Electrospray Ionization and Matrix-Assisted Laser Desorption

Electrospray ionization (ESI)39 and matrix-assisted laser ... potential gating, ions may be guided through the second octupole and trapped in the ICR ...
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Anal. Chem. 1998, 70, 857-864

Electrospray Ionization and Matrix-Assisted Laser Desorption/Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry of Permethylated Oligosaccharides Touradj Solouki,† Bruce B. Reinhold,‡ Catherine E. Costello,‡ Matthew O’Malley, Shenheng Guan,§ and Alan G. Marshall*,§

Center for Interdisciplinary Magnetic Resonance, National High Magnetic Field Laboratory, 1800 East Paul Dirac Drive, Florida State University, Tallahassee, Florida 32310

Mass spectra of fragments of permethylated oligosaccharides are analyzed by Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry. Sustained offresonance irradiation (SORI) collision-induced dissociation (CID), quadrupolar axialization, multiple stages of isolation and dissociation (MSn), and ion remeasurement are exploited for carbohydrate structural analyses. That SORI CID internal energies are adequate for linkage analysis of a permethylated glucose oligomer is demonstrated by identifying ring-opened fragment ions from MALDI-generated mass-isolated and collisionally activated ions. Ion remeasurement and axialization techniques enhance the sensitivity of ion fragmentation analysis. Multiple stages of isolation and dissociation of ion fragments (MSn) provide for structural analysis of an electrospray-ionized permethylated lacto-N-fucopentaose isomer (LNFP II). Compared to MS2 spectra taken with a triple quadrupole, FT-ICR MSn (n > 2) provides more extensive characterization of the parent molecular structure than is available from a single stage of ion isolation and dissociation (MS2). Biological carbohydrates constitute a diverse group of polymers playing various roles in cellular processes, ranging from energy storage (glycogen) or structural support (cellulose, chitin) to signaling, adhesion, and protein modifications.1,2 Biological carbohydrates range from simple monosaccharides to megadalton homopolymers, from precisely structured signaling oligosaccharides to complicated mixtures of carbohydrate-modified proteins and lipids. The study of carbohydrate biology, or glycobiology, has seen enormous growth in the past decade, and with this growth comes an increasing emphasis on analytical tools capable of molecular resolution into structure/function relationships. †Present

address: IIT Research Institute, 10 West 35th St., Chicago, IL 60616. Spectrometry Resource, Boston University School of Medicine, Boston, MA 02118. § Member of the Department of Chemistry, Florida State University, Tallahassee, FL 32310. (1) Karlsson, K.-A. Annu. Rev. Biochem. 1989, 58, 309-350. (2) Varki, A. Glycobiology 1993, 3, 97-130. ‡Mass

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The principal difficulty in structural identification of carbohydrate oligomers is the large number of isomers arising both from variation in linkage positions between monomer residues and from the possibility of multiple linkages to a single residue (branching), e.g., more than 1012 structures for a reducing hexasaccharide!3 Hence, for a straight chain oligomer, structural identification requires the sequence of glycosyl linkage types, i.e., the linkage position and anomeric configuration, as well as the sequence of monomer residues analogous to amino acid (protein) or nucleic acid (DNA, RNA) oligomers. Multiple linkages allow for multiple connection topologies, and biological carbohydrates generally exhibit complicated branching structures as well as linear oligomers. Minor differences in linkage types or branching topology may have only subtle effects on the molecule’s overall physical properties (hydrophobicity, hydrodynamic radius, or solution ionization) but may have profound effects on the local molecular geometry and, hence, on the biological activity. Along with the structural isomers that arise through the variation in the types and locations of glycosidic bonds, individual monosaccharides such as glucose, galactose, and mannose are, themselves, structural isomers. For many of the carbohydrates derived from eukaryotic cells, the number of different monosaccharides is not very large; for prokaryotic cells, the monosaccharide diversity itself can pose a severe challenge.4 Carbohydrates are often modified by sulfates or phosphates or otherwise acylated, and both the nature and position of the modification on the residue and the position of the modified residue within the oligomer may need to be determined. Further, many of the structurally interesting biological carbohydrates are glycoconjugates, e.g., glycoproteins, glycolipids, or, in the case of glycophosphatidyl inositol anchors, glycolipoproteins. Aspects of this association (degree of glycosylation, chemical nature of the linked lipids) are generally sought along with details of the carbohydrate structure. Conjugation also presents an additional dimension of structural isomers, in terms of the specific location of a carbohydrate on a glycoprotein. The combination of conjugation and isomeric structural complexity has prevented the development of standard sequencing methodologies (3) Laine, R. A. Glycobiology 1994, 4, 759-767. (4) Reinhold, B. B.; Hauer, C. R.; Plummer, T. H.; Reinhold, V. N. J. Biol. Chem. 1995, 270, 13197-13203.

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for carbohydrates, as have long been available for polypeptide or nucleic acid analysis. All of the existing analytical techniques applied to glycobiology invariably suffer the usual problems of biological analyses: vanishingly small sample amount, conjugation or chemical modification, and unresolved, complex mixtures. Many of these problems are well suited to mass spectrometry: fast atom bombardment (FAB),5,6 laser desorption (LD) ionization,7 matrixassisted laser desorption/ionization (MALDI),8,9 and electrospray ionization (ESI)10 have all dramatically impacted carbohydrate mass analysis. The ability of these ionization methods to generate intact ions from larger molecules and the constant need to increase sensitivity has shifted analytical methodology away from “wet lab” chemical degradation and MS analysis of the degradation products toward tandem mass spectrometry. Tandem MS has greatly extended the sensitivity and structural resolution of mass analysis applied to carbohydrates.5,10-14 However, the diversity of structural isomers so characteristic of carbohydrates remains a particular challenge for all types of mass analysis, and for the vast majority of biological oligosaccharides the complete structural characterization is not possible by tandem MS alone. The rapid recent development of both Paul and Penning ion trap mass spectrometers is attributable to their suitability for coupling to MALDI and ESI ion sources. Trapped ion instruments offer many inherent advantages over sector and quadrupole beam mass spectrometers.15-26 However, it is the ability of the ion trap devices to select particular parent ion(s), fragment them, detect the product ions, and then repeat the isolation/fragmentation (5) Dell, A. Adv. Carbohydr. Chem. Biochem. 1987, 45, 19-72. (6) Suzuki, S.; Kakehi, K.; Honda, S. Anal. Chem. 1996, 68, 2073-2083. (7) Spengler, B.; Dolce, J. W.; Cotter, R. J. Anal. Chem. 1990, 62, 1731-1737. (8) Stahl, B.; Steup, M.; Karas, M.; Hillenkamp, F. Anal. Chem. 1991, 63, 14631466. (9) Whittal, R. M.; Palcic, M. M.; Hindsgaul, O.; Li, L. Anal. Chem. 1995, 67, 3509-3514. (10) Reinhold, V. N.; Reinhold, B. B.; Costello, C. E. Anal. Chem. 1995, 67, 1772-1784. (11) Egge, H.; von Nicolai, H.; Zilliken, F. FEBS Lett. 1974, 39, 341-344. (12) Dell, A.; Reason, A. J.; Khoo, K. H.; Ranico, M.; McDowell, R. A.; Morris, H. R. Methods Enzymol. 1994, 230, 108-132. (13) Egge, H.; Peter-Katalinic, J. Mass Spectrom. Rev. 1987, 6, 331-393. (14) Lemoine, J.; Fournet, B.; Despeyroux, D.; Jennings, K. R.; Rosenberg, R.; de Hoffmann, E. J. Am. Soc. Mass Spectrom. 1993, 4, 197-203. (15) Guan, S.; Marshall, A. G. Int. J. Mass Spectrom. Ion Processes 1995, 146/ 147, 261-296. (16) Buchanan, M. V.; Hettich, R. L. Anal. Chem. 1993, 65, 245A-259A. (17) McLafferty, F. W. Acc. Chem. Res 1994, 27, 379-386. (18) March, R. E.; Todd, J. F. J. Practical Aspects of Ion Trap Mass Spectrometry. Vol. 1. Fundamentals of Ion Trap Mass Spectrometry; CRC Press: Boca Raton, FL, 1995. (19) March, R. E.; Todd, J. F. J. Practical Aspects of Ion Trap Mass Spectrometry. Vol. 2. Ion Trap Instrumentation; CRC Press: Boca Raton, FL, 1995. (20) Cooks, R. G.; Kaiser, R. E., Jr. Acc. Chem. Res 1990, 23, 213-219. (21) McLuckey, S. A.; Van Berkel, G. J.; Goeringer, D. E.; Glish, G. L. Anal. Chem. 1994, 66, 689A-696A. (22) Wilkins, C. L. In Fourier Transform Mass Spectrometry; Wilkins, C. L., Ed. Trends Anal. Chem. 1994, 13, 223-251. (23) Vartanian, V. H.; Anderson, J. S.; Laude, D. A. Mass Spectrom. Rev. 1995, 14, 1-19. (24) Marshall, A. G. In Fourier Transform Ion Cyclotron Resonance Mass Spectrometry; Marshall, A. G., Ed. Int. J. Mass Spectrom. Ion Processes 1996, 137/138, 410 pp. (25) Marshall, A. G.; Guan, S. Rapid Commun. Mass Spectrom. 1996, 10, 18191823. (26) Dienes, T.; Pastor, S. J.; Schu ¨ rch, S.; Scott, J. R.; Yao, J.; Cui, S.; Wilkins, C. L. Mass Spectrom. Rev. 1996, 15, 163-211.

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processes that provides the most dramatic new path for addressing the difficult analytical problems of carbohydrate biology. The objectives of this paper are thus twofold. First, we outline the role of MSn in the analysis of carbohydrate structural isomers. Second, we examine some of the fundamental issues in the instrumental implementation of MSn in FT-ICR MS. Although both Paul (quadrupole) and Penning (ICR) ion traps are capable of MSn and are, therefore, likely to play a major role in carbohydrate analyses, the nondestructive nature of FT-ICR MS detection (with high sensitivity) makes ICR analysis potentially more versatile for sequential degradation. MSn. Two significant advantages of sequential dissociation (MSn) techniques over single-stage tandem MS (metastable or CID analyses of ions formed in the ion source) are that (a) dissociation of an ion fragment can produce new types of product ions that cannot be observed in a single-stage dissociation of the molecular ion precursor and (b) product ions, and the specific structural features designated by these ions (e.g., linkage types), may be identified not just by their mass but also by virtue of the hierarchy of ion fragmentation. Hierarchical ion fragmentation is especially significant in carbohydrate analyses, in which ion fragments, like molecular ions, are often isobaric. These issues are illustrated in this report by the sequential dissociation of the oligosaccharide, permethylated lacto-N-fucopentaose II (LNFP II), by ESI FT-ICR MSn. Ion Control. FT-ICR MS is often cited for its high resolution capability. Here, the focus is the degree of selectivity and flexibility by which the ion motion can be controlled to perform sophisticated experiments. In MSn, one requires that specified subsets of an ion population be dissociated or ejected. It is well understood that mass selectivity in CID and ion ejection are generally provided by controlling the frequency of applied ac electric field27,28 to excite the cyclotron motion of mass-selected ions.29,30 Nondestructive ICR detection allows for multiple detection (remeasurement) of the ions31,32 and, therefore, improves sensitivity.33 However, the off-axis displacement of the center of an ion cyclotron orbit (i.e., the magnetron radius) increases exponentially with time due to collisions, so that ions are continually drifting radially out of the trap.34 Ion axialization by cyclotron-resonant quadrupolar excitation in the presence of collisional damping35,36 reverses magnetron radial expansion and, therefore, greatly improves remeasurement efficiency.31,37 Axial(27) Marshall, A. G.; Wang, T.-C. L.; Ricca, T. L. J. Am. Chem. Soc. 1985, 107, 7893-7897. (28) Guan, S.; Marshall, A. G. Int. J. Mass Spectrom. Ion Processes 1996, 157/ 158, 5-37. (29) Gord, J. R.; Freiser, B. S. Anal. Chim. Acta 1989, 225, 11-24. (30) Solouki, T.; Pasa-Tolic, L.; Jackson, G. S.; Guan, S.; Marshall, A. G. Anal. Chem. 1996, 68, 3718-3725. (31) Speir, J. P.; Gorman, G. S.; Pitsenberger, C. C.; Turner, C. A.; Wang, P. P.; Amster, I. J. Anal. Chem. 1993, 65, 1746-1752. (32) Williams, E. R.; Henry, K. D.; McLafferty, F. W. J. Am. Chem. Soc. 1990, 112, 6157-6162. (33) Solouki, T.; Marto, J. A.; White, F. M.; Guan, S.; Marshall, A. G. Anal. Chem. 1995, 67, 4139-4144. (34) Guan, S.; Huang, Y.; Xin, T.; Marshall, A. G. Rapid Commun. Mass Spectrom. 1996, 10, 1855-1859. (35) Savard, G.; Becker, S.; Bollen, G.; Kluge, H.-J.; Moore, R. B.; Schweikhard, L.; Stolzenberg, H.; Wiess, U. Phys. Lett. A 1991, 158, 247-252. (36) Guan, S.; Kim, H. S.; Marshall, A. G.; Wahl, M. C.; Wood, T. D.; Xiang, X. Chem. Rev. (Washington, D.C.) 1994, 94, 2161-2182. (37) Wood, T. D.; Ross, C. W., III; Marshall, A. G. J. Am. Soc. Mass Spectrom. 1994, 5, 900-907.

ization and remeasurement offer immediate gains in sensitivity compared to a single measurement;33 moreover, axialization increases ion retention during multiple stages of ion isolation and dissociation.30 Because the instrumental techniques used for sequential dissociation (sustained off-resonance irradiation (SORI)38 collision-induced dissociation (CID), SWIFT ejection, ion measurement) also promote magnetron expansion as an unwanted byproduct, axialization becomes a crucial component of the ion control required for MSn. We, therefore, examine axialization and remeasurement for SORI CID of permethylated maltoheptaose oligomers. ESI and MALDI Mass Analysis of Permethylated Oligosaccharides. Electrospray ionization (ESI)39 and matrixassisted laser desorption/ionization (MALDI)30,40 techniques provide effective means of generating peptide, protein, and oligosaccharide molecular ions for FT-ICR mass spectral analysis. Hillenkamp and co-workers demonstrated the use of MALDI in the analysis of oligosaccharides,8 and many investigators have subsequently applied MALDI to carbohydrate and glycoconjugate MS. MALDI imparts considerable internal energy during ionization, as evidenced by the rapid growth of carbohydrate applications in which the postsource decay (PSD) of metastable ions in a timeof-flight (TOF) mass analyzer is exploited for differentiating sequence isomers. Ion metastability in MALDI has also been exploited by Lebrilla and co-workers, who showed that extensive oligosaccharide fragmentation is observed in MALDI FT-ICR mass spectra compared to linear MALDI-TOF mass spectra of similar compounds for which only intact molecular ions are observed.41 Lebrilla et al. also used MALDI FT-ICR relative dissociation thresholds to detect branched oligosaccharides.42 An ESI ion source coupled with an FT-ICR mass analyzer17,43 generates ions with much less internal energy than MALDI, and metastable decay associated with the ionization is rarely observed. However, electrospray ionization provides multiply charged ions and thus promotes molecular dissociation in ion fragmentation experiments.10 Permethylation of oligosaccharides confers several advantages for MS analysis. Permethylation can increase the structural detail obtained through ion fragmentation spectrometry. Cleavage of the glycosidic bond connecting monosaccharide residues is the principal dissociation pathway in almost all cases of ion fragmentation spectrometry for native or derivatized carbohydrates. For the branched structures common to oligosaccharides, one often needs to distinguish multiple residue losses in a single cleavage from losses of the same number and types of residues, but in multiple glycosidic cleavages. Collisional-induced dissociation (CID) of permethylated carbohydrates, in contrast to native molecules, distinguishes multiple from single glycosidic cleavages

by specific mass shifts. In some cases, linkage analysis is possible by fragmenting permethylated molecules.10,14,44 Permethylation and other types of derivatization increase the sensitivity of both MALDI and ESI6,9,10

(38) Gauthier, J. W.; Trautman, T. R.; Jacobson, D. B. Anal. Chim. Acta 1991, 246, 211-225. (39) Senko, M. W.; Hendrickson, C. L.; Pasa-Tolic, L.; Marto, J. A.; White, F. M.; Guan, S.; Marshall, A. G. Rapid Commun. Mass Spectrom. 1996, 10, 1824-1828. (40) Solouki, T.; Emmett, M. R.; Guan, S.; Marshall, A. G. Anal. Chem. 1997, 69, 1163-1168. (41) Carrol, J. A.; Penn, S. G.; Fannin, S. T.; Wu, J.; Cancilla, M. T.; Green, M. K.; Lebrilla, C. B. Anal. Chem. 1996, 68, 1798-1804. (42) Penn, S. G.; Cancilla, M. T.; Lebrilla, C. B. Anal. Chem. 1996, 68, 23312339. (43) Smith, R. D.; Loo, J. A.; Ogorzalek Loo, R. R.; Busman, M.; Udseth, H. R. Mass Spectrom. Rev. 1991, 10, 359-451.

(44) Reinhold, B. B.; Chan, S.-Y.; Reuber, L.; Walker, G. C.; Reinhold, V. N. J. Bacteriol. 1994, 176, 1997-2002. (45) Fievre, A.; Solouki, T.; Marshall, A. G.; Cooper, W. T. Energy Fuels 1997, 11, 554-560. (46) Chowdhury, S. K.; Katta, V.; Chait, B. T. Rapid Commun. Mass Spectrom. 1990, 4, 81-87. (47) Gabrielse, G.; Haarsma, L.; Rolston, S. L. Int. J. Mass Spectrom. Ion Processes 1989, 88, 319-332. (48) Beu, S. C.; Laude, D. A., Jr. Int. J. Mass Spectrom. Ion Processes 1992, 112, 215-230. (49) Senko, M. W.; Hendrickson, C. L.; Emmett, M. R.; Shi, S. D.-H.; Marshall, A. G. J. Am. Soc. Mass Spectrom. 1997, 8, 970-976. (50) Ciucanu, I.; Kerek, F. Carbohydr. Res. 1984, 131, 209.

EXPERIMENTAL SECTION Electrospray Ionization. ESI FT-ICR mass spectra were acquired with a home-built FT-ICR mass spectrometer equipped with a 9.4-T superconducting magnet described elsewhere.39,45 Briefly, ions are produced with an external electrospray source.46 The electrosprayed ions pass through a 0.8-mm-diameter skimmer prior to their entrance into a first, 60-cm-long, rf-only octupole ion guide. A second octupole, 200 cm in length, guides the ions into a 9.4-cm-diameter cylindrical (∼30.4 cm long) open-ended three-section Penning trap.47,48 By applying a dc voltage to each endcap of the first octupole, we allow ions to accumulate in this (linear trap) beam segment for 0.1-30 s; then, by appropriate potential gating, ions may be guided through the second octupole and trapped in the ICR cell. The use of the first octupole as a linear trap results in an increased number of ions confined in the Penning trap, and these ions form a tighter and better-axialized ion packet.49 A 30-cfm rotary pump (Varian, Lexington, MA) and three 1100 L/s hybrid turbo-drag pumps (Balzers, Hudson, NH) provide differential pumping of the vacuum system to maintain an operating base pressure of ∼2 × 10-8 Torr in the Penning trap. The instrument parameters are controlled by an Odyssey data system (Finnigan Corp., Madison, WI). Argon collision gas was introduced into the ICR ion trap at ∼2 × 10-5 Torr via a pulsed valve (General Valve, Fairfield, NJ). ESI triple-quadrupole mass spectra were obtained with a Quattro II (Micromass, Beverly, MA). Matrix-Assisted Laser Desorption/Ionization. MALDI FTICR mass spectra were acquired with an FTMS-2000 Fourier transform ion cyclotron resonance mass spectrometer (Finnigan Corp., Madison, WI) equipped with a 3-T superconducting magnet, dual 1.875-in. cubic Penning traps, and an Odyssey data system. Laser desorption/ionization was performed with a cartridge-type pulsed N2 laser (Laser Science, Inc., Model VSL-33ND, Newton, MA) operated at a wavelength of 337.1 nm with a pulse width of 3 ns (laser power density, ∼106 W cm-2).30 All reported MALDI sustained off-resonance irradiation (SORI)38 collision-induced dissociation (CID) MSn tandem mass spectra were obtained in the source compartment of the dual cubic ion trap. Sample Preparation. The oligosaccharide samples were purchased from commercial sources (Oxford Glycosystems, Oxford, England) and were methylated by a modification of the method of Ciucanu and Kerek.50 Briefly, vacuum-desiccated samples (1-20 µg) were dissolved in 50-200 µL of an NaOH/

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DMSO suspension prepared by vortex mixing of DMSO and dry, finely ground NaOH pellets. After 1 h at room temperature, 1050 µL of methyl iodide was added, and the solution was incubated for 1 h at room temperature with occasional vortex mixing. Samples were partitioned by adding 1 mL of chloroform, and the suspension was back-extracted twice with 2-3 mL of 30% acetic acid. The chloroform layer was dried, and the samples were stored at -20 °C. For the ESI experiments, the permethylated oligosaccharides were dissolved in 6:4 methanol/0.1 mM NaOH. These solutions were generally at micromolar concentration and were directly sprayed into the mass spectrometer at a flow rate of 0.3-0.6 µL/min. For all reported MALDI experiments, 2,5dihydroxybenzoic acid (DHB) served as the matrix. A 0.1 M stock solution of DHB matrix was prepared fresh daily in water acidified with 0.1% (v/v) trifluoroacetic acid. MALDI FT-ICR mass spectra were obtained at a typical matrix/analyte/alkali metal ratio of ∼1000:1:100. Approximately 1 µL of the solution mixture containing analyte, alkali metal salt (e.g., Na and Li salts), and matrix was applied to the solids insertion probe tip and allowed to dry in air before insertion into the mass spectrometer. MSn Experiments. The experimental event sequences for successive MALDI MSn experiments are published elsewhere.30 Briefly, we used stored-waveform inverse Fourier transform (SWIFT)27,28 radial ejection to remove parent ions of all but selected m/z ratio(s); the mass-selected ions are (re-)detected32 following chirp excitation. Parent ions are dissociated by collisional activation using SORI with an applied dipolar field 800 Hz below the reduced ion cyclotron frequency of the parent ion. In MALDI experiments with the 3-T instrument, azimuthal quadrupolar excitation,36 consisting of a broadband SWIFT waveform, covers the frequency (hence mass) range of interest and serves to axialize the product ions, which are then detected by chirp excitation, and the ion signal is Fourier transformed to yield an MS2 magnitude-mode mass spectrum. To retain the lighter mass ions in the MALDI CID experiments, we used a higher dc trapping potential (∼8 V) during the SORI and axialization events, restored to 2 V during the excitation and detection events. Argon was pulsed into the ICR chamber during the CID event and nitrogen during the remeasurement event. In the CID experiments at 9.4 T, 9 V was applied to each end cap electrode during SORI and 2 V during the measurement. Although the open cylindrical trap used for the ESI experiments was not configured for azimuthal quadrupolar irradiation, the combination of high field (9.4 T) and high vacuum resulting from gated ion injection reduced the rate of magnetron expansion so that electrosprayed ions of even moderate m/z ≈ 1000 could be held inside the trap for several minutes prior to dipolar excitation/detection. However, remeasurement based only on collisional damping of the cyclotron motion (i.e., no quadrupolar axialization) was only moderately effective for singly charged parent ions (>1000 Da) and was ineffective for their lower-mass fragments. For MS2 experiments with permethylated LNFP II with the triple-quadrupole mass analyzer, the collision cell was offset by 30 V, and the doubly charged (by sodium cationization) quasimolecular ion at m/z 561.8 was selectively transmitted by the first quadrupole. The collision cell pressure was 1-2 mTorr of argon. 860 Analytical Chemistry, Vol. 70, No. 5, March 1, 1998

Figure 1. Ion fragmentation mass spectrum of natriated and permethylated lacto-N-fucopentaose II (LNFP II), produced by an ESI triple-quadrupole mass spectrometer.

Figure 2. ESI 9.4-T FT-ICR MS2 spectrum (SORI CID) of singly natriated permethylated LNFP II. The ESI-generated singly charged parent ions at m/z 1100 were isolated and then dissociated to yield the MS2 spectrum.

RESULTS AND DISCUSSION ESI Triple-Quadrupole MS2 of Permethylated Lacto-Nfucopentaose Isomer (LNFP II). In view of the substantial prior experience with tandem MS of permethylated oligosaccharides from triple-quadrupole (QQQ) mass spectrometers,10 it is important to understand that SORI CID in an FT-ICR mass analyzer largely reproduces the dissociation pathways observed by QQQ CID. The ion fragmentation spectrum of natriated and permethylated LNFP II obtained from a QQQ mass spectrometer is shown in Figure 1. The parent ion is a doubly charged molecule carrying two sodium cations at m/z 561.8 (singly natriated molecular ion at m/z 1100.5). The SORI CID spectrum of the singly natriated LNFP II, acquired interactively,51 obtained by ESI FT-ICR at 9.4 T is shown in Figure 2. The m/z value, 561.8 (Figure 1), is consistent with a composition of deoxyhexose (dHex), three hexoses (Hex) and an N-acetylhexosamine (HexNAc). In general, two features of the oligosaccharide’s structure may be addressed by tandem mass spectrometry. The first and most accessible feature is the sequence and branching structure or, more accurately, the connection topology, which is obtained from glycosidic cleavages. The second feature is the glycosidic linkage positions (linkage analysis), which may be inferred from particular cross-ring cleavages. Anomeric configurations and monosaccharide identification are only rarely obtained from tandem MS.52 (51) Guan, S.; Marshall, A. G. Anal. Chem. 1997, 69, 1-4.

Chart 1

The well-known tendency of HexNac residues to direct fragmentation to their reducing side12 and the prominent fragment ions at m/z 463 and 660 would be expected of a B/Y fragment pair10 arising from a glycosidic cleavage on the reducing side of the HexNAc residue and carrying a single sodium cation (Chart 1). In the QQQ mass spectrum (Figure 1), the m/z 660 and 463 fragment pair is incremented and decremented (respectively) by 204 u, forming a pair at m/z 864 and 259. We therefore infer another glycosidic B/Y pair, indicating a methylated Hex residue on the reducing side of the HexNAc. In the FT-ICR SORI CID MS2 spectrum (Figure 2), the m/z 259 peak is also observed, but at low magnitude. Hence, a cursory inspection of the glycosidic cleavages strongly suggests a (dHex, Hex, HexNAc)-Hex-Hex topology, in which the connection among the residues at the nonreducing end is not defined. Based on the triple-quadrupole data, one would like to extend this simple topological analysis to the nonreducing terminal trisaccharide (dHex, Hex, HexNAc)- by assigning ions at m/z 259 and 229 as glycosidic nonreducing terminal Hex and dHex C1-type ions. From this assignment, one would draw the conclusion that the HexNAc is a branched residue. The problem is that the glycosidic fragments on the nonreducing side of the HexNAc are C1 ions, and a hexose C1 fragment at m/z 259 is isobaric with a hexose Y1 fragment. Therefore, a nonreducing terminal hexose cannot be unambiguously identified from this spectrum. A substantial difference between the SORI and triple-quadrupole CID spectra is the relative abundances of ions at the low m/z end of the spectrum, due to differences in the kinetics of internal energy deposition and ion loss through ion-neutral scattering. In quadrupole CID, all of the available energy is first deposited into ion translational motion and then rapidly converted into internal energy by collisions at relatively high pressure (1 mTorr) in the collision cell. Fragment ions, vibrationally excited by initial collisions, retain a large fraction of the incident momentum and undergo secondary fragmentation in subsequent collisions. In FT-ICR SORI CID, the parent ion is periodically accelerated and decelerated in the presence of the neutral gas through many cycles of the applied off-resonance dipolar electric ac field. The neutral gas is usually admitted as a pulse, and the resultant time-dependent pressure is difficult to measure but is generally less than the pressure in the collision cell of a triple quadrupole. Relative to quadrupole CID conditions, ion translational energy in the FT-ICR experiment remains small, the collision rate is low, and the heating period is much longer. Once a fragment ion forms, it is no longer driven by the applied field, and second generation fragments are less likely to form. Ion loss rate in FT-ICR will also affect the relative magnitudes of fragment

Figure 3. ESI 9.4-T FT-ICR MS3 spectrum of LNFP II by sustained off-resonant irradiation (SORI) collision-induced dissociation (CID) of product fragment ions of the isolated B2 fragment ions at m/z 660. After dissociation of the singly charged m/z 1100 parent ions, the product ions at m/z 660 were isolated and then dissociated to yield the MS3 spectrum.

signals. In SORI CID, ion loss represents a combination of axial ejection and magnetron expansion by means of ion-neutral collisions, and both mechanisms predict a higher rate of ion elimination for ions of lower mass.34,36,53 Consequently, the SORI CID spectrum of the m/z 1100 parent (Figure 2) shows almost no monosaccharide fragments. The m/z 660 ion was isolated after dissociation of the m/z 1100 parent and was then dissociated in turn to form an MS3 FT-ICR mass spectrum (Figure 3), from which the m/z 259 ion may be associated unambiguously with a nonreducing terminal hexose. The glycosidic fragments at m/z 472, 259, 442, and 424 are associated with the branched topology, whereas the absence of glycosidic fragments at m/z 415, 433, or 268 militates against a linear structure (Chart 2). Moreover, a significant feature of the MS3 spectrum (Figure 3) is the appearance of fragmentation pathways not observed in the dissociation of the molecular ion (Figure 2). Although we have not examined linkage isomers to LNFP II by this method, it (52) Richter, W. J.; Muller, D. R.; Domon, B. Methods Enzymol. 1990, 193, 607623. (53) Riegner, D. E.; Laude, D. A., Jr. Int. J. Mass Spectrom. Ion Processes 1992, 120, 103-116.

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Figure 4. MALDI 3.0-T FT-ICR positive-ion source-trap mass spectra of isolated [M + Li]+ permethylated maltoheptaose (top) and its argon SORI CID product ions (Y2, Y3, Y4, Y5, Y6) after 36 ion remeasurements (bottom). The most prominent fragment ions are series of reducing end glycosidic fragment ions (Y type), but lowabundance ring-opened fragment ions such as 3,5A5 are also observed.

Chart 2

Figure 5. MALDI 3.0-T FT-ICR source-trap oligosaccharide mass spectra. Top: Isolation of Y2 disaccharide product ions argon SORI CID of the isolated [M + Li]+ permethylated maltoheptaose. Bottom: Additional glycosidic cleavage to yield Y1 monosaccharide fragment ions produced by argon SORI CID of isolated Y2 ions. (Note that ringopened fragment ions are not observed.)

is clear that the lack of fragmentation of the nonreducing terminal trisaccharide a priori precludes identifying structural isomers of this part of the molecule by ion fragmentation analysis. MALDI FT-ICR MS of Permethylated Maltoheptaose. Figure 4 shows MALDI FT-ICR and mass-isolated (by SWIFT) positive-ion-mode mass spectra of lithiated permethylated maltoheptaose ([M + Li]+) (top) and its argon SORI CID product ions (Y2, Y3, Y4, Y5, Y6) after 36 ion remeasurements (bottom). After argon SORI CID, the isolated [M + Li]+ (top) parent ions dissociate to produce several fragment ions; hence, the product ion mass spectrum (bottom) should exhibit a lower S/N value relative to the parent ion mass spectrum, even for perfect ion retention. However, fragment ion remeasurements can compensate for the loss of signal and provide an important tool for detecting low-abundance ion fragments that would be virtually undetectable from a single measurement. In Figure 4 (bottom), the glycosidic ions Y6 to Y2 define the monomer sequence masses (204 u) in the oligomer. However, the Y1 sequence ion is not observed. The most prominent fragment ions are the series of reducing-end glycosidic fragment ions (Y-type ions at m/z 447, 651, 855, 1059, and 1263); the 204-u mass increment between subsequent major peaks corresponds to the mass of each of the permethylated hexose units that form

the oligosaccharide. Note that all of the observed Y ions retain the lithium cation. It should be noted that, under these MALDI conditions, laser-induced fragment ions are also observed; we show only the SWIFT isolated parent ion and its products. As for LNFP II, this sequence ion (Y2 fragment) may be generated by an additional stage of ion isolation and fragmentation. However, direct isolation and dissociation of the Y2 fragment would not be optimal in this case, because the Y6, Y5, Y4, and Y3 glycosidic ions each contain the Y2 fragment. If SORI CID is applied first to the Y6 ion, and then to the Y5, and continuing in sequence to the Y3 ion, the abundance of the Y2 ion (which is a product of dissociating the other ions) is increased. The accumulated Y2 ions may then be isolated and dissociated by SORI CID, to yield the Y1 ion (hence, the complete sequence) as the sole product (Figure 5). The mass-selective activation by SORI CID allows for the collisional disassembly of a molecule that is not a hierarchy of isolation, dissociation, isolation, dissociation, and so on. Circumstances in which the molecular ion produces a number of product ions of distinct m/z, each of which contains the specific moiety (residues, linkages, etc.) under study, are not uncommon in oligosaccharide fragmentation analysis. The sequence of glycosidic linkages may also be obtained by SORI CID from linkage-specific cross-ring fragments. It has been observed that Na cationization improves the extent of cross-ring fragmentation with permethylated oligosaccharides;10,14,44 hence, we performed linkage analysis by collisions of the natriated molecular ion. The glycosidic (1-4) linkage is identified from its 3,5An fragments, e.g., the 4-position of the glycosidic bond between the second and third residues from the nonreducing end requires the identification of a 3,5A3 fragment at m/z 533 (Figure 6). Ring-opened fragments from this experiment are much less

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Figure 6. SWIFT-isolated (400 e m/z e 540) argon CID fragment ions from isolated [M + Na]+ permethylated maltoheptaose from single (top) and multiple (bottom) data acquisitions. Remeasurement of the same ion packet enhances the S/N ratio; hence, ring-opened fragment ions that are not observable in a single-measurement experiment may be identified. Nonreducing end ring-opened fragment ions, such as 3,5A3, that define the 4-linkage of permethylated maltoheptaose are present (see vertical scale-expanded segment).

abundant than the glycosidic fragments and require a much higher signal-to-noise ratio for identification. A single measurement of the SORI CID product ions from the isolated [M + Na]+ ion failed to identify the linkage type because the signal-to-noise ratio was inadequate to identify the 3,5An fragments; as an alternative to repeating the entire sequence of ion generation and dissociation, we simply remeasured (excitation, detection, cooling, and axialization cycle) the product ions already generated, and the 3,5A3 fragment could easily be identified after 80 remeasurement cycles (Figure 6). Low Mass Fragment Ion Axialization. Figure 7 shows data for normalized ion signal magnitude for the trisaccharide fragment ions from SORI CID of the [M + Na]+ permethylated maltoheptaose parent ion as a function of number of remeasurement cycles. After 100 remeasurement cycles (at 2 × 10-7 Torr N2 for axialization), remeasurement of the fragment ions at m/z 667 approached 100%. Without axialization (and all other conditions fixed), the signal disappeared almost completely after about 35 remeasurements. However, during the first 20 remeasurement cycles with axialization, the ion signal magnitude still dropped sharply. Ions with initially large magnetron radii will be converted to ions with large cyclotron radii and, thus, may be lost by collisional scattering and z-ejection.53 Space charge may also distort the static electric potential in the trap, and a certain fraction of the ion population may have to be ejected before the effective trapping potential is strong enough to retain the ions. Pulses of Ar and N2 gas were used as the neutral “buffer” gases for SORI CID and remeasurement, respectively. From simple kinematics, ion-neutral collisions with Ar are expected to result in greater internal energy than collisions with N2 for damping of a given initial ion velocity. For SORI CID, the object is to

Figure 7. FT-ICR mass spectral peak magnitude for successive remeasurements of the same Y3 trisaccharide fragment ions from [M + Na]+ permethylated maltoheptaose parent ions, reported as a fraction of the peak magnitude for the first measurement. Top: Axialization after each successive remeasurement. Bottom: No axialization. Following SORI at 5 × 10-6 Torr of Ar to produce the Y3 ions, remeasurement efficiency for the axialized fragment ions at m/z 667 was 99.22% after 100 remeasurement cycles at 2 × 10-7 Torr of N2. Trapping voltage was increased during the high-pressure event in order to improve ion remeasurement efficiency of low-mass fragment ions.

maximize the internal energy by periodically relaxing the translational energy, whereas for remeasurement, the object is simply to relax the translational energy (largely, cyclotron amplitude) to zero. A lighter target gas or a heavier incident ion results in smaller scattering angles for the ion (zero scattering angle corresponds to scattering along the ion’s initially forward direction), and acquiring less transverse momentum from the collision results in less z-ejection. Thus, helium gas pulses during axialization would have been better than nitrogen pulses, but our vacuum pumps do not pump helium efficiently. The trapping during the high-pressure SORI event was increased from 2 to 8 V to improve further the efficiency of retaining fragment ions. If the trapping voltage was held at 2 V during the SORI CID process, fragment retention decreased drastically. These results are in concert with previous studies.30-33,54 Finally, it should be mentioned that the large fluctuations in ion population (and, presumably, ion energetics and spatial distribution) per laser shot and the vacuum system configuration (internal MALDI source, 3-T magnetic field, ∼10-V maximum trapping dc potential, cryogenic pumps) made it difficult to study ion control systematically. Future studies will utilize a different instrument configuration that is more optimized for this type of experiment. SUMMARY ESI and MALDI FT-ICR both potentially increase the structural resolution provided by ion fragmentation mass spectroscopy of oligosaccharides. As for MS2, mass selection is important in defining the element (molecular ion, ion fragment) being analyzed in the presence of a complex mixture of ions. Secondary fragmentation of product fragment ions generates new dissociation pathways not observed in tandem MS and aids in structural (54) Pitsenberger, C. C.; Easterling, M. L.; Amster, J. I. Anal. Chem. 1996, 68, 3732-3739.

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analysis of both the fragment and its implications for the parent structure. Mass-selective dissociation of multiple fragment ions can accumulate specific target fragments for subsequent isolation and dissociation, for increased sensitivity compared to a strict hierarchical lineage of isolation and dissociation. These features are significant for the whole spectrum of molecular analyses by mass spectrometry but are especially relevant to oligosaccharide analysis, with its characteristic problems of structural isomers and large variations in the kinetics of dissociation pathways (i.e., ringopening versus glycosidic). Extended protocols for ion manipulation, including both multiple SORI CID and remeasurement cycles,

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require methods such as axialization to prevent ion loss during the experiment. ACKNOWLEDGMENT This work was supported by NSF (CHE-93-22824), NIH (GM54045, GM-31683, RR-10888), Florida State University, and the National High Magnetic Field Laboratory in Tallahassee, FL. Received for review June 2, 1997. Accepted December 11, 1997. AC970562+