Strategy for identifying circulating fragments of insulin-like growth factor binding proteins in a hemofiltrate...

A differentiated strategy was established to isolate circulating forms of the six human insulin-like growth factor binding proteins (IGFBPs). As starting material we used our peptide bank, a comprehensive blood plasma peptidoma generated from human blood filtrate. The peptides were initially identified in the fractions of the hemofiltrate peptide bank by their immunoreactivity, their capacity to bind the insulin-like growth factors (IGFs), and their molecular masses determined by polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS). Fractions revealing both immunoreactivity and IGF-binding capacity were analyzed by direct sequencing of immunoreactive bands from a Coomassie-stained gel. Further purification of the IGFBP peptides was performed by consecutive chromatographic steps guided by sensitive MALDI-MS. Using this strategy, different fragments of IGFBP-3, -4, and -5 were identified and a fragment of IGFBP-4 was purified to homogeneity.