Quantitative analysis of protein phosphorylation status and protein kinase activity on microarrays using a novel fluorescent phosphorylation sensor dye.
Ultrasensitive detection of minute amounts of phosphorylated proteins and peptides is a key requirement for unraveling many of the most important signal transduction pathways in mammalian systems. Protein microarrays are potentially useful tools for sensitive screening of global protein expression and post-translational modifications, such as phosphorylation. However, the analysis of signaling pathways has been hampered by a lack of reagents capable of conveniently detecting the targets of protein kinases. Historically, phosphorylation detection methods have relied upon either radioisotopes ((gamma-(32)P)ATP(gamma-(33)P)ATP labeling) or phosphoamino acid-selective antibodies. Both of these methods suffer from relatively well-known shortcomings. In this study, a small molecule fluorophore phosphosensor technology is described, referred to as Pro-Q Diamond dye, which is capable of ultrasensitive global detection and quantitation of phosphorylated amino acid residues in peptides and proteins displayed on microarrays. The utility of the fluorescent Pro-Q Diamond phosphosensor dye technology is demonstrated using phosphoproteins and phosphopeptides as well as with protein kinase reactions performed in miniaturized microarray assay format. Instead of applying a phosphoamino acid-selective antibody labeled with a fluorescent or enzymatic tag for detection, a small, fluorescent probe is employed as a universal sensor of phosphorylation status. The detection limit for phosphoproteins on a variety of different commercially available protein array substrates was found to be 312-625 fg, depending upon the number of phosphate residues. Characterization of the enzymatic phosphorylation of immobilized peptide targets with Pro-Q Diamond dye readily permits differentiation between specific and non-specific peptide labeling at picogram to subpicogram levels of detection sensitivity.
Martin
Karen
K
Proteomics Section, Molecular Probes Inc., 29851 Willow Creek Road, Eugene, OR 97402, USA.
Steinberg
Thomas H
TH
Cooley
Laurie A
LA
Gee
Kyle R
KR
Beechem
Joseph M
JM
Patton
Wayne F
WF
D004305:Dose-Response Relationship, Drug; D005456:Fluorescent Dyes / Q000494:pharmacology*; D005898:Glass; D009842:Oligopeptides / Q000737:chemistry; D010455:Peptides; D010750:Phosphoproteins / Q000737:chemistry; D010766:Phosphorylation; D040081:Protein Array Analysis / Q000379:methods*; D011494:Protein Kinases / Q000737:chemistry*; D011499:Protein Processing, Post-Translational; D014443:Tyrosine / Q000737:chemistry
D005456:Fluorescent Dyes; D009842:Oligopeptides; D010455:Peptides; D010750:Phosphoproteins; D014443:Tyrosine; C037642:kemptide; D011494:Protein Kinases