Anal. Chem. 1995, 67,4205-4209
Enddabel, Free=SolutionCapillary Electrophoresis of Highly Charged Oligosaccharides Jan Sudor and Milos V. Novotny* Department of Chemistry, Indiana University, Blmmington, Indiana 47405
The effect of fluorescent tags on the separation of negatively charged oligosaccharides, derived from a partia& hydrolyzed K-carrageenan,was studied. When the chargeto-friction ratio of oligosaccharides is increased by the end-label, the migration order is &om smaller to larger oligomers, and the resolution of larger oligomers could be improved by using a sieving medium. The migration order can be entirely reversed when the charge-to-friction ratio of the solute is decreased by the end-label. The experimental electrophoretic mobilities obtained in this work are in excellent agreement with the recently reported theoretical model (Mayer, P.; Slater, G. W.; Drouin, G. Anal. Chem. 1994, 66, 1777-1780). The maximum number of separated oligomers (M-) as a function of applied voltage and injection time was also studied, but no strong dependencies were found. Resolution between small oligomers could be significantly improved by following this separation principle. The electrophoretic mobility of any solute is proportional to its charge-to-frictionratio, and it is well known that this ratio is constant and independent of the solute size for uniformly charged polyelectrolytes’ such as DNA. To separate such polyelectrolytes by means of electrophoresis, the common remedy has been to use a sieving medium (a permanent gel or a polymer solution): in high-performance capillary electrophoresis (HPCE), this strategy has worked well for the separation of polynucleotide^,^-^ amino acid homopolymer~,5>~ and negatively charged oligosaccharide~.~Yet another strategy for separation may involve imparting additional charge or friction to the solute to alter its charge-to-frictionratio. In fact, Mayer et alV8 recently proposed a physical model and derived theoretically a set of conditions for high-performance DNA sequencing in gel-free medium, in which uniformly charged biopolymers are end-labeled with a molecular moiety (e.g., a homogeneous protein). They have called this approach “end-labeled free-solution electrophoresis” (ELFSE). The fundamental strategy for analyzing oligosaccharides and other glycoconjugates by HPCE (for a review, see refs 9 and 10) (1) Hermans, J. J. J. Polym. Sci. 1955,18, 529-534. (2) Cohen, A S.; Najarian, D. R.; Paulus, A; Guttman, A; Smith, J. A; Karger, B. L. Proc. Natl. Acad. Sci. U S A . 1988,85,9660-9663. (3) Swerdlow, H.; Wu, S.: Harke, H.; Dovichi, N. J.J. Chromaton. 1990,516. 61-67. (4) Grossman, P. D.; Soane, D. S. Biopolymers 1991,31,1221-1228. (5) Dolnik, V.; Novotny, M. V.; Chmelik, J. Biopolymers 1993,33, 1299-1306. (6) Dolnik, V.; Novotny, M. V. Anal. Chem. 1993,65, 563-5137, (7) Liu. J.; Shirota, 0.; Novotny, M. V. Anal. Chem. 1992,64, 973-975. (8) Mayer, P.; Slater, G. W.; Drouin, G. Anal. Chem. 1994,66, 1777-1780. 0003-2700/95/0367-4205$9.00/0 0 1995 American Chemical Society
OH Figure I. Structure of a K-carrageenan disaccharide unit.
happens to involve end-labeling with a fluorescent moiety. For the sake of sensitive detection by laser-induced fluorescence 0, various oligosaccharideswere labeled with CBQCA7or ANTS and other fluorescent label~.~l-’~ Separation of their derivatives was accomplished electrophoretically in either concentrated gels7 or free buffer media.l1-’3 The end-labeled oligosaccharides with a uniform charge thus seemed appropriate for testing the model by Mayer et al.,8 albeit in a very different molecular weight range than the proposed DNA sequence. Of the available oligosaccharides, we have chosen here the mixtures of partially hydrolyzed K-carrageenan [a highly charged polyelectrolyte with a linear galactan backbone which is known to contain approximately one sulfate group per disaccharide unitI4 (Figure 1)I. Of the fluorescence tags, we have selected one which increases the charge-tofriction ratio of the solute (ANTS) and another which decreases this ratio (6aminoquinoline). It should also be noted that the hydrodynamic and electrostatic interactions between the solute and reagents as well as conformationalheterogeneity of the endlabels (which seems to be a problem when working with large end-labels, such as streptavidin, avidin, etc.) were neglected in this study. First, we investigated the effect of the fluorescent tag’s nature on the electromigration behavior of carrageenan oligomers. By changing the charge and friction at the end-labelingsite, we could reverse the electromigration order. The measured electrophoretic mobilities have been further correlated with the ELBE theory: and the effects of applied voltage and injection time were also studied. (9) Novotny, M. V.; Sudor, J. Electrophoresis 1993,14, 373-389. (10) El Rassi, 2.; Nashabeh, W. High performance capillary electrophoresis of carbohydrates and glycoconjugates. In Carbohydrate Analysis: High Pecformance Liquid Chromatography and Capillary Electrophoresis; El Rassi, Z., Ed.; Elsevier Science: Amsterdam, 1994; pp 447-514. (11) Chiesa, C.; Horvath, C. J. Chromatogr. 1993,645, 337-352. (12) Stefansson, M.; Novotny, M. V. Anal. Chem. 1994,66, 1134-1140. (13) Nashabeh, W.; El Rassi, Z.J. Chromatogr. 1992,600,279-287. (14) Yalpani, M. Polysaccharides: Synthesis, Modifications and Structure/Property Relations; Elsevier: Amsterdam, 1988.
Analytical Chemistty, Vol. 67, No. 22, November 15, 1995 4205
EXPERIMENTAL SECTION
Apparatus. A homebuilt capillary zone electrophoretic (CZE) system, described previou~ly,'~ was used in all experiments. A high-voltage power supply (Spellman High Voltage Electronics, Plainview, NY) capable of delivering 0-40 kV was employed.The separation capillaries, 30-60-cm effective length (40-7km total length; 50-pm id.; 363pm o.d.), were purchased from Polymicro Technologies (Phoenix,AZ). The inner surface of the separation capillary was modified by the attachment of a linear polyacrylamide.16 The capillary was enclosed in a Plexiglas box with an interlock safety system. On-column fluorescence measurements were carried out with a helium-cadmium laser (Model 56X, Omnichrome, Chino, CA) used as a light source (5mW output power at 325 nm). The incident laser beam was aligned to its optimum by adjusting the position of the collecting optics between the optical cell and the detector. Fluorescence emission (r495 nm) was collected through a 60@pmfiber optic placed at a right angle to the incident laser beam. Signals isolated by a long-pass, low-fluorescence filter with the cutoff wavelength at 495 nm (Oriel, Stradford, CT) were monitored with a R928 photomultiplier tube (Hamamatsu Photonics KK, Toyooka Vill., Iwata-Gun, Shizuoka Prefecture, Japan) and amplified with a Model 128A lock-in ampliier (EG&G Princeton Applied Research, Princeton, NJ). The electrophoretic mobilities were calculated from 3-5 replicate measurements. The relative standard deviation of all collected data was better than f2%. Chemicals. Citric acid was purchased from EM Science (Cherry Hill, NJ), sodium hydroxide and hydrochloric acid were from Fisher Scientific (Fair Lawn, NJ), and acetic acid (glacial) was from Malliickrodt (Paris, KY). Acrylamide, ammonium persulfate, and Tris buffer [tris(hydroxymethyl)aminomethanel were received from Bio-Rad Laboratories (Hercules, CA). carrageenan and D-glucose-&sulfate were received from Sigma (St. Louis, MO), and sodium cyanoborohydride was from Aldrich (Milwaukee,WI). 8Aminonaphthalene-l,3,&trisulfonicacid (ANTS) and &aminoquinoline were the products of Molecular Probes, Inc. (Eugene, OR). The linear polyacrylamide (Mwxz (5-6) x 106) was purchased from Polysciences, Inc. (Warington, PA). Sample Preparation. Partially hydrolyzed Kmageenan and D-glucose-&sulfate were derivatized through the Schiff base formation between the aromatic amine of a reagent and the aldehyde form of a sugar, followed by reduction of the Schiff base to a stable product. The concentrationsof the reagents were 3070 mM in 3% (w/w) acetic acid.17 A 10-mg amount of the sugar was dissolved in 1 mL of 0.1 M HCl and mixed with 100 pL of reagent. The mixture was heated to 95 "C for 2 min before addition of 50 pL of 2 M sodium cyanoborohydride and then derivatized at 95 "C for 3 h. The samples were stable when stored at -20 "C before analysis. The hydrodynamic and electrokinetic techniques of sample introduction were employed. RESULTS AND DISCUSSION
Figure 2 compares the separation of the oligomers, derivatized with ANTS, from the partially hydrolyzed Kcarrageenan in (A) a gel-free medium and (B) 1%linear polyacrylamide (Mw = (5-6) x lo6), both recorded at pH = 3. It is clear that the migration (15) Liu, J.; Hsieh, Y:A; Wiesler, D.; Novotny, M. V. Anal. Chem. 1991,63, 408-412.
(16) Hjerten, S. J. Chromafogr. 1985,347, 191-198. (17)Jackson, P. Biochem. J. 1990,270,705-713.
4206 Analytical Chemistry, Vol. 67,No. 22, November 75,7995
8
7
9
time
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Figure 2. Separation of the ANTS-derivatized oligosaccharides derived from a partially hydrolyzed K-carrageenan. Numbers 5 and 10 above the peaks correspond to deca- and eicosasaccharide, respectively. Capillary: 60-cm effective length (70-cm total); 50-pm i.d. (363ym 0.d.); coated with a linear polyacrylamide. Buffer: (A) 25 mM sodium citrate, pH = 3.0; hydrodynamic injection, Ah = 10 cm, injection time, 10 s. (B) 25 mM sodium citrate, pH = 3.0; 1% linear polyacrylamide (4 (5-6) x 1Oe); electrokinetic injection, 10 s at 100 V/cm. Applied voltage: 350 V/cm (24 kV).
10
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Figure 3. Separation of 6-aminoquinoline-derivatized oligosaccharides derived from a partially hydrolyzed K-carrageenan. Conditions were the same as in Figure 2.
proceeds from smaller to larger oligomers and that the separation of larger oligosaccharides is improved in the polymer solution in comparison to the freebuffer condition. This is a well-known phenomenon, as the separation mechanisms in a polymer solution could be accounted for by the Ogston sieving model (for the large oligomers on which the label has little effect). In the case of oligosaccharides, the end-labeling happens to be a sensible remedy to detection problems?JO In most cases, the fluorescence tag is likely to have a different charge-to-friction ratio than the monomeric unit in an oligosaccharide structure and can, therefore, change the electrophoretic mobility of small oligomers significantly. This is actually seen in Figure 3 4 where the same mixture of sulfated oligosaccharides, now derivatized with a slightly positive (at pH = 3) reagent, &amin~quinoline,'~ is displayed (for better clarity of presentation, only a zoom of the entire run is shown in this figure). The migration order now starts with large oligomers (the unresolved "hump" at the beginning) and proceeds to smaller ones. This occurs because the reagent has decreased
the chargeto-friction ratio of the small oligomers significantly more than for the larger mixture components. We further demonstrate ( F i r e 3B) that the resolution of larger oligosaccharides deteriorates upon the presence of a sieving medium in the capillary. This is because the faster (larger) oligomers are now being retarded by the polyacrylamide medium more than the slower (smaller) oligomers. Using the end-labeling strategy for a gel-free medium, the following equation becomes applicable+
1P
I
!i
lo
where p and 5 are the charge and friction of one monomer unit, and B and a are the charge and friction of the end-label (normalized to the charge and/or friction of the monomer), respectively. M is the number of monomers, and po = p / 5 is the electrophoreticmobility of the polyelectrolyte without an end-label. The limiting cases of eq 1can be expressed as follows: (2)
(3) where j 3 and ~ aR are the charge and friction of the end-structure (not normalized to the charge and friction of the solute). For the case of M = 0 (eq 2), one obtains an electrophoretic mobility of the end-label which is proportional to its chargetofriction ratio. In the second case, when M >> /3 and M x =a (eq 3) (the charge as well as the friction of an oligomer are much larger than that of the end-label), one obtains the electrophoreticmobility of a polyelectrolyte not innuenced by the end-label (which is constant and size-independent). We have further used the ELFSE model of Mayer et a1.* to describe the above experiments. First, the migration times of oligosaccharides (derivatized with ANTS and/or Baminoquinoline) were measured and recalculated into the electrophoretic mobilities (in Figure 4, the electrophoretic mobilities are plotted as their absolute values). These experiments were done in 25 mM Tris-acetate buffer (PH = 8.1) to simplify the system. It is reasonable to assume that the reduced Schiff base is not protonated at such a high pH; therefore, the charges on the end-labels are given solely by their functional groups (Le., -3 for ANTS and 0 for 6aminoquinoline). To identify the separated peaks, the sample was spiked with D-glucose6sulfate. As seen in Figure 4, D-glucos&sulfate migrated before the first peak (for ANTS label) and between the first and the second peaks (for 6aminoquinoline). This proves that the first peak of the sample has a smaller chargeto-friction ratio than the monosaccharidewith one negative charge. Moreover, it is known from the literature14that the K-carrageenan contains approximately one sulfate group per disaccharide unit (Figure l),and the charge increases linearly with the number of disaccharide units. Consequently, it seems plausible that the first peak in the sample is a disaccharide, and that disaccharide (with one sulfate group) behaves as a monomeric unit. It is also important to mention that Mm, for ANTSderivatized oligosaccharides increased through elevated pH, and consequently, M,, for 6aminoquinolinederivatizedoligosaccharidesdecreased. This seems to be a logical consequence of the charge-to-frictionratio changes of end-labels at increasing pH, while the chargeto-friction
0
5
15
10
20 90
100
M
Flgure 4. Dependence of the electrophoretic mobility on the
monomeric number (nn) of oligosaccharides derived from a partially hydrolyzed K-carrageenan. Capillary: 30-cm effective length (40-cm total).Buffer: 25 mM Tris-acetate (pH = 8.1). Applied voltage: 400 V/cm (16 kV). 0 , ANTS-derivatized oligosaccharides; 0, 6-aminoquinoline-derivatized oligosaccharides. The lines are the values calculated using eq 1: a = 2.02, p = 3 (ANTS); a = 1.023, p = 0 (6-aminoquinoline);and po = 31.93 x low5cm2N.s. D-Glucose6-sulfate derivatized with ANTS; 0, ~-glucose-6-sulfatederivatized with 6-aminoquinoline.
*,
ratio of the solute remained constant (see also eqs 4 and 5 of ref 8). Another important parameter that can be obtained experimentally is the prvalue. We have estimated po from the migration time of the unseparated hump (Figures 2A and 3A: po = 31.93 x cm2/V*sf 3.06%, which is the average value for both reagents). To calculate p(w, we must also know the p- and U-values. In the case of &aminoquinoline,j3 = 0 (PH = 8.1), and a can be easily obtained from a linear form of eq 1: po/pcM,= a / M
+1
(4)
From the dependence of po/p(w on the reciprocal of M,we obtained the intercept, which was very close to unity (0.984), and the slope was 1.023 (a= 1.023, R2 = 0.997). In the case of ANTS derivatized oligosaccharides,we rearranged eq 1into the following form:
and from the intercept (extrapolating M to zero) of the dependence B(uo/p(~Jvs M,we further obtained a (a = 2.02, knowing that /3 = 3). By using the values for a, ,B, and pol we calculated next the
electrophoreticmobilities from eq 1and compared them with our experimental data (Figure 4); the points are experimental data, and the lines are the corresponding calculated mobilities. The values of po are indicated for a large M (-100). We observe excellent agreement between the experimental data and the mobilities calculated from the ELFSE model (Figure 4). Going back to Figures 1 and 2 (at pH = 3.0) and using the U-values obtained from eqs 4 and 5, the experimental electrophoretic mobilities were made to fit with the calculated values using once again eq 1. The values of p found for the best fit were Analytical Chemistry, Vol. 67, No. 22, November 15, 1995
4207
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2.7 for ANTS and -0.6 for 6-aminoquinoline F i e 5), indicating the charge of ANTS to be approximately -2.7, and that for &aminoquinoline, +0.6 (at pH = 3.0). The electrophoretic mobilities of oligosaccharidesobtained in 1%linear polyacrylamide did not agree with the calculated values because, besides the ELFSE principle, the Ogston sieving model is also involved in the separation mechanism Figure 5). Next, we tested the limiting behavior of the ELFSE model as described by Mayer et alS8This model predicts for M >> a and a small initial width of the bands, WO,that M m , =V3(V= EL, where E is the applied electric field strength and L is the effective length of the separation capillary [see eq 4, ref 81). M,, was measured as a function of applied voltage (in kv) (Figure 6), and the exponents were obtained from a log-log plot (the slope is 0.15 for ANTSderivatized and 0.07 for 6-aminoquinoline-derivatized oligosaccharides, while the theoretical value is l/3). We assume that there is only a slight dependence of Mmw on the applied voltage in this case, concluding that M is not much larger than a. This dependence should be more important when larger endlabels are used, but this does not seem to be particularly pertinent to a typical oligosaccharide analysis. In the second limiting case (for small molecules and large WO), M,, = I . U O ~ / ~ (see eq 5 of ref 8), we measured M,, as a function of injection time (in s). Because the amount of injected sample was not a linear function of injection time, we measured the area of a selected peak (the second peak in the sample, corresponding to tetrasaccharide) and plotted it logarithmically as a function of injection time [the slope was 0.96 (F= 0.998) for ANTS and 0.84 (F= 0.996) for 6-aminoquinolinel. Consequently, the injection times were recalculated according to the slopes (corrected injection time = injection timeslop). Interestingly, the slope for 6aminoquinolinederivatized oligosaccharides was smaller than that for ANTSderivatized oligosaccharides. This is most probably due to the different viscosities of these two samples. The molar concentration of 6-aminoquinoline in the sample during derivatization was approximately twice that of ANTS. Because both 4208 Analytical Chemistry, Vol. 67, No. 22,November 15, 1995
' 2
'
10
30 V / kV
M
Figure 5. Dependence of the electrophoretic mobilities on the monomeric number ( M ) of the oligosaccharides derived from a partially hydrolyzed K-carrageenan. 0, ANTS-derivatized oligosaccharides; 0, 6-aminoquinoline-derivatizedoligosaccharides. Conditions were the same as in Figure 2A. Corresponding lines are the calculated values using eq 1: a = 2.02, /3 = 2.7 (ANTS); a = 1.023, /3 = -0.6 (6-aminoquinoline);pco= 31.93 x cm2N-s. +, ANTSderivatized oligosaccharides; 0, 6-aminoquinoline-derivatized oligosaccharides. Conditions were the same as in Figure 26.
'
Flgure 6. Dependence of Mmax on the applied voltage (kV). 0, ANTS-derivatized oligosaccharides; 0, 6-aminoquinoline-derivatized oligosaccharides. Capillary: 30-cm effective length (40-cm total). The slopes are 0.15 (ANTS) and 0.07 (6-aminoquinoline). +, Measured current @A) (slope = 1.2). Other conditions were the same as in Figure 2A. 100
I
i
I 1
3
10
100
corrected injection time / sec
Figure 7. Dependence of Mmax on injection time. Applied voltage: 400 V/cm (16 kV). The slopes are -0.15 (ANTS) and -0.005 (6aminoquinoline). Ah = 10 cm. Other conditions were the same as in Figure 6.
reagents are weak bases, the hydrogen ions were more easily neutralized by &aminoquinoline,and the corresponding carrageenan sample was less hydrolyzed than the sample using ANTS. Once again, we obtained the slopes from the log-log plot of M,, vs corrected injection time (Figure 7). No strong dependence was visible [slope was -0.15 (for ANTS, the less viscous solution) and -0.005 (for &aminoquinoline)]. The measurements were done on the capillary with a total length of 40 cm. We noticed that M,, was more dependent on injection time in shorter capillaries (e.g., 20cm total length), but the absolute values of exponent were still much smaller than '/z (results not shown). It should be noted that, due to the high sensitivity of the LIF technique and a higher viscosity of sample compared to the buffer used, the injection time should not significantly influence Mma. We experienced a strong dependence of Mm on the migration distance (the effective length of a separation capillary, L). Nevertheless, as the experimental results obtained here were slightly influenced by different values of ut0 for varying capillary
25
20
CONCLUSIONS
J
lo i
e
I
0 0
0
0 ' 0
'
'
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"
5
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15
A M Figure 8. Resolution between the neighboring oligosaccharides. K),where tm is the (Resolution is defined as Ri,,= 2(fm,j - fm,i)/( migration time and Y is the peak width at the baseline). Note that resolution between the first and second peaks is shown at AM = 1, between the second and the third peaks at AM = 2, etc. 0 , ANTSderivatized oligosaccharides; 0, 6-aminoquinoline derivatized oligosaccharides. Other conditions were the same as in Figure 2A.
+
lengths, we find that the values of M,, are typically proportional to L1/3-L1/2. Finally, we were interested in the effect of reagents on resolution between neighboring oligosaccharide molecules. This dependence is shown in Figure 8. Note that resolution between the first and the second peaks is shown at position one (on the x-axis), resolution between the second and third peaks at position 2, etc. As seen in Figure 8, resolution between small oligosaccharides derivatized with Gaminoquinolineis significantly better than that for the oligosaccharides labeled by ANTS. This could be explained by a greater difference in the charge-to-friction ratio between Gaminoquinoline and a disaccharide unit than the corresponding relation for ANTS and a disaccharide unit. However, resolution between the adjacent oligosaccharides decreases relatively fast in both cases and vanishes for larger oligomers.
We have shown that the charge-to-friction ratio due to an endlabel has a significant effect on the electrophoretic mobility of fluorescently labeled, charged oligosaccharides. By changing a charge and/or friction of the end-label, the migration order can be reversed. This principle was hypothetically described elsewhere! and a theoretical model was derived for the sake of improved DNA sequencing in gel-free media. We have shown here experimentally that the same model is applicable to additional cases of the oligomers with a constant chargeto-friction ratio (independent of their size) which are labeled with an end-label whose charge-to-friction ratio is different from that of the oligomers. In all likelihood, the range of resolved components and their resolution can be adjusted by molecular design of appropriate end-labels. Since the fluorescence tags in oligosaccharide analysis are rather small, Mmawdoes not depend on the applied voltage. In addition, we have not experienced a strong dependence of M,, on injection time, which supports the idea that, through using LIF detection and a higher sample viscosity, the values of tug are small, and M,, is not limited by injection time. However, we experienced some dependence of M,, on the effective length of a separation capillary. ACKNOWLEDGMENT This research was supported by Grant No. CHE-9321431from the National Science Foundation and a grant-in-aid from Astra/ Hassle, Molndal, Sweden. The authors thank Anastasia Crowell for her information on derivatization of K-carrageenan, which proved very useful in this work.
Received for review July 14, 1995. Accepted September 8, 1995.@ AC950701S
@
Abstract published in Adoance ACS Abstracts, October 15, 1995.
Analytical Chemistry, Vol. 67, No. 22, November 15, 1995
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