Capillary zone electrophoresis for analysis of phytochelatins and other thiol peptides in complex biological samples derivatized with monobromobimane.
A new method to improve the analysis of phytochelatins and their precursors (cysteine, gamma-Glu-Cys, and glutathione) derivatized with monobromobimane (mBrB) in complex biological samples by capillary zone electrophoresis is described. The effects of the background electrolyte pH, concentration, and different organic additives (acetonitrile, methanol, and trifluoroethanol) on the separation were studied to achieve optimum resolution and number of theoretical plates of the analyzed compounds in the electropherograms. Optimum separation of the thiol peptides was obtained with 150 mM phosphate buffer at pH 1.60. Separation efficiency was improved when 2.5% v/v methanol was added to the background electrolyte. The electrophoretic conditions were 13 kV and capillary dimensions with 30 cm length from the inlet to the detector (38 cm total length) and 50 microm inner diameter. The injection was by pressure at 50 mbar for 17 s. Under these conditions, the separation between desglycyl-peptides and phytochelatins was also achieved. We also describe the optimum conditions for the derivatization of biological samples with mBrB to increase electrophoretic sensitivity and number of theoretical plates. The improved method was shown to be simple, reproducible, selective, and accurate in measuring thiol peptides in complex biological samples, the detection limit being 2.5 microM glutathione at a wavelength of 390 nm.
Perez-Rama
Mónica
M
Laboratorio de Microbiología, Facultad de Ciencias, Universidade da Coruña, La Coruña, Spain.
Torres Vaamonde
Enrique
E
Abalde Alonso
Julio
J
D001894:Borohydrides; D001643:Bridged Bicyclo Compounds; D000460:Chlorophyta / Q000378:metabolism; D004151:Dipeptides / Q000302:isolation & purification; D019075:Electrophoresis, Capillary / Q000379:methods*; D005978:Glutathione / Q000302:isolation & purification; D008623:Mercaptoethanol; D008667:Metalloproteins / Q000302:isolation & purification*; D010455:Peptides / Q000302:isolation & purification; D054811:Phytochelatins; D019163:Reducing Agents; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013438:Sulfhydryl Compounds / Q000302:isolation & purification*; D013439:Sulfhydryl Reagents
D001894:Borohydrides; D001643:Bridged Bicyclo Compounds; D004151:Dipeptides; D008667:Metalloproteins; D010455:Peptides; D019163:Reducing Agents; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; D008623:Mercaptoethanol; C025364:sodium borohydride; D054811:Phytochelatins; D005978:Glutathione; C017341:gamma-glutamylcysteine; C028381:monobromobimane