一種微量制備型凝膠電泳裝置的研制
發(fā)布時間：2018-09-18 11:20
【摘要】：蛋白質(zhì)混合物的高效分離對蛋白質(zhì)以及蛋白質(zhì)組的定性和定量分析及應(yīng)用具有重要意義,但由于蛋白質(zhì)種類多,動態(tài)范圍寬,其分離和制備面臨巨大挑戰(zhàn)。目前,蛋白質(zhì)組學(xué)中常用的蛋白質(zhì)混合物高效分離技術(shù)主要包括高效液相色譜和電泳技術(shù)。電泳技術(shù)相較于高效液相色譜,其優(yōu)勢是分離度高,并且可以得到等電點、相對分子質(zhì)量信息,其劣勢是費時費力,自動化程度低,且難以用于蛋白質(zhì)的制備。制備電泳技術(shù)是在制備電泳裝置中對目標(biāo)物質(zhì)進(jìn)行分離、純化和收集制備的一種技術(shù)。在過去的幾十年里,制備電泳技術(shù)和制備型電泳裝置取得了很大的進(jìn)展,其制備量也從毫克級的蛋白質(zhì)逐漸減少到微克級的蛋白質(zhì),可滿足蛋白質(zhì)組學(xué)研究需求�，F(xiàn)已商品化的制備型電泳裝置分為毫克級制備型電泳裝置和微克級制備型電泳裝置。毫克級制備型電泳裝置多為早期的裝置,其不足是樣品分離和收集的耗時很長(通常在10 h以上),回收率低,收集的過程中需不斷補(bǔ)充洗脫液,實驗過程較為繁瑣,且只能用于毫克級以上的蛋白質(zhì)樣品的制備;近年來,微克級制備型電泳裝置的發(fā)展較為迅速,但也存在諸如實驗過程繁瑣、樣品分離度不夠高等問題。為此,我們設(shè)計制作了一套微量級制備型凝膠電泳裝置,用于實驗室的微量級快速、簡便的蛋白質(zhì)分離與制備,其制備過程為:首先將微克級的蛋白質(zhì)按照分子量大小進(jìn)行分離,然后轉(zhuǎn)移凝膠至洗脫收集裝置中對蛋白質(zhì)進(jìn)行洗脫收集。對此套制備型凝膠電泳裝置的性能測試結(jié)果表明,該裝置可以簡便、高效、高回收率地完成蛋白質(zhì)樣品的分離制備。本論文由三章組成。第一章介紹了現(xiàn)有的分離分析技術(shù)在蛋白質(zhì)組學(xué)中的發(fā)展與現(xiàn)狀,尤其是對制備型電泳方法與技術(shù)做了詳細(xì)的概述。同時,第一章還包括本研究的內(nèi)容、所要解決的具體問題以及研究意義。在第二章,介紹了我們設(shè)計并制作的一套制備型凝膠電泳與紫外檢測聯(lián)用裝置。該裝置通過將改進(jìn)的平板凝膠電泳裝置與外部氣體壓縮泵、高壓儲液罐、紫外檢測器、電腦以及餾分收集器相連,實現(xiàn)了在線完成蛋白樣品組分的分離、檢測與回收的目標(biāo)。在對裝置的性能測試與評價實驗中,采用馬心肌紅蛋白和牛血清白蛋白作為標(biāo)準(zhǔn)蛋白混合物,考察了該裝置分離性能,紫外吸收光譜結(jié)果表明成功分離了這兩個蛋白。盡管如此,在實驗中我們發(fā)現(xiàn)該裝置存在有許多問題:整個實驗過程耗時過長,裝置漏液現(xiàn)象嚴(yán)重,正極處產(chǎn)生氣泡現(xiàn)象對檢測和收集產(chǎn)生影響,而且裝置在加工中成型慢,加工周期長,這些都是我們進(jìn)一步改進(jìn)備型凝膠電泳設(shè)計時需要解決的問題。在第三章,介紹了在制備型凝膠電泳與紫外檢測聯(lián)用裝置基礎(chǔ)上的一種新型制備型凝膠電泳裝置。為了解決制備型凝膠電泳與紫外檢測聯(lián)用裝置存在的諸多問題,我們將裝置由在線檢測改為了離線檢測,并基于3D打印技術(shù)和數(shù)字控制加工技術(shù),設(shè)計了制作簡單,成型快,周期短,精度高的制備型凝膠電泳分離-洗脫聯(lián)用裝置。按照蛋白質(zhì)分離與洗脫方向的不同,可分為橫向洗脫和垂直洗脫制備型凝膠電泳分離-洗脫聯(lián)用兩種裝置。通過對設(shè)計制作的制備型凝膠電泳分離-洗脫聯(lián)用裝置的機(jī)械強(qiáng)度、防漏液與操作性能測試,對符合要求的裝置進(jìn)一步進(jìn)行了蛋白樣本分離、洗脫回收、分離度、回收率、洗脫時間以及洗脫樣本的液相色譜-質(zhì)譜分析等測試。在優(yōu)化設(shè)計和性能測試的基礎(chǔ)上,確定定型的制備型凝膠電泳裝置為垂直洗脫的制備型凝膠電泳分離-洗脫聯(lián)用裝置。蛋白混合物分離實驗結(jié)果表明制備型凝膠電泳分離裝置不僅能夠?qū)Φ鞍踪|(zhì)樣本進(jìn)行高效分離,而且具有高的重復(fù)性;在采用制備型凝膠電泳洗脫裝置進(jìn)行的洗脫實驗中,由于蛋白樣本垂直于凝膠厚度方向洗脫,因此,在最佳洗脫條件下,洗脫時間少于20分鐘,碳酸酐酶的回收率高達(dá)91.8%,實現(xiàn)了快速、高回收率的洗脫,優(yōu)于同類型的商品化的制備型凝膠電泳裝置。將垂直洗脫的制備型凝膠電泳分離-洗脫聯(lián)用裝置用于標(biāo)準(zhǔn)蛋白混合物以及酵母全蛋白的分離與制備,其分離度和分離效率均優(yōu)于商品化的制備型凝膠電泳裝置。將該裝置作為預(yù)分離手段,與液相色譜-質(zhì)譜聯(lián)用,對10μg的牛血清白蛋白進(jìn)行分離、洗脫后進(jìn)行FASP(filter aided proteome preparation)酶解與經(jīng)過該分離裝置分離后進(jìn)行膠內(nèi)酶解做了對比,結(jié)果表明,經(jīng)該裝置分離、洗脫和FASP酶解的方法最多鑒定到94個特異性肽段,平均蛋白質(zhì)序列覆蓋率為90.12%;經(jīng)該分離裝置分離后進(jìn)行膠內(nèi)酶解,最多鑒定到70個特異性肽段,平均蛋白質(zhì)序列覆蓋率為86%。同樣地,我們對20μg的酵母蛋白提取物也做了上述對比實驗,結(jié)果表明,相同條件下使用該裝置分離、洗脫和FASP酶解的方法鑒定到的肽段數(shù)目、序列覆蓋率大于50%的蛋白數(shù)目以及打分值大于1000的蛋白數(shù)目均遠(yuǎn)遠(yuǎn)多于利用該裝置分離后進(jìn)行膠內(nèi)酶解所鑒定到的相應(yīng)的肽段數(shù)目和蛋白數(shù)目。以上實驗說明使用該裝置進(jìn)行分離、洗脫后進(jìn)行FASP酶解的方法更有利于蛋白質(zhì)樣品的鑒定,可有效解決凝膠電泳分離后膠上酶切時蛋白質(zhì)提取效率低的問題。我們在垂直洗脫的制備型凝膠電泳分離-洗脫聯(lián)用裝置的基礎(chǔ)上,加入了半導(dǎo)體制冷片等部件,完成了一整套制備型凝膠電泳系統(tǒng)的制作。該系統(tǒng)可作為蛋白質(zhì)樣品預(yù)分離手段,大大降低蛋白質(zhì)樣本的復(fù)雜度,也可幫助去除高豐度蛋白,發(fā)現(xiàn)低豐度蛋白。另外,該裝置也有助于“自上而下”(“Top-down”)的蛋白質(zhì)組學(xué)研究策略的應(yīng)用。
[Abstract]:Efficient separation of protein mixtures is of great significance for qualitative and quantitative analysis and application of proteins and proteomes, but the separation and preparation of protein mixtures are facing great challenges due to the variety and dynamic range of proteins. Electrophoresis technology, compared with high performance liquid chromatography, has the advantages of high separation, isoelectric point and relative molecular mass information. Its disadvantages are time-consuming, laborious, low automation and difficult to be used in protein preparation. Preparatory electrophoresis technology is to separate, purify and collect the target substances in preparation electrophoresis equipment. Preparatory electrophoresis and preparative electrophoresis devices have made great progress in the past few decades. The amount of preparation has been gradually reduced from milligram to microgram, which can meet the needs of proteomics research. Most of the preparative electrophoresis devices at the milligram level are early ones. The shortcomings are that the separation and collection of samples take a long time (usually more than 10 hours), the recovery rate is low, and the elution needs to be continuously supplemented during the collection process. The experimental process is complicated and can only be used for the preparation of protein samples above the milligram level. In recent years, the development of microgram level preparative electrophoresis device has developed rapidly. But there are also problems such as tedious process and insufficient sample separation. Therefore, we designed and produced a set of microscale preparative gel electrophoresis device for rapid and simple protein separation and preparation in the laboratory. The microgram level proteins were separated according to their molecular weight, and then transferred to the elution collection device for elution and collection. The performance test results of this set of gel electrophoresis device showed that the device could be simple, efficient and high recovery to complete the separation and preparation of protein samples. This paper consists of three chapters. The first chapter introduces the development and current situation of separation and analysis techniques in proteomics, especially the methods and techniques of preparative electrophoresis. At the same time, the first chapter also includes the contents of this study, the specific problems to be solved and the significance of the study. Preparative gel electrophoresis and UV detection device. The device achieves the goal of separation, detection and recovery of protein sample components by completing the improved flat gel electrophoresis device with external gas compression pump, high pressure liquid storage tank, UV detector, computer and fraction collector. In the experiment, horse myoglobin and bovine serum albumin were used as standard protein mixture, and the separation performance of the device was investigated. The results of UV absorption spectrum showed that the two proteins were successfully separated. The phenomenon of bubble formation at the positive pole has an impact on the detection and collection, and the device is slow in forming and long processing cycle. These are the problems to be solved when we further improve the design of preparative gel electrophoresis. In the third chapter, a new type of preparative coagulant based on the combination of preparative gel electrophoresis and ultraviolet detection is introduced. In order to solve the problems existing in the combination of preparative gel electrophoresis and ultraviolet detection, we have changed the device from on-line detection to off-line detection. Based on 3D printing technology and digital control processing technology, we have designed a simple gel electrophoresis separation elution combination which is simple in production, fast in formation, short in cycle and high in precision. According to the difference of protein separation and elution direction, it can be divided into two devices: lateral elution and vertical elution, preparative gel electrophoresis separation elution combination. Through the design of the mechanical strength, leak proof liquid and operation performance of the preparative gel electrophoresis separation and elution device, the device that meets the requirements is further carried out. Separation, elution and recovery, separation, recovery, elution time, and liquid chromatography mass spectrometry analysis of elution samples were carried out. Based on the optimization design and performance test, the established gel electrophoresis device was established as a vertical elution gel electrophoresis separation elution unit. The results show that the preparative gel electrophoresis separation device can not only efficiently separate protein samples, but also has high repeatability. In the elution experiment using the preparative gel electrophoresis elution device, the elution time is less than 20 minutes under the best elution conditions, because the protein sample is eluted vertically in the direction of the gel thickness. The recovery rate of carbonic anhydrase was as high as 91.8%, and the elution of fast and high recovery rate was faster than that of the same type of commercialized gel electrophoresis device. The vertical elution gel electrophoresis separation elution combination device was used for the separation and preparation of standard protein mixture and yeast whole protein, and its separation and separation efficiency were excellent. In a commercialized preparative gel electrophoresis device, the device was used as a pre separation method, and 10 mu g of bovine serum albumin was separated by liquid chromatography-mass spectrometry. After elution, the enzymatic hydrolysis of FASP (filter aided proteome preparation) was carried out and compared with that obtained by the separation device. Separation, elution and FASP enzymatic hydrolysis methods identified up to 94 specific peptides with an average protein sequence coverage of 90.12%. After isolation, 70 specific peptides were identified with an average protein sequence coverage of 86%. Similarly, we compared the above results with the 20 mg yeast protein extract. The results showed that under the same conditions, the number of peptides, the number of proteins with a sequence coverage of more than 50% and the number of proteins with a score of more than 1000 were much more than the number of peptides and the number of proteins identified by in-gel enzymatic hydrolysis. The above experiments show that the method of FASP hydrolysis is more conducive to the identification of protein samples, and can effectively solve the problem of low protein extraction efficiency when gelatin electrophoresis is used for enzyme digestion. A complete set of preparative gel electrophoresis system has been completed. The system can be used as a pre separation method for protein samples, greatly reducing the complexity of protein samples, and also helping to remove high abundance protein and finding low abundance protein. In addition, the device also helps to "top-down" ("Top-down") protein. The application of omics research strategy.
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：Q503
本文編號：2247747
[Abstract]:Efficient separation of protein mixtures is of great significance for qualitative and quantitative analysis and application of proteins and proteomes, but the separation and preparation of protein mixtures are facing great challenges due to the variety and dynamic range of proteins. Electrophoresis technology, compared with high performance liquid chromatography, has the advantages of high separation, isoelectric point and relative molecular mass information. Its disadvantages are time-consuming, laborious, low automation and difficult to be used in protein preparation. Preparatory electrophoresis technology is to separate, purify and collect the target substances in preparation electrophoresis equipment. Preparatory electrophoresis and preparative electrophoresis devices have made great progress in the past few decades. The amount of preparation has been gradually reduced from milligram to microgram, which can meet the needs of proteomics research. Most of the preparative electrophoresis devices at the milligram level are early ones. The shortcomings are that the separation and collection of samples take a long time (usually more than 10 hours), the recovery rate is low, and the elution needs to be continuously supplemented during the collection process. The experimental process is complicated and can only be used for the preparation of protein samples above the milligram level. In recent years, the development of microgram level preparative electrophoresis device has developed rapidly. But there are also problems such as tedious process and insufficient sample separation. Therefore, we designed and produced a set of microscale preparative gel electrophoresis device for rapid and simple protein separation and preparation in the laboratory. The microgram level proteins were separated according to their molecular weight, and then transferred to the elution collection device for elution and collection. The performance test results of this set of gel electrophoresis device showed that the device could be simple, efficient and high recovery to complete the separation and preparation of protein samples. This paper consists of three chapters. The first chapter introduces the development and current situation of separation and analysis techniques in proteomics, especially the methods and techniques of preparative electrophoresis. At the same time, the first chapter also includes the contents of this study, the specific problems to be solved and the significance of the study. Preparative gel electrophoresis and UV detection device. The device achieves the goal of separation, detection and recovery of protein sample components by completing the improved flat gel electrophoresis device with external gas compression pump, high pressure liquid storage tank, UV detector, computer and fraction collector. In the experiment, horse myoglobin and bovine serum albumin were used as standard protein mixture, and the separation performance of the device was investigated. The results of UV absorption spectrum showed that the two proteins were successfully separated. The phenomenon of bubble formation at the positive pole has an impact on the detection and collection, and the device is slow in forming and long processing cycle. These are the problems to be solved when we further improve the design of preparative gel electrophoresis. In the third chapter, a new type of preparative coagulant based on the combination of preparative gel electrophoresis and ultraviolet detection is introduced. In order to solve the problems existing in the combination of preparative gel electrophoresis and ultraviolet detection, we have changed the device from on-line detection to off-line detection. Based on 3D printing technology and digital control processing technology, we have designed a simple gel electrophoresis separation elution combination which is simple in production, fast in formation, short in cycle and high in precision. According to the difference of protein separation and elution direction, it can be divided into two devices: lateral elution and vertical elution, preparative gel electrophoresis separation elution combination. Through the design of the mechanical strength, leak proof liquid and operation performance of the preparative gel electrophoresis separation and elution device, the device that meets the requirements is further carried out. Separation, elution and recovery, separation, recovery, elution time, and liquid chromatography mass spectrometry analysis of elution samples were carried out. Based on the optimization design and performance test, the established gel electrophoresis device was established as a vertical elution gel electrophoresis separation elution unit. The results show that the preparative gel electrophoresis separation device can not only efficiently separate protein samples, but also has high repeatability. In the elution experiment using the preparative gel electrophoresis elution device, the elution time is less than 20 minutes under the best elution conditions, because the protein sample is eluted vertically in the direction of the gel thickness. The recovery rate of carbonic anhydrase was as high as 91.8%, and the elution of fast and high recovery rate was faster than that of the same type of commercialized gel electrophoresis device. The vertical elution gel electrophoresis separation elution combination device was used for the separation and preparation of standard protein mixture and yeast whole protein, and its separation and separation efficiency were excellent. In a commercialized preparative gel electrophoresis device, the device was used as a pre separation method, and 10 mu g of bovine serum albumin was separated by liquid chromatography-mass spectrometry. After elution, the enzymatic hydrolysis of FASP (filter aided proteome preparation) was carried out and compared with that obtained by the separation device. Separation, elution and FASP enzymatic hydrolysis methods identified up to 94 specific peptides with an average protein sequence coverage of 90.12%. After isolation, 70 specific peptides were identified with an average protein sequence coverage of 86%. Similarly, we compared the above results with the 20 mg yeast protein extract. The results showed that under the same conditions, the number of peptides, the number of proteins with a sequence coverage of more than 50% and the number of proteins with a score of more than 1000 were much more than the number of peptides and the number of proteins identified by in-gel enzymatic hydrolysis. The above experiments show that the method of FASP hydrolysis is more conducive to the identification of protein samples, and can effectively solve the problem of low protein extraction efficiency when gelatin electrophoresis is used for enzyme digestion. A complete set of preparative gel electrophoresis system has been completed. The system can be used as a pre separation method for protein samples, greatly reducing the complexity of protein samples, and also helping to remove high abundance protein and finding low abundance protein. In addition, the device also helps to "top-down" ("Top-down") protein. The application of omics research strategy.
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：Q503
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