Development of an integrated chromatographic system for on-line digestion and characterization of phosphorylated proteins.

The development of an integrated chromatographic system for complete phosphoprotein analysis is described. The digestion of phosphoproteins with trypsin- or pronase-based monolithic bioreactors is carried out on-line with selective enrichment on a TiO(2) trap and separation of the produced phosphopeptides by reversed-phase liquid chromatography-multiple mass spectrometry (RPLC/MS(n)). A detailed study on the selective extraction of peptides with different degrees of phosphorylation on TiO(2) cartridges is discussed. This analytical strategy has been optimized using beta-casein as a standard phosphoprotein, and then applied to the identification of phosphorylation sites in insulin-like grow factor-binding protein 1 (IGFBP-1) isolated from amniotic fluid.