Use of a small reporter molecule to determine cell-surface proteins by capillary electrophoresis and laser-induced fluorescence: use of 5-SAENTA-x8f for quantitation of the human equilibrative nucleoside transporter 1 protein.
The human equilibrative nucleoside transporter 1 protein (hENT1) is a major mediator of cellular entry of nucleosides and anticancer nucleoside drugs; its assay is important in understanding and diagnosing chemotherapy resistance. Here we present a novel assay for quantifying hENT1 using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). A cellular population is treated with 5'-S-(2-aminoethyl)-N6-(4-nitrobenzyl)-5'-thioadenosine-x8-fluorescein (5-SAENTA-x8f), which binds with high affinity and specificity to the hENT1 protein. The cells are washed to remove excess reagent, lysed, and centrifuged, and the supernatant is analyzed by CE-LIF with the use of an internal standard. Accuracy was evaluated by comparing the capillary electrophoresis results with those obtained by flow cytometry; the results were highly correlated, r = 0.96. The relative standard deviation of the hENT1 assay was 10%, determined from nine independent assays of the same cell line, which is 3 times superior to results obtained in a flow cytometry assay. The detection limit for 5-SAENTA-x8f was 4300 molecules injected into the capillary.
Li
Nan
N
Department of Chemistry, University of Alberta, Edmonton, Canada.
Cook
Lillian
L
Santos
Cheryl
C
Cass
Carol E
CE
Mackey
John R
JR
Dovichi
Norman J
NJ
D000241:Adenosine / Q000031:analogs & derivatives* / Q000737:chemistry*; D000345:Affinity Labels; D019075:Electrophoresis, Capillary; D033721:Equilibrative Nucleoside Transporter 1; D005434:Flow Cytometry; D005453:Fluorescence; D006801:Humans; D007202:Indicators and Reagents; D007834:Lasers; D008565:Membrane Proteins / Q000032:analysis*; D026901:Membrane Transport Proteins / Q000032:analysis*; D013872:Thionucleosides / Q000737:chemistry*; D014407:Tumor Cells, Cultured
D000345:Affinity Labels; D033721:Equilibrative Nucleoside Transporter 1; D007202:Indicators and Reagents; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; C438649:SLC29A1 protein, human; D013872:Thionucleosides; C066257:5'-S-(2-aminoethyl)-N(6)-(4-nitrobenzyl)-5'-thioadenosine; D000241:Adenosine