亲和组合分组系统的研究及在蛋白组学中应用

发布时间：2018-03-06 11:07

  本文选题：亲和组合分组　切入点：串联亲和　出处：《上海交通大学》2009年博士论文　论文类型：学位论文

【摘要】：细胞中含有成千上万种不同分子量、相对丰度、酸碱性和疏水性分布范围很宽的各种结构或功能蛋白，用现有仪器手段分析如此复杂的生物样品，就需要一个有效的方法分离、简化复杂的组织蛋白样品，提高质谱对蛋白的检出率。本论文开发了一套基于亲和层析的组合分组系统对组织样品进行简化分组，分组后分别进行trypsin蛋白酶消化和LC-MS/MS分析，分组后通过质谱分析能够鉴定出更多的蛋白信息。本论文开发出串联亲和与级联亲和两种亲和组合分组系统，并应用于大鼠肝细胞溶质和小鼠睾丸蛋白组学研究中。 将不同的氨基酸、小肽或氨基化合物连接到Sepharose固相基质上构建了本实验室小分子化学配体库，这些亲和配体的结构差异导致了不同的蛋白-配体非键相互作用，从而能够特异亲和吸附不同的蛋白组。分析不同亲和配体的蛋白结合图谱，找出蛋白条带分布差异明显、吸附性能适中的亲和配体，如果这些差异亲和配体的吸附蛋白图谱的条带叠加能够覆盖原始样品电泳图谱的全蛋白谱，就可以作为亲和组合分组系统的备选配体。本论文筛选到的主要差异亲和配体为：A1-4、A6、A7-56、A8-54、A11-70、A15、A17-56、A25-35、A29-32和A84。 大鼠肝细胞溶质组织匀浆蛋白（Fraction0）通过A7-56、A84、A11-70、A6、A29-32五柱串联亲和组合系统分配后得到5个吸附组分（Fraction1~Fraction5）和1个流穿组分（Fraction6）。通过LC-MS/MS分析，Fraction0中鉴定出371个非冗余的可信蛋白组（1450个特异多肽），而6个串联亲和组分Fraction1~Fraction6中分别鉴定出289、123、152、279、154和60个非冗余的可信蛋白组。统计合并剔除重复部分后，6个串联亲和分配亚组分中总共鉴定得到665个非冗余的可信蛋白组（2413个特异多肽），是串联亲和分组前蛋白鉴定数目的1.8倍。665个大鼠肝鉴定蛋白中，430个蛋白只出现在特异的某一组分中而不与其他组分重叠，比例高达64.7%，，这充分展示了串联亲和分组系统中各亲和配体蛋白吸附特性的差异，通过这些差异配体的串联组合实现了复杂样品的简化分组。对鉴定蛋白的生物信息分析发现，串联亲和分组系统对各种极端特性蛋白都有较好的敏感性，665个鉴定蛋白中有61个极端尺寸蛋白(MW10kD, or100kD)，14个极端等电点蛋白(pI4.3, or10)，55个疏水蛋白（GRAVY正值）和41个膜蛋白。 大鼠肝细胞溶质组分（F0）通过A15~A8-54~A11-70三级联亲和分组系统：第一级分配得到F1-1和F1-2两个组分，第二级得到F2-1~F2-4四个组分，第三级得到F3-1~F3-8八个组分。LC-MS/MS分析，F0中鉴定得到391个非冗余的可信蛋白组，F1-1和F1-2中鉴定出499个蛋白组（提高27.6%），F2-1~F2-4中鉴定出616个蛋白组（相比第一级提高了23.4%），F3-1~F3-8中鉴定出738个蛋白组（相比第二级提高了19.8%）。大鼠肝细胞溶质通过三级联亲和分组系统总共鉴定出859个非冗余的可信蛋白组，是未分组前鉴定蛋白数目的2.2倍。鉴定的859个大鼠肝蛋白中，有75个分子量20kDa的蛋白，73个分子量100kDa蛋白，72个疏水蛋白和49个膜蛋白。 以成熟的A15~A8-54~A11-70三级联亲和分组系统对小鼠睾丸蛋白组学进行了研究，级联亲和分组后的8个组分中共鉴定得到1378个非冗余的可信蛋白组，是分组前鉴定蛋白数目的2.6倍，是文献报道的2-DE-MS方法鉴定的最大小鼠睾丸蛋白数目的2.7倍。1378个鉴定蛋白中，尺寸最小的是只有817Da的小肽，最大的高达630.35kDa，12个蛋白分子量10kDa，169个蛋白分子量100kDa，38个蛋白pI4.5，51个蛋白pI10，有93个疏水蛋白和81个膜蛋白，这都超出了现有小鼠睾丸蛋白组学研究中获得极端特性蛋白的极限。我们还对所有鉴定蛋白的亚细胞器定位和蛋白功能定义进行了归类分析，其中有310个蛋白（22.5%）亚细胞器定位信息未知，221个蛋白在Uniprot数据库没有GO分子功能定义，分析找出了16个与生精及精子发育相关的蛋白。 我们的研究结果表明：串联和级联两种亲和组合系统都能够快速实现对复杂组织样品的预分组，分组后大大提高了质谱分析的蛋白检出率。亲和组合分组系统相比2-DE和2D-LC分配方法具备无可比拟的优越性：简单快速（可以节省至少2/3时间），亲和配体容易制备、稳定性好（耐酸、碱和高压处理），不需要特别高昂的设备，对不同组织样品可以采用标准化统一的分组系统，非常适于大规模蛋白组学研究。
[Abstract]:With tens of thousands of different molecular weight, the relative abundance of various cells, protein structure or function of pH and hydrophobicity distribution of a wide range of biological samples, analysis of such a complex with existing instruments, it needs an effective separation method, simplifying the complex tissue protein samples, improve the detection rate of protein mass spectrometry. This paper presents a packet system based on a combination of affinity chromatography of tissue samples to simplify the analysis of trypsin packet, LC-MS/MS packet and protease digestion respectively, after grouping by mass spectrometry to identify more protein information. This paper developed two kinds of cascade tandem affinity and affinity affinity combination group system, and applied research to solute and mice liver cells of rats testis proteome.
The different amino acids, peptides or amino compounds connected to Sepharose solid phase matrix constructed in this laboratory small molecule ligand library, these affinity ligand structural differences lead to different protein ligand noncovalent interaction, thereby protein group specific affinity adsorption different. Analysis of different affinity ligand protein binding map, find out protein band distribution, affinity ligand adsorption medium, if the full spectrum of protein adsorption protein profiles of these differences in affinity ligand bands superimposed to cover the original sample electrophoresis, it can be used as alternative ligand affinity system. The main differences between the groups in the screening of the affinity ligand: A1-4, A6. A7-56, A8-54, A11-70, A15, A17-56, A25-35, A29-32 and A84.
The homogenate of rat liver cells (Fraction0) by tissue solute A7-56, A84, A11-70, A6, A29-32 five column tandem affinity combination system distribution obtained after the adsorption of 5 components (Fraction1~Fraction5) and 1 (Fraction6) flow through the components. By LC-MS/MS analysis, Fraction0 identified 371 non redundant trusted protein group (1450 and 6 specific peptides), tandem affinity fractions of Fraction1~Fraction6 were identified in 289123152279154 protein group trusted and 60 non redundant. Statistics with excluding repeat part, 6 tandem affinity subfraction distribution total identified 665 non redundant protein group (2413 specific and credible is the number of tandem affinity peptides), protein identification before grouping 1.8 times.665 rat liver protein, 430 protein only appeared in a group of specific and not overlap with other components, the proportion is as high as 64.7%, which fully demonstrated the string Linkingaffinity differences in affinity adsorption characteristics of ligand protein packet system, through a series combination of these differences can simplify the complex ligand grouping samples. Bioinformatics analysis of the identified protein found in tandem affinity grouping system has good sensitivity to the extreme properties of protein, there are 61 extreme size protein identification of 665 proteins (MW10kD, or100kD), the 14 extremes of the isoelectric point of protein (pI4.3, or10), 55 hydrophobic protein (GRAVY positive) and 41 membrane proteins.
The big rat liver cytosolic fractions (F0) affinity group system through A15~A8-54~A11-70 three cascade: the first is to get the F1-1 and F1-2 distribution of two components, second grade F2-1~F2-4 and four components, third F3-1~F3-8 eight components,.LC-MS/MS analysis, F0 identified 391 non redundant protein trusted group, F1-1 2 and F1- identified 499 proteins (27.6% increase), F2-1~F2-4 identified 616 protein group (compared to the first level increased by 23.4%), F3-1~F3-8 identified 738 protein group (compared to second level increased by 19.8%). Rat liver cytosol through three cascaded affinity grouping system we identified 859 a non redundant protein trusted group, is 2.2 times the number of packets before the identification of proteins. The identification of 859 rat liver protein, 75 20kDa protein, 73 100kDa proteins, 72 proteins and 49 hydrophobic membrane proteins.
With the mature A15~A8-54~A11-70 three cascade system on mouse testis protein affinity group group was studied, the 8 groups after the cascade affinity of the identified 1378 non redundant protein trusted group, is 2.6 times the number of packets before the identification of proteins, is 2.7 times the number of 2-DE-MS.1378 protein identification methods reported in the literature the largest mouse testis protein, the smallest size is only 817Da small peptide, the largest up to 630.35kDa, 12 proteins with molecular weight of 10kDa, 169 protein molecular weight 100kDa, 38 protein pI4.5,51 protein pI10, 93 protein and 81 hydrophobic membrane proteins, which are beyond the existing mouse testis protein group to obtain the extreme characteristics of protein research. We also define the subcellular localization of all identified proteins and protein functions are classified and analyzed, of which 310 (22.5%) protein sub cellular localization. Unknowns, 221 proteins have no GO function definition in the Uniprot database, and found 16 proteins related to spermatogenesis and sperm development.
Our results show that two kinds of affinity and tandem cascade combination system can quickly realize the pre grouping of complex tissue samples, after grouping has improved the detection rate of mass spectrometry analysis of the protein. The compatible combination packet system compared to 2-DE and 2D-LC allocation method have advantages: simple and fast (There is nothing comparable to this can save at least 2/3 time). Affinity ligands, easy preparation, good stability (acid, alkali and high pressure treatment), do not need special expensive equipment, packet system on different tissue samples can adopt a unified standard, is very suitable for large-scale proteomics research.

【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R346


本文编号：1574576