| Proteomics becomes a new frontier of life science in post-genome era, and will play vital role in elucidation of fundamental life process. Blood plasma contains rich information about the health status of the body, and as one of the most complicated proteomes is especially challenging, and thus mostly concerned. Presently,2dimensional electrophoresis (2-DE) and mass spectrometry (MS) are the main core isolation and identification methods for complex protein mixtures. However, reliable analysis is difficult to achieve due to the existence of high abundance proteins and low abundance proteins, and preparative isolation of small amount sample is crucial. A chip free flow electrophoresis (FFE) in isoelectric focusing (IEF) format is introduced for preparative isolation of complex proteins from small amount of samples, aiming the above problems.Firstly, the former established chip FFE-IEF system was improved. The separation channel was defined by a double tape, and isolation of the electrodes from the separation channel was easily implemented by agarose gel. The pH gradient established in the channel with ampholyte was characterized by multiple methods, including fluorescent dye, acid/base color indicators and direct imaging.Secondly, with the improved chip system, preparative separation of model proteins (Hb and cytochrome C) was successfully achieved. The system was further used in the separation of a human blood plasma sample, and both the removal of high abundance proteins and enrichment of low abundance protein were well demonstrated as witnessed by SDS-PAGE. Lastly, chip FFE-IEF without ampholyte was tried by in-situ monolithic immobilization of pH gradient in the separation bed. Preliminary results showed that the chip FFE was good in permeability, and IEF without ampholyte is possible.This study shows that chip FFE IEF is especially suitable for fast micro-preparative separation of complex proteins, and may find wide applications both in research and in clinical analysis. |
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