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  本文選題：親和組合分組　切入點：串聯(lián)親和　出處：《上海交通大學(xué)》2009年博士論文　論文類型：學(xué)位論文

【摘要】：細(xì)胞中含有成千上萬種不同分子量、相對豐度、酸堿性和疏水性分布范圍很寬的各種結(jié)構(gòu)或功能蛋白，用現(xiàn)有儀器手段分析如此復(fù)雜的生物樣品，就需要一個有效的方法分離、簡化復(fù)雜的組織蛋白樣品，提高質(zhì)譜對蛋白的檢出率。本論文開發(fā)了一套基于親和層析的組合分組系統(tǒng)對組織樣品進(jìn)行簡化分組，分組后分別進(jìn)行trypsin蛋白酶消化和LC-MS/MS分析，分組后通過質(zhì)譜分析能夠鑒定出更多的蛋白信息。本論文開發(fā)出串聯(lián)親和與級聯(lián)親和兩種親和組合分組系統(tǒng)，并應(yīng)用于大鼠肝細(xì)胞溶質(zhì)和小鼠睪丸蛋白組學(xué)研究中。 將不同的氨基酸、小肽或氨基化合物連接到Sepharose固相基質(zhì)上構(gòu)建了本實驗室小分子化學(xué)配體庫，這些親和配體的結(jié)構(gòu)差異導(dǎo)致了不同的蛋白-配體非鍵相互作用，從而能夠特異親和吸附不同的蛋白組。分析不同親和配體的蛋白結(jié)合圖譜，找出蛋白條帶分布差異明顯、吸附性能適中的親和配體，如果這些差異親和配體的吸附蛋白圖譜的條帶疊加能夠覆蓋原始樣品電泳圖譜的全蛋白譜，就可以作為親和組合分組系統(tǒng)的備選配體。本論文篩選到的主要差異親和配體為：A1-4、A6、A7-56、A8-54、A11-70、A15、A17-56、A25-35、A29-32和A84。 大鼠肝細(xì)胞溶質(zhì)組織勻漿蛋白（Fraction0）通過A7-56、A84、A11-70、A6、A29-32五柱串聯(lián)親和組合系統(tǒng)分配后得到5個吸附組分（Fraction1~Fraction5）和1個流穿組分（Fraction6）。通過LC-MS/MS分析，F(xiàn)raction0中鑒定出371個非冗余的可信蛋白組（1450個特異多肽），而6個串聯(lián)親和組分Fraction1~Fraction6中分別鑒定出289、123、152、279、154和60個非冗余的可信蛋白組。統(tǒng)計合并剔除重復(fù)部分后，6個串聯(lián)親和分配亞組分中總共鑒定得到665個非冗余的可信蛋白組（2413個特異多肽），是串聯(lián)親和分組前蛋白鑒定數(shù)目的1.8倍。665個大鼠肝鑒定蛋白中，430個蛋白只出現(xiàn)在特異的某一組分中而不與其他組分重疊，比例高達(dá)64.7%，，這充分展示了串聯(lián)親和分組系統(tǒng)中各親和配體蛋白吸附特性的差異，通過這些差異配體的串聯(lián)組合實現(xiàn)了復(fù)雜樣品的簡化分組。對鑒定蛋白的生物信息分析發(fā)現(xiàn)，串聯(lián)親和分組系統(tǒng)對各種極端特性蛋白都有較好的敏感性，665個鑒定蛋白中有61個極端尺寸蛋白(MW10kD, or100kD)，14個極端等電點蛋白(pI4.3, or10)，55個疏水蛋白（GRAVY正值）和41個膜蛋白。 大鼠肝細(xì)胞溶質(zhì)組分（F0）通過A15~A8-54~A11-70三級聯(lián)親和分組系統(tǒng)：第一級分配得到F1-1和F1-2兩個組分，第二級得到F2-1~F2-4四個組分，第三級得到F3-1~F3-8八個組分。LC-MS/MS分析，F(xiàn)0中鑒定得到391個非冗余的可信蛋白組，F(xiàn)1-1和F1-2中鑒定出499個蛋白組（提高27.6%），F(xiàn)2-1~F2-4中鑒定出616個蛋白組（相比第一級提高了23.4%），F(xiàn)3-1~F3-8中鑒定出738個蛋白組（相比第二級提高了19.8%）。大鼠肝細(xì)胞溶質(zhì)通過三級聯(lián)親和分組系統(tǒng)總共鑒定出859個非冗余的可信蛋白組，是未分組前鑒定蛋白數(shù)目的2.2倍。鑒定的859個大鼠肝蛋白中，有75個分子量20kDa的蛋白，73個分子量100kDa蛋白，72個疏水蛋白和49個膜蛋白。 以成熟的A15~A8-54~A11-70三級聯(lián)親和分組系統(tǒng)對小鼠睪丸蛋白組學(xué)進(jìn)行了研究，級聯(lián)親和分組后的8個組分中共鑒定得到1378個非冗余的可信蛋白組，是分組前鑒定蛋白數(shù)目的2.6倍，是文獻(xiàn)報道的2-DE-MS方法鑒定的最大小鼠睪丸蛋白數(shù)目的2.7倍。1378個鑒定蛋白中，尺寸最小的是只有817Da的小肽，最大的高達(dá)630.35kDa，12個蛋白分子量10kDa，169個蛋白分子量100kDa，38個蛋白pI4.5，51個蛋白pI10，有93個疏水蛋白和81個膜蛋白，這都超出了現(xiàn)有小鼠睪丸蛋白組學(xué)研究中獲得極端特性蛋白的極限。我們還對所有鑒定蛋白的亞細(xì)胞器定位和蛋白功能定義進(jìn)行了歸類分析，其中有310個蛋白（22.5%）亞細(xì)胞器定位信息未知，221個蛋白在Uniprot數(shù)據(jù)庫沒有GO分子功能定義，分析找出了16個與生精及精子發(fā)育相關(guān)的蛋白。 我們的研究結(jié)果表明：串聯(lián)和級聯(lián)兩種親和組合系統(tǒng)都能夠快速實現(xiàn)對復(fù)雜組織樣品的預(yù)分組，分組后大大提高了質(zhì)譜分析的蛋白檢出率。親和組合分組系統(tǒng)相比2-DE和2D-LC分配方法具備無可比擬的優(yōu)越性：簡單快速（可以節(jié)省至少2/3時間），親和配體容易制備、穩(wěn)定性好（耐酸、堿和高壓處理），不需要特別高昂的設(shè)備，對不同組織樣品可以采用標(biāo)準(zhǔn)化統(tǒng)一的分組系統(tǒng)，非常適于大規(guī)模蛋白組學(xué)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.

【學(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2009
【分類號】：R346


本文編號：1574576