CE separation of proteins and yeasts dynamically modified by PEG pyrenebutanoate with fluorescence detection.
The optimized protocols of the bioanalytes separation, proteins and yeasts, dynamically modified by the nonionogenic tenside PEG pyrenebutanoate, were applied in CZE and CIEF with the acidic gradient in pH range 2-5.5, both with fluorescence detection. PEG pyrenebutanoate was used as a buffer additive for a dynamic modification of proteins and/or yeast samples. The narrow peaks of modified analytes were detected. The values of the pI's of the labeled proteins were calculated using new fluorescent pI markers in CIEF and they were found to be comparable with pI's of the native compounds. As an example of the possible use of the suggested CIEF technique, the mixed cultures of yeasts, Candida albicans, Candida glabrata, Candida kefyr, Candida krusei, Candida lusitaniae, Candida parapsilosis, Candida tropicalis, Candida zeylanoides, Geotrichum candidum, Saccharomyces cerevisiae, Trichosporon asahii and Yarrowia lipolytica, were reproducibly focused and separated with high sensitivity. Using UV excitation for the on-column fluorometric detection, the minimum detectable amounts of analytes, femtograms of proteins and down to ten cells injected on the separation capillary, were estimated.
Horká
Marie
M
Institute of Analytical Chemistry, Academy of Sciences of the Czech Republic, Brno, Czech Republic. horka@iach.cz
Růzicka
Filip
F
Holá
Veronika
V
Slais
Karel
K
D002175:Candida / Q000302:isolation & purification; D019075:Electrophoresis, Capillary / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D005848:Geotrichum / Q000302:isolation & purification; D007525:Isoelectric Focusing / Q000379:methods*; D011092:Polyethylene Glycols / Q000737:chemistry* / Q000494:pharmacology; D011506:Proteins / Q000302:isolation & purification*; D012441:Saccharomyces cerevisiae / Q000302:isolation & purification; D014250:Trichosporon / Q000302:isolation & purification; D025062:Yarrowia / Q000302:isolation & purification; D015003:Yeasts / Q000187:drug effects / Q000302:isolation & purification*
D005456:Fluorescent Dyes; D011506:Proteins; D011092:Polyethylene Glycols