Anal. Chem. 2005, 77, 5998-6003
Separation and Analysis of Nanomole Quantities of Heparin Oligosaccharides Using On-Line Capillary Isotachophoresis Coupled with NMR Detection Albert K. Korir, Valentino K. Almeida, Douglas S. Malkin,† and Cynthia K. Larive*,‡
Department of Chemistry, University of Kansas, 1251 Wescoe Hall Drive, Lawrence, Kansas 66045
Glycosaminoglycans (GAGs) are important in a number of biological processes and are structurally altered in many pathological conditions. The complete determination of GAG primary structures has been hampered by the lack of sensitive and specific analytical techniques. Nuclear magnetic resonance spectroscopy (NMR) is a powerful tool for GAG structure elucidation despite its relatively poor limits of detection. Solenoidal microcoils have greatly enhanced the mass limits of detection of NMR, enabling the on-line coupling of microseparation and concentration techniques such as capillary isotachophoresis (cITP), which can separate and concentrate analytes by 2-3 orders of magnitude. We have successfully used cITP coupled with on-line NMR detection to separate and concentrate nanomole quantities of heparin oligosaccharides. This sensitive on-line measurement approach has the potential to provide new insights into the relationships between biological function and GAG microstructures. Heparin and the related glycosaminoglycan (GAG), heparan sulfate, are linear microheterogeneous polysaccharides that are highly sulfated and therefore highly negatively charged. GAGs are located primarily on cell surfaces or in the extracellular matrix where they participate in many biological processes and are structurally altered in several pathological conditions. Heparin is used clinically to prevent blood coagulation and is abundant in granules of mast cells.1 Pharmaceutical heparin is primarily composed of trisulfated disaccharides, the major component being (1f4)-O-(2-O-sulfo-R-L-idopyranosyluronic acid)-(1f4)-O-(2-deoxy2-sulfamido-6-O-sulfo-R-D-glucopyranose). Variation in levels of sulfation and the presence of minor constituents such as β-Dglucuronic acid and N-acetylglucosamine contribute to the structural heterogeneity. Many of these sequence microheterogeneities are responsible for the wide range of biological actions mediated by heparin.2 * To whom correspondence should be addressed. Phone: (951) 827-2990. Fax: (951) 827-4713. E-mail:
[email protected]. † Current address: Bowling Green State University, Bowling Green, Ohio 43403. ‡ Current address: Department of Chemistry, University of California, Riverside, CA 92521. (1) Nader, H. B.; Dietrich, C. P. In Heparin Chemical and Biological Properties, Clinical Applications; Lane, D. A., Lindahl, U., Eds.; CRC Press: Boca Raton, FL, 1989; pp 81-96. (2) Rabenstein, D. L. Nat. Prod. Rep. 2002, 19, 312-331.
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Elucidation of the relationships between biological function and the microstructure of these biopolymers remains a challenge, mainly due to the difficulty in determining the fine structure of sulfated GAGs.3 Heparin and heparan sulfate interact with hundreds of different proteins, including growth factors, cytokines, serine proteases, and lipoprotein lipases, many of which are important in development and cancer.4 However, the question of exactly which oligosaccharide sequences and patterns of sulfation are important for binding to a particular protein has been difficult to answer due to the challenges of analyzing the primary structures of GAGs.5 Thus far, the primary sequences of relatively few heparin and heparan sulfate oligosaccharides have been determined. A limitation of structure elucidation and biological function studies of GAGs has been the lack of sufficiently sensitive and specific analytical techniques. Characterization of intact GAGs is difficult due to their large size and structural heterogeneity. Consequently, studies to elucidate the structure of heparin and heparan sulfate employ experimental strategies that involve enzymatic or chemical depolymerization of the polysaccharide followed by one or more separation steps and eventually characterization of the pure oligosaccharide fragments. These fragments are usually size-fractionated before subjecting the fragments of uniform size to strong anionic exchange (SAX) HPLC to separate the heterogeneous mixtures into pure oligosaccharides.6-8 Further purification can also be achieved using capillary electrophoresis (CE), which is a particularly effective separation method for analysis of GAG digests owing to their strongly anionic character. In fact, there has been a growing use of CE as the technique of choice in separation of GAGs and GAG-derived oligosaccharides due to its high resolving power and flexibility in separation order.9-11 Enzymatically derived GAG oligosaccharides consist of unsaturated saccharides that strongly absorb at 232 nm thus (3) Kinoshita, A.; Sugahara, K. Anal. Biochem. 1999, 269, 367-378. (4) Hari, S. P.; McAllister, H.; Chuang, W.-L.; Christ, M. D.; Rabenstein, D. L. Biochemistry 2000, 39, 3763-3773. (5) Vive`s, R. R.; Pye, D. A.; Salmivirta, M.; Hopwoods, J. J.; Lindahl, U.; Gallagher, J. T. Biochem. J. 1999, 339, 767-773. (6) Chuang, W.; McAllister, H.; Rabenstein, D. L. J. Chromatogr., A 2001, 932, 65-74. (7) Murata, K.; Murata, A.; Yoshida, K. J. Chromatogr., B 1995, 670, 3-10. (8) Karamanos, N. K.; Vanky, P.; Tzanakakis, G. N.; Tsegenidis, T.; Hjerpe, A. J. Chromatogr., A 1997, 765, 169-179. (9) Mao, W.; Thanawiroon, C.; Linhardt, R. J. Biomed. Chromatogr. 2002, 16, 77-94. (10) Ruiz-Calero, V.; Puignou, L.; Galceran, M. T. J. Chromatogr., A 1998, 828, 497-508. 10.1021/ac050669u CCC: $30.25
© 2005 American Chemical Society Published on Web 08/20/2005
allowing ultraviolet (UV) detection.12 Limits of detection can be improved using laser-induced fluorescence.13,14 While UV and laser-induced fluorescence provide sensitive detection, they are not sufficient for the structure elucidation of GAGs. Recent developments in mass spectrometry (MS) ionization methods have significantly advanced the characterization of GAGs using this technique.15-21 The challenges that must be addressed, however, include developing depolymerization protocols that provide MS amenable samples, interpretation of complex spectra, and the unambiguous identification of isomers. Despite the advances in the field of MS, in many cases, a combination of nuclear magnetic resonance (NMR) spectroscopy and MS data is required to elucidate primary structure of unknown compounds. NMR is an important tool for oligosaccharide structure elucidation and provides structural information complementary to that obtained through MS analysis, allowing complete elucidation of the primary structure where multiple isomers are possible.22 NMR spectroscopy can be used to determine the chemical fine structure of GAGs, often permitting assignment of all protons.23 For complex NMR spectra, chemometric tools can be used to extract chemical information using the NMR signals as fingerprints for characterization and quantification.24 NMR spectroscopy has been used to study the solution conformation of heparin-derived hexasaccharides to provide better structural models for larger heparin molecules.25 The technique is thought to be the most accurate method for direct quantification of the iduronic and glucuronic acid content in a saccharide sequence.26 One- and two-dimensional 1H NMR experiments have been used to elucidate the structure of heparin-derived oligosaccharides obtained from partial enzymatic depolymerization.27,28 Around 0.1 µmol of purified oligosaccharide is the minimum amount required for such analysis. NMR suffers from relatively poor limits of detection, which hinders the analysis of less abundant GAG oligosaccharide components. Development of more sensitive analytical methods for sequencing very small quantities of heparin and HS-derived (11) Desai, U. R.; Wang, H.; Ampofo, S. A.; Linhardt, R. J. Anal. Biochem. 1993, 213, 120-127. (12) Militsopoulou, M.; Lecomte, C.; Bayle, C.; Couderc, F.; Karamanos, N. K. Biomed. Chromatogr. 2003, 17, 39-41. (13) Militsopoulou, M.; Lamari, F. N.; Hjerpe, A.; Karamanos, N. K. Electrophoresis 2002, 23, 1104-1109. (14) Kinoshita, A.; Sugahara, K. Anal. Biochem. 1999, 269, 367-378. (15) Shinohara, Y.; Furukawa, J.; Niikura, K.; Miura, N.; Nishimura, S. Anal. Chem. 2004, 76, 6989-6997. (16) Zaia, J.; Costello, C. E. Anal. Chem. 2001, 73, 233-239. (17) Chai, W.; Luo, J.; Lim, C. K.; Lawson, A. M. Anal. Chem. 1998, 70, 20602066. (18) Juhasz, P.; Biemann, K. Carbohydr. Res. 1995, 270, 131-147. (19) Desaire, H.; Leary, J. A. J. Am. Soc. Mass Spectrom. 2000, 11, 916-920. (20) Saad, O. M.; Leary, J. A. Anal. Chem. 2003, 75, 2985-2995. (21) McClellan, J. E.; Costelo, C. E.; O’Connor, P. B.; Zaia, J. Anal. Chem. 2002, 74, 3760-3771. (22) Yamada, S.; Sakamato, K.; Tsuda, H.; Yoshida, K.; Sugiura, M.; Sugahara, K. Biochemistry 1999, 38, 838-847. (23) Toida, T.; Linhardt, R. J. Trends Glycosci. Glycotechnol. 1998, 10, 125-136. (24) Ruiz-Calero, V.; Saurina, J.; Galceran, M. T.; Puignou, S. H. L. Anal. Bioanal. Chem. 2002, 373, 259-265. (25) Guerrini, M.; Raman, R.; Venkataraman, G.; Torri, G.; Sasisekharan, R.; Casu, B. Glycobiology 2002, 12, 713-719. (26) Loganathan, D.; Wang, H. M.; Mallis, L. M.; Linhardt, R. J. Biochemistry 1990, 29, 4362-4368. (27) Mikhailov, D.; Mayo, K. H.; Pervin, A.; Linhardt, R. J. Biochem. J. 1996, 315, 447-454. (28) Toida, T.; Hileman, R. E.; Smith, A. E.; Vlahova, P. I.; Linhardt, R. J. J. Biol. Chem. 1996, 271, 32040-32047.
oligosaccharides is essential for progress in this field. One approach for improving the mass detection limits of NMR uses solenoidal microcoil probes with nanoliter to microliter detection volumes.29-33 Although they improve the sensitivity of analysis for mass-limited samples, microcoil NMR probes have poorer concentration detection limits compared to probes with a traditional Helmholtz coil design. The use of capillary isotachophoresis (cITP) to concentrate charged analytes prior to NMR detection is an effective strategy for improving the detection limits of microcoil probes.34 cITP is an electrophoretic sample stacking technique that separates and concentrates charged analytes even in the presence of a large excess of neutral sample matrix.35 In cITP, analytes are separated according to their electrophoretic mobilities by applying a high electric field (10-30 kV) across a capillary containing a discontinuous buffer system composed of a leading electrolyte (LE) and a trailing electrolyte (TE). Through careful selection of LE and TE, the separation conditions can be optimized to concentrate analytes by 2-3 orders of magnitude. This focusing characteristic of cITP enables on-line concentration improving the effective concentration sensitivity of NMR detection.36 In addition, on-line cITP NMR can potentially provide unique insights into the separation process.37 All the cITP NMR results reported thus far have been studies of cationic analytes. Because of the high negative charges of GAGs, the anionic mode of cITP is better suited for the analysis of these compounds. The major challenges of conducting anionic cITP NMR experiments are the development of suitable buffer systems and overcoming electroosmotic flow (EOF), which opposes the migration of anions and degrades the ability of cITP to separate and focus the analyte bands. To overcome this limitation, we have used commercially available zero-EOF capillaries. We present in this paper an analytical method employing cITP directly coupled with on-line microcoil NMR detection for the separation and analysis of nanomole quantities of negatively charged heparin oligosaccharides. Figure 1 shows the structures of the oligosaccharides used in this study. EXPERIMENTAL SECTION Materials and Reagents. The zero-EOF capillaries were purchased from MicroSolv Technology Corp. (Eatontown, NJ). The heparin oligosaccharides used in this study were in the sodium salt form. The heparin-derived disaccharides, R-δUA-2S[1f4]-GlcNS-6S (IS) and R-δUA-2S[1f4]-GlcNS (IIIS), as well as the chemicals, imidazole and 2-morpholinoethanesulfonic acid (29) Olson, D. L.; Peck, T. L.; Webb, A.; Magin, R. L.; Sweedler, J. V. Science 1995, 270, 1967-1970. (30) Lacey, M. E.; Subramanian, R.; Olson, D. L.; Webb, A. G.; Sweedler, J. V. Chem. Rev. 1999, 99, 3133-3152. (31) Olson, D. L.; Lacey, M. E.; Sweedler, J. V. Anal. Chem. 1998, 70, 257A264A. (32) Webb, A. G. Prog. Nucl. Magn. Reson. Spectrosc. 1997, 31, 1-42. (33) Wolters, A. M.; Jayawickrama, D. A.; Sweedler, J. V. Curr. Opin. Chem. Biol. 2002, 6, 711-716. (34) Kautz, R. A.; Lacey, M. E.; Wolters, A. M.; Foret, F.; Webb, A. G.; Karger, B. L.; Sweedler, J. V. J. Am. Chem. Soc. 2001, 123, 3159-3160. (35) Wolters, A. M.; Jayawickrama, D. A.; Larive, C. K.; Sweedler, J. V. Anal. Chem. 2002, 74, 2306-2313. (36) Jayawickrama, D. A.; Sweedler, J. V. Anal. Bioanal. Chem. 2004, 378, 15281535. (37) Wolters, A. M.; Jayawickrama, D. A.; Larive, C. K.; Sweedler, J. V. Anal. Chem. 2002, 74, 4191-4197.
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Figure 1. Structure of (a) the heparin disaccharides and (b) tetrasaccharide used in the study.
(MES), were purchased from Sigma (St. Louis, MO). Other heparin-derived saccharides, R-δUA-2S[1f4]-GlcNS (IIIS*), R-δUA2S[1f4]-GlcNAc (IIIA), R-δUA-2S[1f4]-GlcNS-6S (IS), and its corresponding tetrasaccharide, were purchased from V-Labs, Inc. (Covington, LA). The asterisk in IIIS* distinguishes only the source of the supplier since the compound is structurally identical to IIIS. NaOD, DCl, and low paramagnetic D2O were purchased from Cambridge Isotope Laboratories (Andover, MA). NMR Microcoil Probes. The details of the probe design have previously been described.38,39 Briefly, the solenoidal microcoil constructed in our laboratory consisted of a 50-µm polyurethanecoated wire wrapped around a polyimide sleeve with inner and outer diameters of 370 and 430 µm, respectively. By carefully winding seven equally spaced turns around the sleeve, a 1-mm microcoil with coil diameter of 430 µm was constructed for use at 600 MHz. The capillary used in the cITP experiments was 180 cm long with an inner diameter of 180 µm. When this capillary is inserted into the polyimide sleeve supporting the microcoil, an observe volume of 25 nL is obtained. The coil is enclosed in a plastic bottle filled with a fluorocarbon fluid with a magnetic susceptibility closely matching that of copper. cITP Procedure. The instrumental cITP NMR setup is similar to that used in previous studies.38,39 An LE consisting of 160 mM DCl and 80 mM imidazole and a TE containing 160 mM MES, both at pD 6.9, were used. The pD of the leading and trailing electrolytes was adjusted using NaOD. The analyte (2.5 nmol in 10 µL) was hydrodynamically injected such that it would focus just prior to the detection coil of the NMR probe. A detailed description of a typical cITP NMR operation has been reported previously.34,35 Solutions of the analytes were prepared in 85% D2O and 15% TE. The samples were hydrodynamically injected into the capillary at a height of 8 cm giving an injection rate of 1.25 µL/min. The injection protocol involved first filling the capillary with LE, followed by an 8-min injection of sample, and finally injecting 8 min of TE. The LE and the TE capillary ends were placed in their respective buffer vials and electrical connections (38) Cardoza, L. A.; Almeida, V. K.; Larive, C. K.; Graham, D. W. Trends Anal. Chem. 2003, 22, 766-775. (39) Almeida, V. K.; Larive, C. K. Magn. Reson. Chem., in press.
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Figure 2. Results of the on-line cITP NMR spectra for the disaccharide R-δUA-2S[1f4]-GlcNS (IIIS). Each spectrum was obtained by coaddition of eight FIDs with time resolution of 11.2 s.
to a high-voltage supply made via platinum electrodes. A potential of 15 kV was used in the experiment producing running currents ranging from 70 to 35 µA during the course of the cITP analysis. NMR Acquisition. On-line cITP NMR experiments were conducted using a Varian Unity spectrometer operating at 599.741 MHz. The spectrometer was set to acquire an array of 1H NMR spectra using a 45° pulse, a spectral width of 7196.8 Hz, and an acquisition of time of 1.40 s. Each spectrum was obtained by coadding eight FIDs giving a time resolution of 11.2 s. Line broadening equivalent to 2.0 Hz and zero-filling to 128K points were applied prior to Fourier transformation. Static spectra of the LE yielded line widths between 2.2 and 2.6 Hz although the line widths were found to be broader (4-5 Hz) during on-line cITP NMR analysis. RESULTS AND DISCUSSION The results of on-line cITP focusing and NMR analysis of the heparin disaccharide IIIS are shown in Figure 2. Spectra a and b, measured before the focused sample band entered the NMR microcoil, contain only the LE resonances of imidazole and the solvent HOD, which is detected throughout. Spectra c-f contain the disaccharide resonances detected as the focused sample band passed through the microcoil. These spectra are basically a time profile of the focused analyte band. Spectra g and h contain primarily the resonances of the TE components, imidazole, and MES. At the time period during which spectrum g was acquired, the analyte band began to leave the detection coil; therefore, this
Figure 4. 1H NMR spectra of the disaccharides (a) IIIS and (b) IS obtained by postacquisition coaddition of the cITP NMR spectra for each analyte.
Figure 3. Results of the on-line cITP NMR spectra for the disaccharide R-δUA-2S[1f4]-GlcNAc (IIIA).
spectrum is a time average of the sample and TE resonances. The splitting of the imidazole resonance at 7.60 ppm in spectrum g reflects the presence of a diffusional boundary between the focused analyte band and the TE. Although the LE and TE were originally prepared at the same pD (6.9), during the course of the experiment, electrolytic reduction of hydronium ion at the cathode will raise the TE pD altering the ratio of protonated and deprotonated imidazole. Within the pD range of pKa ( 1, the 1H chemical shift of imidazole is very sensitive to solution pD; therefore, this resonance can be used as an internal indicator of the actual pD of the cITP buffers. Figure 3 shows on-line cITP NMR spectra obtained for the analysis of another disaccharide, IIIA. This disaccharide, R-δUA2S[1f4]-GlcNAc, is structurally similar to IIIS except that it is N-acetylated instead of being N-sulfated (Figure 1). Similarly to what was observed in Figure 2, spectra a and b contain the resonances of the LE, while spectra c-g are a time profile of the focused IIIA band. Spectrum g again is a time average of the sample/TE interface, with the TE pD jump detected most clearly for this sample by the change in imidazole chemical shift between spectra g and h. The acetyl proton signal at 2.1 ppm in the analyte spectra c-g clearly distinguishes IIIA from IIIS. Comparison of the integrals of the acetyl and imidazole protons indicated that the cITP process concentrated the analytes by a factor of 200. To determine the ability of cITP NMR experiments to focus larger oligosaccharides, studies were also carried out using the tetrasaccharide composed of the disaccharide IS. Although the same number of moles of the tetrasaccharide was injected, the
signal-to-noise ratio of the tetrasaccharide spectra was poorer than those obtained for the disaccharides. Signal averaging is commonly used to improve the signal-tonoise ratio of NMR spectra. However, to have sufficient time resolution to profile the analyte band in our on-line cITP NMR experiments, only a few FIDs were coadded for each spectrum. It is possible to recoup the signal-to-noise improvements that were sacrificed in favor of time resolution by postacquisition coaddition of the analyte FIDs. For example, the 1H NMR spectrum of IIIS resulting from postacquisition coaddition of spectra in Figure 2c-f is shown in Figure 4a. A similar cITP NMR experiment was performed for the heparin disaccharide IS with the results obtained by postacquisition coaddition of the analyte spectra shown in Figure 4b. The 1H NMR spectra in Figure 4 are distinctly different despite the fact that the only structural difference between these disaccharides is the presence of a sulfate group in IS instead of a hydrogen atom in IIIS. Although the spectral features in the coadded 1H NMR spectra are useful in distinguishing the disaccharides, they do not provide sufficient information for complete structure elucidation. Work is underway to implement stopped-flow experiments to facilitate 2D NMR analysis, which would greatly enhance structure elucidation using this technique. For cITP NMR to be most useful for oligosaccharide analyses it should be capable of separating mixture components as well as concentrating analytes for on-line NMR detection. Figure 5 shows the on-line cITP NMR separation of a mixture of the disaccharides IS, IIIS*, and IIIA. In this experiment, the mixture containing 2.5 nmol of each disaccharide was hydrodynamically injected into the capillary for cITP NMR analysis. Although these disaccharides have similar masses, they have different charges (IS ) -4, IIIS* ) -3, and IIIA ) -2) and, therefore, different electrophoretic mobilities. On the basis of their mass/charge ratios, disaccharide IS would be expected to be on the leading edge of the cITP sample band, and disaccharide IIIA, with the lowest mobility, would be expected to focus at the TE interface. This prediction is largely born out in the experimental results. Spectra 5b-d contain the resonances of the disaccharide IS, spectra e-g contain the resonances of the disaccharide IIIS*, and spectra i and j contain Analytical Chemistry, Vol. 77, No. 18, September 15, 2005
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Figure 6. 1H NMR spectra of (a) the tetrasaccharide corresponding to IS and (b) the IS disaccharide obtained by postacquisition coaddition of the cITP NMR spectra for each analyte.
Figure 5. On-line cITP NMR separation of IS, IIIS*, and IIIA. A profile of the separation can be observed as the sample passes through the solenoidal microcoil (25 nL detection volume).
the resonances of the disaccharide IIIA. Interestingly, we did not expect to observe spectrum h, which differs from the other oligosaccharide spectra due of the presence of a trace impurity. cITP NMR analysis of each disaccharide individually (results not shown) indicated that this impurity was associated with the disaccharide IIIS*, emphasizing the benefits of performing online NMR analysis Current methods of heparin analysis subject the enzymatic depolymerization products to a size-based separation followed by an additional SAX or reversed-phase HPLC separation. While the spectra shown in Figure 5 illustrate the potential of cITP NMR to concentrate and separate nanomole quantites of disaccharides based on charge, direct analyses of depolymerization reaction products solely with cITP NMR will be difficult. This was exemplified by the analysis of disaccharide, IS, and its corresponding tetrasaccharide. Figure 6 shows the postacquisition coadded spectra from cITP NMR analyses of the tetrasaccharide and IS, respectively. The spectra obtained were not easily distinguishable because of the structural similarities of the two oligomers. In addition, although the net charges on the compounds are different (tetrasaccharide ) -8; IS ) -4), the mass of the tetrasaccharide is twice that of the disaccharide; therefore, both analytes have similar mass/charge ratios and electrophoretic mobilities. These results demonstrate the capability of cITP as an analytical method for separating and concentrating mass-limited samples of charged anionic analytes, thus allowing detection of 6002 Analytical Chemistry, Vol. 77, No. 18, September 15, 2005
nanomole quantitities of GAG oligosaccharides by NMR. The technique has application in the characterization of rare GAGprotein binding sites, a task that would otherwise be difficult and time-consuming due to the need to isolate and collect sufficient quantities of material for characterization. Although cryogenically cooled probes have been shown to improve concentration sensitivity of NMR experiments up to a factor of 4,40 there is still a need for the development of innovative NMR technology for masslimited samples. This is especially true for high ionic strength aqueous samples, such as those analyzed in this work, for which cryogenically cooled probes provide only modest gains in sensitivity. CONCLUSION The results presented here demonstrate the potential of cITP NMR for the on-line separation and analysis of oligosaccharides. NMR spectra were acquired in real time using sample quantities ∼2 orders of magnitude less than would be used with conventional NMR probes. In addition, the power of on-line analysis is demonstrated both for mixtures of disaccharides with differing charges and for a sample, IIIS*, containing an unintended impurity. Although the spectra were recorded at relatively high sensitivity, it is noteworthy that solvent and buffer impurities are not detected, thus emphasizing the power of the technique for the analysis of mass-limited samples. For oligosaccharides containing components with similar electrophoretic mobilities, an additional separation step, for example, size fractionation, will generally be needed prior to cITP NMR analysis. Further studies are underway to evaluate the utility of cITP NMR for analysis of more complex mixtures such as those derived from enzymatic digestion of GAGs. By dramatically reducing the quantity of sample required for NMR analysis, this sensitive on-line measurement approach has the potential to provide new insights into the relationships between biological function and microstructures of GAGs. (40) Spraul, M.; Freund, A. S.; Nast, R. E.; Withers, R. S.; Maas, W. E.; Corcoran, O. Anal. Chem. 2003, 75, 1546-1551.
ACKNOWLEDGMENT C.K.L gratefully acknowledges financial support from National Science Foundation, CHE 0213407. D.S.M. gratefully acknowledges support from the NSF REU site grant CHE-0244041. The authors thank Professors Jonathan V. Sweedler and Andrew G. Webb and their research associates at the University of Illinois
for assistance in the design and construction of the NMR microcoil probe used in this research. Received for review April 18, 2005. Accepted July 24, 2005. AC050669U
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