Restricted Proteolysis and LC-MS/MS To Evaluate the Orientation of Surface-Immobilized Antibodies.

ABSTRACT: The molecular orientation of antibodies immobilized on solid surfaces plays a significant role in the sensitivity of immunoassays and efficiency of protein isolation using antibody-decorated nanoparticles. Optimally, nearly all antibody binding sites should be available to bind. Here we report for the first time an LC-MS/MS approach to probe antibody orientation directly, utilizing sterically restricted proteolysis. Trypsin-decorated magnetic beads (MBs, 1.5 μm) were much larger than average antibody-free areas (55 × 55 nm) of oriented antibodies on MBs, restricting proteolysis to mainly Fab regions. Randomly attached antibodies on MB surfaces served as controls. The tryptic-hydrolyzed peptides were quantified using LC-MS/MS peptide analysis as markers for average positions of Fc and Fab o

SUBMITTER: Shen M 

PROVIDER: S-EPMC7159037 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature