Yu Kenneth H, Rustgi Anil K, Blair Ian A
Division of Hematology/Oncology, Center for Cancer Pharmacology, and Genomics Institute, University of Pennsylvania, 421 Curie Boulevard, Philadelphia, PA 19104-6160, USA.
J Proteome Res. 2005 Sep-Oct;4(5):1742-51. doi: 10.1021/pr050174l.
The purpose of this study was to develop techniques for identifying cancer biomarkers in human serum using differential in-gel electrophoresis (DIGE), and characterizing the protein biomarkers using tandem mass spectrometry (MS/MS). A major problem in profiling protein expression by DIGE comes from the presence of high concentrations of a small number of proteins. Therefore, serum samples were first chromatographed using an immunoaffinity HPLC column (Agilent Technologies), to selectively remove albumin, immunoglobulins, transferrin, haptoglobin, and antitrypsin. Serum samples from three individuals with pancreatic cancer and three individuals without cancer were compared. Serum samples were processed using the immunoaffinity column. Differential protein analysis was performed using DIGE. A total of 56 protein spot-features were found to be significantly increased and 43 significantly decreased in cancer serum samples. These spot features were excised, trypsin digested, and analyzed by MALDI/TOF/TOF (4700 Proteomics Analyzer, Applied Biosystems). We identified 24 unique proteins that were increased and 17 unique proteins that were decreased in cancer serum samples. Western blot analysis confirmed increased levels of several of these proteins in the pancreatic cancer serum samples. In an independent series of serum samples from 20 patients with pancreatic cancer and 14 controls, increased levels of apolipoprotein E, alpha-1-antichymotrypsin, and inter-alpha-trypsin inhibitor were found to be associated with pancreatic cancer. These results suggest that affinity column enrichment and 2-D DIGE can be used to identify numerous proteins differentially expressed in serum from individuals with pancreatic cancer.
本研究的目的是开发利用差异凝胶电泳（DIGE）在人血清中鉴定癌症生物标志物的技术，并使用串联质谱（MS/MS）对蛋白质生物标志物进行表征。通过DIGE分析蛋白质表达的一个主要问题来自少数高浓度蛋白质的存在。因此，首先使用免疫亲和高效液相色谱柱（安捷伦科技公司）对血清样本进行色谱分离，以选择性去除白蛋白、免疫球蛋白、转铁蛋白、触珠蛋白和抗胰蛋白酶。比较了三名胰腺癌患者和三名非癌症患者的血清样本。血清样本使用免疫亲和柱进行处理。使用DIGE进行差异蛋白质分析。发现癌症血清样本中共有56个蛋白质斑点特征显著增加，43个显著减少。这些斑点特征被切除、胰蛋白酶消化，并通过基质辅助激光解吸电离飞行时间串联质谱（MALDI/TOF/TOF，应用生物系统公司的4700蛋白质组分析仪）进行分析。我们鉴定出癌症血清样本中增加的24种独特蛋白质和减少的17种独特蛋白质。蛋白质印迹分析证实了胰腺癌血清样本中其中几种蛋白质的水平升高。在来自20名胰腺癌患者和14名对照的独立系列血清样本中，发现载脂蛋白E、α-1-抗糜蛋白酶和α-胰蛋白酶抑制剂水平升高与胰腺癌有关。这些结果表明，亲和柱富集和二维DIGE可用于鉴定胰腺癌患者血清中差异表达的多种蛋白质。
