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J Biomol Tech. 2007 February; 18(1): 25–26.
PMCID: PMC2291898

P75-T Selective Detection of Phosphoproteins and Subsequent Enrichment of Constituent Phosphopeptides for Phosphorylation Site Analysis by Mass Spectrometry


Protein phosphorylation is crucial to the global regulation of the intricate biochemical networks of cell signaling pathways, allowing protein functional activity, stability, association into multi-subunit complexes, and subcellular localization to be integrated and tuned for generation of highly specific biological responses crucial to fundamental cellular functions. An antibody-free process workflow is described involving orthogonal phosphomonoester selective binding strategies. First, complex protein samples, such as rat liver cytosol proteins, are separated by conventional gel electrophoresis and a fluorophore-conjugated alkoxide-bridged dinuclear zinc complex is employed to selectively highlight phosphoproteins via binding to the phosphomonoester dianion moieties of serine, threonine, and tyrosine residues at neutral pH. Interaction with other anionic residues, including carboxylate residues on proteins, is insignificant. As little as 1 ng of phosphoprotein is detectable by this method using standard charge-coupled device camera- or laser-scanner-based imaging systems. Then, phosphoprotein bands are excised and subjected to proteolytic digestion. Constituent phosphopeptides are subsequently purified using titanium dioxide thin-film-coated magnetic beads at acidic pH. Phosphopeptides are eluted at alkaline pH and directly characterized by MALDI-TOF or tandem mass spectrometry, without chemical modification by methyl esterification. Phosphopeptides can readily be identified from as little as 78 fmol of starting material, with minimal contamination of samples by acidic peptides, as is often encountered using conventional trivalent ferric- or gallium-based metal ion affinity approaches. The high affinity and capacity titanium dioxide-based purification method is also suitable for the direct enrichment of phosphopeptides from human serum, which could lead to new approaches for biomarker discovery from biological fluids.

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