Enhanced sensitivity employing zwitterionic and pI balancing dyes (Z-CyDyes) optimized for 2D-gel electrophoresis based on side chain modifications of CyDye fluorophores. New tools for use in proteomics and diagnostics.
The CyDye family of fluorescent dyes is currently the overwhelming choice for applications in proteomic analysis, using two-dimensional difference gel electrophoresis (2D-DIGE). Protein labeling with CyDyes is hampered by protein precipitation and gel smearing when used above minimal labeling. The solubility of labeled protein may be improved by introducing water solubilizing groups on the dye such as cysteic acids. However, addition of a negatively charged functionality will have the undesired effect of shifting the pI in relation to the unlabeled protein. These limitations have been addressed through the synthesis of highly water-soluble and pI balancing zwitterionic CyDye fluorophores (Z-CyDyes). The new dyes feature a cysteic acid motif, a titratable amine functionality and a NHS activated ester group. In side by side 2D-DIGE comparisons of Z-CyDyes and CyDyes, the new dyes significantly enhanced protein spot volume and the number of spots that were detected. Z-CyDyes have the potential to enhance the depth of proteome coverage and provide a general strategy for improving the performance of protein tagging reagents.
Department of Chemistry and Biochemistry, Montana State University , PO Box 173400, Bozeman, Montana 59717-3400, United States.
Mark G
MG
Epstein
Benjamin D
BD
Reeves
Walid S
WS
Maaty
David
D
Fouchard
Edward A
EA
Dratz
Brian
B
Bothner
Paul A
PA
Grieco
D019843:Archaeal Proteins / Q000032:analysis*; D002232:Carbocyanines / Q000032:analysis; D003545:Cysteine / Q000031:analogs & derivatives*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D005456:Fluorescent Dyes / Q000032:analysis*; D040901:Proteomics / Q000379:methods*; D012995:Solubility; D013194:Staining and Labeling / Q000379:methods; D048229:Sulfolobus solfataricus / Q000737:chemistry*; D014867:Water / Q000737:chemistry
D019843:Archaeal Proteins; D002232:Carbocyanines; D005456:Fluorescent Dyes; C089187:cyanine dye 3; C085321:cyanine dye 5; D014867:Water; D003545:Cysteine