一种微量制备型凝胶电泳装置的研制
发布时间：2018-09-18 11:20
【摘要】：蛋白质混合物的高效分离对蛋白质以及蛋白质组的定性和定量分析及应用具有重要意义,但由于蛋白质种类多,动态范围宽,其分离和制备面临巨大挑战。目前,蛋白质组学中常用的蛋白质混合物高效分离技术主要包括高效液相色谱和电泳技术。电泳技术相较于高效液相色谱,其优势是分离度高,并且可以得到等电点、相对分子质量信息,其劣势是费时费力,自动化程度低,且难以用于蛋白质的制备。制备电泳技术是在制备电泳装置中对目标物质进行分离、纯化和收集制备的一种技术。在过去的几十年里,制备电泳技术和制备型电泳装置取得了很大的进展,其制备量也从毫克级的蛋白质逐渐减少到微克级的蛋白质,可满足蛋白质组学研究需求。现已商品化的制备型电泳装置分为毫克级制备型电泳装置和微克级制备型电泳装置。毫克级制备型电泳装置多为早期的装置,其不足是样品分离和收集的耗时很长(通常在10 h以上),回收率低,收集的过程中需不断补充洗脱液,实验过程较为繁琐,且只能用于毫克级以上的蛋白质样品的制备;近年来,微克级制备型电泳装置的发展较为迅速,但也存在诸如实验过程繁琐、样品分离度不够高等问题。为此,我们设计制作了一套微量级制备型凝胶电泳装置,用于实验室的微量级快速、简便的蛋白质分离与制备,其制备过程为:首先将微克级的蛋白质按照分子量大小进行分离,然后转移凝胶至洗脱收集装置中对蛋白质进行洗脱收集。对此套制备型凝胶电泳装置的性能测试结果表明,该装置可以简便、高效、高回收率地完成蛋白质样品的分离制备。本论文由三章组成。第一章介绍了现有的分离分析技术在蛋白质组学中的发展与现状,尤其是对制备型电泳方法与技术做了详细的概述。同时,第一章还包括本研究的内容、所要解决的具体问题以及研究意义。在第二章,介绍了我们设计并制作的一套制备型凝胶电泳与紫外检测联用装置。该装置通过将改进的平板凝胶电泳装置与外部气体压缩泵、高压储液罐、紫外检测器、电脑以及馏分收集器相连,实现了在线完成蛋白样品组分的分离、检测与回收的目标。在对装置的性能测试与评价实验中,采用马心肌红蛋白和牛血清白蛋白作为标准蛋白混合物,考察了该装置分离性能,紫外吸收光谱结果表明成功分离了这两个蛋白。尽管如此,在实验中我们发现该装置存在有许多问题:整个实验过程耗时过长,装置漏液现象严重,正极处产生气泡现象对检测和收集产生影响,而且装置在加工中成型慢,加工周期长,这些都是我们进一步改进备型凝胶电泳设计时需要解决的问题。在第三章,介绍了在制备型凝胶电泳与紫外检测联用装置基础上的一种新型制备型凝胶电泳装置。为了解决制备型凝胶电泳与紫外检测联用装置存在的诸多问题,我们将装置由在线检测改为了离线检测,并基于3D打印技术和数字控制加工技术,设计了制作简单,成型快,周期短,精度高的制备型凝胶电泳分离-洗脱联用装置。按照蛋白质分离与洗脱方向的不同,可分为横向洗脱和垂直洗脱制备型凝胶电泳分离-洗脱联用两种装置。通过对设计制作的制备型凝胶电泳分离-洗脱联用装置的机械强度、防漏液与操作性能测试,对符合要求的装置进一步进行了蛋白样本分离、洗脱回收、分离度、回收率、洗脱时间以及洗脱样本的液相色谱-质谱分析等测试。在优化设计和性能测试的基础上,确定定型的制备型凝胶电泳装置为垂直洗脱的制备型凝胶电泳分离-洗脱联用装置。蛋白混合物分离实验结果表明制备型凝胶电泳分离装置不仅能够对蛋白质样本进行高效分离,而且具有高的重复性;在采用制备型凝胶电泳洗脱装置进行的洗脱实验中,由于蛋白样本垂直于凝胶厚度方向洗脱,因此,在最佳洗脱条件下,洗脱时间少于20分钟,碳酸酐酶的回收率高达91.8%,实现了快速、高回收率的洗脱,优于同类型的商品化的制备型凝胶电泳装置。将垂直洗脱的制备型凝胶电泳分离-洗脱联用装置用于标准蛋白混合物以及酵母全蛋白的分离与制备,其分离度和分离效率均优于商品化的制备型凝胶电泳装置。将该装置作为预分离手段,与液相色谱-质谱联用,对10μg的牛血清白蛋白进行分离、洗脱后进行FASP(filter aided proteome preparation)酶解与经过该分离装置分离后进行胶内酶解做了对比,结果表明,经该装置分离、洗脱和FASP酶解的方法最多鉴定到94个特异性肽段,平均蛋白质序列覆盖率为90.12%;经该分离装置分离后进行胶内酶解,最多鉴定到70个特异性肽段,平均蛋白质序列覆盖率为86%。同样地,我们对20μg的酵母蛋白提取物也做了上述对比实验,结果表明,相同条件下使用该装置分离、洗脱和FASP酶解的方法鉴定到的肽段数目、序列覆盖率大于50%的蛋白数目以及打分值大于1000的蛋白数目均远远多于利用该装置分离后进行胶内酶解所鉴定到的相应的肽段数目和蛋白数目。以上实验说明使用该装置进行分离、洗脱后进行FASP酶解的方法更有利于蛋白质样品的鉴定,可有效解决凝胶电泳分离后胶上酶切时蛋白质提取效率低的问题。我们在垂直洗脱的制备型凝胶电泳分离-洗脱联用装置的基础上,加入了半导体制冷片等部件,完成了一整套制备型凝胶电泳系统的制作。该系统可作为蛋白质样品预分离手段,大大降低蛋白质样本的复杂度,也可帮助去除高丰度蛋白,发现低丰度蛋白。另外,该装置也有助于“自上而下”(“Top-down”)的蛋白质组学研究策略的应用。
[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.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q503
本文编号：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.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q503
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