Identification of altered protein expression and post-translational modifications in primary colorectal cancer by using agarose two-dimensional gel electrophoresis.
OBJECTIVE
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).


METHODS
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 beta 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.
Tomonaga
Takeshi
T
Department of Molecular Diagnosis (F8), Graduate School of Medicine, Chiba University, Chiba, Japan. tomonaga@faculty.chiba-u.ac.jp
Matsushita
Kazuyuki
K
Yamaguchi
Seiko
S
Oh-Ishi
Masamichi
M
Kodera
Yoshio
Y
Maeda
Tadakazu
T
Shimada
Hideaki
H
Ochiai
Takenori
T
Nomura
Fumio
F
D000368:Aged; D000369:Aged, 80 and over; D000595:Amino Acid Sequence; D015179:Colorectal Neoplasms / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers; D004587:Electrophoresis, Agar Gel; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010446:Peptide Fragments / Q000276:immunology; D011499:Protein Processing, Post-Translational; D020133:Reverse Transcriptase Polymerase Chain Reaction
D017931:DNA Primers; D009363:Neoplasm Proteins; D010446:Peptide Fragments