【摘要】 本文建立了一种基于强阳离子交换填料/强阴离子交换填料(SCX/SAX)混合填料的集成化蛋白质组学样品前处理方法.本工作将前期已发展的离心式集成化蛋白质组学样品前处理技术SISPROT中的SCX填料替换成SCX和SAX混合填料,以溶菌酶和牛血清白蛋白为模型蛋白,研究了3种pH(3,7.4,12)条件下蛋白质在SCX/SAX混合填料上的保留行为;应用BCA方法对SCX/SAX混合填料的比例进行了优化,并应用SDS-PAGE法对酶解步骤中pH变化引起的蛋白质丢失情况进行了系统的考察.实验结果发现,在pH 7.4条件下,质量比为1:1的SCX/SAX混合填料的富集效率最佳,蛋白质富集容量为180μg,是SCX填料富集容量的两倍左右;且酶解步骤的pH变化过程不会影响蛋白质在SCX/SAX混合填料上的保留.最后应用改进的SISPROT方法对少量肠癌组织样品进行了集成化蛋白质组学样品前处理和分析,并与传统的基于SCX填料的SISPROT方法进行了对比研究.结果表明,蛋白质鉴定量、特异性肽段数量和肽段谱图匹配数量均与基于SCX填料的SISPROT技术相当,证实了该方法作为蛋白质反应器的可靠性.鉴于该方法可以实现在生理pH条件下进行样品前处理,且显著提高了上样容量和减少了样品损失,该方法有望成为一种更为通用的集成化蛋白质组学样品前处理技术.更多还原

【Abstract】 An integrated technology for preparing proteomics samples was developed based on SCX/SAX mixed beads.Unlike previously reported SISPROT strategies,SCX/SAX mixed beads were used to replace SCX beads.A mixture of BSA and LYZ was chosen as the model protein to evaluate the protein retention behavior of the SCX/SAX beads at pH of 3,7.4,and 12.The mass ratio of SCX/SAX was optimized using the BCA method.Further,the protein loss caused by pH replacement during digestion was systematically investigated by the SDS-PAGE method.The results indicated that at a pH of 7.4,the optimized mass ratio of SCX/SAX mixed beads was 1:1,and the loading capacity of SCX/SAX mixed beads with the mass ratio of 1:1 was 180 μg,which is almost twice that of SCX beads.Furthermore,pH replacement during digestion did not affect the protein retention behavior of SCX/SAX mixed beads.The improved SISPROT method was used for processing a limited quantity of colon cancer tumor samples.For comparison,the traditional SISPROT method based on SCX beads was used.The results indicated that the number of protein identification,unique peptides and peptide spectrum matching obtained from the two methods were comparable.These results confirmed the applicability of the method as a proteomics reactor.The method is expected to be a more generic sample preparation technology because it can be operated at physiological pH and exhibits significantly improved loading capacity and reduced sample loss compared with those of the traditional SISPROT method.更多还原