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.
Temporini
C
C
Dipartimento di Chimica Farmaceutica, Università di Pavia, Via Taramelli 12, Pavia, Italy. caterina.temporini@unipv.it
Dolcini
L
L
Abee
A
A
Calleri
E
E
Galliano
M
M
Caccialanza
G
G
Massolini
G
G
D000653:Amniotic Fluid / Q000737:chemistry; D019149:Bioreactors; D002364:Caseins / Q000737:chemistry; D002853:Chromatography, Liquid / Q000379:methods; D005260:Female; D006801:Humans; D018970:Insulin-Like Growth Factor Binding Protein 1 / Q000737:chemistry; D009862:Online Systems / Q000295:instrumentation*; D010748:Phosphopeptides / Q000302:isolation & purification; D010750:Phosphoproteins / Q000737:chemistry* / Q000378:metabolism; D011247:Pregnancy; D053719:Tandem Mass Spectrometry; D014357:Trypsin / Q000378:metabolism
D002364:Caseins; C032231:IGFBP1 protein, human; D018970:Insulin-Like Growth Factor Binding Protein 1; D010748:Phosphopeptides; D010750:Phosphoproteins; D014357:Trypsin