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

作者：Temporini C^*; Dolcini L; Abee A; Calleri E; Galliano M; Caccialanza G; Massolini G

来源：Journal of Chromatography A, 2008, 1183(1-2): 65-75.

DOI：10.1016/j.chroma.2007.12.091

• phosphoproteins
• integrated analytical system
• localization of phosphorylation sites
• TiO2
• on-line SPE
• on-line digestion
• phosphopeptides
• IGFBP-1

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/MS''). 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 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.

• 出版日期2008-3-7