La Jolla Institute for Immunology, La Jolla, CA, USA.
Methods Mol Biol. 2021;2271:343-359. doi: 10.1007/978-1-0716-1241-5_24.
Analytical size-exclusion chromatography (SEC) is a powerful technique that separates proteins based on their hydrodynamic radii. This approach can provide some rudimentary information about the molecular weight of proteins, but results are also influenced by the in-solution protein conformation and hydrophobicity. SEC also can be affected by nonspecific interactions with the column matrix that influence protein separation. Light scattering (LS) is an absolute and highly accurate measurement of protein molecular weight. Coupling analytical size-exclusion chromatography with multiangle light scattering (SEC-MALS) yields a more robust and accurate method for determining multiple biophysical parameters of proteins while avoiding SEC artifacts. This union of two techniques can help determine the absolute molecular stoichiometry, homo- and heteroassociation of sample components, the nature of protein conjugates, and the molar mass of single molecules and multisubunit complexes. In this chapter, we provide several examples of analysis of glycosylated protein conjugates to showcase the power of SEC-MALS.
分析型尺寸排阻色谱（SEC）是一种强大的技术，可根据蛋白质的流体力学半径将其分离。该方法可以提供有关蛋白质分子量的一些基本信息，但结果也受到溶液中蛋白质构象和疏水性的影响。SEC 还可能受到与柱基质的非特异性相互作用的影响，这些相互作用会影响蛋白质的分离。光散射（LS）是蛋白质分子量的绝对和高度准确的测量方法。将分析型尺寸排阻色谱与多角度光散射（SEC-MALS）相结合，可以在避免 SEC 假象的情况下，更稳健、更准确地测定蛋白质的多个生物物理参数。这两种技术的结合可以帮助确定样品成分的绝对分子化学计量、同型和异型缔合、蛋白质缀合物的性质以及单分子和多亚基复合物的摩尔质量。在本章中，我们提供了一些分析糖基化蛋白缀合物的示例，以展示 SEC-MALS 的强大功能。
