The Design And Study Of A Novel Microscale Preparative Gel Electrophoresis Apparatus

The separation of protein mixtures is of great importance to protein and proteome researchand applications. Due to the variety and wide dynamic range of proteins, the fractionation of the protein samples is a big challenge. Typically, two main techniques are used in protein fractionation including high performance liquid chromatography(HPLC) and electrophoresis. Compared with HPLC, electrophoresis has the advantages of high resolution, the ability to acquire protein information such as isoelectric point and molecular weight, but it also has the disadvantages of laboriousness, low automation extent and inability to be applied in protein preparation. Preparative electrophoresis is one category of protein fractionation techniques, and targeted proteins can be separated, purified or fractionated under the existence of electric field. Over the past few decades,preparative electrophoresis, especially preparative electrophoresis apparatuses has made considerable progress. Nowadays the proteomics begin to focus on microscale samples and the separation targets of preparative electrophoresis are changing from milligrams of proteins to micrograms of proteins. The commercial preparative electrophoresis apparatuses mainly include milligram-scale preparative electrophoresis apparatuses and microgram-scale preparative electrophoresis apparatuses. Most of the milligram-scale preparative electrophoresis apparatuses were developed at early stage. The complete separation and collection of all of the fractions required approximately 10 h using these apparatuses. Furthermore, they all suffer from low recovery and the concentration of the electroeluate was too low to be recycled for microscale samples. During the past decades, microgram-scale preparative electrophoresis apparatuses have progressed a lot. However, among the commercially available microscale preparative electrophoresis apparatuses, many suffer from low recovery and low resolution, and they are either time-consuming or laborious. In order to solve those problems, we have developed a set of preparative gel electrophoresis system aimed at a simple and fast microscale protein fractionation.Microscale protein samples are separated on the gel according to their molecular weights and then the gel is transferred to the elution apparatus for a fast elution of protein fractions. Our tests showed that this apparatus could accomplish protein fractionation with high efficiency and high recovery.This thesis consists of 3 chapters. The first chapter introduces the development of separation and analytical techniques in the proteomics. We summarized the pre-fractionation methods and thepreparative electrophoresis techniques. Meanwhile, the first chapter also includes the background,content and meanings of our research.The second chapter introduces a device of preparative gel electrophoresis combined with UV detector. This device connected a gel slab with an external pump, a high-pressured reservoir, a UV detector, a computer and a fraction collector in sequence and achieved the purpose of protein separation, detection and collection on-line. In the performance test and evaluation of the device,we utilized myoglobin and bovine serum albumin mixture as model proteins and conducted a separation in this device. Finally we witnessed two strong absorption peaks in the UV spectrum.Nevertheless, there were still many problems in this device. The experiment was laborious and time-consuming; the device suffered from severe leakage and there were bubbles in the anode which disrupted the separation, detection and collection; the device was inconvenient in the manufacture, and these urge us to design a new preparative electrophoresis device.In the third chapter we introduce an improved apparatus based on the device of preparative gel electrophoresis combined with UV detector. In order to solve the many problems in the device mentioned in Chapter two, we changed the online detection into offline, and utilized 3D printing and CNC machining in the design and manufacture for our apparatus. Our preparative gel electrophoresis separation-elution apparatuses could be classified into two categories: the transverse elution apparatuses and the perpendicular elution apparatuses. After the manufacture,these apparatuses were tested in mechanical strength, anti-leakage and operability. The apparatuses that passed the examination were further investigated in protein separation, protein elution, resolution, recovery, elution time and used for the HPLC-MS analysis as a prefraction step.Finally, a preparative gel electrophoresis separation-elution apparatus with perpendicular elution in which the separation and elution were conducted in two components was evaluated as the best one. It achieved high resolution, high recovery and high reproducibility in the separation tests and in the elution tests. The elution time was less than 20 min and the recovery was as high as 91.8%in the recovery test. The results indicated that this apparatus was better than the commercial preparative gel electrophoresis apparatuses. The preparative gel electrophoresis separation-elution apparatus with perpendicular elution was also tested in the fractionation of a standard protein mixture and a crude yeast extract. The experimental results with high resolution were acquired and compared with the commercial preparative gel electrophoresis apparatuses, our apparatus performed better. In the experiment of the combination of perpendicular elution apparatus and HPLC-MS, 10 μg of bovine serum albumin were separated, eluted and FASP digested in prior to HPLC-MS analysis. In comparison, 10 μg of bovine serum albumin was separated in our apparatus and followed by an in-gel digestion; the digest was then analyzed by HPLC-MS. Theresults showed that 94 unique peptides were identified and the average sequence coverage was90.12%. 70 peptides were identified and the average sequence coverage was 86% when in gel digestion was used after electrophoresis of the bovine serum albumin sample. Another experiment was carried out in order to compare the elution-FASP method with in-gel digestion. 20 μg of crude yeast extract was separated, eluted and FASP digested in prior to HPLC-MS analysis, and in comparison, 20 μg of crude yeast extract were separated, in-gel digested prior to HPLC-MS analysis. The results showed that the identified peptides, proteins(sequence coverage>50% or score>1000) using the elution-FASP method were much larger than those using the in-gel digestion method. These results suggest that better identification results could be achieved through separation, elution and FASP digestion using our apparatus.In addition, in the preparative gel electrophoresis separation-elution apparatus with perpendicular elution, we added the semiconductor refrigeration sheets and assembled a whole preparative gel electrophoresis system. This system can be used in protein fractionation, reducing the complexity of protein samples, helping to remove high-abundance proteins and discovering some low-abundance proteins. This system can also serve as an alternative used in the “top-down”proteomics.