Abstract
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.
Original language | English |
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Pages (from-to) | 2157-2162 |
Number of pages | 6 |
Journal | Rapid Communications in Mass Spectrometry |
Volume | 19 |
Issue number | 15 |
DOIs | |
State | Published - 2005 |