We have compared two different solubilization buffers, we also evaluated protein precipitation with ethanol (EtOH) and optimized 2-DE conditions for human myeloma proteins. Myeloma cell line ARH-77 was used to in the pilot study. Proteins from myeloma cell line ARH-77 were separated by 2-DE using a 17 cm pH 3 10 nonlinear immobilized pH gradient strips in the first dimension. Separation in the seconddimension was carried out on the Protean II Cell (Bio-Rad) using a 12% SDS-polyacrylamide gel. Separated proteins were stained with ProteoSilver Plus (Sigma). Spot detection, quantification, and alignment were performed with the PDQuest software (version 7.3.0., Bio-Rad). Results: Two concentrations of the detergent CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) in the solubilization buffer were tested, 2% and 4%. Higher concentration of CHAPS showed a greater ability to solubilize proteins. The most efficient solubilization was obtained with lysis buffer containing 7 M urea, 2 M thiourea, 4% CHAPS, 60 mM DTT (dithiothreitol), 0.8% carrier ampholytes and 0.003% BPB (bromphenol blue). About 1448 spots were visualized in the gel using this lysis buffer. Many proteins (206 spots) were lost after the EtOH precipitation though.
Conclusions: Initial extraction and solubilization are key factors of proteomic analysis. The successful solubilization and 2-DE separation of proteins will be a valuable tool for further study of the mechanisms of therapy resistance in purified plasma cells of patients with MM and further study of the biomarkers for prediction of progression of MGUS to overt myeloma.