Development of an integrated chromatographic system for on-line digestion and characterization of phosphorylated proteins |
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Authors: | Temporini C Dolcini L Abee A Calleri E Galliano M Caccialanza G Massolini G |
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Institution: | Dipartimento di Chimica Farmaceutica, Università di Pavia, Via Taramelli 12, Pavia, Italy. caterina.temporini@unipv.it |
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Abstract: | 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. |
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