Characterization of human insulin-like growth factor-binding proteins by two-dimensional polyacrylamide gel electrophoresis and Western ligand blot analysis

The insulin-like growth factor (IGF)-binding proteins (IGFBPs) from adult human serum, amniotic fluid, and cerebrospinal fluid were analyzed by a modified two-dimensional gel electrophoresis followed by Western ligand blotting. The samples were subjected to immobilized pH gradient isoelectric focusing in the first dimension, followed by nondenaturing SDS-PAGE in the second dimension and autoradiography after ligand blotting with [I-125]IGF-I Or [I-125]IGF-II. The identity of the binding proteins was confirmed by immunoblotting and immunoprecipitation with specific antibodies. Using this method, all six human high affinity IGFBPs could be clearly separated from each other according to their molecular mass and isoelectric points (pI). All IGFBPs exhibited a variety of specific pi isoforms, which presumably represent posttranslational modifications. In adult human serum, glycosylated IGFBP-3 is found as a broad band of spots with molecular masses of 41 and 45 kDa and a pi in the range of 4.8-8.2. The two IGFBP-3 bands could be reduced to a single 36-kDa band after deglycosylation (pI 6-9). Furthermore, the specific spots for IGFBP-2 (33 kDa; pi 6.2-7.1) and deglycosylated IGFBP-4 (24 kDa; pi 6.3, 6.5, and 6.8) were found with their expected molecular masses. Additionally, the diffuse bands around 30 kDa, found in one-dimensional Western ligand blotting, could be clearly separated into distinct groups of specific spots representing IGFBP-1 (30 kDa; pi 4.0-4.8), IGFBP-6 (30 kDa; pi 4.8-5.8), glycosylated IGFBP-4 (29 kDa; pi 6.1 and 6.3), and IGFBP-5 (30/31 kDa; pi 6.4-8). As expected, IGFBP-6 was visible only when IGF-II was used as radioligand. In conclusion, two-dimensional gel electrophoresis followed by Western ligand blotting allows identification of all six high affinity IGFBPs with their isoforms on the basis of their characteristic molecular masses and pi, especially in the range of 30 kDa. This technique can be rapidly performed with small amounts of complex biological fluids and is a powerful tool for the detection and analysis of posttranslational modifications of IGFBPs.