Amino acid analysis in five pooled single plant cell samples using capillary electrophoresis coupled to laser-induced fluorescence detection.
In this study 21 amino acid standards, samples of pure phloem sap and samples of pooled mesophyll cells were derivatized with fluorescein isothiocyanate, separated by capillary electrophoresis and detected with laser-induced fluorescence at 488 nm. Two different background electrolytes, a sodium borate buffer containing sodium dodecyl sulfate and a sodium borate buffer containing alpha-cyclodextrin, were used for the separation. Using the sodium dodecyl sulfate buffer, 14 amino acid standards could be separated, spiking identified 12 amino acids in pure phloem sap and 13 amino acids in pooled mesophyll cells. With the alpha-cyclodextrin containing background electrolyte, a resolution of 20 amino acid standards could be attained, 17 amino acids in pure phloem sap and 10 amino acids in mesophyll cells could be assigned. Leucine and isoleucine comigrated in both buffer systems. All separations were performed with a voltage of +20 kV and completed within 30 min. The detection limits obtained were in the fmol range for the sodium dodecyl sulfate and in the pmol range for the alpha-cyclodextrin background electrolyte. Compared to the one published capillary electrophoresis-based method for the determination of amino acids from few plant cells, the procedure described here allows very high sensitivity due to the use of laser-induced fluorescence detection and opens the possibility to dilute and measure pl samples with an fully automated, commercially available CE system.
Arlt
K
K
Max-Planck-Institut für Molekulare Pflanzenphysiologie, Department Willmitzer, Golm, Germany.
Brandt
S
S
Kehr
J
J
D000596:Amino Acids / Q000032:analysis*; D028464:Cucurbita / Q000737:chemistry* / Q000166:cytology; D019075:Electrophoresis, Capillary / Q000379:methods*; D007834:Lasers; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods*
D000596:Amino Acids