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Sensitive, Laser-Assisted Determination of Complex Oligosaccharide Mixtures Separated by Capillary Gel Electrophoresis at High Resolution Jinping Liu, Osamu Shirota, and Milos V. Novotny* Department of Chemistry, Indiana University, Bloomington, Indiana 47405 INTRODUCTION Scientific interest in complex carbohydrates has been rapidly increasing due to their multilateral biological roles in glycoproteins, proteoglycans, and various natural polysaccharide forms. It is commonly perceived that substantial progress in this wide-ranging area of carbohydrate biochemistry and physiology has been primarily hampered by the lack of effective analytical methodology for resolving complex mixtures, often available only a t low sample concentrations. Traditional separation and measurement techniques, such as gas chromatography, gel filtration, or various forms of highperformance liquid chromatography, often lack an adequate resolving power to distinguish numerous oligomers and isomeric species that may be present in a sample of interest. In addition, the general lack of detectable moieties within a typical carbohydrate structure further complicates the task of high-sensitivity measurements.' Although the scope of carbohydrate analysis has recently been enhanced by an effective application of ion-exchange principles and pulsed amperometric detection?* such improvement still falls short of the general needs in carbohydrate analysis. We report here the use of capillary gel electrophoresis with laser-induced fluorescence detection as a new and extremely powerful approach to the analysis of complex oligosaccharide mixtures. The virtues and potential of capillary electrophoresis (CE) have recently been explored with complex oligonucleotide proteins (see ref 9 for a review), and a variety of small molecules. We have recently reported CE separations of the fluorescent derivatives of reducing neutral sugars'O and amino sugars" in the open tubular format; however, the unfavorable mass-to-charge ratio of higher oligosaccharides prevented their effective resolution. This paper describes substantial improvements in this regard, made using a series of oligomers derived from polygalacturonic acid.
EXPERIMENTAL SECTION Fused silica capillaries (50-pm i.d. x 30-cm length; 23-cm effective separation length) were filled with poly(acry1amide) gels at high concentrations after deactivation of the capillary wall. A progressive polymerization (isotachophoretic)method, developed in this laboratory,12was used to prepare such columns. Highly concentrated gels (up to 30% T and 3% C)13were found essential to success with complex samples of oligosaccharides. The gel-fded columns were conditioned with the operating buffer (0.1 M Tris/0.25 M boric acid/:! mM EDTA; pH = 8.48) prior to their use in separation. High voltage, applied for a few seconds during sampling, was used as the principal means of introducing adequate samples of the analyzed carbohydrates. The electric field applied during the reported separation was 234 V/cm, with a measured The electrophoresis apparatus used in this study current of 6.5 4. was described previ0us1y.l~ On-column fluorescence measurements were performed with the previously described detections system14 featuring an Argon-ion laser (Model 543-AP, from Omnichrom, Chino, CA) operated at the wavelength of 457 nm as the light source. Optical window was made by removing the polyimide coating for a short section of the fused silica capillary before filling the column with *To whom all correspondence should be addressed.
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Flgura 1. Separatbn of oilgo" derlved from an autoclave hydroiysls of poly(galacturon1c ackl). The numbers lndlcate estlmated degree of polymerlzatlon repeated by a monosaccharlde unh. Capillary: 5 G ~ m i.d. X 3 2 t m (23-cm effective length). Poly(acry1amMe) gel concenisatkm 18% 1,3% C. Buffer: 0.1 M TrlsIO.25 M boratel2 mM EDTA (pH = 8.48). Electromigration injection: 5 kV, 25 s. Applied electric flekl: 234 Vlcm (0.5 MA).
a gel matrix. The incident laser beam was aligned to ita optimum position of focus (maximum sensitivity) by adjusting the positioner holding the capillary. A 600-~mfiber-optic mounted at a right angle to the incident beam was employed to collect fluorescence emieaion at 550 nm. The p i t i o n e n holding the column and fiber were fine-adjusted by monitoring the fluorescence signal that originated from a test sample. Poly(galacturonic acid) was obtained from Sigma (St. Louis, MO). Prior to derivatization with 3-(4-carboxybenzoyl)-2-quinolinecarboxaldehyde(CBQCA),14 poly(galacturonic acid) was subjected to hydrolysis in an autoclave (at 120 "C for 50 min) and reductively aminated.1° The fluorogenic reagent, CBQCA, was synthesized in this laboratory, as reported previou~ly.~~
RESULTS AND DISCUSSION In order to detect carbohydrates a t high sensitivity, it is essential to attach a fluorophore to the molecules of interest. For the oligosaccharides with a reducing end group, as is the case for oligogalacturonic acid, the reductive amination is followed by the reaction with 3-(4-carboxybenzoyl)-2quinolinecarboxaldehyde (CBQCA) (see Scheme I). Although this strategy can be employed for a number of saccharides, poly(galacturonic acid) serves here to demonstrate the potential of CE with laser-induced fluorescence detection in this type of analysis. The sample of poly(ga1acturonic acid) was
0003-2700/92/0364-0973$03.00100 1992 American Chemical Society
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Flgure 2. Correlatlon of mlgratlon times with degree of poiymerlzatlon (data were extracted from Figure 1).
Scheme I
coon subjected to hydrolysis in an autoclave to form a series of oligomers. The dominant structural feature of the polymer are expected to be the linear 1-4-a-linked Dgalacturonic acid units, so that a partial hydrolysis should yield a mixture of oligosaccharides with a wide range of chain lengths. Since only the terminal sugar molecule is tagged with CBQCA, highly sensitive detection becomes essential. Within a wall-treated, gel-filled capillary, practically no electroosmotic flow occurs. Electromigration of the labeled oligosaccharides depends solely on their electrophoretic mobility, determined by their size and charge. However, their separation occurs due to the gel network, as shown in Figure 1. While unprecedented resolution of the mixture components is clearly indicated, the minimum detectable quantities down to the low attomole mol) range have also been observed. Approximately an 85-fmol aliquot of the original polysaccharide has resulted in Figure 1, giving this new methodology a sensitivity several orders of magnitude greater than the best results reported p r e v i ~ u s l y . ~While J ~ the only standard compound available to use was the galacturonic acid monomer, we assigned tentatively the degrees of polymerization to the regularly occurring oligosaccharide peaks and plotted them against the electrophoretic migration times (Figure 2). Such a curve is not unlike those produced in gel CE of oligonucleotide homologues.16
While the example of poly(galacturonic acid) presented here is intended to demonstrate the great potential of gel CE in the oligosaccharide analysis, we were also successful in resolving highly complex mixtures of oligosaccharides originating from both plant and animal sources. For example, an enzymatic hydrolyzate of a hyaluronic acid sample, derivatized and separated in a fashion similar to the galacturonic acid shown above, yielded at least 40 resolved peaks (up to 80-mer mixtures) and revealed a heterogeneous nature of the sample." Similar results were obtained with certain heparin-like polymers and chondroitin sulfate. Highly concentrated gels appear essential to success in separating large oligosaccharides. Further improvements in gel technology are currently being sought.
ACKNOWLEDGMENT This work was supported by Grant No. GM 24349 from the National Institute of General Medical Sciences, U.S.Department of Health and Human Services, and a grant-in-aid from Astra Hbsle Pharmaceutical Company, Molndal, Sweden. REFERENCES (1) Chaplin, M. F.,Kennedy, J. F., Eds. Carboh~drrateAnalysis: A PrracNa/ Approach; IRL Press: Oxford-Washington, 1986. (2) Rockiln, R. D.; Pohl, C. A. J . L/q. Chromtogr. 1983, 6, 1577-1590.
ANALYTICAL CHEMISTRY, VOL. 64, (3) Hardy, M. R.; Townsend, R. R.; Lee, Y. C. Anal. Bbchem. 1988, 770, 54-62. (4) Wang, W. T.; Zopf, D. A. cerbohya.Res. 1989, 789, 1-11. (5) Kdzumi, K.; Yubota, Y.; Tanimoto, T.; Okada. Y. J . Chrometogr. 1989, 464. 365-373. (6) Hotchklss, A. T., Jr.; Hicks, K. B. Anal. B b c t ” . 1990, 784, 200-206. (7) Cohen, A. S.; Najarlan, D. R.; Pauius, A.; (kMman, A.; Smith, J. A.; Karger, 8. L. m.Net/. Aced. SCi. U . S . A . 1988, 85, 9660. (6) t)roseman, H.; Luckey, J. A.; Kostichka, A. J.; D’Cunha, J.; Smith. L. M. A M . them. 1990, 62, $00-903. (9) NovOtny, M.; Cobb, K. A.; Uu,J. Elecbophoresls 1990, 7 7 , 735-749. (IO) Liu, J.; Shlrota, 0.; Wlesler, D.; Novotny, M. Roc. NeH. Aced. Sci. U.S.A 1991, 88, 2302-2306. (11) Uu, J.; Shhota, 0.; Novotny, M. Anal. Chem. 1991, 63, 413-417. (12) Doink, V.; Cobb, K. A.; Novotny, M. J . Mlcrocol. Sep. 1991, 3, 155-159.
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(13) T% and C% designation used here was defined by HJerten (Arch. BlocMm. Bbphys. 1982, 7 , 147) for the characteristics of poly(acryiamide) gels. T% represents the total concentration, or the number of grams of a monomer added to 100 mL of water, while C% is the concentratbn of a cross-linker N,N’-methylenebis(acrylamMe). (14) Liu, J.; Hsleh, Y.-A.; Wlesler, D.; Novotny, M. Anal. Chem. 1991, 63, 406-412. (15) Maness, N. 0.; Mort, A. J. Anal. Chem. 198g, 784, 248-254. (16) Pauius. A.; Qassmann, E.; FleM, M. J. Electrophoresis 1990, 7 7 . 702-708. (17) Liu, J.; Dolnik, V.; Hsieh, Y.-Z.; Novotny, M. A n d . Chem., in press.
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RECEIVED for review September 30,1991. Accepted January 21, 1992.
