Langmuir. 2021 Dec 28;37(51):14856-14865. doi: 10.1021/acs.langmuir.1c02644. Epub 2021 Dec 14.
Silica passivating agents have shown great success in minimizing nonspecific protein binding to glass surfaces for imaging and microscopy applications. Amine-derivatized surfaces are commonly used in conjugation with amide coupling agents to immobilize peptides/proteins through C-terminal or side-chain carboxylic acids. In the case of the single-molecule fluorosequencing of peptides, attachment occurs via the C-terminus and nonspecific surface binding has previously been a source of error in peptide identification. Here, we employ fluorosequencing as a high-throughput, single-molecule sensitivity assay to identify and quantify the extent of nonspecific binding of peptides to amine-derivatized surfaces. We show that there is little improvement when using common passivating agents in combination with the surface derivatizing agent 3-aminopropyl-triethoxysilane (APTES) to couple the peptides to the modified surface. Furthermore, many xanthene fluorophores have carboxylic acids in the appended phenyl ring at positions ortho and meta or ortho and para, and the literature shows that conjugation through the ortho position is not favored. Because xanthene-derived fluorophores are commonly used for single-molecule applications, we devised a novel assay to probe the conjugation of peptides via their fluorophores relative to their C-termini on silane-derivatized surfaces. We find significant attachment to the ortho position, which is a warning to those attempting to immobilize fluorophore-labeled peptides to silica surfaces via amide coupling agents. However, eliminating all amines on the surface by switching to 3-azidopropyl-triethoxysilane (AzTES) for coupling via copper-catalyzed azide-alkyne cycloaddition (CuAAC) and omitting additional passivation agents allowed us to achieve a high level of C-terminally bound peptides relative to nonspecifically or ortho-phenyl-bound, fluorophore-labeled peptides. This strategy substantially improves the specificity of peptide immobilization for single-molecule fluorosequencing experiments.
硅烷钝化剂在最小化玻璃表面非特异性蛋白质结合方面取得了巨大成功，可用于成像和显微镜应用。胺衍生表面通常与酰胺偶联剂一起使用，通过 C 末端或侧链羧酸将肽/蛋白质固定在表面上。在肽的单分子荧光测序中，附着发生在 C 末端，非特异性表面结合以前是肽鉴定中的一个误差源。在这里，我们采用荧光测序作为一种高通量、单分子灵敏度测定法，以确定和量化肽与胺衍生表面非特异性结合的程度。我们发现，当使用常见的钝化剂与表面衍生化试剂 3-氨丙基三乙氧基硅烷 (APTES) 结合使用时，将肽偶联到修饰表面上，改善效果不大。此外，许多香豆素荧光团在附加的苯基环的邻位和间位或邻位和对位都有羧酸，文献表明通过邻位进行偶联不受青睐。由于香豆素衍生的荧光团通常用于单分子应用，因此我们设计了一种新的测定法，以探测肽相对于其 C 末端在硅烷衍生表面上的荧光团偶联。我们发现与 C 末端相比，在邻位的附着显著增加，这对于那些试图通过酰胺偶联剂将荧光标记的肽固定在硅表面上的人来说是一个警告。然而，通过切换到 3-叠氮丙基三乙氧基硅烷 (AzTES) 用于通过铜催化的叠氮-炔环加成 (CuAAC) 偶联，并省略其他钝化剂，我们能够实现相对于非特异性或邻位-苯结合的、荧光标记的肽的高 C 末端结合肽的水平。这种策略大大提高了肽固定在单分子荧光测序实验中的特异性。
