Characterization of phosphoproteins from electrophoretic gels by nanoscaleFe(III) affinity chromatography with off-line mass spectrometry analysis

Detailed characterization of phosphoproteins as well as other post-translat ionally modified proteins is required to fully understand protein function and regulatory events in cells and organisms. Here we present a mass spectr ometry (MS) based experimental strategy for the identification and mapping of phosphorylation site(s) using only low-picomole amounts of phosphoprotei n starting material. Miniaturized sample preparation methods for MS facilit ated localization of phosphorylation sites in phosphoproteins isolated by p olyacrylamide gel electrophoresis. Custom made, nanoscale immobilized Fe(II I) affinity chromatography (Fe(III)-IMAC) columns were employed for enrichm ent of phosphorylated peptides from crude peptide mixtures prior to off-lin e analysis by matrix-assisted laser desorption/ionization (MALDI) MS or nan oelectro-spray tandem mass spectrometry (MS/MS). An optimized and sensitive procedure for alkaline phosphatase treatment of peptide mixtures was imple mented, which in combination with nano-scale Fe(lll)-IMAC and MALDI-MS allo wed unambiguous identification of phosphopeptides by observation of 80 Da m ass shifts. Nafioelectrospray MS/MS was used for phosphopeptide sequencing for exact determination of phosphorylation sites. The advantages and limita tions of the experimental strategy was demonstrated by enrichment, identifi cation and sequencing of phosphopeptides from the model proteins ovalbumin and bovine beta -casein isolated by gel electrophoresis. Furthermore, an au tophosphorylation site at Ser-3 in recombinant human casein kinase-2 beta s ubunit was determined. The potential of miniaturized Fe(lll)-IMAC and MALDI -MS for characterization of in vivo phosphorylated proteins was demonstrate d by identification of tryptic phosphopeptides derived from the human p47/p hox phosphoprotein isolated by two-dimensional gel electrophoresis.