【作者】 唐新科；

【导师】 王贤纯； 梁宋平；

【作者基本信息】 湖南师范大学 ， 生物化学与分子生物学， 2004， 硕士

【摘要】 为探索蛋白质组学研究中3种不同方法鉴定蛋白质的准确性、可靠性及简便性和蛋白质序列的手工解析法在蛋白质化学中的应用，利用高效液相色谱(RP-HPLC)与电喷雾串联质谱(ESI-MS／MS)联用技术分别分析模式蛋白分子(牛血清白蛋白，BSA)及合成多肽粗产物。在此基础上，并以小鼠肝脏为材料经SDS-PAGE分离后用联用技术分析其膜蛋白质组。通过联用技术直接对模式蛋白分子的胰蛋白酶酶解产物进行分离和测定，从而获得系列BSA酶解片段的一级(MS)和二级(MS／MS)质谱数据，经分析软件处理后，分别通过不同处理和不同参数设置，用3种不同方法通过网上蛋白质数据库进行蛋白质搜寻鉴定。结果显示，三种搜寻法都能正确地鉴定该蛋白质，其中以利用MS数据的肽质量指纹谱搜寻法(PMF法)较为快捷方便，但鉴定结果易受数据处理和数据库搜寻鉴定时参数设置等因素影响；利用未解析MS／MS数据(raw MS／MS data)的搜寻法可在较宽的搜寻参数变化范围内获得明确的鉴定结果；而借助从头测序(de novo sequencing)结果的序列搜寻法(sequence query)则显示出更高的专一性，利用较少酶解片段数据就能得到稳定和明确的鉴定结果，对搜寻参数变化的影响很小。同时就酶解条件、数据处理和搜寻参数设置对蛋白质鉴定结果的影响展开了详细的讨论，为蛋白质组学研究中的数据处理和库搜寻鉴定积累可借鉴的资料。然后以相同的联用技术直接分析用芴甲氧羰基(Fmoc)固相多肽合成方法手工偶联合成的一个模型十三肽(Lys-Lys-Glu-Ser-Asp-Phe-Leu-Met-Phe-Val-Tyr-Leu-Val，Mr=1617.8)粗产品。RP-HPLC结果显示：合成粗产物中含有1个主成分，2个次要成分和多个微量成分；与之联用的电喷雾质谱同步准确地测定出各成分的分子量(m／z)并自动对各主要成分的化学结构进行了串联质谱分析。结果证明，粗产物中的主成分即为目标十三肽，另外几个主要副产物为十三肽的残缺肽或氧化肽。对色谱和生物质谱联用技术分析合成多肽的意义和副产物形成的可能机制进行了讨论。最后以小鼠肝脏为材料，运用亚细胞分离技术(蔗糖密度梯度离心)分级分离质膜，再用两性离子去垢剂(CHAPS)、非离子去垢剂(NP一40)和离液剂(ehaotropes如硫脉、尿素等)组合，综合处理细胞质膜得到膜蛋白，经SDS一PAGE分离、考马斯亮兰染色、胶内原位酶解，再通过毛细管高效液相色谱(capLC)和电喷雾串联质谱(ESI一MS/MS)进行色谱分离与质谱鉴定，得到的质谱数据文件运用MASCOT软件分析。结果鉴定了512个蛋白，其中有91个蛋白重复出现在不同条带上(共重复135次)，所以实际鉴定出377种不同(NCBI中91号不同)蛋白质。其中膜蛋白有88种，包括内膜蛋白和整合膜蛋白等类型。它们的分子量范围为10152Da一255882Da，pl范围为4.86一9.5。并对质膜分离进行了较为详细的讨论。更多还原

【Abstract】 In order to investigate the accuracy, simplification and reliability of protein identification with three different methods in proteomics and the application of de novo sequencing manually in protein chemistry, The enzymatic digest of bovine serum albumin(BSA), used as a model protein, and the crude products of solid phase synthesis of the tridecapeptide were directly analyzed by reversed-phase high performance liquid chromatography (rpHPLC)/electrospray tandem mass spectrometry (ESI-MS/MS). On the basis of the above application, the membrane proteins of mouse liver separated by SDS-PAGE were analyzed using capillary liquid chromatography(capLC) coupled with tandem mass spectrometry(MS/MS). The resulting MS and MS/MS data, after being processed with analytical software under different parameters, were used for the protein identification through database search with three different methods. All the three methods could identify the protein correctly. Peptide mass fingerprint (PMF) was the simplest and most direct, with its search results being easily influenced by parameter changes during the data process and database search. Using raw MS/MS data could obtain correct identification result in wider change range of database search parameters. The Sequence Query (SQ) utilizing the de novo sequencing results could make steady and definite identification based on the data of a few peptides, which was hardly influenced by the main database search parameter changes. The effects of enzymatic digestion, data process and database search parameters on the identification of proteins were discussed in detail, which provided references for the proteomics researches. The crude product ofsolid phase synthesis of the tridecapeptide was investigated by using rpHPLC-ESI-MS/MS. One major component, two minor components and several trace level components were detected on the HPLC chromatogram. The accurate molecular weights of each components were determined. The amine acid sequences of the main components were analyzed by MS/MS synchronously. The results showed that the major component of the crude product was the desired tridecapeptide and the main byproducts were derived from the oxidation or deformity of the desired peptide. The significance of the application of biological mass spectrometry coupled with HPLC to synthetic peptide analysis and the possible origins of the by-products were discussed. The mouse liver plasma membrane (PM) was purified using subcellular fractionation and treated with some detergents such as CHAPS, NP-40, thiourea. The plasma membrane proteins obtained by one-dimensional (ID) gel electrophoresis were digested in-gel and identified by electrospray ionization mass spectrometry coupled with capLC. The data files were used to search the NCBI database with the MS/MS ion search option of the Mascot search programme at www.matrixscience.com. 88 out of 377 identified were plasma membrane proteins, which were composed of membrane, inner membrane and integral membrane. The molecular weight range of membrane proteins identified is from 10152Da to 255882Da, and pI is from 4.86 to 9.5. The plasma membrane fractionation were discussed in detail.更多还原