Improved separation of fluorogenic derivatized intact proteins with high resolution and efficiency using a reversed-phase liquid chromatographic system.
Ichibangase Tomoko,Nakata Katsunori,Imai Kazuhiro
Biomedical chromatography : BMC
Although the efficient separation of intact protein mixtures is extremely difficult, reversed-phase chromatography is an important technique for performing quantitative, accurate and reproducible proteomics analyses. Here, we show that, despite the operating constraints of conventional high-performance liquid chromatography, such as column temperature, operating pressure and separation time, comprehensive separation of fluorogenic derivatized intact proteins could be achieved with high resolution and separation efficiency. First, amylin was chosen as a model peptide and used to estimate the separation efficiency with respect to column temperature and flow rate, as indicated by peak capacity. Then, an extract of human primary hepatocytes was used to model complex component mixtures and the separation conditions were optimized. The effects of mobile-phase pH, the separation time and the column length were also investigated. Consequently, more than 890 peaks could be separated efficiently in the extract, which is 1.5-fold greater than when using conventional conditions. Finally, it was demonstrated that both longer separation time and column length contributed greatly to the effective separation of the protein mixture. These results are expected to provide insights into the separation of intact proteins.
10.1002/bmc.3172
New methods of protein purification. Displacement chromatography.
Galaev I Y
Biochemistry. Biokhimiia
This review discusses a new method for protein purification, displacement chromatography. Elution of proteins bound to an adsorbent is carried out in displacement chromatography by a substance, the so-called "displacer", which has a higher affinity for the chromatographic matrix than any of the adsorbed proteins. The latter are "pushed out" from the matrix by the displacer and form a displacement train when moving along the column. The component with the weakest affinity moves first, and the one with the highest affinity moves last in the train. The concentration of the component in the displacement zone is determined by the intersection of the operating line and binding isotherm of the given component. The shape of the displacement zone is close to rectangular. A more powerful resolving force is created compared to other methods of selective elution, especially at high column loadings, when nearly the whole working capacity of the column is used. Independence of component concentration in the displacement zone on its content in the feed allows significant concentrating of the purified protein during displacement chromatography. Examples of application of displacement chromatography for the separation of model protein systems and purification of proteins from crude extracts are discussed as well as methods of column regeneration.