【作者】 李晓海；

【导师】 钱小红；

【作者基本信息】 中国人民解放军军事医学科学院 ， 药物分析学， 2004， 博士

【副题名】国际人类蛋白质组组织血浆蛋白质组合作研究项目

【摘要】 血浆/血清蛋白质由于其组成中包含了来源于细胞、组织、器官的蛋白信息,具有非同寻常的生物学复杂性,而且又是临床常见的用于疾病诊断与治疗的重要生物样本,因此对血浆蛋白组成的深入研究具有极其重要的生物学及医学价值,也正如此,国际人类蛋白质组组织首先启动了人血浆蛋白质组国际合作研究项目。 我们在参与并进行HUPO PPP项目初期研究中,先后对Bio-rad Aurum亲合柱,Agilent MARS亲合柱,制备溶液等电聚焦法Rotofor和有机溶剂沉淀法进行了考查,比较了各种方法的应用特点及局限性;在此基础上,我们选择了重复性好、去除高丰度蛋白选择性较强的Agilent MARS亲合柱用于HUPO PPP项目参考血浆和血清样品的处理方法,为完成HUPO PPP标准血浆/血清样品的分析鉴定及比较多种蛋白质组研究技术策略在血清蛋白质组研究中的应用准备了条件。 在已选定的利用亲合色谱法去除血浆六中高丰度蛋白基础上,选择高通量二维毛细管液相色谱离子阱串联质谱联用技术作为鉴定分析HUPO PPP标准血浆和血清样品的技术策略。在不断优化蛋白酶切方法及改进质谱数据扫描采集方法的基础上,改善了HUPO PPP提供的标准血浆和血清样品的蛋白鉴定结果,保证了分析结果重复性,完成了HUPO血浆蛋白质组研究项目中血浆及血清样品的分析工作:每个样品平均鉴定110余种蛋白质,蛋白鉴定水平处于所有参与实验室中等水平。 2003年在蒙特利尔召开的HUPO PPP项目阶段讨论会上,不同实验室之间提交的血浆/血清蛋白鉴定结果的重叠率很低。我们分析不同实验室蛋白鉴定结果间的低重叠率可部分归咎于所采取的不同样品处理方法以及不同的蛋白分离鉴定技术策略。为了系统比较不同样品制备方法和所采用的不同分离鉴定技术策略对血清蛋白鉴定结果的影响,我们采取了五种不同或相似的蛋白鉴定策略,对经过同一亲合色谱方法去除六种高丰度蛋白的HUPP PPP标准血清样品—中国人血清样品进行了系统分析。所比较的技术策略包括:2DE策略;整体蛋白预分离策略;在线“鸟枪法”蛋白鉴定策略;离线“鸟枪法”蛋白鉴定策略和离线“鸟枪法”纳升级电喷雾鉴定策略;通过比较五个不同技术策略蛋白鉴定结果来比较它们各自的应用特点、优点及局限性。每条技术路线鉴定的非冗余蛋白数分别是78,179,131,224和330。在全部560个血清蛋白的鉴定结果中,被五种技术共同鉴定的蛋白仅有37个,反映了不同技术策略蛋白鉴定结果具有明显的互补性。蛋白鉴定结果存在明显差异的原因主要是样品制备过程,肽混合物色谱分离效率及质谱检测灵敏度等存在明显差异。 在实际应用中,每个技术策略都有其各自的优点及局限性。2DE策略能提供更多的有关血清蛋白异构体的信息及蛋白的相对定量结果。蛋白二维色谱预分离更多还原

【Abstract】 Of all the proteomes, the proteins of the blood are perhaps of the greatest biological, medicinal importance. Serum or plasma is of unusual biological complexity, reflecting its communication with all cells, tissues and organs. Thus, the task of the HUPO Plasma Proteome Project (PPP) has been distributed among collaborating labs around the world.During the first stage of participating in and carrying out the HUPO PPP research, a varies of serum protein preparation methods for depleting high abundant proteins or fractionating proteins, which were Bio-rad Aurum Serum kit, Agilent multiple affinity removal system(MARS), preparative solution IEF (Rotofor) and organic solvent precipitation, were investigated and their applicable characteristics and limits were compared and summarized. On this basis, the Agilent MARS column with better reproducibility and specificity for depletion of high abundant proteins was applied for the sample preparation of HUPO PPP reference plasma or serum samples, which laid the solid ground for completing the analysis of the reference samples and comparing multiple proteomic techniques for their application in the research of serum protoeme.Based on the selection of MARS column for the depletion of six high a bundant proteins in plasma or serum samples, the high throughput two dimensional capillary liquid chromatography combined with electrospray ion trap MS/MS was established for the analysis of HUPO PPP reference plasma and serum samples. With optimized protein digestion method and improved mass spectra acquiring approach, the protein identification result of the reference samples was obviously improved and reproducible result was obtained. In average, about 110 proteins were identified in each of the reference samples. The protein identification level was medium among all the participating labs.At the workshop of HUPO PPP held in Montreal in 2003, the preliminary data sets submitted from different labs had little overlap in proteins identified. The lack of overlap can be attributed in part to the different technological strategies or methods of sample preparation used. In a systematic approach to this issue, we here compare the results from five different techniques using the same HUPO PPP reference serum specimen with the six proteins of highest abundance depleted by affinity chromatography. The approaches compared were: (1) Intact protein fractionation by anion exchange chromatography (WAX) followed by 2-dimensional electrophoresis (2-DE) and MALDI-TOF-MS-MS for protein identification (2DE strategy); (2) Intact protein fractionation by 2D-HPLC and then coupled with solution digestion of each fraction and micro-capillary RP-HPLC microESI-MS-MS identification (protein prefractionation strategy); (3) Digestion of mixed proteins by trypsin followed by automated online micro-capillary 2D-HPLC with ion trap micro-ESI-MS/MS; (online shotgun strategy); (4) Same as 3) with the SCX step performed offline (offline shotgun strategy) and (5) Same as 4) with the SCX fractions reanalysed by optimised nanoRP-HPLC-nanoESI-MS-MS. (offline shotgun-nanospray strategy). The protein identification results of each strategy were compared and their particular features were summarized. All five approaches yielded complementary sets of protein identifications. The total number of unique proteins identified by each of these five approaches was 1) 78, 2) 179, 3) 131, 4) 224, and 5) 330, respectively. In all 560 unique proteins were identified, only 37 proteins were identified by all five approaches which reflecting the complementary protein identification result from all these five strategies. The result could be attributed to obvious variations in the sample preparation process, differences in peptide separation efficiency, and the sensitivity of the mass spectrometer.In practice, the 2-DE approach yielded more information on the pi-altered isoforms of some serum proteins and the relative abundance of identified proteins. The protein 2D-HPLC prefractionation strategy slightly improved the capacity to detect proteins of lower abundance. Optimising the separation at the peptide level and improving the detection sensitivity of ESI-MS/MS were more effective than fractionation of intact proteins in increasing the total number of proteins identified with extended dynamic range . This is very important for the serum proteome research. The results also indicate that by increasing the number of well-separated fractions collected in offline SCX and optimization of RP-HPLC-ESI-MS/MS, more serum proteins of low abundance can be identified, such as Coagulation factor IX precursor (ug/mL), L-selectin precursor, and Hepatocyte growth factor-like protein precursor, identified through high-confidence MS-MS spectra of their peptides.All three strategies had similar distribution characteristics of mass range, isoelectric point and hydrophobicity of the identified proteins. It does appear that 2-D HPLC of intact proteins works best for proteins representing extremes in these properties while 2DE strategy is difficult to profiling the proteins with low molecular weight, or proteins with extremely acidic, basic or high hydrophobic characters.At last, when the whole dataset was compared with the high confident proteins list of HUPO PPP(N=3020), about 257 unique proteins were overlapped, 139 proteins of which were identified with 2 or more peptides; While compared with the reported non redundant list, totally 169 unique proteins were overlapped, 117 of which were identified with 2 or more peptides.更多还原