|Home | About | Journals | Submit | Contact Us | Français|
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.