2D DIGE for the Analysis of RAMOS Cells Subproteomes

Abstract

Overexpression of human polμ in a Burkitt’s lymphoma-derived B cell line (RAMOS), in which somatic hypermutation (SHM) is constitutive, induced an increase in somatic mutations in the parental cell line (Nucleic Acids Res 32:5861–5873, 2004). To further study Polμ implications in SHM, a dominant-negative (DN) mutant of Polμ (Polμ-DN) was generated which showed moderated overexpression of the Polμ-DN protein. The subcellular prefractionation was used to improve the detection of low-abundance proteins contained in membrane/organelles and nuclei, which are efficiently separated from high-abundance proteins commonly found in the cytosol that might otherwise hamper analysis. Two-dimensional (2D) difference gel electrophoresis (DIGE) is a technique for comparative proteomics, which improves the reproducibility and reliability of differential protein expression analysis between samples. The standard sample included in every gel (Cy2) comprises equal amounts of each sample to be compared, and thus improves the accuracy of protein quantification between samples from different gels, allowing accurate detection of small differences in protein levels. The combination of this techniques allowed the detection in Fraction F2 (membrane/organelles) of 2,111 spots, 55 of them with significant variation (19 increased and 36 decreased in a ratio >2.0 or <−2.0), and in Fraction F3 (nuclear) of 2,416 spots, 80 of them with significant variation (51 increased and 29 decreased in a ratio >1.5 or <−1.5).