Shaw Jared B, Harvey Sophie R, Du Chen, Xu Zhixin, Edgington Regina M, Olmedillas Eduardo, Saphire Erica Ollmann, Wysocki Vicki H
Department of Chemistry, University of Nebraska, Lincoln, Nebraska 68588, United States.
Native Mass Spectrometry Guided Structural Biology Center, Ohio State University, Columbus, Ohio 43210, United States.
ACS Cent Sci. 2024 Jul 26;10(8):1537-1547. doi: 10.1021/acscentsci.4c00461. eCollection 2024 Aug 28.
We illustrate the utility of native mass spectrometry (nMS) combined with a fast, tunable gas-phase charge reduction, electron capture charge reduction (ECCR), for the characterization of protein complex topology and glycoprotein heterogeneity. ECCR efficiently reduces the charge states of tetradecameric GroEL, illustrating Orbitrap / measurements to greater than 100,000 /. For pentameric C-reactive protein and tetradecameric GroEL, our novel device combining ECCR with surface induced dissociation (SID) reduces the charge states and yields more topologically informative fragmentation. This is the first demonstration that ECCR yields more native-like SID fragmentation. ECCR also significantly improved mass and glycan heterogeneity measurements of heavily glycosylated SARS-CoV-2 spike protein trimer and thyroglobulin dimer. Protein glycosylation is important for structural and functional properties and plays essential roles in many biological processes. The immense heterogeneity in glycosylation sites and glycan structure poses significant analytical challenges that hinder a mechanistic understanding of the biological role of glycosylation. Without ECCR, average mass determination of glycoprotein complexes is available only through charge detection mass spectrometry or mass photometry. With narrow / selection windows followed by ECCR, multiple glycoform / values are apparent, providing quick global glycoform profiling and providing a future path for glycan localization on individual intact glycoforms.
我们展示了采用快速、可调谐的气相电荷减少方法——电子捕获电荷减少（ECCR）的基质辅助激光解吸电离质谱（nMS）在蛋白质复合物拓扑结构和糖蛋白异质性表征方面的应用。ECCR能有效降低十四聚体GroEL的电荷态，使Orbitrap质谱仪的测量分辨率大于100,000。对于五聚体C反应蛋白和十四聚体GroEL，我们将ECCR与表面诱导解离（SID）相结合的新型装置降低了电荷态，并产生了更多拓扑结构信息丰富的碎片。这是首次证明ECCR能产生更接近天然状态的SID碎片。ECCR还显著改善了对高度糖基化的严重急性呼吸综合征冠状病毒2（SARS-CoV-2）刺突蛋白三聚体和甲状腺球蛋白二聚体的质量和聚糖异质性测量。蛋白质糖基化对结构和功能特性很重要，在许多生物学过程中发挥着关键作用。糖基化位点和聚糖结构的巨大异质性带来了重大分析挑战，阻碍了对糖基化生物学作用的机制理解。没有ECCR时，糖蛋白复合物的平均质量测定只能通过电荷检测质谱法或质量光度法进行。通过ECCR后的窄质量选择窗口，多个糖型质量值变得明显，可提供快速的整体糖型分析，并为在单个完整糖型上进行聚糖定位提供了未来途径。
