PDF(Pubmed)
Abstract：
We present a hydrogen/deuterium exchange workflow coupled to tandem mass spectrometry (HX-MS2) that supports the acquisition of peptide fragment ions alongside their peptide precursors. The approach enables true auto-curation of HX data by mining a rich set of deuterated fragments, generated by collisional-induced dissociation (CID), to simultaneously confirm the peptide ID and authenticate MS1-based deuteration calculations. The high redundancy provided by the fragments supports a confidence assessment of deuterium calculations using a combinatorial strategy. The approach requires data-independent acquisition (DIA) methods that are available on most MS platforms, making the switch to HX-MS2 straightforward. Importantly, we find that HX-DIA enables a proteomics-grade approach and wide-spread applications. Considerable time is saved through auto-curation and complex samples can now be characterized and at higher throughput. We illustrate these advantages in a drug binding analysis of the ultra-large protein kinase DNA-PKcs, isolated directly from mammalian cells.
摘要：
我们提出了与串联质谱（HX-MS2）耦合的氢/氘交换工作流程，该流程支持肽片段离子及其肽前体的获取。该方法通过挖掘一组丰富的氘代片段来实现HX数据的真正自动管理，由碰撞诱导解离(CID)产生，以同时确认肽ID并验证基于MS1的氘代计算。片段提供的高冗余支持使用组合策略的氘计算的置信度评估。该方法需要在大多数MS平台上可用的数据独立采集(DIA)方法，直接切换到HX-MS2。重要的是,我们发现HX-DIA能够实现蛋白质组学级别的方法和广泛的应用。通过自动管理节省了相当多的时间，现在可以表征复杂的样品，并以更高的吞吐量。我们在超大型蛋白激酶DNA-PKcs的药物结合分析中说明了这些优势，直接从哺乳动物细胞中分离。
