The way to immobilize a protein onto a solid surface is the cornerstone for developing many bioanalytical assays which are applied in protein functional assay, protein-ligand interaction analysis and drug screening. Most immobilization approaches are established for soluble or cytosolic proteins, however, when it comes to transmembrane proteins (TMPs) like G protein-coupled receptors (GPCRs), they are challenged, and innovation is needed. In the last decade, site-specific immobilization strategies taking advantages of click chemistry and bioorthogonal conjugation have been developed, which have improved the sensitivity, selectivity, and repeatability of the protein based analytical methods. In this review, we focus on the immobilization of TMPs, because they constitute the largest pharmacological targets in clinics and serve as a paradigm to develop new analytical methods for bioanalysis. In particular, we summarize the progresses of site-specific immobilization made for establishing biosensors, bioassays, and chromatographic methods to pursue drug-protein interaction analysis and library screening.

将蛋白质固定在固体表面的方法是开发许多应用于蛋白质功能测定、蛋白质-配体相互作用分析和药物筛选的生物分析测定的基石。大多数固定化方法都是针对可溶性或胞质蛋白建立的，然而，当涉及 G 蛋白偶联受体 (GPCR) 等跨膜蛋白 (TMP) 时，它们面临挑战，需要创新。在过去的十年中，利用点击化学和生物正交共轭的优势的位点特异性固定策略得到了发展，提高了基于蛋白质的分析方法的灵敏度、选择性和可重复性。在这篇综述中，我们专注于 TMP 的固定化，因为它们构成了临床中最大的药理学靶标，并作为开发生物分析新分析方法的范例。特别是，我们总结了为建立生物传感器、生物测定和色谱方法进行药物-蛋白质相互作用分析和文库筛选而进行的位点特异性固定化的进展。