Enhancement of phosphoprotein analysis using a fluorescent affinity tag and mass spectrometry
Article first published online: 29 JUN 2005
Copyright © 2005 John Wiley & Sons, Ltd.
Rapid Communications in Mass Spectrometry
Volume 19, Issue 15, pages 2157–2162, 15 August 2005
How to Cite
Stevens, S. M., Chung, A. Y., Chow, M. C., McClung, S. H., Strachan, C. N., Harmon, A. C., Denslow, N. D. and Prokai, L. (2005), Enhancement of phosphoprotein analysis using a fluorescent affinity tag and mass spectrometry. Rapid Commun. Mass Spectrom., 19: 2157–2162. doi: 10.1002/rcm.2027
- Issue published online: 29 JUN 2005
- Article first published online: 29 JUN 2005
- Manuscript Revised: 23 MAY 2005
- Manuscript Accepted: 23 MAY 2005
- Manuscript Received: 3 MAR 2005
- National Center for Research Resources, Bethesda, MD, USA. Grant Number: RR 016780
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.