Capillary sodium dodecyl sulfate-DALT electrophoresis of proteins in a single human cancer cell.
Capillary Sodium dodlecyl sulfate (SDS)-DALT an (abbreviation for Dalton) electrophoresis was applied to analysis of proteins in single HT29 human colon adenocarcinoma cells. A vacuum pulse was employed to introduce a single cell into the coated capillary. Once the cell was lysed, proteins were denatured with SDS, fluorescantly labeled with 3-(2-furoyl)-quinoline-2-carboxaldehyde (FQ), and then separated by using 8% pullulan as the sieving matrix. This method offers a few advantages for single-cell protein analysis. First, it provides reproducible separation of single-cell proteins according to their size. Based on comparison with the migration time of standard proteins, most components from a single HT29 cancer cell have molecular masses within the range of 10-100 kDa. Second, as a one-dimensional separation method, it gives fairly good resolution for proteins. Typically, around 30 protein components of a single HT29 cell were resolved, indicating that this method has similar peak capacity to SDS-polyacrylamide gel electrophoresis (PAGE). Third, this method shows high detection sensitivity and wide dynamic range, which is important because of the wide range of protein expression in living systems. Detection limits for standard proteins ranged from 10(-10) to 10(-11) M. Finally, this method provides much higher speed than classical gel electrophoresis methods, and it provides automated anlysis of cellular proteins at the single-cell level; the separation is complete in 30 min and the entire analysis takes approximately 45 min.
Hu
S
S
Department of Chemistry, University of Washington, Seattle 98195-1700, USA.
Zhang
L
L
Cook
L M
LM
Dovichi
N J
NJ
D000230:Adenocarcinoma / Q000737:chemistry; D003110:Colonic Neoplasms / Q000737:chemistry; D019075:Electrophoresis, Capillary / Q000379:methods; D005456:Fluorescent Dyes; D006801:Humans; D008970:Molecular Weight; D009363:Neoplasm Proteins / Q000032:analysis*; D009369:Neoplasms / Q000737:chemistry*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured
D005456:Fluorescent Dyes; D009363:Neoplasm Proteins