Label-free quantification in ion mobility-enhanced data-independent acquisition proteomics
Label-free quantification in ion mobility-enhanced data-independent acquisition proteomics
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DOI：
10.1038/nprot.2016.042
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发表时间：
2016-04-01
期刊：
影响因子：
14.8
通讯作者：
Tenzer, Stefan
中科院分区：
文献类型：
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作者：
Distler, Ute;Kuharev, Joerg;Tenzer, Stefan
无偏数据独立采集（DIA）策略在定量蛋白质组学领域越来越受欢迎。将离子迁移率分离（IMS）集成到DIA工作流程中为液相色谱-质谱（LC-MS）提供了额外的分离维度，并增加了DIA方法可实现的分析深度。在这里，我们提供了一个详细的协议，无标记的定量蛋白质组学工作流程的基础上，离子迁移率增强DIA，它使前体离子漂移时间与碰撞能量，以提高前体裂解效率。该方案包括所有主要步骤的详细描述，包括仪器设置、过滤器辅助样品制备、LC-IMS-MS分析和数据处理。我们的方案可以处理任何复杂性的蛋白质组样品，并且它可以在完整细胞裂解物中多次运行高达5，600种蛋白质的高度可重复和准确的基于前体强度的无标记定量。根据待分析样品的数量，该方案从蛋白水解消化到数据评估至少需要3天。
Unbiased data-independent acquisition (DIA) strategies have gained increased popularity in the field of quantitative proteomics. The integration of ion mobility separation (IMS) into DIA workflows provides an additional dimension of separation to liquid chromatography-mass spectrometry (LC-MS), and it increases the achievable analytical depth of DIA approaches. Here we provide a detailed protocol for a label-free quantitative proteomics workflow based on ion mobility-enhanced DIA, which synchronizes precursor ion drift times with collision energies to improve precursor fragmentation efficiency. The protocol comprises a detailed description of all major steps including instrument setup, filter-aided sample preparation, LC-IMS-MS analysis and data processing. Our protocol can handle proteome samples of any complexity, and it enables a highly reproducible and accurate precursor intensity-based label-free quantification of up to 5,600 proteins across multiple runs in complete cellular lysates. Depending on the number of samples to be analyzed, the protocol takes a minimum of 3 d to complete from proteolytic digestion to data evaluation.