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 TiO2 trap and separation of the produced
phosphopeptides by reversed-phase liquid chromatography-multiple mass spectrometry (RPLC/MSn). A detailed study on the selective extraction
of peptides with different degrees of phosphorylation on TiO2 cartridges is discussed. This analytical strategy has been optimized using -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.