激光捕获显微切割技术与蛋白质组学在结肠癌中的应用
Application of laser capture microdissection and proteomics in colon cancer.
作者信息
Lawrie L C, Curran S, McLeod H L, Fothergill J E, Murray G I
机构信息
Department of Pathology, University of Aberdeen, Foresterhill, Aberdeen, AB25 2ZD, UK.
出版信息
Mol Pathol. 2001 Aug;54(4):253-8. doi: 10.1136/mp.54.4.253.
AIMS
Laser capture microdissection is a recent development that enables the isolation of specific cell types for subsequent molecular analysis. This study describes a method for obtaining proteome information from laser capture microdissected tissue using colon cancer as a model.
METHODS
Laser capture microdissection was performed on toluidine blue stained frozen sections of colon cancer. Tumour cells were selectively microdissected. Conditions were established for solubilising proteins from laser microdissected samples and these proteins were separated by two dimensional gel electrophoresis. Individual protein spots were cut from the gel, characterised by mass spectrometry, and identified by database searching. These results were compared with protein expression patterns and mass spectroscopic data obtained from bulk tumour samples run in parallel.
RESULTS
Proteins could be recovered from laser capture microdissected tissue in a form suitable for two dimensional gel electrophoresis. The solubilised proteins retained their expected electrophoretic mobility in two dimensional gels as compared with bulk samples, and mass spectrometric analysis was also unaffected.
CONCLUSION
A method for performing two dimensional gel electrophoresis and mass spectrometry using laser capture microdissected tissue has been developed.
目的
激光捕获显微切割技术是一项最新进展，它能够分离特定细胞类型以便后续进行分子分析。本研究描述了一种以结肠癌为模型，从激光捕获显微切割组织中获取蛋白质组信息的方法。
方法
对经甲苯胺蓝染色的结肠癌冰冻切片进行激光捕获显微切割。选择性地显微切割肿瘤细胞。确定了从激光显微切割样品中溶解蛋白质的条件，并用二维凝胶电泳分离这些蛋白质。从凝胶上切下各个蛋白质点，通过质谱进行表征，并通过数据库搜索进行鉴定。将这些结果与从平行运行的大块肿瘤样品中获得的蛋白质表达模式和质谱数据进行比较。
结果
能够从激光捕获显微切割组织中以适合二维凝胶电泳的形式回收蛋白质。与大块样品相比，溶解的蛋白质在二维凝胶中保留了预期的电泳迁移率，质谱分析也未受影响。
结论
已开发出一种使用激光捕获显微切割组织进行二维凝胶电泳和质谱分析的方法。
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