Oligosaccharide Structures Studied by Hydrogen−Deuterium

Anal. Chem. 2006, 78, 5302-5308

Oligosaccharide Structures Studied by Hydrogen-Deuterium Exchange and MALDI-TOF Mass Spectrometry Neil P. J. Price*

USDA-ARS-NCAUR, Bioproducts and Biocatalysis Research Unit, 1815 North University Street, Peoria, Illinois 61604

Hydrogen-deuterium exchange matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (HXMALDI-TOF MS) is reported for the determination of exchangeable protons in diverse oligosaccharide and glycoconjugate structures. The method has broad application for determining carbohydrate structure and conformation and to the study of carbohydrate-ligand interactions. The proton exchange process has been optimized to maximize the forward deuterium exchange and to suppress the well-known problem of back-exchange and is suitable for the analysis of all exchangeable proton types in carbohydrates. This has been validated for several diverse carbohydrate structures, including series of maltoand xylopyranose oligosaccharides; r- and β-cyclodextrins; a nonreducing tetrasaccharide, stachyose; an Nacetylamide-containing oligosaccharide, chitotetraose; and a tertiary hydroxyl-containing antibiotic glycoconjugate, erythromycin. In recent years, several studies have been reported in which mass spectrometry (MS) was used to investigate H/D exchange behavior of water-soluble peptides and proteins.1-10 From the observed H/D exchange patterns, information can be extracted on protein folding11-14 and noncovalent complex formation.15 The * To whom correspondence should be addressed. Phone: (309) 681-6246. Fax: (309) 681-6040. E-mail: [email protected]. (1) Smith, D. L.; Deng, Y.; Zhang, Z. J. Mass Spectrom. 1997, 32, 135-146. (2) Figueroa, I. D.; Russell, D. H. J. Am. Soc. Mass Spectrom. 1999, 10, 719731. (3) Busenlehner, L. S.; Armstrong, R. N. Arch. Biochem. Biophys. 2005, 433, 34-46. (4) Hoofnagle, A. N.; Resing, K. A.; Ahn, N. G. Annu. Rev. Biophys. Biomol. Struct. 2003, 32, 1-25. (5) Engen, J. R.; Smith, D. L. Anal. Chem. 2001, 73, 256-265. (6) Mandell, J. G.; Falick, A. M.; Komives, E. A. Anal. Chem. 1998, 70, 39873995. (7) Zhang, Z.; Smith, D. L. Protein Sci. 1993, 2, 522-531. (8) Yan, X.; Watson, J.; Ho, P. S.; Deinzer, M. L. Mol. Cell Proteomics 2004, 3, 10-23. (9) Englander, S. W. Annu. Rev. Biophys. Biomol. Struct. 2000, 29, 213-238. (10) Bai, Y.; Milne, J. S.; Mayne, L.; Englander, S. W. Proteins 1993, 17, 7586. (11) Konermann, L.; Simmons, D. A. Mass Spectrom. Rev. 2003, 22, 1-26. (12) Katta, V.; Chait, B. T. Rapid Commun. Mass Spectrom. 1991, 5, 214-217. (13) Englander, J. J.; Del Mar, C.; Li, W.; Englander, S. W.; Kim, J. S.; Stranz, D. D.; Hamuro, Y.; Woods, V. L., Jr. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 7057-7062. (14) Maity, H.; Maity, M.; Krishna, M. M.; Mayne, L.; Englander S. W. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 4741-4746.

5302 Analytical Chemistry, Vol. 78, No. 15, August 1, 2006

technique relies on the replacement of exchangeable protons that are exposed to deuterated solvent, typically D2O. The most readily exchangeable positions are back-exchanged by lowering the pH, and this same process greatly slows the back-exchange process for the peptide amide protons. This results in a selective labeling of the surface peptide amides exposed to solvent, leaving those buried in the protein interior unexchanged. The H/D exchange patterns can be observed by proton NMR or, with much greater sensitivity, by mass spectrometry. Hydrogen-deuterium exchange matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (HX-MALDI-TOF MS, HX ) proton exchange) has been particularly useful for this purpose, and various studies have demonstrated the unique capability for examining dynamics and structural changes associated with enzyme catalysis.2,6,13 Although HX-MALDI-TOF MS is gaining considerable attention for probing the noncovalent structure of proteins and peptides, the technique has not as yet been applied to carbohydrates. This is because unlike peptide amide protons, the exchange of carbohydrate anomeric and hydroxyl protons is too rapid to be measured by most techniques. For NMR analysis, the hydroxyl groups on sugars are generally fully exchanged in D2O to simplify the spectra. Since these spectra are usually recorded in liquid D2O, the process of proton back-exchange does not occur, and spectra are obtained of the fully H/D-exchanged substrates.16 However, despite the availability of recent NMR and MS techniques, the structural assignment of complex carbohydrates remains a challenging problem. This paper describes the optimization of the deuterium exchange process for application to carbohydrates. The influence of low temperature, inert atmosphere, dehydrating agents, acidic and basic additives, and matrix composition have been assessed, resulting in an optimized HX-MALDI-TOF MS technique suitable for the analysis of all exchangeable proton types in carbohydrates. This has been applied to several diverse carbohydrate structures, including series of linear glucose oligosaccharides up to dp 11, containing either R-1,4 linkages (malto-oligosaccharides) or alternating R1,3-R-1,6-linkages (alternan oligosaccharides); a series of β-1,4-linked xylopyranose oligosaccharides; R- and β-cyclodextrins; a nonreducing tetrasaccharide, stachyose; an N-acetylamidecontaining oligosaccharide, chitotetraose; and a tertiary hydroxyl(15) King, D.; Lumpkin, M.; Bergmann, C.; Orlando, R. Rapid Commun. Mass Spectrom. 2002, 6, 1569-1574. (16) Wagner, G.; Stassinopoulou, C. I.; Wuthrich, K. Eur. J. Biochem. 1984, 145, 431-436. 10.1021/ac052168e CCC: $33.50

© 2006 American Chemical Society Published on Web 07/04/2006

Table 1. Compiled MALDI-TOF MS Data on Deuterium-Exchanged Malto-oligosaccharides native massesa

exchanged massesa

sugar

molecular formulas

exchangeable Hs

calcd

obsd

calcd

obsd

maltotriose maltotetraose maltopentaose maltohexaose maltoheptaose maltooctaose maltononaose maltodecaose maltoundecaose

C18H32016 C24H42021 C30H52026 C36H62031 C42H72036 C48H82041 C54H92046 C60H102051 C66H112056

11 14 17 20 23 26 29 32 35

527.159 689.211 851.264 1013.317 1175.370 1337.423 1499.476 1661.528 1823.581

527.056 689.169 851.261 1013.326 1175.363 1337.403 1499.472 1661.548 1823.566

538.227 703.298 868.369 1033.441 1198.512 1363.583 1528.655 1693.726 1858.797

538.136 703.223 868.333 1033.435 1198.526 1363.591 1528.653 1693.698 1858.725

a Monoisotopic mass of sodium adduct ions, [M + Na]+, and the corresponding exchanged ions [M(2H )+Na]+. The spectra are shown in n Figure 1 and in the Supporting Information.

containing antibiotic glycoconjugate, erythromycin. Essential features of the technique are the use of a mixed oxalic acid/2,5dihydrobenzoic acid matrix to reduce back-exchange and the inclusion of xylo-oligosaccharides as internal standards to monitor the completeness of the exchange process and to assess the degree of back-exchange. METHODS AND MATERIALS Source of Carbohydrates and Chemicals. Malto-oligosaccharide series and alternan oligosaccharides were a gift from Dr. Greg Cote (NCAUR-ARS, Peoria, IL). Xylo-oligosaccharides were from Suntory Corporation, Osaka, Japan. Chitotetraose was obtained from Seikagaku America, East Falmouth, MA. Stachyose, cyclodextrins, and erythromycin A were obtained from SigmaAldrich, St. Louis, MO. Other reagents, including matrix chemicals for MALDI-TOF MS, were of the highest obtainable purity available. Optimized Proton-Deuterium Exchange Reaction. Typically, the exchange reactions were undertaken by mixing the carbohydrate sample (0.2-0.3 mg) and the matrix (2,5-dihydrobenzoic acid, 0.5 mg; oxalic acid, 0.5 mg) in deuterated water (200 µL, 99.9% D) containing 10% deuterated acetonitrile (99.8% D). The acetonitrile was necessary to ensure the complete solubility of the matrix. The solutions were lyophilized and redissolved in D2O (100 µL, 99.99% D) plus D3-acetonitrile (25 µL, 99.8%) immediately prior to spotting 0.5 µL onto an ice-cold, stainless steel target. The target was stored in an airtight polyethylene container at -20 °C over Dryrite and after 24 h was transferred to the spectrometer inlet. To minimize the condensation of atmospheric water onto the cold target, this transfer was made with the inlet to the mass spectrometer enclosed inside a nitrogen-flushed glovebox. MALDI-TOF MS spectra were accumulated using 80 laser pulses as described below. During optimization, solid acids or bases (citric, ascorbic, oxalic, carbonate), ion exchange beads, or drying agents (calcium sulfate, magnesium sulfate, molecular sieves, or deuterated cupric sulfate) were included in the exchange reactions. Anhydrous cupric sulfate was deuterated by rehydration with D2O to provide CuSO4.72H2O. Matrix-Assisted Laser Desorption/Ionization-Time-ofFlight (MALDI-TOF) Mass Spectrometry. MALDI-TOF mass spectra were recorded on a Bruker-Daltonic Omniflex instrument operating in reflecton mode. Ion source 1 was set to 19.0 kV, and source 2, to 14.0 kV, with lens and reflector voltages of 9.20 and

20.00 kV, respectively. A 200-ns pulsed ion extraction was used with matrix suppression up to 200 Da. The instrument was calibrated externally on a dp series of malto-oligosaccharides. Excitation was at 337.1 nm, typically at 60% of 150 µJ maximum output, and 80 shots were accumulated. Nonexchanged samples were typically dried under a lamp on a conventional 7 × 7 stainless steel target. In some optimization experiments, the target was covered with PTFE tape to provide a nonconducting, ion-free surface. RESULTS Optimization of the Proton Exchange Process. Malto- and Xylo-oligosaccharides. The initial aim was to optimize the deuterium exchange of hydroxyl groups on oligosaccharides and to minimize the back-exchange process. The extent of exchange was assessed from the isotopomer clusters observed by MALDITOF mass spectrometry for a well-defined series of maltooligosaccharides. In positive ion mode, the nonexchanged maltooligosaccharides are characterized by a series of [M + Na]+ ions that are characteristic of the number of glucose residues (i.e., the degree of polymerization). For example, maltopentaose (dp 5) is characterized by m/z 851.3; maltohexaose (dp 6), by m/z 1013.3; maltoheptaose (dp 7), by m/z 1175.4; etc. (Figure 1, Table 1). Terminal and internal glucose residues have four and three exchangeable protons, respectively, and fully exchanged masses are, therefore, described by the mass series MD ) MH + (3n + 2), where n is the degree of polymerization. Prior to optimization, these fully exchanged ions were difficult to obtain because of the process of back exchange, and in general, series of isotopomer clusters were observed for each component oligosaccharide arising from partial deuterium exchange. The problem of back-exchange was found to be significantly improved by the inclusion of oxalic acid with the 2,5-dihydrobenzoic acid matrix. This is evident from the proton exchange of the maltooligosaccharide series either with or without the oxalic acid (Figure 1). Hence, after deuterium exchange [M(2Hn) + Na]+, ions due to maltopentaose, maltohexaose, and maltoheptaose are at m/z 868.3, 1033.4, and 1198.5, respectively (Figure 1, Table 1). These data are consistent with calculated masses for the corresponding fully exchanged oligosaccharides, and the mass differences (∆) reflect the total number of exchangeable hydroxyl groups (∆ ) 17, 20, and 23, respectively, for dp 5, 6, and 7). It is, therefore, possible to determine the total number for exchangeable Analytical Chemistry, Vol. 78, No. 15, August 1, 2006

5303

Figure 1. HX-MALDI-TOF MS analysis of malto-oligosaccharides. Spectra A, nonexchanged [M + Na]+ ions for maltopentaose (dp 5, m/z 851.3), maltohexaose (dp 6, m/z 1013.3), and maltoheptaose (dp 7, 1175.4). Mass differences correspond to one glucose residue (162 mass units). Minor peaks at m/z 867.3, 1029.3, and 1191.4 are [M + K]+ ions. Spectra B, D2O-exchanged without oxalic acid. Spectra C, D2O-exchanged with oxalic acid. m/z 868.3 (dp 5), 1033.4 (dp 6), and 1198.5 (dp 7) correspond to fully exchanged [M(2Hn) + Na]+ ions. The mass difference (∆) corresponds to the total number of exchangeable protons, 3n + 2, where n ) number of hexose residues. Full spectra are included in the Supporting Information. 5304 Analytical Chemistry, Vol. 78, No. 15, August 1, 2006

protons for each oligosaccharide from maltotriose (dp 3, exchangeable protons ) 11) to maltoundecaose (dp 11, exchangeable protons ) 35) (Table 1). Similar results were obtained with xylo-oligosaccharides (Figure 2) and alternan oligosaccharides (data not shown), indicating that deuterium exchange occurs irrespective of the glycosidic linkages. It is known that the isotopic exchange of peptide amide protons is both acid- and base-catalyzed and that the rate constant, kex, is minimized as the pH approaches 2.5. Careful control of the pH or lowering of the temperature is necessary to slow the process of back-exchange. Indeed, improving HX-MS by reducing the back-exchange process has been the subject of several papers.6,19-21 For carbohydrates, the exchange rates for the primary and secondary hydroxyl protons are expected to be much faster than for amide protons, and it was therefore also necessary to keep the exchanged samples free from external sources of moisture. For example, fully deuterated [M(2Hn) + Na]+ ions were obtained for xylopentaose (dp 5, m/z 713.1), xylohexaose (dp 6, m/z 847.1), and xyloheptaose (dp 7, m/z 981.0), with the mass difference between the native and deuterium-exchanged masses (∆) corresponding to the total number of exchangeable protons (Figure 2). However, after exposure to ambient atmospheric moisture (18 h, room temperature) the fully exchanged xylo-oligosaccharides were shown to be substantially back-exchanged by protons (Figure 2.). These data show that for the xylo-oligosaccharides, the forward deuterium exchange process goes to completion and that any partially exchanged species observed arise as a result of backexchange with moisture in the surrounding environment. Deuterium-Exchange MALDI-TOF MS of r- and β-Cyclodextrins. The deuterium exchange reaction was used to analyze exchangeable protons in several other diverse carbohydrate structures (Figures 3 and 4). Cyclodextrins (CDs) are closed, circular, R-1,4-linked D-glucopyranose oligosaccharides, analogous to linear malto-oligosaccharides but lacking the anomeric reducing terminus.22 They are widely used in industry because of their ability to form inclusion compounds with a variety of substrates in aqueous solution. Cyclomaltoheptaose (β-CD) has seven glucose residues and is characterized by a flattened torus shape with a narrow rim composed of primary 6-OH groups and a wide rim of secondary OHs. The secondary OH groups on adjacent residues form seven intramolecular O2-H-O3′ hydrogen bonds that impart rigidity to the structure. The protons involved in these hydrogen bonds are readily observed by NMR spectroscopy and are more deshielded than “free” protons.22 The six-membered cyclomaltohexaose (R-CD) has a similar structure, but because of steric distortion at one glucosyl residue, only four O2-H-O3′ hydrogen bonds can form, rather than the expected six. The CD structures also gives rise to a relatively hydrophobic cavity formed by the ring of glycosidic oxygens and two rings of CH groups and that is involved in the formation of noncovalent inclusion complexes.22,23 (17) Price, N. P. Anal. Chem. 2004, 76, 6566-6574. (18) Seymour, F. R.; Plattner, R. D.; Slodki, M. E. Carbohydr. Res. 1975, 44, 181-198. (19) Kipping, M.; Schierhorn, A. J. Mass Spectrom. 2003, 38, 271-276. (20) Wang, L.; Smith, D. L. Anal. Biochem. 2003, 314, 46-53. (21) Figueroa, I. D.; Torres, O.; Russell, D. H. Anal. Chem. 1998, 70, 45274533. (22) Schneider, H.-J.; Hacket, F.; Rudiger, V. Chem. Rev. 1998, 98, 1755-1785.

Figure 3. HX-MALDI-TOF MS of cyclodextrins. Top panel, R-cyclodextrin (m/z 995.2 ) [M + Na]+, m/z 1011.7 ) [M + K]+). Lower panel, β-cyclodextrin (m/z 1157.3 ) [M + Na]+, m/z 1173.3 ) [M + K]+). Proton exchange was undertaken in D2O with 2,5-dihydrobenzoic and oxalic acids and included xylo-oligosaccharides as internal standards. Deuterium-exchanged ions [M(2H17) + Na]+ and [M(2H20) + Na]+ are at m/z 1012.3 and 1177.4, respectively. The observed number of exchanged protons (∆) is indicated.

Figure 2. HX-MALDI-TOF MS of xylo-oligosaccharides. [M + Na]+ ions for xylopentaose (dp 5, m/z 701.2), xylohexaose (dp 6, m/z 833.2), and xyloheptaose (dp 7, m/z 965.2). Mass differences correspond to one xylose residue (132 mass units). Spectra A, fully exchanged [M(2Hn) + Na]+ ions, m/z 713.1, 847.1, and 981.0. Spectra B, extent of back-exchange after 18 h of exposure to ambient environment. Spectra C, nonexchanged controls. The mass difference (∆) corresponds to the total number of exchangeable protons, 2n + 2, where n ) number of pentose residues.

The hydroxy protons of R- and β-cyclodextrin were deuteriumexchanged in the presence of a series of xylo-oligosaccharides as internal standards (Figure 3.). Complete proton exchange was observed for the internal standards, generating the expected series of ions for deuteroxylose oligosaccharides, as shown in Figure 2. Fully exchanged sodium adduct ions were also observed for Rand β-cyclodextrin, at m/z 1013.3 and 1178.4, respectively; however, the base peaks were centered around [M(2H17) + Na]+ (m/z 1012.3) and [M + (2H20) + Na]+ (m/z 1177.4) (Figure 3). This corresponds to M + 17 and M + 20 exchanged protons for R-CD and β-CD, respectively, and indicate that one hydroxyl position is recalcitrant to deuterium exchange (Figure 3.). An investigation by Verma et al. of the exchangeable protons of β-CD by fast atom bombardment MS identified a M + 21 ion ([M(2H21) (23) Verma, S.; Pomerantz, S. C.; Sethi, S. K.; McCloskey, J. A. Anal. Chem. 1986, 58, 2898-2902.

Analytical Chemistry, Vol. 78, No. 15, August 1, 2006

5305

+ 2H]+, m/z 1157) as the major isotopomer.23 However, Sethi et al. noted that the use of either 94.6 or 96.6% 2H deuterioglycerol as the matrix is sufficient to shift the calculated base peak for exchanged β-CD from m/z 1156 to m/z 1158.24 The kinetics for hydrogen-deuterium exchange of protonated cyclodextrins in the gas phase have been determined by Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry.25 Exchange rates for the protonated CDs were slowed by >10-fold when a small amine “guest” occupied the inclusion site. Exchange rates were very fast, and 7-8 protons were exchanged for R-CD (i.e., ∼40% exchanged), and 15-16 protons for β-CD (i.e., 75% exchanged) within a 2-min time course. However, unlike the present MALDI-TOF study, it was noted that the HX-FTICR MS signals deteriorated significantly before the full extent of the exchange could be determined.25 Bekiroglu et al. have used NMR to measure and compare the proton exchange rate constants (kex) for CDs and maltoheptaose, the open form of β-CD.26 The kex values for the primary 6-OH groups are typically 5-6-fold faster than for secondary 2-OH and 3-OH. The exchange rate constants for the secondary OH groups of the CDs are also considerably smaller than for open-chain amylose (KR-CD ) 0.75, Kβ-CD ) 0.75, Kamylose ) 0.85), and this has been attributed to their involvement in intramolecular O2H-O3′ hydrogen bonding.22 By comparison, the exchange rates for the anomeric R1-OH and β1-OH hydroxyl protons of maltoheptaose are more than a magnitude greater.26 Deuterium Exchange MALDI-TOF MS of Stachyose. Stachyose (R-D-Gal-(1 f 6)-R-D-Gal-(1 f 6)-R-D-Glc-(1 f 2)-β-D-Fru) is a nonreducing tetrasaccharide from tubers of Japanese artichoke (Stachys sieboldii).27 It is structurally related to sucrose and raffinose in that it lacks a reducing sugar and contains an R,β1,2-linked D-Glc-D-Fru motif. Following MALDI-TOF MS, nonexchanged molecular ions ([M + Na]+ and [M + K]+) were evident for stachyose at m/z 689.1 and 705.1, respectively (Figure 4A), arising from the monoisotopic mass of 666.2 mass units for the neutral oligosaccharide. Following deuterium exchange, the [M + Na]+ peak was shifted to give a cluster of isotopomer peaks centered around m/z 702.3 (Figure 4A). This ion corresponds to [M(2H13) + Na]+ and is evidence for the exchange of 13 protons. A peak due to the fully exchanged stachyose ion, [M(2H14) + Na]+, was also evident at m/z 703.3, but at lower intensity that the +13 ion (Figure 4A). The adjacent peak at m/z 713.3 indicates that the xylopentaose internal standard was fully exchanged under these conditions. The observed 13 exchanged protons for stachyose is in apparent conflict with the 14 actively exchangeable sites in this molecule. As with the cyclodextrins, the recalcitrant, nonexchanged proton may occupy an environment that excludes D2O solvent, or it may be involved in a hydrogen bond of sufficient strength so as to resist exchange. For the structurally related sucrose disaccharide, an interresidue O2(Glc)-H-O1(Fru) hydrogen bond has been demonstrated to exist in aqueous solution.28 However, this interaction is transient and could only be observed (24) Sethi, S. K.; Smith, D. L.; McCloskey, J. A. Biochem. Biophys. Res. Commun. 1983, 112, 126-131. (25) Kellersberger, K. A.; Dejsupa, C.; Liang, Y.; Pope, R. M.; Dearden, D. V. Int. J. Mass Spectrom. 1999, 193, 181-195. (26) Bekiroglu, S.; Kenne, L.; Sandstrom, C. J. Org. Chem. 2003, 68, 16711678. (27) Greutert, H.; Keller, F. Plant Physiol. 1993, 101, 1317-1322.

5306 Analytical Chemistry, Vol. 78, No. 15, August 1, 2006

Figure 4. HX-MALDI-TOF MS of stachyose (A), chitotetraose (B), and erythromycin (C). The lower spectrum in each panel shows nonexchanged [M + Na]+ and M + K]+ ions (A, m/z 689.1 and 705.1; B, 853.2; and C, 738.5, 754.5, and 756.5). In the upper panels, ∆ is the observed number of protons exchanged by treatment with 2,5DHB-oxalic acid in D2O. Erythromycin (C) is predominantly present as its 6,12-ketal form (m/z 738.5 and 754.5) with three exchangeable protons, giving rise to m/z 741.5 and 757.5. The nonketal form (m/z 756.5) has the expected five exchangeable protons (exchanged m/z 761.5). The fully exchanged xylo-oligosaccharide internal standards (Xyl-5 ) m/z 713.3, and Xyl-6 ) m/z 846.8) are evident in panels A and B, respectively, and were assigned by reference to Figure 2.

Figure 5. Exchangeable protons for erythromycin (A) and 6,12-anhydroerythromycin (B). The tertiary 6-OH and 12-OH groups of anhydroerythromycin form ketal bonds to the 9-keto group, reducing the number of exchangeable protons by two. The exchangeable protons are italicized in bold, and position 6, 9, and 12 are as indicated.

under supercooled conditions.28,29 The torsional angles describing the (1 f 2) linkage for sucrose are remarkably similar to stachyose (but less so for raffinose),30 and a comparison of the crystallographic structures of R-D-Glc-(1 f 2)-β-D-Fru-containing oligosaccharides shows that it is only in sucrose and stachyose that the interresidue O2(Glc)-H-O1(Fru) hydrogen bond can exist.31 Noticeably, melezitose (R-D-Glc-(1 f 3)-β-D-Fru-(2 f 1)R-D-Glc), which lacks the O2(Glc)-H-O1(Fru) hydrogen bond, has be studied by deuterium-exchange fast atom bombardment MS with perdeuterioglycerol and is reported to undergo complete exchange.24 This is consistent with the present HX-MALDI-TOF MS data and suggests that it is the O2(Glc)-H-O1(Fru) hydrogen bonded proton of stachyose that is resistant to deuterium exchange. Deuterium Exchange MALDI-TOF MS of Chitotetraose. Deuterium exchange of proteins and peptides has established that amide protons are substantially more stable than hydroxyl protons. Chitotetraose is an amide-containing oligosaccharide consisting of four β-1,4-linked N-acetylglucosaminyl residues. It has a total of 14 exchangeable protons, including 4 amide protons, 4 primary hydroxyls, 5 secondary hydroxyls, and the anomeric 1-OH of the reducing residue. The MALDI-TOF mass spectrum of chitotetraose was characterized by the expected [M + Na]+ ion at m/z 853.2 (Figure 4B). Deuterium-exchanged chitotetraose generated a cluster of ions, of which the base peak (m/z 866.8) corresponds to the fully exchanged [M(2H14) + Na]+ ion (Figure 4B). Hence, the four amide protons on each N-acetylglucosaminyl residue of chitotetraose are exchangeable, analogous to the amide exchange of peptides. This is support for the general applicability of the method for other amide-containing sugars, such as the biologically important glycoaminoglycan, glycolipids, and N-linked glycoproteins. In this respect, one classic hydrogen-tritium exchange study of a hyaluronic acid tetrasaccharide, in which an acetamide hydrogen bond was identified based on exchange rate kinetics, has been reported.32 Deuterium Exchange MALDI-TOF MS of Erythromycin. The carbohydrate-containing antibiotic erythromycin A is char-

acterized by two unusual structural features.33,34 First, it is one of few natural products to contain two tertiary hydroxyl groups, at positions 6 and 12 of its macrolide ring (Figure 5). HX-MALDITOF MS might, therefore, determine whether these tertiary hydroxyls are amenable to deuterium exchange. Second, erythromycin is able to form intramolecular ketal bonds from the 6,12 tertiary hydroxyls to the position 9 ketone group. Hence, this 6,12anhydro form (called anhydroerythromycin A) was expected to contain two exchangeable protons less than the “free” erythromycin molecule (Figure 5). The MALDI-TOF MS spectrum indicated that erythromycin A was predominantly present in the 6,12-anhydro form, as characterized by molecular adduct ions [M + Na]+ and [M + K]+ at m/z 738.5 and 754.5, respectively (Figure 4.C.). An additional [M + Na]+ ion at m/z 756.5 is assigned to the native erythromycin A ketone. Following deuterium exchange, the anhydroerythromycin ions are shift by three mass units to m/z 741.5 and 757.5 (Figure 4C). This is indicative of the three deuterium exchanges presumed to occur on the free 11-OH group in the macrolide ring and on the hydroxyl group in each of the cladinose and desosamine sugar residues (Figure 5). Because the C-6 and C-12 hydroxy groups are also exchangeable for the nonketal form, erythromycin A was expected to undergo five deuterium exchanges to give a predicted [M(2H5) + Na]+ ion. Although the spectrum is complex in this region due to overlap with the [M(2H3) + K]+ for exchanged anhydroerythromycin, the predicted [M(2H5) + Na]+ ion is observed as a small peak at m/z 761.5 (Figure 4C). Thus, the tertiary hydroxyl groups of erythromycin are freely exchangeable, and it was possible to enumerate the proton exchangeable groups that distinguish erythromycin and 6,12-anhydroerthyromycin.

(28) Sheng, S.; van Halbeek, H. Biochem. Biophys. Res. Comm. 1995, 215, 504510. (29) French, A. D.; Kelterer, A.-M.; Cramer, C. J.; Johnson, G. P.; Dowd, M. K. Carbohydr. Res. 2000, 326, 305-322. (30) Gilardi, R. D.; Flippen, J. L. J. Am. Chem. Soc. 1975, 97, 6264-6266. (31) Jeffrey, G. A.; Huang, D.-B. Carbohydr. Res. 1991, 210, 89-104.

(32) Oberholtzer, J. C.; Englander, S. W.; Horwitz, A. F. FEBS Letts. 1983, 158, 305-309. (33) Mordi, M. N.; Pelta, M. D.; Boote, V.; Morris, G. A.; Barber, J. J. Med. Chem. 2000, 43, 467-474. (34) Tsuji, K.; Robertson, J. H. Anal. Chem. 1971, 43, 818-821. (35) Hunt, D. F.; Sethi, S. K. J. Am. Chem. Soc. 1980, 102, 6953-6963.

DISCUSSION H/D exchange has long been used in mass spectrometry to probe protein structures, although this is the first reported use of HX-MALDI-TOF MS for carbohydrates. It is generally believed that proton affinities must be similar for exchange to occur35,36

Analytical Chemistry, Vol. 78, No. 15, August 1, 2006

5307

and typically within 25 kcal/mol of that of 2H2O.25 The assumption is that the forward exchange reaction goes to completion when the sample is dissolved in excess D2O.36 Earlier work on gas-phase deuterium exchange has suggested that the exchange occurs via a sequential mechanism involving the formation of a HD2O+ complex at each position where proton transfer can occur.35-38 The existence of proton-bridged intermediates is inferred from the observation that the exchange rates for adjacent sites are slower than those that could be spanned by a bridging proton.25 An important observation is that H/D exchange in the solid phase occurs on the order of days, whereas in the gas-phase exchange is close to the collision limited rate.26,39,40 For the MALDI-TOF MS experiment, the sample must be cocrystallized onto the target with the matrix. Hence, the process of backexchange that occurs on the MS target is far slower than the forward exchange reaction because the diffusion of water through the crystalline matrix becomes rate-limiting. In the present study on oligosaccharides, the back-exchange was found to be reduced by oxalic acid, and this was crucial for obtaining spectra of fully exchanged ions. Oxalic is a relatively strong diprotic acid (pKa1 ) 5.6 × 10-2), and in its presence in D2O solution, the weaker 2,5-dihydrobenzoic acid (2,5-DHB) matrix is in its fully deuterated conjugate acid form. Indeed, [M(2H3) + 2H]+ and [M(2H3) + Na]+ ions for deuterium-exchanged 2,5-DHB were evident at m/z 159.0 and 180.0, respectively (data not shown). Hence, this may act to buffer the system so that any back-exchange with atmospheric moisture occurs predominantly on the 2,5-DHB matrix rather than on the exchanged hydroxyl groups of the carbohydrate. In summary, the HX-MALDI-TOF MS methodology importance to the study of protein conformation has been optimized for use (36) Scheiner, S.; Cuma, M. J. Am. Chem. Soc. 1996, 118, 1511-1521. (37) Freiser, B. S.; Woodin, R. L.; Beauchamp, J. L. J. Am. Chem. Soc. 1975, 97, 6893-6894. (38) Ausloos, P.; Lias, S. G. J. Am. Chem. Soc. 1981, 103, 3641-3647. (39) Green, M. K.; Lebrilla, C. B. Mass Spectrom. Rev. 1997, 16, 53-71. (40) Amado, A. M.; Ribeiro-Claro, P. J. A. J. Chem. Soc., Faraday Trans. 1997, 93, 2387-2390.

5308

Analytical Chemistry, Vol. 78, No. 15, August 1, 2006

with oligosaccharide sugars. Carbohydrate samples are fully exchanged in the presence of deuterated water. The crystallized matrix-sample complexes necessary for obtaining MALDI-TOF MS spectra undergo slow back-exchange, but this process is sufficiently blocked by the inclusion of oxalic acid to obtain spectra of fully exchanged sugars. The technique has been applied to several diverse carbohydrate structures, and amide; carboxy; and primary, secondary, and tertiary hydroxy protons are all fully exchanged, providing a new tool for the characterization of complex carbohydrates. Furthermore, the potential for controlling the back-exchange of matrix-substrate cocrystals and the use of defined internal standards to assess the extent of the exchange process should facilitate kinetic exchange studies crucial to understanding carbohydrate folding, binding studies, and ligand interactions. Abbreviations. m.u., mass units; dp, degree of polymerization; NMR, nuclear magnetic resonance. ACKNOWLEDGMENT Thanks are due to Mr. Jim Nicholson for technical support and to my colleagues Drs. Shuqun Sheng and Frank Momany for discussion. Deborah Palmquist is thanked for the statistical analysis, and Dr. Greg Cote, for the gift of alternan oligosaccharides. This work was supported by NIH grant U01 DE016267-02. Mention of trade names or commercial products in this paper is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review December 8, 2005. Accepted June 6, 2006. AC052168E