Identification of Proteins by LC-MALDI System (1)[Analysis of Samples Obtained from Frozen Tissue Sections]

Introduction

We conducted proteome analysis using a two- dimensional gel electrophoresis system and frozen tissue sections obtained by laser microdissection (LMD). Two-dimensional gel electrophoresis is a technique that is recognized for its excellent reproducibility and its ability to quantitate. Therefore, it is still the most widespread analytical method in proteomics. Its ability to identify several thousand proteins from a single electrophoretic run and allow the results to be registered in a database, as well as the low initial cost investment represent just a few of its many benefits. In addition, thanks to powerful database functionality and advancements in mass spectrometry, protein identification has become incredibly simplified. LMD refers to the technique in which a laser beam is used to selectively cut out target cells present on a tissue section under an optical microscope. Up to now, conducting proteomic analysis of cancerous tissue has been limited to the use of techniques that assume the cancerous tissue contains only cancer cells or techniques that involve the separation of cancerous tissue from non-cancerous tissue under the naked eye. With experiments burdened with this level of analysis accuracy, some margin of error can be expected in the analytical results due to the inclusion of proteins from non-target cells, and reduced contrast of protein expression. The use of LMD permits isolation of a uniform cell cluster from a mixed tissue sample, and offers the added benefit of enabling the analysis of gene- and protein-expression specific to those cells. Despite the difficulty in collecting a large number of cells, it was shown that two-dimensional gel electrophoresis could be conducted from a very small quantity of cells using fluorescent dye (SYPRO Ruby). Furthermore, identification of the phosphorylation site of protein was demonstrated. Here, the sample consisted of oral squamous cell carcinoma isolated from normal mucosal epithelium of human tongue tissue by LMD.

April 21, 2010 GMT