The potential biological and clinical significance of selenium binding protein 1 (SBP1) has been suggested in various types of cancer. To evaluate the role of SBP1 and reveal the molecular basis for its function, we examined the SBP1 protein complex. A gene transfection assay revealed that overexpression of SBP1 promoted proliferation and migration of A549 lung adenocarcinoma cells. Halo-tag-based affinity purification coupled with liquid chromatography-tandem mass spectrometry identified 23 components of the SBP1 protein complex. The functional classification of these 23 proteins suggests that the SBP1 complex participates in critical biological events including cell structure, protein translation, stress response, chaperone, and apoptosis. Moreover, the SBP1 complex includes several proteins that are aberrantly expressed in cancers. These finding indicate that SBP1 may function coordinately with these multiple proteins to facilitate cancer progression. A comprehensive study of the multiple proteins associated with SPB1 together with an examination of individual proteins will be required to elucidate the roles of aberrant SBP1 regulation in cancer progression.
A procedure for the direct coupling of affinity chromatography and capillary isoelectric focusing in a single capillary is described. A unified capillary device was made to implement this procedure. The inner surface of a fused-silica capillary was tandemly coated with a chelating polymer, polyiminodiacetate, at the inlet side and with a neutral polymer, polydimethylacrylamide, at the outlet side. After loading a fluorescence-labeled recombinant Fab with a hexahistidine tag, a model sample, the device was rinsed with a high-salt buffer and then with a carrier ampholyte solution to fill the device. The bound Fab was eluted by filling the nickel-chelate column with an anode solution, 100 mM phosphoric acid. A positive voltage was applied at the chelate column side with a pressure at the same side, so as to slightly overwhelm the anodic electroosmosis produced in the acidified chelate column and gradually move the focused protein bands through a stationary fluorescence detector. The mixture of the labeled recombinant Fab at variable concentration and labeled bovine serum albumin at 50 nM was analyzed with the capillary device. A linear relationship between the peak area and the concentration was demonstrated for the Fab at 3.2 pM to 10 nM. The coefficients of variation for the peak area and detection time at 10 nM were 4.3% and 4.4%, respectively. The coupled procedure described here allows total transfer of the specifically adsorbed proteins from the affinity column to the capillary for isoelectric focusing without any compromise in separation efficiency. Removal of excess salts and concentration of dilute samples are also attractive features of the coupled procedure that can provide a new option for the analysis of charge variants of a protein in biological samples.