Instrumentation for medium-throughput two-dimensional capillary electrophoresis with laser-induced fluorescence detection.
In two-dimensional capillary electrophoresis, a sample undergoes separation in the first dimension capillary by sieving electrophoresis. Fractions are periodically transferred across an interface into a second dimension capillary, where components are further resolved by micellar electrokinetic capillary electrophoresis. Previous instruments employed one pair of capillaries to analyze a single sample. We now report a multiplexed system that allows separation of five samples in parallel. Samples are injected into five first-dimension capillaries, fractions are transferred across an interface to 5 second-dimension capillaries, and analyte is detected by laser-induced fluorescence in a five-capillary sheath-flow cuvette. The instrument produces detection limits of 940 +/- 350 yoctomoles for 3-(2-furoyl)quinoline-2-carboxaldehyde labeled trypsin inhibitor in one-dimensional separation; detection limits degrade by a factor of 3.8 for two-dimensional separations. Two-dimensional capillary electrophoresis expression fingerprints were obtained from homogenates prepared from a lung cancer (A549) cell line, on the basis of capillary sieving electrophoresis (CSE) and micellar electrophoresis capillary chromatography (MECC). An average of 131 spots is resolved with signal-to-noise greater than 10. A Gaussian surface was fit to a set of 20 spots in each electropherogram. The mean spot width, expressed as standard deviation of the Gaussian function, was 2.3 +/- 0.7 transfers in the CSE dimension and 0.46 +/- 0.25 s in the MECC dimension. The standard deviation in spot position was 1.8 +/- 1.2 transfers in the CSE dimension and 0.88 +/- 0.55 s in the MECC dimension. Spot capacity was 300.
Zhu
Cuiru
C
Department of Chemistry, University of Washington, Seattle, Washington 98195-1700, USA.
He
Xinya
X
Kraly
James R
JR
Jones
Megan R
MR
Whitmore
Colin D
CD
Gomez
David G
DG
Eggertson
Michael
M
Quigley
Wes
W
Boardman
Anna
A
Dovichi
Norman J
NJ
D001679:Biogenic Amines / Q000032:analysis*; D045744:Cell Line, Tumor; D020374:Chromatography, Micellar Electrokinetic Capillary; D019075:Electrophoresis, Capillary / Q000295:instrumentation*; D005453:Fluorescence; D005663:Furans / Q000032:analysis; D006801:Humans; D007834:Lasers; D008175:Lung Neoplasms; D011506:Proteins / Q000032:analysis*; D011804:Quinolines / Q000032:analysis; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000295:instrumentation*; D014361:Trypsin Inhibitors / Q000032:analysis
D001679:Biogenic Amines; D005663:Furans; D011506:Proteins; D011804:Quinolines; D014361:Trypsin Inhibitors; C063756:3-(2-furoyl)quinoline-2-carbaldehyde