CE separation of proteins and yeasts dynamically modified by PEG pyrenebutanoate with fluorescence detection

Abstract

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.

Separation of similar yeast strains by IEF techniques

“…The isoelectric points of most MOs lie within the pH range reaching from 1.5 to 4.5 [24,25] and/or in the case of studied yeasts, C. albicans and C. parapsilosis, C. tropicalis biofilm-negative , in the pH range from 2 to 4 [26]. The reproducible results and the smooth pH gradient in the pH range were achieved at CIEF by means of the segmental injection [21,22].…”

“…Initially CIEF was based on the knowledge of the isoelectric points of the clinically important yeasts, C. albicans (pI 2.8) and C. parapsilosis (À) (pI 3.8) and C. tropicalis (pI 2.5), determined previously [26]. Preliminary CIEF of tested yeasts (8 Â 10 7 cell/mL), C. dubliniensis and C. parapsilosis (+) and C. tropicalis (+) in the pH gradient in the pH range 3-10 was performed before the optimization of the experimental conditions.…”

“…biofilm-negative strains. It may be achieved by the selection of appropriate additives to buffer solutions [26,27,[30][31][32][33] such as PEG [19,21,26,34,[36][37][38][39]. The peak shape can also be improved by the use of permanent capillary coating [39].…”

“…We suppose that pI's of C. dubliniensis and C. albicans, as well as biofilm-positive and biofilm-negative strains, can be very similar. The pH gradient in the required pH range [26] will be necessary to modify for better separation and identification of the Candida strains according to their pI's.…”

“…The reproducible results and the smooth pH gradient in the pH range were achieved at CIEF by means of the segmental injection [21,22]. Some difficulties, such as the adsorption of MOs onto the capillary wall [26][27][28][29], are necessary to solve with respect to the different surface properties of the separated cells especially biofilm-positive and. biofilm-negative strains.…”