title: Strategies and solid-phase formats for the analysis of protein and peptide phosphorylation employing a novel fluorescent phosphorylation sensor dye.
abstract: Protein kinases represent one of the largest families of regulatory enzymes, with more than 2,000 of them being encoded for by the human genome. Many cellular processes are regulated by the reversible phosphorylation of proteins and upwards of 30% of the proteins comprising the eukaryotic proteome are likely to be phosphorylated at some point during their existence. In the past, analysis of global protein phosphorylation has been accomplished through radiolabelling of samples with inorganic (32P or [gamma-32)P] ATP. The approach is limited to specimens amenable to radiolabelling and poses certain safety and disposal problems. Alternatively, immunodetection with antibodies to the common phosphoamino acids may be employed, but the antibodies are relatively expensive and exhibit limited specificity and a certain degree of cross-reactivity. Pro-Q Diamond dye is a new fluorescent phosphosensor technology suitable for the detection of phosphoserine-, phosphothreonine- and phosphotyrosine-containing proteins directly in isoelectric focusing gels, SDS-polyacrylamide gels and two-dimensional gels. Additionally, the technology is appropriate for the detection of phosphoproteins or phosphopeptides arrayed on protein chips or affixed to beads. Dye-stained proteins and peptides can be excited with a laser-based light source of 532 or 543 nm or with a xenon-arc lamp-based system equipped with appropriate band pass filters. Alternatively, ultraviolet light of about 302 nm may be employed, providing that sufficiently long exposure times are used to collect the fluorescence signal. Pro-Q Diamond dye emits maximally at approximately 580 nm. The fluorescence-based detection technology is easy to conduct, cost effective and allows rapid large-scale screening of protein and peptide phosphorylation in a variety of solid-phase assay formats.
authors.affiliation: Molecular Probes, Inc., Eugene, Oregon 97402, USA. wayne.patton@probes.com
authors.forename: Karen
authors.initials: K
authors.lastname: Martin
authors.forename: Thomas H
authors.initials: TH
authors.lastname: Steinberg
authors.forename: Terrie
authors.initials: T
authors.lastname: Goodman
authors.forename: Birte
authors.initials: B
authors.lastname: Schulenberg
authors.forename: Jason A
authors.initials: JA
authors.lastname: Kilgore
authors.forename: Kyle R
authors.initials: KR
authors.lastname: Gee
authors.forename: Joseph M
authors.initials: JM
authors.lastname: Beechem
authors.forename: Wayne F
authors.initials: WF
authors.lastname: Patton
mesh_terms: D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism; D008863:Microspheres; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D010748:Phosphopeptides / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D010750:Phosphoproteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D040081:Protein Array Analysis; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
chemical_list: D005456:Fluorescent Dyes; D010455:Peptides; D010748:Phosphopeptides; D010750:Phosphoproteins; D011506:Proteins