Physical and Theoretical Chemistry, Department of Chemistry, University of Oxford, South Parks Road, Oxford, OX1 3TA, UK.
Angew Chem Int Ed Engl. 2020 Jun 26;59(27):10774-10779. doi: 10.1002/anie.202001578. Epub 2020 Apr 2.
Interactions between biomolecules control the processes of life in health and their malfunction in disease, making their characterization and quantification essential. Immobilization- and label-free analytical techniques are desirable because of their simplicity and minimal invasiveness, but they struggle with quantifying tight interactions. Here, we show that mass photometry can accurately count, distinguish by molecular mass, and thereby reveal the relative abundances of different unlabelled biomolecules and their complexes in mixtures at the single-molecule level. These measurements determine binding affinities over four orders of magnitude at equilibrium for both simple and complex stoichiometries within minutes, as well as the associated kinetics. These results introduce mass photometry as a rapid, simple and label-free method for studying sub-micromolar binding affinities, with potential for extension towards a universal approach for characterizing complex biomolecular interactions.
生物分子之间的相互作用控制着健康状态下生命过程的进行，而它们在疾病中的功能失常则表明需要对其进行特征描述和定量分析。由于其具有简单性和微创性，因此固定化和无标记的分析技术是可取的，但它们在定量分析紧密相互作用方面存在困难。在这里，我们表明，质量光度法可以在单分子水平上准确地计数、按分子质量区分，从而揭示混合物中不同未标记生物分子及其复合物的相对丰度。这些测量在几分钟内即可确定平衡状态下简单和复杂化学计量比的四个数量级的结合亲和力，以及相关的动力学。这些结果表明，质量光度法是一种快速、简单和无标记的方法，可用于研究亚微摩尔亲和力，有可能扩展为一种通用方法来表征复杂的生物分子相互作用。
