摘要
蛋白质组学的研究是集分析化学、分子生物学、生物信息学等相关学科研究领域中的热门、前沿课题。本论文利用目前较为先进的CPLL、蛋白质组学技术,建立了CPLL应用于比较蛋白质组学的研究方法。开展了鸡蛋蛋清中低丰度蛋白质组在孵化过程中的比较蛋白质组学研究。利用肽配体库技术、二维电泳技术、LTQ质谱检测、MALDI-TOF二级质谱检测等技术,考察了鸡蛋蛋清低丰度蛋白质组的变化特征。对鸡胚蛋孵化前期、胚胎快速发育期的蛋清蛋白质组、差异低丰度蛋白质的生物学功能进行了相关研究,主要内容摘要如下：
利用低丰度蛋白质研究中的最新技术方法,肽配体库技术(Combinatorial peptide ligand libraries, CPLL)研究比较蛋白质组学研究中的适用性,并对该方法适用于蛋清低丰度蛋白质富集过程进行方法优化。通过考察pH7.4和pH8.8, PBS和非PBS缓冲体系、HOS和urea CHAPS不同洗脱顺序下,肽配体库对蛋清低丰度蛋白质丰度均衡化的影响。利用肽配体较高效的富集蛋清低丰度蛋白质的使用条件为非PBS缓冲体系,urea CHAPS直接洗脱。分离溶菌酶条带的条件为：先HOS后urea CHAPS洗脱顺序。利用CPLL-LTQ线性四级杆离子阱检测技术,对5个差异条带进行质谱检测能够检测到47种蛋清蛋白质。该研究突破CPLL目前利用的局限性,有望将低丰度蛋白质种类特征作为鸡蛋品质检测的评价标准之一。利用检测蛋清中特征低丰度蛋白质识别有关蛋品的应用思路。
利用二维电泳技术,探明蛋清中低丰度蛋白质组二维电泳谱图谱信息数据库。建立了蛋清中低丰度蛋白质组在二维电泳谱图上的分子量、等电点坐标数据库。将蛋清蛋白质pH4-7、0-170KDa二维电泳图谱划分为17个具有代表性的质谱鉴定区。将新鲜蛋清各个区域检测蛋白质,建立数据、蛋清蛋白点坐标数据、各个蛋白点坐标的质谱数据信息,为鸡蛋蛋清蛋白质组的研究提供了数据标准。该数据库为以蛋清蛋白质组学、比较蛋清蛋白质组学研究为基础的蛋白质研究,提供参考基准。
利用CPLL、二维电泳、基质辅助激光解吸-飞行时间二级质谱(Matrix-assisted laser desorption/ionization-time of flight, MALDI-TOF)研究孵化早期蛋清低丰度蛋白质组变化。考察了受精鸡蛋早期孵化过程,伴随胚胎发育,蛋清蛋白的溶液特征。利用CPLL观察到的蛋清蛋白质组的变化特点。研究发现孵化早期,ovoinhibitor卵抑制剂蛋白前体、鸡簇蛋白的前体、载脂蛋白D前体、和细胞外脂肪酸结合蛋白前体,相对丰度值含量显著增加(P<0.01or p<0.05)。同时,鸡簇蛋白的前体,在两个区域,一个范围为较大的分子量范围,另一个高pH值的范围。此外,卵清蛋白、卵转铁蛋白,位于高pH区域的新坐标。针对这些发现,对这一阶段中低丰度蛋白质的功能进行推测,对这些低丰度蛋白质可能参与的生物反应进行分析。
基于上述研究,针对胚胎发育过程中蛋清存留的最后一天,即孵化16d进行蛋清蛋白质组学特征研究。扩大二维电泳图的观测范围,研究并分析蛋清蛋白质在被胚胎全部吞食完毕的过程中,蛋白质组经过肽配体富集前后变化。研究发现,胚胎大量吞食蛋白质的过程中,蛋清蛋白质并非简单营养及能量的供给者。随着胚胎的生长,蛋白质含量逐天下降,蛋清蛋白质的种类则逐天增多。相对丰度上升的蛋白点涉及Serpin Family; Clusterin Family; Transferrin Family和bacterial permeability-increasing protein (BPI) family; Lysozyme family5个蛋白质家族。相对丰度值显著性下降的点涉及Serpin Family, Transferrin Family和bacterial permeability-increasing protein (BPI) family3个蛋白质家族：。
开展了胚胎快速增长阶段,蛋清蛋白质组的变化规律的研究。利用CPLL2-DE、 MALDI-TOF MS/MS技术针对孵化过程中蛋清蛋白质组的变化研究中发现：相对于孵化7d,蛋清蛋白质组中有88个蛋白点经过肽配体库富集后得到检测,并表现出与7d同样处理方式下,相对丰度值显著性增多的变化状态。16个蛋白点只出现在蛋清蛋白质被完全吸收后的最后1d的二维电泳谱图中,且未在其他对照试验组中发现。对三个蛋白质家族：uPAR/Ly-6超家族,组氨酸磷酸酶家族和OLFML3中的5个蛋白质的分析和研究发现,这些蛋白很可能参与胚胎神经系统发育,并发挥其生物学功能。该研究还说明,蛋清蛋白质在保护受精鸡蛋胚胎发育的过程中,不仅为鸡胚个体成熟提供营养,其功能性包括保护胚胎微环境,蛋清蛋白质似乎以系统性统筹调节的方式,稳定鸡蛋内部,使鸡胚个体最大程度实现成活可能性等。
最后,利用比较二维电泳ImageMaster, V7.0操作环境、NCBI数据库、GO分析、KEGG通路分析数据库,比较孵化过程中各阶段蛋清蛋白质组的变化规律。发生变化蛋清中低丰度蛋白质的分子功能主要分为6种,分布在细胞质13个位置区域内。变化的蛋清蛋白质的功能性覆盖：离子结合蛋白,脂质结合蛋白,酶调节活性蛋白,水解酶活性,作用于糖基键蛋白,磷酸酶活性蛋白,酶结合蛋白。至少11个蛋清低丰度蛋白参与的这23个生物发育过程中,至少有5个低丰度蛋白质参与运输过程。鸡胚发育16d检测到的lysophosphatidic acid phosphatase type6参与核黄素代谢通路Riboflavin metabolism-Gallus gallus (chicken)拟合≥75%。
Study of Proteomics included the collection of Analytical Chemistry, Molecular Biology, Bioinformatics and other related frontier disciplines in the area of research. This work study on the changes of hen egg white proteome during specific biological processes by utilizes classical proteomics research tools. Comparative proteomic investigation of low-abundance proteins from hen egg white was explored during different incubations, using combinatorial peptide ligand libraries, two-dimensional electrophoresis, LTQ mass spectrometry, MALDI-TOF tandem mass spectrometry detection technology. To carry out this work was benefit in deep understanding of proteomic changes in low abundance proteins during chicken embryo development and biological function of low-abundance proteins from chicken egg white.
The thesis was divided into eight chapters, the main contents of each chapters are summarized as follows:
Establishment of a high throughput technique platform including combinatorial peptide ligand libraries technology with LTQ identification, found an applicability of the method in experimental basis for comparative proteomics investigations. Low-abundance proteins enrichment process optimized by examining the type of pH buffering system, different order of elution and egg white protein sample with different contents of buffer salt. Higher efficiency of combinatorial peptide ligand libraries in low-abundance proteins were obtained under free pretreated condition. LTQ linear quadrupole ion trap detection technology was performed in discrepancy SDS-PAGE bands identification. Combinatorial peptide ligand libraries technology with LTQ identification was suggested as a breakthrough currently used in limited basic research to better serve "foodomics" research. Feature detection in identifying low-abundance proteins from chicken egg white characterized as an evaluation of egg quality testing standards in applications.
Applications of comparative proteomics in relative low-abundance proteins from hen egg white under chicken embryonic early development using combinatorial peptide ligand libraries technology, two-dimensional electrophoresis and matrix-assisted laser desorption-time of flight tandem mass spectrometry (Matrix-assisted laser desorption/ionization-time of flight, MALDI-TOF). For early hatching embryo has not yet formed a large number of features with the ability to absorb the egg white protein, egg white proteome changes relatively stable. It was found that relative abundance values of many protein precursor protein were significantly (p<0.01or<0.05) increase during early embryonic development phase. Meanwhile, it was found that some protein precursors were identified as new location coordinates on the two-dimensional electrophoresis maps. Thus, these biological function of low-abundance proteins were discussed and proposed.
Exploration of low-abundance proteome from fertilized egg white on the last day of residue under chicken embryonic development. Expanding the two-dimensional electrophoresis of the observation area of low-abundance proteins comparing before and after combinatorial peptide ligand libraries treatment. In addition, comparative proteomic analysis of low-abundance egg white proteins during the rapid embryonic growth were curried out. It was found that the proteome of low-abundance proteins in incubation day16were more complexity in compare incubation day7.
Relative abundance increase of proteins involved in five protein family, they were: Serpin Family; Clusterin Family; Transferrin Family and bacterial permeability-increasing protein (BPI) family; Lysozyme family. Relative abundance values were significantly decreased involved three protein families, they are:Serpin Family, Transferrin Family and bacterial permeability-increasing protein (BPI) family. According to such experiments, proposed and designed Chapter Ⅵ and Chapter Ⅶ of research ideas and content. It was found that these proteins may also play an important biological nervous system during embryonic development learning effect. Therefore, the proteome of low-abundance in hen egg residue egg white was different between early phase of embryonic development and the rapid growth period of embryonic development.
Comparative proteomic analysis of low-abundance egg white proteins during the rapid embryonic growth period using peptide ligand library technology, two-dimensional electrophoresis, MALDI-TOF mass spectrometry techniques. The protein composition increased and the amount of albumin solids diminished in the residual egg white. There was a significant increase in the relative abundance of88protein spots (P<0.01and<0.05) over16days of incubation, representing11proteins from9protein families were identified. Moreover,16protein spots appear only in the egg white protein is completely absorbed after the last day of the two-dimensional electrophoresis spectra in controlled trials and not found in other groups. Of the three protein families:uPAR/Ly-6super family, histidine phosphatase family and OLFML3family5proteins. In addition, clusterin precursors were observed over a wide range of pH values and tenp protein increased continuously during embryonic development. Several of these dysregulated proteins were confirmed at the transcriptional level.
Low-abundance proteins from two control groups were compared across different incubators in order to better understand the function of these proteins in egg whites. The relative increase in abundance of these differentially expressed proteins during the rapid embryonic growth period was different compared to store-incubated eggs, suggesting the pivotal role of hen egg white during embryonic development. These findings provide insights into regulating and supporting embryonic development during embryogenesis.
Regularity analysis on low-abundance proteomic of chicken egg white during the whole process of embryo development based on PDQuest, NCBI data, GO and KEGG. Changes in the molecular function of low-abundance proteins in egg white is mainly divided into six species, while distributed in the cytoplasm13location area. Functional changes in egg white proteins covering:ion-binding protein, a lipid-binding protein, regulating the activity of the enzyme protein, hydrolyze activity, the key role in protein glycosylation, protein phosphatase activity, the enzyme-binding protein. These23process at least11egg whites biological development of low-abundance proteins involved in at least five low-abundance proteins involved in the transport process. Chick embryos detected the first16days of lysophosphatidic acid phosphatase type,,6metabolic pathways involved in riboflavin riboflavin metabolism-Gallus (chicken) fit≥75%. The study found that the combinatorial peptide ligand library technology advantaged in the exploration of low-abundance proteome changes in obvious. However, proteomic information may also missed due to limit of technology.
Summarize multi-faceted research and explore multiple perspectives of variation of egg white proteome, low-abundance proteome through chicken embryonic development on the day0,3,7,10,14,16d hatching process. In addition, summary of shortage during experiments. Finally, prospects for further research and development of research strategy on chicken egg white, egg white protein and low-abundance proteome.
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