Characterization of the osteogenic stromal cell line MN7: identification of secreted MN7 proteins using two-dimensional polyacrylamide gel electrophoresis, western blotting, and microsequencing

Abstract

Proteins secreted by the osteogenic stromal cell line MN7 were analyzed using two-dimensional polyacrylamide gel electrophoresis (PAGE), western blotting, immunodetection, and microsequencing. Trichloroacetic acid-precipitated proteins from the conditioned medium of MN7 cell cultures, harvested at different times of growth, were dissolved in denaturing and reducing sample buffer and separated in the first dimension according to isoelectric point and in the second dimension according to molecular weight. Protein patterns were visualized using silver staining. Among the 350 separated protein spots, we identified type I collagen, bone sialoprotein, osteonectin, and cathepsin B by western blotting and immunodetection using polyclonal antibodies. Osteocalcin could not be detected in the conditioned medium of MN7 cells. Furthermore, 15 MN7-specific protein spots were localized after comparison with two-dimensional PAGE patterns from the conditioned medium of the nonosteogenic stromal cell lines MM1 and MV1. Microsequencing of the internal peptides of five selected spots revealed three known proteins, namely the carboxyl-terminal propeptide of the alpha 2 chain of collagen type I, cathepsin L, and the tissue inhibitor of metalloproteinases-2, an 18 kilodalton peptide fragment from osteopontin that has not previously been described, and a novel glycosylated 85 kD protein with an average isoelectric point of 5.7. All identified proteins did not vary in presence between the different time points analyzed by two-dimensional PAGE. The use of two-dimensional PAGE to investigate the secreted proteins of MN7 cells will enable us to establish a complete protein data base of extracellular osteoblast-specific proteins. Furthermore, two-dimensional PAGE in combination with other techniques is a fast and accurate method for the identification of novel proteins that could function as markers in osteoblast differentiation and/or bone formation.