Department of Chemistry, University of Nevada, 1664 N. Virginia Street, Reno, NV 89557, USA.
Analyst. 2023 Mar 27;148(7):1534-1542. doi: 10.1039/d2an01682f.
Mass spectrometry (MS)-based proteomics workflows of intact protein ions have increasingly been utilized to study biological systems. These workflows, however, frequently result in convoluted and difficult to analyze mass spectra. Ion mobility spectrometry (IMS) is a promising tool to overcome these limitations by separating ions by their mass- and size-to-charge ratios. In this work, we further characterize a newly developed method to collisionally dissociate intact protein ions in a trapped ion mobility spectrometry (TIMS) device. Dissociation occurs prior to ion mobility separation and thus, all product ions are distributed throughout the mobility dimension, enabling facile assignment of near isobaric product ions. We demonstrate that collisional activation within a TIMS device is capable of dissociating protein ions up to 66 kDa. We also demonstrate that the ion population size within the TIMS device significantly influences the efficiency of fragmentation. Lastly, we compare CIDtims to the other modes of collisional activation available on the Bruker timsTOF and demonstrate that the mobility resolution in CIDtims enables the annotation of overlapping fragment ions and improves sequence coverage.
基于质谱（MS）的完整蛋白质离子蛋白质组学工作流程已越来越多地用于研究生物系统。然而，这些工作流程通常会导致复杂且难以分析的质谱。离子淌度谱（IMS）是一种很有前途的工具，可以通过其质量-电荷比和大小-电荷比来分离离子，从而克服这些限制。在这项工作中，我们进一步描述了一种新开发的方法，即在捕获离子淌度谱（TIMS）装置中使完整的蛋白质离子发生碰撞解离。解离发生在离子淌度分离之前，因此，所有的产物离子都分布在淌度维度上，便于对近等电点的产物离子进行归属。我们证明，TIMS 装置内的碰撞激活能够使高达 66 kDa 的蛋白质离子解离。我们还证明，TIMS 装置内的离子群体大小显著影响碎裂效率。最后，我们将 CIDtims 与 Bruker timsTOF 上可用的其他碰撞激活模式进行了比较，并证明 CIDtims 中的淌度分辨率能够注释重叠的片段离子并提高序列覆盖率。
