Govorukhina Natalia, Horvatovich Peter, Bischoff Rainer
Centre of Pharmacy Analytical Biochemistry, University of Groningen, Antonius, Groningen, The Netherlands.
Methods Mol Biol. 2008;484:67-77. doi: 10.1007/978-1-59745-398-1_5.
In this chapter we describe a method to analyze human serum with the goal of discovering disease-related changes in the serum proteome. The methodology is based on the removal of the six most abundant serum proteins by immunoaffinity chromatography. This step is followed by trypsin digestion and reversed-phase high-performance liquid chromatography (HPLC) coupled on-line to mass spectrometry (MS) using either a capillary HPLC or a microfluidics chip HPLC system. The obtained, highly complex data sets are processed and statistically analyzed to discover significant differences between groups of samples. The complete analytical procedure will be described with serum samples, to which a given amount of horse heart cytochrome c has been added as well as with serum samples from early stage cervical cancer patients prior to and after therapy. The use of reversed-phase HPLC to separate serum proteins at 80 degrees C with subsequent analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in order to lower the concentration sensitivity will also be briefly described.
在本章中，我们描述了一种分析人血清的方法，目的是发现血清蛋白质组中与疾病相关的变化。该方法基于通过免疫亲和色谱法去除六种最丰富的血清蛋白。此步骤之后是胰蛋白酶消化，然后使用毛细管高效液相色谱或微流控芯片高效液相色谱系统将反相高效液相色谱（HPLC）与质谱（MS）在线联用。对获得的高度复杂数据集进行处理和统计分析，以发现样本组之间的显著差异。将用添加了一定量马心血红蛋白c的血清样本以及早期宫颈癌患者治疗前后的血清样本描述完整的分析程序。还将简要描述使用反相HPLC在80摄氏度下分离血清蛋白，随后通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）进行分析以降低浓度灵敏度的方法。
