A Method Developed To Fractionate Intact Proteins Based On Capillary Electrophoresis

With the completion of genome sequencing,human realize that genome alone is not enough to fully reflect body state,revealing of life activities rules depends on proteomics research.By the combination of multidimensional chromatography or electrophoresis methods,human have been able to identify over 1,000 kinds of protein,but many proteins are still undetectable,one important reason is that protein concentration in serum differ significantly,some of the low abundance proteins can be easily masked by high abundance proteins,resulting in difficult detection of them.In this paper we present the concept of the velocity gap mode of capillary electrophoresis(VGCE),by which a good resolution no longer rely solely on an increase in the effective length of a capillary or a change between different separation modes,it can be easily achieved by the VG effect.In this experiment,the optimum separation conditions was chosen by circular dichroism spectroscopy(CD)combined with conventional capillary electrophoresis.Then the relationship between sample injection volume and resolution was examined,a negative correlation between resolution and injection volume was obtained,providing a theoretical basis for the the removal of high abundance proteins by VGCE.Based on the above theory,we used standard proteins mixed at different ratio(lysozyme: BSA = 1: 30,1: 80,1: 120,1: 150)to simulate low abundance protein and high abundance protein.In conventional capillary electrophoresis,lysozyme signal cannot be detected because of the mask of BSA.While in VGCE,"cut big and stay small" removed most of BSA,lysozyme signal appeared and it could even be baseline resolved with the remaining fraction of BSA.In addition to applications in the removal of high abundance proteins,VGCE can also be used to enlarge resolution between proteins.For a mixture of good resolution(lysozyme,?-lactoglobulin,ribonuclease A),VG effect can make resolution more than 10 times larger;for a partially resolved mixture(lysozyme,BSA),as most of pure product was fractionated out by VGCE "cut big and stay small" or "multiple cutting",the sample zone width as well as mixing degree decrease compared with original sample,and resolution of each sample zone greatly increased;for a mixture that only has very little separation tendency(?-lactoglobulin and BSA),VGCE "multiple cutting" can also be applied to improve resolution.