Anal. Chem. 2000, 72, 4603-4607
Capillary Isoelectric Focusing of Yeast Cells Yufeng Shen, Scott J. Berger, and Richard D. Smith*
Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, Washington 99352
In the present work, capillary isoelectric focusing (CIEF) methods were developed for the separation and identification of yeast cells. Yeast cells (∼4-µm diameter) cultured to various phases of growth were shown to be reproducibly resolved by CIEF using 100-µm-i.d. fused-silica capillaries coated with hydroxypropyl methylcellulose. Separation efficiencies corresponding to peak capacities of >4000 were obtained. The suitable cell concentration range for obtaining repeatable elution in CIEF separations was found to be quite low (100-µm i.d. is significantly influenced by flow resulting from a difference in reservoir levels (i.e., gravitational forces), we used 100-µm-i.d. capillaries coated with HPMC in this study. Figure 1 shows the repeatable CIEF elution behavior observed for yeast cells. The cell concentration used was ∼1.0 cell/µL in a capillary column having a volume of 5.1 µL. From the figure, it can be seen that the ∼5 cells in the capillary column were very effectively focused in the 100-µm-i.d. capillary column and eluted with symmetric peaks (peak asymmetry of ∼1). The migration time reproducibility is limited (∼4-min deviation existed during successive runs of ∼35min elution time period or ∼10% difference in elution time). Cell Concentration Range for CIEF. We found that the cell concentration is a critical factor affecting the reproducibility of CIEF analysis. Assuming that cells were uniformly distributed in solution, the number of cells loaded on a capillary column of known volume can be estimated. Figure 2 shows CIEF elution profiles obtained for different cell concentrations in a 100 µm i.d. × 65 cm capillary column. When the cell concentration was less than 2.4 cells/µL (the cell number was less than 12 cells in the capillary column), the migration of focused cells was reproducibly observed with a difference of ∼10% in migration time, which is in the same range as described in Figure 1. During the elution of the sample, some nonreproducible sharp peaks of small intensity were occasionally observed. The reason for this is not clear. When the cell concentration was increased to >3 cells/µL
or >15 cells in the capillary column, no peaks were observed. The solution was checked under microscopy prior to CIEF, and no aggregates or precipitates were observed. Since the number of cells analyzed should not have been sufficient to cause blockages of the 100-µm-i.d column, it is likely that the nonelution of the cells resulted in the formation of large aggregates or precipitates, which were strongly adsorbed on the capillary inner wall. The overloaded column could not be effectively regenerated by washing with either water or the Pharmalyte solution. Cell Separation and Characterization Based upon Surface Isoelectric Point Using CIEF. The pI is a characteristic property of amphoteric molecules and the scale can be readily established using a calibration with standard pI markers such as dyes6 and various commercially available proteins. In CIEF of cells, the measured pI values reflect the cell surface charge properties, which are determined by specific cell surface molecular properties and their higher-order structures. This means that cell surface pI measurement could be a simple, direct approach to identify cells or changes in their status. In preliminary experiments, it was found that CIEF electropherograms of standard protein pI markers contained very sharp impurity peaks. These sharp peaks resulted from unsolved species that disappeared after filtering the standard protein solution through 0.2-µm filters and can thus be attributed to large particles. Figure 3 shows the reproducibility of pI measurements for yeast cells obtained during the stationary phase of the cell growth Analytical Chemistry, Vol. 72, No. 19, October 1, 2000
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Table 1. Isoelectric Points Measured by CIEF for Yeast Cell Cultured to Different Densitiesa OD600 ) 3
OD600 ) 12
OD600 ) 30
1 2 3 4 5
5.2 5.3 5.2 5.1 5.1
6.0 6.0 6.1 6.0 6.1
6.4 6.4 6.5 6.4 6.5
av pIb ∆pImaxc RSD, %
5.2 0.2 1.3
6.0 0.1 0.7
6.4 0.1 0.6
run
a The experimental conditions are the same as in Figure 4. b Average pI value. c Maximum deviation of pI measurements.
Figure 3. Yeast cell pI characterization with standard protein pI markers using CIEF. Conditions: sample solution containing 1% (v/v) of Pharmalyte (pH 3-10), yeast cells cultured at OD600 ) 30 with a concentration of 1.0 cell/µL and 0.003 µg/µL of carbonic anhydrase I (pI 6.6) and carbonic anhydrase II (pI 5.9), respectively. Other conditions are the same as given in Figure 1. (A) standard pI markers; (B-D) standard pI markers + cell sample.
culture (OD600 ) 30). Two protein pI markers were reproducibly separated when analyzed with cells. It should also be noted that several irreproducible sharp peaks with low intensities were also observed during these runs. The major peak due to the yeast cells was reproducibly observed. However, the observed pI was not exactly the same in each separation (i.e., the major peak was observed at pI of 6.3 ( 0.1). The uncertainty of (0.1 pI unitprobably results from formation of relatively small cell aggregates in the highly focused zones, which possess slight differences in aggregate geometry. Even though some uncertainty exists in the pI measurement for cells, the difference in charge properties on yeast cell surfaces can be much more significant than this uncertainty. Table 1 gives pI measurements for cells obtained from early log (OD600 ) 3), mid log (OD600 ) 12), and stationary (OD600 ) 30) phases of cell growth. The surface charge characteristics of yeast clearly shift from acidic toward neutral pI with increasing cell culture densities. This pI shift is unlikely to be explained by a single mechanism or biochemical pathway, as the adaptation process for stationaryphase survival is highly complex.8 Differences of up to 1.2 pH units were found for the pI of yeast cells at different culture stages 4606 Analytical Chemistry, Vol. 72, No. 19, October 1, 2000
Figure 4. High-efficiency CIEF separation for a mixture of yeast cells cultured to difficult cell densities. Conditions: sample solution containing 1% (v/v) of Pharmalyte (pH 3-10), yeast cells cultured at OD600 ) 30 (stationary phase) with a concentration of 1.0 cell/µL, yeast cells cultured at OD600 ) 12 (mid log) with a concentration of 1.0 cell/µL, and yeast cells cultured at OD600 ) 3 (early log) with a concentration of 0.4 cell/µL. The lower figure shows the pI-based peak assignments using carbonic anhydrase I (pI 6.6, 0.003 µg/µL) and carbonic anhydrase II (pI 5.9, 0.003 µg/µL) as markers. Other conditions are the same as given in Figure 1.
even though they retain the same average diameter. Figure 4 shows an electropherogram for the CIEF analysis of a mixture of yeast cells cultured at various stages. Extremely narrow peaks (∼2 s at base) were observed. The separation efficiency was estimated using peak capacity under isoelectric focusing conditions.9 Using the linear Pharmalyte with a pH range of 3-10, two pI standard proteins with pI values of 6.6 and 5.9 were separated with a time period of ∼9.5 min. The peak capacity (8) Werner-Washburne, M.; Braun, E.; Johnston, G. C.; Singer, R. Microbiol. Rev. 1993, 383-401.
(resolution of unity) of this separation is estimated to be >4000 [1.5 × (10 - 3)/(6.6 - 5.9) × 9.5 × 60/2]. CIEF as a separation and identification technique should be easily expandable to other biological systems (e.g., mammalian cells) with the inclusion of appropriate nonionic osmotic stabilizing agents. Extending the range of particle sizes resolved by CIEF may be particularly useful for the study of tumor cells (and potential cancer chemotherapeutics), which are known to undergo large restructuring of their surfaces, including the overexpression and shedding of highly immunosuppressive acidic glycosphingolipids.10-12 CONCLUSIONS We have shown that yeast cells can be reproducibly resolved by CIEF in 100-µm-i.d. capillaries coated with HPMC. Extremely high column efficiencies (peak capacities of >4000) have been achieved for CIEF cell separations. Successful CIEF separations of cells were found to require limited cell concentrations. The (9) Giddings, J. C. United Separation Science; John Wiley & Sons: New York, 1991; p 181. (10) Dabelsteen, E. J. Pathol. 1996, 179, 358-369. (11) Kiessling, L. L.; Gordon, E. Chem. Biol. 1998, 5, R49-R62. (12) McKallip, R.; Li, R.; Ladisch, S. J. Immunol. 1999, 163, 3718-3726.
present studies were limited to yeast cell concentrations of less than 3 cells/µL or 15 cells in a 100 µm i.d. × 65 cm capillary column. We have found that the cell surface pI values can be determined using standard protein pI markers (small molecules compared with cells) with an uncertainty of (0.1 pI units during typical cell pI measurements. Yeast cells cultured to different cell densities displayed distinctive pI differences of up to 1.2 pI units, a difference easily discriminated using high-efficiency CIEF. We expect that such separations will be practical for a wide range of microorganisms and cell types and that differences in pI values will allow separations based upon the phenotype or physiological state of individual cells. ACKNOWLEDGMENT This work was supported by the Office of Environmental and Biological Research, U.S. Department of Energy. Pacific Northwest National Laboratory is operated by Battelle Memorial Institute for the United States Department of Energy through Contract DEACO6-76RLO 1830. Received for review February 7, 2000. Accepted July 19, 2000. AC000144W
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