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...

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Bibliographic Details
Published in:Rapid communications in mass spectrometry 2005-01, Vol.19 (15), p.2157-2162
Main Authors: Stevens Jr, Stanley M., Chung, Alfred Y., Chow, Marjorie C., McClung, Scott H., Strachan, Camille N., Harmon, Alice C., Denslow, Nancy D., Prokai, Laszlo
Format: Article
Language:English
Subjects:
Affinity Labels
Animals
Caseins - analysis
Cattle
Chromatography, Affinity - methods
Chromatography, High Pressure Liquid
Fluorescence
Peptide Mapping - methods
Serine - analysis
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - methods
Threonine - analysis
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Description
Summary: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. Copyright © 2005 John Wiley & Sons, Ltd.
ISSN:0951-4198
1097-0231
DOI:10.1002/rcm.2027