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J Biomol Tech. 2007 February; 18(1): 50.
PMCID: PMC2291977

P143-M Microarray-Generated Phosphopeptide Selectivity Profiles for Fluorescent Conjugates of an Artificial Phosphomonoester-Binding Small Molecule; Implications for the Global Enrichment and Detection of Phosphopeptides and Phosphoproteins


Reliable identification and characterization of protein kinase substrates is of paramount importance to deciphering cellular signaling pathways. Previously, an artificial phosphomonoester-binding molecule, 1,3-bis[bis(pyridin-2-ylmethyl)amino]propan-2-olato Zn2 complex (a.k.a. Phos-tag) was shown to bind to phenylphosphate under physiological conditions with a Kd of 25 nM. A fundamental challenge in chemical biology is efficiently performing comprehensive profiling of the activity of artificial chemical structures, such as the Phos-tag molecule, against the hundreds or even thousands of biological targets they could potentially interact with. A peptide microarray-based phosphorylation site screening assay was thus performed to determine the binding selectivity of fluorophore-conjugated Phos-tag molecule, based upon direct detection of binding through readout of fluorescent signal generated by the reporter group. Epitope mapping was performed on a comprehensive list of phosphorylation sites known to be expressed in the human proteome, including serine, threonine and tyrosine phosphorylated residues. The collected data was evaluated using a combination of different in-house software packages resulting in scores for each amino acid residue relative to the phosphorylation site. The study demonstrates that fluorophore-conjugated Phos-tag exhibits very broad substrate specificity and does not appear to depend upon a specific sequence surrounding the targeted phosphorylation site. This relatively low degree of sequence restriction at the phosphorylation recognition site suggests that Phos-tag is broadly suitable for universal enrichment and detection of phosphorylation sites on phosphoproteins and phosphopeptides. The fluorescent Phos-tag technology is easy to perform, cost effective and should allow rapid large-scale screening of protein and peptide phosphorylation using a variety of solid-phase assay formats.

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