RESUMO
Protein analysis (using either specific protein quantitation by methods such as HPLC and immunoassays or structural analysis by methods such as LC-MS) usually requires significant sample preparation, including quantitative purification of the target protein from complex sample matrices and potentially enzymatic treatment or labeling. We have developed platform for high-throughput microchromatography, capable of running 96 or more small volume samples in parallel, producing from 10 pg to 100 µg of purified protein from each sample. The platform is based on disposable cartridge devices with 5 µL packed bed of resin. The cartridges may be operated as spin columns or run on a modified 96-channel liquid handler with ultra-low dead volume syringes that directly connect to the cartridges, providing very precisely controlled positive-displacement flow control. A major application is quantitative purification of target proteins using affinity or physical chromatography. Using large diameter nonporous beads, standard microplate enzyme-linked immunosorbent assay reagents can be used to perform 30-min immunoassays. Enzymatic digestion methods have also been developed on the system for application in glycan profiling.