Enhancement of phosphoprotein analysis using a fluorescent affinity tag and mass spectrometry.
A fluorescent affinity tag (FAT) was synthesized and was utilized to selectively modify phosphorylated serine and threonine residues via beta-elimination and Michael addition chemistries in a 'one-step' reaction. This labeling technique was used for covalent modification of both phosphoproteins and phosphopeptides, allowing identification of these molecular species by fluorescence imaging after solution- or gel-based separation methods. In addition to the strong fluorescence of the rhodamine tag, a commercially available antibody can be used to enrich low-abundance post-labeled phosphopeptides present in complex mixtures. Application of this methodology to phosphorylation-site mapping has been evaluated for a phosphoprotein standard, bovine beta-casein. Initial results demonstrated low femtomole detection limits after fluorescence image analysis of FAT-labeled proteins or peptides.
Stevens
Stanley M
SM
University of Florida, Protein Chemistry Core Facility, ICBR, Gainesville, FL 32610-0156, USA. sstevens@biotech.ufl.edu
Chung
Alfred Y
AY
Chow
Marjorie C
MC
McClung
Scott H
SH
Strachan
Camille N
CN
Harmon
Alice C
AC
Denslow
Nancy D
ND
Prokai
Laszlo
L
D000345:Affinity Labels; D000818:Animals; D002364:Caseins / Q000032:analysis*; D002417:Cattle; D002846:Chromatography, Affinity / Q000379:methods*; D002851:Chromatography, High Pressure Liquid; D005453:Fluorescence; D010449:Peptide Mapping / Q000379:methods*; D012694:Serine / Q000032:analysis; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013912:Threonine / Q000032:analysis
D000345:Affinity Labels; D002364:Caseins; D013912:Threonine; D012694:Serine