Characterizing protein G B1 orientation and its effect on immunoglobulin G antibody binding using XPS, ToF-SIMS, and quartz crystal microbalance with dissipation monitoring.

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.