Pan Hua, Klein Shelby M, Gunaratne Akalanka, Jarrold Martin F, Clemmer David E
Department of Chemistry, Indiana University, 800 Kirkwood Avenue, Bloomington, Indiana 47401, United States.
Anal Chem. 2025 Jan 21;97(2):1419-1425. doi: 10.1021/acs.analchem.4c06038. Epub 2025 Jan 7.
Charge detection mass spectrometry (CD-MS) is used to monitor the dissociation of large (300 kDa to 20 MDa) protein complexes in droplets heated with a 10.6 μm CO laser. In this approach, electrospray ionization (ESI) is used to produce charged droplets containing macromolecular complexes. As the droplets travel from the ESI capillary tip to the entrance of the CD-MS instrument, they pass through a variable-power laser field, where they are rapidly heated and dissociate to produce fragments. The approach is illustrated for three model systems: glutamate dehydrogenase (GDH), a 334 kDa hexameric protein complex, which dissociates into protein monomers, dimers, and tetramers; the ∼3 MDa = 3, and ∼4 MDa = 4 hepatitis B virus VLPs (virus-like particles) that produce a distribution of protein dimer clusters; and the ∼20 MDa = 7 human papillomavirus VLP, which dissociates primarily into small capsid protein clusters that are not well-resolved by CD-MS. The fragments produced by in-droplet activation provide information that is useful for characterizing the structures of the intact antecedent complexes. A discussion of the advantages and current limitations of this approach is presented.
电荷检测质谱法（CD-MS）用于监测在10.6μm CO激光加热的液滴中大型（300 kDa至20 MDa）蛋白质复合物的解离。在这种方法中，电喷雾电离（ESI）用于产生含有大分子复合物的带电液滴。当液滴从ESI毛细管尖端移动到CD-MS仪器的入口时，它们会穿过一个可变功率的激光场，在那里它们会被快速加热并解离产生碎片。该方法通过三个模型系统进行说明：谷氨酸脱氢酶（GDH），一种334 kDa的六聚体蛋白质复合物，可解离为蛋白质单体、二聚体和四聚体；约3 MDa（n = 3）和约4 MDa（n = 4）的乙型肝炎病毒病毒样颗粒（VLPs），可产生蛋白质二聚体簇的分布；以及约20 MDa（n = 7）的人乳头瘤病毒VLP，其主要解离为小衣壳蛋白簇，CD-MS无法很好地分辨这些簇。液滴内活化产生的碎片提供了有助于表征完整前体复合物结构的信息。本文讨论了该方法的优点和当前局限性。
