2D Differential In-gel Electrophoresis for the Identification of Esophageal Scans Cell Cancer-specific Protein Markers

Abstract

The reproducibility of conventional two-dimensional (2D) gel electrophoresis can be improved using differential in-gel electrophoresis (DIGE) Proteomics (1) includes the systematic cataloging of protein expression on a large scale, providing complementary information to that obtained from mRNA profiling by microarray (2, 3). Such studies could lead to the molecular characterization of cellular events associated with cancer progression, cellular signaling, and developmental stages (4 -7). Proteomics studies of clinical tumor samples have led to the identification of cancer-specific protein markers, which provide a basis for developing new methods for early diagnosis and early detection and clues to understand the molecular characterization of cancer progression (5, 8 -10).,A mainstay of conventional proteomics is high resolution 2D 1 gel electrophoresis (11, 12) followed by protein identification using mass spectrometry (13-15). The state of the art 2D gel system can be loaded with a few milligrams of protein and separates thousands of protein spots (5, 16). Although the technique has been widely used and successfully applied in a variety of biological systems, several technical limitations exist. Because of subtle changes in experimental conditions, the protein expression patterns on a single 2D gel usually cannot be fully duplicated, which makes it difficult to find the proteins changed between gels and to quantify the changes in protein expression. Although a comparison of protein expression profiles from regular 2D gel electrophoresis can be carried out with the assistance of various software programs, it typically requires some computerized justification of 2D gel images so that two images can be superimposed and compared. These difficulties limit the speed and accuracy of quantitation of protein spots in 2D gel electrophoresis.The differential in-gel electrophoresis (DIGE) technique recently introduced by Amersham Biosciences, Inc. is aimed at improving reproducibility. The concept of DIGE was originally developed by Minden and colleagues (17). To analyze the samples in DIGE, two pools of protein extracts are labeled covalently with fluorescent cyanine dyes, Cy3 and Cy5, re-

An approach to the proteomic analysis of a breast cancer cell line (SKBR‐3)

“…As was shown in a study of human growth hormone in plasma, additional strategies are necessary, such as the use of sample prefractionation steps, so that the presence of multiple peptides can be demonstrated by this approach [23]. Zhou et al [13] confirmed protein identifications by Western blots of cell extracts, although such an approach is dependent on the availability of antibodies of appropriate specificity. Another approach is to use multiple enzyme digests of replicate proteomic analyses to enhance the sequence coverage of low level proteins in complex samples [14].…”

“…While this is a model system, the authors believe that the sample preparation protocol reported here would be applicable to clinical samples. The sample is substantially less than the number of cells reported by Zhou [13] where 250 000 cells from epithelial tissue were collected by LCM, but with the increased recoveries of the shotgun sequencing approach vs. the 2-D gel studies it was possible to characterize a number of potential tumor markers.…”

“…One approach to proteomic studies is termed shotgun sequencing in which the components of a proteomic sample are digested with a suitable protease and the resulting peptides are then resolved by chromatography and detected by mass spectrometry (LC-MS/MS) [9][10][11][12]. A popular alternative approach is to use 2-D gels to achieve much higher resolution of such complex mixtures, but the lower recovery from gels can preclude the analysis of very small samples [12,13]. Such gels are unique, however, in their ability to store low level purified protein samples and to distinguish isoform patterns resulting from post-translational modifications and thus it is the goal of several laboratories to integrate the information from these different approaches [12].…”

“…Such gels are unique, however, in their ability to store low level purified protein samples and to distinguish isoform patterns resulting from post-translational modifications and thus it is the goal of several laboratories to integrate the information from these different approaches [12]. Zhou et al [13] recently used 2-D gels with differential staining to compare esophageal carcinoma cells and normal epithelial cells. While this study identified over 1000 spots from a sample of 250 000 cells, the protein identification data was limited to annexin I, tumor rejection antigen (gp96) and a-tubulin.…”

“…One promising new approach uses laser capture microdissection (LCM) to collect specific cell types from a tissue sample [13][14][15][16][17]. In the study reported here we have evaluated the potential of this approach to collect small samples containing 5 to 10 000 cells by the application of LCM to the SKBR-3 breast cancer cell line.…”