Li Chen, Tan Ye-Xiong, Ai Jian-Hua, Zhou Hu, Li Su-Jun, Zhang Lei, Xia Qi-Chang, Wu Jia-Rui, Wang Hong-Yang, Zeng Rong
Research Center for Proteome Analysis, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.
Methods Mol Biol. 2008;428:193-208. doi: 10.1007/978-1-59745-117-8_11.
Laser capture microdissection (LCM) is a powerful tool that enables the isolation of specific cell types from tissue sections, overcoming the problem of tissue heterogeneity and contamination. We combined the LCM with isotope-coded affinity tag (ICAT) technology and two-dimensional liquid chromatography to investigate the qualitative and quantitative proteomes of hepatocellular carcinoma (HCC). The effects of three different histochemical stains on tissue sections have been compared, and toluidine blue stain was proved as the most suitable stain for LCM followed by proteomic analysis. The solubilized proteins from microdissected HCC and non-HCC hepatocytes were qualitatively and quantitatively analyzed with two-dimensional liquid chromatography tandem mass spectrometry (2D-LC-MS/MS) alone or coupled with cleavable isotope-coded affinity tag (cICAT) labeling technology. A total of 644 proteins were qualitatively identified and 261 proteins were unambiguously quantified. These results showed that the clinical proteomic method using LCM coupled with ICAT and 2D-LC-MS/MS can carry out not only large-scale but also accurate qualitative and quantitative analysis.
激光捕获显微切割（LCM）是一种强大的工具，能够从组织切片中分离出特定的细胞类型，克服了组织异质性和污染的问题。我们将LCM与同位素编码亲和标签（ICAT）技术及二维液相色谱相结合，以研究肝细胞癌（HCC）的定性和定量蛋白质组。比较了三种不同组织化学染色对组织切片的影响，结果证明甲苯胺蓝染色是LCM后续蛋白质组分析最合适的染色方法。对显微切割的HCC和非HCC肝细胞中的可溶性蛋白质，单独或结合可裂解同位素编码亲和标签（cICAT）标记技术，采用二维液相色谱串联质谱（2D-LC-MS/MS）进行定性和定量分析。共定性鉴定出644种蛋白质，明确量化了261种蛋白质。这些结果表明，使用LCM结合ICAT和2D-LC-MS/MS的临床蛋白质组学方法不仅可以进行大规模分析，还能进行准确的定性和定量分析。
