Department of Pharmaceutical Chemistry & Bioanalytics, Institute of Pharmacy, Kurt-Mothes-Str. 3, D-06120 Halle (Saale), Germany.
Center for Structural Mass Spectrometry, Kurt-Mothes-Str. 3, D-06120 Halle (Saale), Germany.
Anal Chem. 2021 Aug 24;93(33):11442-11450. doi: 10.1021/acs.analchem.1c01317. Epub 2021 Aug 10.
The combination of cross-linking/mass spectrometry (XL-MS) and ion mobility is still underexplored for conducting protein conformational and protein-protein interaction studies. We present a method for analyzing cross-linking mixtures on a timsTOF Pro mass spectrometer that allows separating ions based on their gas-phase mobilities. Cross-linking was performed with three urea-based MS-cleavable cross-linkers that deliver distinct fragmentation patterns for cross-linked species upon collisional activation. The discrimination of cross-linked species from non-cross-linked peptides was readily performed based on their collisional cross sections. We demonstrate the general feasibility of our combined XL-MS/ion mobility approach for three protein systems of increasing complexity: (i) bovine serum albumin (BSA), (ii) ribosome, and (iii) HEK293T cell nuclear lysates. We identified a total of 623 unique cross-linking sites for BSA, 670 for the ribosome, and 1623 unique cross-links for nuclear lysates, corresponding to 1088 intra- and 535 interprotein interactions and yielding 564 distinct protein-protein interactions. Our results underline the strength of combining XL-MS with ion mobility not only for deriving three-dimensional (3D) structures of single proteins but also for performing system-wide protein interaction studies.
交联/质谱联用（XL-MS）和离子淌度联用技术在用于进行蛋白质构象和蛋白质-蛋白质相互作用研究方面仍未得到充分探索。我们提出了一种在 timsTOF Pro 质谱仪上分析交联混合物的方法，该方法允许根据其气相迁移率分离离子。交联反应使用了三种基于脲的 MS 可切割交联剂，这些交联剂在碰撞激活时会产生不同的交联物种碎裂模式。交联物种与非交联肽的区分可以基于它们的碰撞截面来轻松完成。我们展示了我们的组合 XL-MS/离子淌度方法对于三个具有不同复杂程度的蛋白质系统的一般可行性：（i）牛血清白蛋白（BSA），（ii）核糖体，和（iii）HEK293T 细胞核裂解物。我们总共鉴定了 BSA 的 623 个独特交联位点、核糖体的 670 个交联位点和核裂解物的 1623 个独特交联位点，分别对应 1088 个内蛋白和 535 个外蛋白相互作用，并产生了 564 个不同的蛋白质-蛋白质相互作用。我们的结果强调了将 XL-MS 与离子淌度联用的优势，不仅可以用于推导单个蛋白质的三维（3D）结构，还可以用于进行系统范围的蛋白质相互作用研究。
