Controlling how proteins are immobilized (e.g., controlling their orientation and conformation) is essential for developing and optimizing the performance of in vitro protein-binding devices, such as enzyme-linked immunosorbent assays. Characterizing the identity, orientation, etc., of proteins in complex mixtures of immobilized proteins requires a multitechnique approach. The focus of this work was to control and characterize the orientation of protein G B1, an immunoglobulin G (IgG) antibody-binding domain of protein G, on well-defined surfaces and to measure the effect of protein G B1 orientation on IgG antibody binding. The surface sensitivity of time-of-flight secondary ion mass spectrometry (ToF-SIMS) was used to distinguish between different proteins and their orientation on both flat and nanoparticle gold surfaces by monitoring intensity changes of characteristic amino acid mass fragments. Amino acids distributed asymmetrically were used to calculate peak intensity ratios from ToF-SIMS data to determine the orientation of protein G B1 cysteine mutants covalently attached to a maleimide surface. To study the effect of protein orientation on antibody binding, multilayer protein films on flat gold surfaces were formed by binding IgG to the immobilized protein G B1 films. Quartz crystal microbalance with dissipation monitoring and x-ray photoelectron spectroscopy analysis revealed that coverage and orientation affected the antibody-binding process. At high protein G B1 coverage, the cysteine mutant immobilized in an end-on orientation with the C-terminus exposed bound 443 ng/cm² of whole IgG (H + L) antibodies. In comparison, the high coverage cysteine mutant immobilized in an end-on orientation with the N-terminus exposed did not bind detectable amounts of whole IgG (H + L) antibodies.

中文翻译：

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使用XPS，ToF-SIMS和石英晶体微量天平通过耗散监测来表征蛋白G B1方向及其对免疫球蛋白G抗体结合的影响

控制蛋白质的固定方式（例如，控制其方向和构象）对于开发和优化体外性能至关重要蛋白结合装置，例如酶联免疫吸附测定。表征固定蛋白质复杂混合物中蛋白质的身份，方向等需要多种技术方法。这项工作的重点是在明确的表面上控制和表征蛋白质G B1（蛋白质G的免疫球蛋白G（IgG）抗体结合结构域）的方向，并测量蛋白质G B1方向对IgG抗体结合的影响。飞行时间二次离子质谱（ToF-SIMS）的表面灵敏度用于通过监测特征氨基酸质量片段的强度变化来区分不同的蛋白质及其在平坦和纳米粒子金表面上的取向。不对称分布的氨基酸用于从ToF-SIMS数据计算峰强度比，以确定共价附于马来酰亚胺表面的蛋白G B1半胱氨酸突变体的方向。为了研究蛋白质取向对抗体结合的影响，通过将IgG与固定的蛋白G B1膜结合形成在平坦金表面上的多层蛋白膜。带有耗散监测和X射线光电子能谱分析的石英微天平显示，覆盖范围和方向会影响抗体结合过程。在高蛋白G B1覆盖率下，半胱氨酸突变体固定在末端方向，暴露的C末端为443 ng / cm 通过将IgG结合到固定的蛋白G B1膜上，在平坦的金表面上形成多层蛋白膜。带有耗散监测和X射线光电子能谱分析的石英微天平显示，覆盖范围和方向会影响抗体结合过程。在高蛋白G B1覆盖率下，半胱氨酸突变体固定在末端方向，暴露的C末端为443 ng / cm 通过将IgG结合到固定的蛋白G B1膜上，在平坦的金表面上形成多层蛋白膜。带有耗散监测和X射线光电子能谱分析的石英微天平显示，覆盖范围和方向会影响抗体结合过程。在高蛋白G B1覆盖率下，半胱氨酸突变体固定在末端方向，暴露的C末端为443 ng / cm²个完整的IgG（H + L）抗体。相比之下，固定的高覆盖度半胱氨酸突变体以末端定位方式固定，暴露的N端暴露在外，不能结合可检测量的完整IgG（H + L）抗体。