Liquid Infrared Atmospheric Pressure Matrix-Assisted Laser

Anal. Chem. 2003, 75, 3212-3218

Liquid Infrared Atmospheric Pressure Matrix-Assisted Laser Desorption/Ionization Ion Trap Mass Spectrometry of Sialylated Carbohydrates Christopher E. Von Seggern,† Susanne C. Moyer,†,‡ and Robert J. Cotter*,†

Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, Maryland 21205, and Department of Chemistry, Johns Hopkins University, 3400 North Charles Street, Baltimore, Maryland 21218

A 2.94-µm Er:YAG laser for IR atmospheric pressure matrix-assisted laser desorption/ionization on an ion trap mass spectrometer is used for the analysis of sialylated oligosaccharides. This approach provided the opportunity to utilize liquid matrixes and is effective in determining structural featuresssequence, branching, and linkages of intact, fully sialylated molecular species. Sialic acid residues are nine-carbon sugars that are often found at the nonreducing end of oligosaccharides, glycoproteins, and glycolipids. The proximity of sialic acids to the terminal ends of sugars, as well as their inherent charge, makes them integral in cell-cell interactions.1 Conversely, sialic acids have the ability to prevent cell-cell interactions through their bulky structure and negative charge repulsion.2 Sialic acids also serve to mask nearterminal sugar residues important for cell-cell contact, further inhibiting cellular interactions.3 Sialic acids can also act as ligands for binding proteins. Two main classes of sialic acid binding proteins exist: Siglecs (sialic acid binding Ig-like lectins) and selectins. These binding proteins are integral in cellular attachment and adhesion and are crucial for the function of the immune system.4 Sialic acids are critical for viral adhesion and infection. Influenza A and B, human cytomegalovirus, and adenovirus type 7 infections are inhibited by sialidase treatment, indicating a vital role for sialic acids as attachment and infection determinants.5 Furthermore, sialic acids play key roles in bacterial adhesion and colonization in Escherichia coli, Haemophilus influenzae, and Bordetella pertussis.5 Increasing awareness of the importance of sialic acids as recognition determinants has led to the development of therapeutic agents for the treatment of a variety of ailments ranging from viral infection to cancer.4 * Corresponding author. Phone: (410) 955-3022. Fax: (410) 955-3420. E-mail: [email protected]. † Department of Pharmacology and Molecular Science. ‡ Department of Chemistry. (1) Varki, A. FASEB J. 1997, 11, 248-255. (2) Varki, A. Glycobiology 1993, 3, 97-130. (3) Crocker, P. R.; Varki, A. Immunology 2001, 103, 137-145. (4) Schauer, R. Glycoconjugate J. 2000, 17, 485-499. (5) Schauer, R.; Kelm, S. Int. Rev. Cytol. 1997, 175, 137-240.

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The analysis of carbohydrate structure is a complex problem due to the variation in oligosaccharide substituents as well as the need for branching and linkage information. Mass spectrometric analysis of carbohydrates is attractive due to the high sensitivity of this method. Tandem mass spectrometry of oligosaccharides affords the opportunity to acquire sequence, branching, and linkage information from the resulting fragmentation spectra. To utilize matrix-assisted laser desorption/ionization (MALDI) mass spectrometry for the evaluation of acidic oligosaccharide samples, analysis of the intact species, as well as controlled fragmentation to characterize both the constituents and the linkages, is needed. Thorough analysis of carbohydrates has been demonstrated by MALDI6 and electrospray ionization (ESI), with emphasis placed on structural determination of acidic sugars.7 Although sialic acid-containing carbohydrates have an inherent negative charge, sialic acids are relatively unstable and are likely to decompose in the flight tube of a time-of-flight instrument. In particular, spectra recorded in the reflectron mode tend to display significant postsource decay fragmentation resulting from the loss of sialic acid residues,8-10 requiring many analyses of acidic carbohydrates to be carried out in the linear mode in order to minimize the loss of these groups.11 The internal energy of a MALDI ion directly affects the ion fragmentation. In a vacuum MALDI, parameters such as laser wavelength and power, extraction voltages, and “hot” or “cool” matrixes directly influence the type and degree of ion fragmentation.12 While some of these factors may be exploited to produce fragmentation of a MALDI-generated ion, which reveals structural information, many fragmentation processes result in the elimination of labile groups that are structurally and functionally important. (6) Harvey, D. H. Mass Spectrom. Rev. 1999, 18, 349-451. (7) Wheeler, S. F.; Harvey, D. J. Anal. Chem. 2000, 72, 5027-5039. (8) Harvey, D. J.; Hunter, A. P.; Bateman, R. H.; Brown, J.; Critchley, G. Int. J. Mass Spectrom. 1999, 188, 131-146. (9) Huberty, M. C.; Vath, J. E.; Yu, W.; Martin, S. A. Anal. Chem. 1993, 65, 2791-2800. (10) Talbo, G.; Mann, M. Rapid Commun. Mass Spectrom 1996, 10, 100-103. (11) Tsarbopoulos, A.; Bahr, U.; Pramanik, B. N.; Karas, M. Int. J. Mass Spectrom. Ion Processes. 1997, 169/170, 251-256. (12) Guanghong, L.; Marginean, I.; Vertes, A. Anal. Chem. 2002, 74, 61856190. 10.1021/ac0262006 CCC: $25.00

© 2003 American Chemical Society Published on Web 05/13/2003

Laiko and co-workers recently introduced an atmospheric pressure (AP)-MALDI source that was coupled to an orthogonal acceleration time-of-flight mass spectrometer.13 The development of the AP MALDI source resulted in an ionization method that produces molecular ions with low internal energy and, as a result, minimal fragmentation. This may be due to the thermalization of the AP MALDI ions that occurs due to collisions with ambient gases. Conversely, MALDI ions produced in a vacuum can possess sufficient internal energy to undergo unimolecular dissociation, producing either prompt fragments or metastable decay fragments that complicate spectral interpretation.13,14 AP MALDI offers the advantages typically associated with a MALDI source such as minimum sample cleanup, ease of sample preparation, and easily interpretable singly charged spectra. At the same time, AP MALDI does not require a vacuum at the source and is readily coupled to instruments possessing an atmospheric pressure interface, making it easily interchangeable with other atmospheric pressure sources, such as ESI. A number of groups have now combined AP-MALDI with commercial ion trap mass spectrometers.15-17 Coupling the APMALDI source with an ion trap mass analyzer combines the benefits of MALDI sample preparation and simplicity of spectral analysis resulting from the production of predominantly singly charged ions with the MSn capabilities of the quadrupole ion trap mass spectrometer. This configuration has proven to be useful in obtaining structural information for peptides and protein digests as well as for the identification and characterization of posttranslational modifications.15,16,18-22 Ultraviolet AP-MALDI has been previously applied to the analysis of the neutral oligosaccharide, difucosyllacto-N-tetraose, by Laiko and co-workers13 using an orthogonal acceleration timeof-flight instrument. UV AP-MALDI coupled to an ion trap mass spectrometer was implemented by Creaser et al.15 to acquire MSn spectra of sodium adducts of neutral oligosaccharides and by Moyer and co-workers for the negative mode analyses of sialylated lactoses.19 Recently, Laiko et al. demonstrated the use of liquid water and glycerol matrixes with an infrared optical parametric oscillator laser tuned to 3 µm for the AP-MALDI-ion trap mass spectrometric (13) Laiko, V. V.; Baldwin, M. A.; Burlingame, A. L. Anal. Chem. 2000, 72, 652657. (14) Spengler, B.; Kirsch, D.; Kaufmann, R. J. Phys. Chem. 1992, 96, 96789684. (15) Creaser, C. S.; Reynolds, J. C.; Harvey, D. J. Rapid Commun. Mass Spectrom. 2002, 16, 176-184. (16) Danell, R. M.; Glish, G. L. An Atmospheric Pressure MALDI Probe for Use with ESI Source Interfaces. Proceedings of the 48th ASMS Conference on Mass Spectrometry and Allied Topics, Long Beach, CA, June 2000. (17) Galicia, M. C.; Vertes, A.; Callahan, J. H. Anal. Chem. 2002, 74, 18911895. (18) Laiko, V. V.; Moyer, S. C.; Cotter, R. J. Anal. Chem. 2000, 72, 5239-5243. (19) Moyer, S. C.; Marzilli, L. A.; Woods, A. S.; Laiko, V. V.; Doroshenko, V. M.; Cotter, R. J. Int. J. Mass Spectrom. 2003, 226, 133-150. (20) Moyer, S. C.; Cotter, R. J.; Woods, A. S. J. Am. Soc. Mass Spectrom. 2002, 13 274-283. (21) Marzilli, L. M.; Moyer, S. C.; Cotter, R. J. Analysis of Posttranslational Modifications by Atmospheric Pressure MALDI/Ion Trap Mass Spectrometry. Proceedings of the 49th ASMS Conference on Mass Spectrometry and Allied Topics, Chicago, IL, May 2001. (22) Moyer, S. C.; Cotter, R. J.; Woods, A. S. Atmospheric Pressure MALDI/Ion Trap Mass Spectrometry of Peptide-Peptide Interactions. Proceedings of the 49th ASMS Conference on Mass Spectrometry and Allied Topics, Chicago, IL, May 2001.

(ITMS) analysis of peptides.23 Subsequent work in our laboratory24,25 has focused on the use of liquid IR AP-MALDI-ITMS for the analysis of noncovalent complexes and phosphopeptides. In this work, we describe the use of a 2.94-µm Er:YAG laser for liquid AP-MALDI-ITMS of sialylated oligosaccharides. EXPERIMENTAL SECTION Materials. Disialylmonofucosyllacto-N-hexaose, disialyllactoN-tetraose, LS-tetrasaccharide a, LS-tetrasaccharide b, 3′sialyl-3fucosyllactose, and 6′-sialyllactose were purchased from Glyko, Inc. (Novato, CA) and dissolved to 1 mM concentration in 18MΩ MilliPure water, unless otherwise stated. Glycerol and 3-nitrobenzyl alcohol (NBA) matrixes were acquired from SigmaAldrich (St. Louis, MO), and all materials were used without further purification. Liquid Infrared AP-MALDI Ion Trap Mass Spectrometry. Analyses were carried out on a ThermoFinnigan LCQ Classic (San Jose, CA) quadrupole ion trap mass spectrometer equipped with a modified Mass Technologies, Inc. (Burtonsville, MD) AP-MALDI source. This experimental configuration has been described previously16,17 and is currently equipped with a Bioscope UV+ laser system (Bioptic Lasersysteme, Berlin, Germany) consisting of a Nd:YAG laser (355 nm) and an Er:YAG laser (2940 nm) focused with a sapphire lens (F ) 150 mm). The laser spot size was ∼0.1 mm, measured by matrix ablation. For the experiments described here, the AP-MALDI source was operated in the infrared mode (2940 nm) in order to utilize liquid matrixes that absorb infrared radiation at this wavelength. The Er:YAG laser generated ∼100-ns pulses at 5 Hz with ∼400 µJ/pulse. The laser was run asynchronously with the trapping cycle and spectra were acquired at 300 ms/scan. A voltage of 2.5 kV was applied to the AP-MALDI target plate, and the capillary temperature was set to 200 °C. Samples were prepared by mixing ∼1 µL of liquid matrix (NBA or glycerol) with 1 µL of analyte solution on the AP-MALDI target plate. Aqueous samples were spotted directly on the target for analysis, using water as a matrix. The resulting mixture was then analyzed in liquid form. RESULTS AND DISCUSSION AP MALDI is a “soft” ionization technique located external to the mass spectrometer. This configuration provides the ability to perform collision-induced dissociation (CID) in an ion-trap mass spectrometer on MALDI-generated ions. One advantage that APMALDI has over typical vacuum MALDI is the ability to use liquid matrixes. Liquid samples survive much longer at atmospheric pressure than in a vacuum and consequently can be utilized as matrixes. The wavelength of an Er:YAG laser is 2940 nm, which coincides with the OH and NH stretching frequencies, introducing the possibility of carrying out analyses in liquid water or other (23) Laiko, V. V.; Taranenko, N. I.; Berkout, V. D.; Yakshin, M. A.; Prasad, C. R.; Lee, H. S.; Doroshenko, V. M. J. Am. Soc. Mass Spectrom. 2002, 13, 354-361. (24) Moyer, S. C.; Woods, A. S.; Cotter, R. J. Fragmentation of Phosphotyrosine Containing Peptides by Atmospheric Pressure MALDI Ion Trap Mass Spectrometry. Presented at the 2nd Mass Spectrometry Conference Applied to Biological Warfare Agents, Warwick, U.K., April, 2002. (25) Cotter, R. J.; Moyer, S. C. Atmospheric Pressure MALDI on an Ion Trap Mass Spectrometer. Proceedings of the 50th ASMS Conference on Mass Spectrometry and Allied Topics, Orlando, FL, June 2002.

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Chart 1. Structures of (a) 6′-Sialyllactose, (b) 3′-Sialyl-3-fucosyllactose, (c) LS-tetrasaccharide a, (d) LS-tetrasaccharide b, (e) Disialyllacto-N-tetraose, and (f) Disialylmonofucosyllacto-N-hexaose

liquid matrixes such as glycerol. Thus, the combination of an infrared laser and atmospheric ionization by MALDI provides a novel approach to oligosaccharide analysis with the unique opportunity for obtaining structural information from the collisioninduced dissociation of structures that are intact, including normally labile groups. Spectra were obtained using NBA, glycerol, or water liquid matrixes (Figure 1). A 1-µL sample of the aqueous 6′-sialyllactose (6′-SL) (Chart 1a) solution was mixed with either nitrobenzyl alcohol or glycerol and spotted directly onto the AP-MALDI target plate. 6′-SL was analyzed in the negative ion mode, with the predominant peak in each spectrum corresponding to the intact [M - H]- ion. While all three liquid matrixes produced repeatable results, liquid samples spotted in glycerol were longer-lived and produced spectra with better signal-to-noise ratios than those 3214

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samples analyzed in NBA or liquid water. The 6′-SL sample spotted with glycerol appeared to mix continuously due to the laser pulsing, which could account for the longevity of the sample and the consistency of the spectral scans. While spectra can be obtained directly from aqueous samples, the rapid heating of water by the IR laser results in fast evaporation, and the water samples survive for only a short period of time. In addition, the spectrum resulting from analysis of the sample in water displays a decreased signal-to-noise ratio as compared to the sample in glycerol matrix. Samples spotted in NBA do not evaporate as quickly as aqueous samples; however, they also do not appear to mix as thoroughly as the glycerol spotted samples. The resulting spectra appear similar to glycerol samples, but with lower signal-to-noise ratios. As a result, glycerol was used exclusively for the remainder of the experiments described here.

Figure 2. Negative mode MS/MS spectra of 6′-SL at m/z 632 in glycerol matrix.

Figure 1. Full-scan negative mode spectra of 6′-sialyllactose (6′SL) (40 pmol applied to target) with (a) NBA (b) water, and (c) glycerol matrixes.

The negative mode MS/MS spectrum of a singly sialylated lactose sugar (6′-SL) was obtained (Figure 2a). The fragmentation is described as per the nomenclature established by Domon and Costello.26 The predominant peak resulting from CID of 6′-SL is the cross-ring cleavage 0,2A3 with significant peaks resulting from the C2 fragmentation as well as the loss of sialic acid from the nonreducing end of the oligosaccharide. (26) Domon, B.; Costello, C. Glycoconjugate J. 1988, 5, 397-409.

3′-Sialyl-3-fucosyllactose (3′-S, 3-FL) was analyzed to determine the effect of a fucose linked to a lactose core (Chart 1b). Fullscan analysis yields only an intact molecular ion peak, without the loss of either the sialic acid or the fucose, both labile groups (data not shown). Negative mode MS/MS reveal Z1β and the C1 fragments, indicating a linkage of a fucose to glucose, information unknown from simple molecular weight analysis (data not shown). Structural information can also be gained from oligosaccharides that contain the same constituents, and therefore the same molecular weight, but have different linkage patterns. This is the most interesting challenge since oligosaccharides have no simple oligomeric structure similar to that of peptides or oligonucleotides. The complexity can vary at both the branching points and the carbon linkage location for each monosaccharide, resulting in an overwhelming number of possible conformations. Fragmentation spectra can aid in the determination of differences in branching and linkage. LS-tetrasaccharide a and LS-tetrasaccharide b (LSTa and LSTb) differ only by the location of the sialic acid with respect to their core structure. The sialic acid on LSTa is linked to the terminal galactose at carbon-3, while the sialic acid in LSTb is linked to an internal N-acetylglucosamine (GlcNAc) at carbon-6 (Chart 1c,d). The location of each sialic acid can be determined through MS/MS analysis of the molecular ion. Negative mode analysis results in different fragmentation patterns for each compound (Figure 3b). Fragment ion spectra can be used to determine the composition of larger molecules with similar structures. While the majority of the peaks are the same in each spectrum, subtle differences aid in branching determination. For example, the peak at m/z 452 (Figure 3a) corresponds to a sialic acid residue linked to a hexose, indicative of LSTa. This peak is not present in Figure 3b, indicating a different linkage from sialic acid to the core oligosaccharide. In Figure 3b, the peak at m/z 493 corresponds to a sialic acid linked to a GlcNAc, a structural possibility only for LSTb. Disialyllacto-N-tetraose (DSLNT) has the same structure as LSTa with an additional sialic acid residue linkage as is found in LSTb (Chart 1e). Negative ion mode full scan of DSLNT produces a spectrum with predominantly the intact molecular ion, with a minor amount of sialic acid loss apparent at m/z 997 (Figure 4a). The loss of sialic acid was minimized by the adjustment of the Analytical Chemistry, Vol. 75, No. 13, July 1, 2003

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Figure 3. Negative mode MS/MS spectra of (a) LSTa at m/z 997 and (b) LSTb at m/z 997 in glycerol matrix.

Figure 4. Negative mode spectra of DSLNT. (a) Full-scan and (b) MS3 of m/z 997.

nozzle (capillary)-skimmer voltage, to a potential difference of 5 V. MS/MS analysis resulted in one primary cleavage (Y4R), the loss of a sialic acid residue (data not shown). The Y4R assignment, rather than the Y3β assignment, can be made based on MS3 analysis and is consistent with electrospray data for this compound as reported by Wheeler and Harvey.7 MS3 analysis of the Y4R ion results in a spectrum similar to that of the MS/MS spectrum of LSTb rather than that of LSTa (Figure 4b). MS4 analysis of the C4/Y4R ion revealed more structural information (data not shown). Multiple rounds of CID can increase the number of structurally informative fragments over those obtained by PSD. Positive mode analysis of the sodium adducts of sialylated sugars was performed in order to produce complimentary CID data. Performing analyses in both the positive and negative ion modes is useful in acquiring structural information for oligosaccharides. One advantage of oligosaccharide analysis in glycerol is the ease of glycan cationization with sodium and potassium. Sodium adduction to the sialylated sugars results in the most abundant peaks observed in the positive mode full-scan spectrum. As a result, the [M + Na]+ ion of DSLNT was chosen for positive mode MSn analysis (Figure 5a). As expected, the initial loss in the positive mode MS/MS analysis of DSLNT was a sialic acid cleavage at m/z 1021.3 (Y4R), with a minor peak at m/z 730.3 corresponding to the loss of both sialic acids (Y4R/3b) (Figure 5b). 3216 Analytical Chemistry, Vol. 75, No. 13, July 1, 2003

MS3 analysis of the sodiated Y4R ion of DSLNT at m/z 1021.4 results primarily in the loss of the second sialic acid, producing the sodiated Y4R/3b ion (Figure 5c). Structural information from the core structure of the sugar can be gleaned from the MS4 spectrum (Figure 5d). Cross-ring cleavages aid in linkage determination for the glucose at the reducing end of the sugar. Furthermore, the ordered linkage of sugar residues can be determined from the progressive loss of hexoses, leaving the eventual Gal-GlcNAc or GlcNAc-Gal linkage. Combining the data from both positive and negative mode CID experiments of DSLNT provides information regarding terminal residue linkages, as well as branching and linkage from the reducing end of the oligosaccharide that could not be assessed from the negative mode fragmentation spectra. Furthermore, the longevity of these samples in glycerol provides the opportunity for positive and negative mode analysis of the same spotted sample, with the ability to perform multiple MSn experiments without respotting the analyte. Glycerol viscosity allowed up to 30 min of continuous analysis. Positive mode analysis of sodiated oligosaccharides can be extended to larger sugars, which contain both sialic acid residues and fucose residues (Figure 6). Determination of monomer composition of larger oligosaccharides can be difficult. Disialyl,monofucosyllacto-N-hexaose (DSFLNH) is an oligosaccharide that

Figure 5. Positive mode CID spectra of sodiated DSLNT. (a) Full-scan (b) MS/MS of [M + Na]+ at m/z 1312, (c) MS3 of m/z 1021, and (d) MS4 of m/z 730.

Figure 6. Positive mode CID spectra of sodiated DSFLNH. (a) MS/MS of [M + Na]+ at m/z 1823 and (b) MS4 of m/z 1241.

contains two terminal sialic acid residues as well as a terminal fucose residue (Chart 1f). Both fucose and sialic acids are labile groups and tend to fragment easily by MALDI techniques.6 However, the positive ion mode full-scan spectrum of DSFLNH is dominated by the singly charged sodiated ion, with no apparent loss of either sialic acid or fucose (data not shown). Potassium

adducts as well as sodium and potassium salts are also formed when analysis is carried out in glycerol. However, the sodium adduct ion at m/z 1823 was chosen for MSn analysis because it was the most abundant ion present in the spectrum. As was seen with DSLNT, MS/MS analysis of DSFLNH results in the loss of a sialic acid residue, though it is not possible to identify which Analytical Chemistry, Vol. 75, No. 13, July 1, 2003

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residue is lost first (Figure 6a). MS3 analysis of DSFLNH indicates the loss of the second sialic acid residue (data not shown). Figure 6b is the MS4 spectrum of DSFLNH following the loss of both sialic acids and provides a variety of fragment ions that are structurally informative. Comparison of the fragments obtained from the CID spectra of DSLNT and DSFLNH reveal structural similarities between the two glycans (Figure 6b). CID spectra of both compounds have ions at m/z 610 and 670, corresponding to cross-ring cleavages of the glucose at the reducing end of the sugar. The presence of a peak at m/z 550 indicates a similar core containing two hexoses and a GlcNAc.

The IR AP-MALDI technique combines the production of thermally cooled MALDI ions generated by AP-MALDI with the ability to ionize from liquid samples that are excited at 2940 nm. We have demonstrated the use of liquid matrixes including water, nitrobenzyl alcohol, and glycerol for the analysis of acidic oligosaccharides. Due to the longevity of the glycerol analyzed samples, successive rounds of MSn were performed on sialylated oligosaccharide samples. Data from both positive and negative modes were obtained, providing complimentary fragmentation information. The “soft” ionization produced by AP-MALDI enables the study of intact singly charged sialylated oligosaccharides.

CONCLUSIONS While significant structural information can be obtained from complimentary CID fragmentation of acidic glycans, not all linkage and branching information can be determined. There is still work to be done in the development of methods to increase the performance of MSn experiments for this class of compounds. In higher orders of MSn of these compounds, data become difficult to analyze due to sequential decreases in ion counts and the eventual fragmentation to core structures that may be invariant between different samples. Often there are choices between different identification possibilities that make linkage and overall structure difficult to address.

ACKNOWLEDGMENT Funding for this work is provided by a contract (DABT63-991-0006) to R.J.C. from the Defense Advanced Research Project Agency (DARPA). C.E.V. is supported by a NIH Training Grant in Anti-Cancer Drug Development (CA 09243). Support for S.C.M. is provided by a NSF-GOALI Grant (CHE 9634238).

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Received for review October 4, 2002. Accepted March 28, 2003. AC0262006