Cell cycle-dependent characterization of single MCF-7 breast cancer cells by 2-D CE.
The composition of single MCF-7 breast cancer cells is characterized using 2-D CE. Individual MCF-7 cells were aspirated into a 30 mum inner diameter fused-silica capillary and lysed by contact with an SDS-containing buffer. Proteins and other primary amines were fluorescently labeled on-column using the fluorogenic dye 3-(2-furoyl)quinoline-2-carboxaldehyde. Labeled components were separated first according to molecular weight using capillary sieving electrophoresis (CSE) and then by MEKC. Analytes were detected in a sheath-flow cuvette using LIF. The expression profiles for MCF-7 cellular homogenate and a single MCF-7 cell are compared. As a proof-of-principle investigation, variation in expression was also compared within and between G1 and G2/M cell cycle phases for MCF-7 cells. Following their treatment with the viable nuclear stain Hoechst 33342, MCF-7 cells were sorted by flow cytometry on the basis of their ploidy. Sorted cells were then analyzed by 2-D CE. The degree of variability was >2.5 times larger between cells of different phases than between cells of the same phase. In typical 1 h 2-D CE separations using MCF-7 cells, over 100 components are resolved.
Harwood
Melissa M
MM
Department of Chemistry, University of Washington, Seattle, WA 98195, USA.
Bleecker
Joan V
JV
Rabinovitch
Peter S
PS
Dovichi
Norman J
NJ
D001943:Breast Neoplasms / Q000737:chemistry* / Q000378:metabolism; D002453:Cell Cycle; D020374:Chromatography, Micellar Electrokinetic Capillary; D019075:Electrophoresis, Capillary / Q000379:methods*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D005456:Fluorescent Dyes; D005663:Furans / Q000737:chemistry; D006801:Humans; D008970:Molecular Weight; D009363:Neoplasm Proteins / Q000032:analysis*; D011804:Quinolines / Q000737:chemistry; D014407:Tumor Cells, Cultured
D005456:Fluorescent Dyes; D005663:Furans; D009363:Neoplasm Proteins; D011804:Quinolines; C063756:3-(2-furoyl)quinoline-2-carbaldehyde