry (XL-MS) is a powerful method for the investigation of protein–protein interactions (PPI) from highly complex samples. XL-MS combined with tandem mass tag (TMT) labeling holds the promise of large-scale PPI quantification. However, a robust and efficient TMT-based XL-MS quantification method has not yet been established due to the lack of a benchmarking dataset and thorough evaluation of various MS parameters. To tackle these limitations, we generate a two-interactome dataset by spiking in TMT-labeled cross-linked Escherichia coli lysate into TMT-labeled cross-linked HEK293T lysate using a defined mixing scheme. Using this benchmarking dataset, we assess the efficacy of cross-link identification and accuracy of cross-link quantification using different MS acquisition strategies. For identification, we compare various MS2- and MS3-based XL-MS methods, and optimize stepped higher energy collisional dissociation (HCD) energies for TMT-labeled cross-links. We observed a need for notably higher fragmentation energies compared to unlabeled cross-links. For quantification, we assess the quantification accuracy and dispersion of MS2-, MS3-, and synchronous precursor selection-MS3-based methods. We show that a stepped HCD-MS2 method with stepped collision energies 36-42-48 provides a vast number of quantifiable cross-links with high quantification accuracy. This widely applicable method paves the way for multiplexed quantitative PPI characterization from complex biological systems.

更新日期：2022-03-15

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