Biarsenical-tetracysteine motif as a fluorescent tag for detection in capillary electrophoresis.
Biarsenical dyes complexed to tetracysteine motifs have proven to be highly useful fluorescent dyes in labeling specific cellular proteins for microscopic imaging. Their many advantages include membrane permeability, relatively small size, stoichiometric labeling, high affinity, and an assortment of excitation/emission wavelengths. The goal of the current study was to determine whether the biarsenical labeling scheme could be extended to fluorescent detection of analytes in capillary electrophoresis. Recombinant protein or synthesized peptides containing the optimized tetracysteine motif "-C-C-P-G-C-C-" were labeled with biarsenical dyes and then analyzed by micellar electrokinetic capillary chromatography (MEKC). The biarsenical-tetracysteine complex was stable and remained fluorescent under standard MEKC conditions for peptide and protein separations. The detection limit following electrophoresis in a capillary was less than 3 x 10(-20) mol with a simple laser-induced fluorescence system. A mixture of multiple biarsenical-labeled peptides and a protein were easily resolved. Demonstrating that the label did not interfere with bioactivity, a peptide-based enzyme substrate conjugated to the tetracysteine motif and labeled with a biarsenical dye retained its ability to be phosphorylated by the parent kinase. The feasibility of using this label for chemical cytometry experiments was shown by intracellular labeling and subsequent analysis of a recombinant protein possessing the tetracysteine motif expressed in living cells. The extension of the biarsenical-tetracysteine tag to fluorescent labeling of peptides and proteins in chemical separations is a valuable addition to biochemical and cell-based investigations.
Kottegoda
Sumith
S
Department of Chemistry, University of North Carolina, Chapel Hill, North Carolina 27599-3290, USA.
Aoto
Phillip C
PC
Sims
Christopher E
CE
Allbritton
Nancy L
NL
D020816:Amino Acid Motifs; D003545:Cysteine / Q000737:chemistry; D019075:Electrophoresis, Capillary / Q000379:methods*; D004926:Escherichia coli / Q000737:chemistry / Q000166:cytology / Q000235:genetics / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000032:analysis*; D007834:Lasers; D010455:Peptides / Q000032:analysis* / Q000737:chemistry; D010766:Phosphorylation; D011506:Proteins / Q000032:analysis / Q000737:chemistry; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D005456:Fluorescent Dyes; D010455:Peptides; D011506:Proteins; D011994:Recombinant Proteins; D003545:Cysteine