96-well plate format in conjunction with ultra-high-performance liquid chromatography coupled to orbitrap mass spectrometry for high-throughput screening protein binders from ginseng.

Conventional strategies for screening of protein binders cannot be used for complicated samples such as ligand libraries created by combinatorial chemistry or from natural product extracts. In the current study, we developed a novel method in a competitive binding configuration for screening protein binders from complicated samples by a combination of streptavidin-coated 96-well plate format in conjunction with ultra-high-performance liquid chromatography coupled with Orbitrap mass spectrometry (UHPLC-Orbitrap-MS). The concanavalin A (Con A) modified 96-well plate and lysozyme modified 96-well plate (as control) were incubated with oligosaccharide standards respectively, and the compounds with the decreased peak areas in experimental group compared to those in the control group were detected as binders by UHPLC-ESI-MS. The factors such as incubation time, incubation temperature, and buffer, which might affect the binding affinity and reproducibility were optimized. The potential of the approach is examined using the extracts of Radix ginseng cruda and American ginseng. The relative binding degrees (RBDs) of the detected disaccharides were relatively high in the extracts of Radix ginseng cruda, and those of the trisaccharides were similar in the extracts of the two kinds of ginseng. To our knowledge, it's the first time to reveal the differences and analogies in lectin peanut agglutinin (PNA)-binding capabilities of oligosaccharides between the extracts of radix ginseng cruda and American ginseng, indicating the efficiency of the method for analysis of complicated samples.