Proteome Analysis of Membrane Fractions in Colorectal Carcinomas by Using 2D-DIGE Saturation Labeling

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Abstract
Because of its high content in receptors and signaling proteins, the analysis of membrane fractions is critical for the study of neoplastic diseases as colorectal cancer. Here, we have used the new saturation labeling for 2D-DIGE analysis of the membrane proteome of colorectal cancer mucosal tissues. Samples from 6 patients (tumoral and normal paired biopsies) were included in this study. Twelve analytical gels were performed and considered for the quantitative study and statistical analysis. A spot pattern analysis, by using an unsupervised clustering algorithm, allowed the classification of the samples according to similar expression patterns in tumoral and normal samples. Those proteins whose expression changed significantly (Student’s t-test, p < 0.05) were further digested and characterized by mass spectrometry. Among the differentially expressed proteins: annexin A2, annexin A4, annexin A5, annexin A7, lamin B, calponin 1 and VDAC were analyzed by immunohistochemistry using tissue microarrays. Annexin A2, annexin A4 and VDAC appear as potential markers of interest for colorectal cancer diagnosis and, presumably, therapy. In summary, saturation labeling provides a new and sensitive tool for the analysis of scarce amounts of samples, allowing sample classification and direct identification of deregulated proteins.