Identification of Altered Protein Expression and Post-Translational Modifications in Primary Colorectal Cancer by Using Agarose Two-Dimensional Gel Electrophoresis

Abstract

Purpose: Although numerous proteome studies have been performed recently to identify cancer-related changes in protein expression, only a limited display of relatively abundant proteins has been identified. The aim of this study is to identify novel proteins as potential tumor markers in primary colorectal cancer tissues using a high-resolution two-dimensional gel electrophoresis (2-DE).Experimental Design: 2-DE using an agarose gel for isoelectric focusing was used to compare protein profiling of 10 colorectal cancer tissues and adjacent normal mucosa. Altered expression and post-translational modification of several proteins were examined using Western blot analysis and immunohistochemistry.Results: Ninety-seven proteins of 107 spots (90.7%) that were differentially expressed between matched normal and tumor tissues were identified by mass spectrometry. Among them, 42 unique proteins (49 spots) significantly increased or decreased in the tumors. They include eukaryotic translation initiation factor 4H, inorganic pyrophosphatase, anterior gradient 2 homologue, aldolase A, and chloride intracellular channel 1, whose elevated expression in tumor tissues was confirmed by Western blot analysis and immunohistochemistry. Interestingly, only isoform 1 of two transcript variants of eukaryotic translation initiation factor 4H was greatly up-regulated in most of the tumor tissues.Moreover, post-translational modifications of the prolyl-4-hydroxylase ␤ subunit and annexin A2 also were identified.Conclusions: We identified several novel proteins with altered expression in primary colorectal cancer using agarose 2-DE. This method is a powerful technique with which to search for not only quantitative but also qualitative changes in a biological process of interest and may contribute to the deeper understanding of underlying mechanisms of human cancer.

Biochemical and Molecular Analysis of Superoxide Dismutase in Sordaria fimicola and Aspergillus niger Collected from Different Environments

“…PTMs are means of important mechanisms for the proper functioning of proteins [26]. The interplay between different PTMs such as phosphorylation and glycosylation, phosphorylation and acetylation, and phosphorylation and methylation is known to play a key role in the functional regulation of different proteins [27]. The PTMs were common among the five strains of S. fimicola (Table 8).…”

Gene expression profiling of human HBV- and/or HCV-associated hepatocellular carcinoma cells using expressed sequence tags

“…Our results indicate that transcripts of ANXA2 are overexpressed in HCC, especially in HBV-HCCs compared to the HCVHCCs, although ANXA1 and ANXA5 are slightly overexpressed in HCV-HCCs (data not shown). ANXA2 has been reported to be increased in colorectal cancer (49), but decreased in prostate cancer (50). These reports indicate that these proteins have different functions, such as in tumor progression or tumor suppression, depending on the cancer type or specific stage.…”

High expression of HSP47 in ulcerative colitis-associated carcinomas: proteomic approach

“…Proteomic analysis was carried out using agarose 2-DE according to previously published protocols by Oh-Ishi et al (2000) (Tomonaga et al, 2004). The agarose isoelectric focusing gel for the first dimension (180 mm in length and 3.4 mm in inner diameter) was prepared by stratification of three types of pharmalyte (pH 2.5-5 for acidic, pH 3 -10 and pH 4 -6.5 for neutral, and pH 8 -10.5 for basic solution).…”