Yoctomole analysis of ganglioside metabolism in PC12 cellular homogenates.
We report an ultrasensitive method for the analysis of glycosphingolipid catabolism. The substrate G(M1) and the set of seven metabolites into which it can be degraded (G(A1), G(M2), G(A2), G(M3), LacCer, GlcCer, and Cer) were labeled with the highly fluorescent dye tetramethylrhodamine. CE with LIF detection was used to assay these compounds with 150 +/- 80 yoctomole mass (1 ymol = 10(-24) mol = 0.6 copies) detection limits and 5 +/- 3 pM concentration detection limits. An alignment algorithm based on migration of two components was employed to correct for drift in the separation. The within-day and between-day precision in peak height was 20%, in peak width 15%, and in adjusted migration time 0.03%. After normalization to total sample injected, the RSD in peak height reduced to 2-6%, which approaches the limit set by molecular shot noise in the number of molecules taken for analysis. PC12 cells were incubated with the labeled G(M1). Fluorescent microscopy demonstrated uptake by the cells. CE was used to separate a cellular homogenate prepared from these cells. A set of peaks was observed, which were tentatively identified based on comigration with the standards. Roughly 120 pL of homogenate was injected, which contained a total of 150 zmol of labeled substrate and products. Metabolite that preserves the fluorescent label can be detected at the yoctomole level, which should allow characterization of this metabolic pathway in single cells.
Department of Chemistry, University of Washington, Seattle, WA, USA.
Colin D
CD
Whitmore
Ulf
U
Olsson
E Andreas
EA
Larsson
Ole
O
Hindsgaul
Monica M
MM
Palcic
Norman J
NJ
Dovichi
D000818:Animals; D019075:Electrophoresis, Capillary / Q000379:methods; D005456:Fluorescent Dyes / Q000737:chemistry; D016537:Gangliosidosis, GM1 / Q000378:metabolism*; D016716:PC12 Cells; D051381:Rats; D012235:Rhodamines / Q000737:chemistry
D005456:Fluorescent Dyes; D012235:Rhodamines; C005358:tetramethylrhodamine