High-Energy Collision-Induced

Anal. Chem. 2005, 77, 3274-3283

Laser-Induced Dissociation/High-Energy Collision-Induced Dissociation Fragmentation Using MALDI-TOF/TOF-MS Instrumentation for the Analysis of Neutral and Acidic Oligosaccharides Urs Lewandrowski,† Anja Resemann,‡ and Albert Sickmann*,†

Rudolf-Virchow Center for Experimental Biomedicine, Versbacher Strasse 9, 97078 Wuerzburg, Germany, and Bruker Daltonik GmbH, Fahrenheitstrasse 4, 28359 Bremen, Germany

Producing detailed mass spectrometric fragmentation data of native oligosaccharides for the purpose of basic structure elucidation has become a readily accessible tool since the availability of enhanced technical equipment. In this report, high-energy collision-induced dissociation (heCID) in combination with MALDI-TOF/TOF technology for analysis of native neutral and acidic oligosaccharides is described. By providing complementary data, heCIDMALDI-TOF/TOF adds a variety of valuable cross-ring fragmentation information to the information of glycosidic fragmentation obtained preferably by laser-induced dissociation (LID). We examined parameters influencing fragmentation behavior of bothsacidic and neutrals compounds. Results show a dependency of the fragmentation pattern for the employed matrix as well as the laser intensity provided for the ionization of the analytes and the complexity of the analytes. Due to instrument-specific settings, protonated glycosidic ion series within spectra of sodiated compounds could also be identified. Furthermore, acquired spectra could be readily used to identify compounds by comparison to existing glycan databases such as GlycoSuiteDB and GlycosciencesDB. The results show a better scoring of heCID data sets in comparison to LID-derived data. heCID-MALDI-TOF/TOF analysis in combination with database search algorithms is demonstrated to be suitable for an initial identification/classification of carbohydrates. The analysis of glycans using primarily mass spectrometricderived methods is still gaining importance due to the ongoing progress of instrumental development during the last years. The advantage of mass spectrometry being a rapid method of acquiring data on low abundant molecule species in the femto- to picomole range is of major importance for the analysis of biological samples where the sample amount is often severely limited. Especially given the case of extensive microheterogeneity on individual glycosylation sites, a highly sensitive method for analysis is desirable since the amount of individual carbohydrate species * Corresponding author. Tel. +49(0)93120148730. Fax +49(0)93120148123. E-mail: [email protected]. † Rudolf-Virchow Center for Experimental Biomedicine. ‡ Bruker Daltonik GmbH.

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decreases with increasing complexity. Therefore, an analysis strategy comprising primarily high-sensitivity methods is of advantage. Recently, the analysis of glycans using instruments with advanced MALDI-TOF/TOF design allowed detailed structural analysis of neutral oligosaccharides including linkage analysis of structural isomers.1 Intrinsic to the design of the study has been the generation of so-called cross-ring fragmentation ions (A- and X-ions according to the nomenclature of Domon and Costello2). These ions allow the linkage analysis even of underivatized glycans in contrast to a range of previous approaches, e.g., permethylation followed by MS/MS analysis. Using similar instrumentation Stephens et al.3 employed high-energy collision-induced dissociation (heCID)-MALDI-TOF/TOF in combination with different matrixes (R-cyano-4-hydroxycinnamic acid and 2,5-dihydroxybenzoic acid) for the fragmentation of native and permethylated neutral glycans. Results show among other topics a dependency of the dominating ion series from the employed matrix. 2,5Dihydroxybenzoic acid was demonstrated to be more suitable for the generation of cross-ring cleavages while R-cyano-4-hydroxycinnamic acid favored cleavages of glycosidic bonds. Beside A- and X-ions, the so-called glycosidic cleavage ions (B-/C-fragments including the nonreducing end of the carbohydrate structure and Y-/Z-ions for the reducing end) are among the most common fragments observed so far with most MALDIMS systems. The occurrence of cross-ring fragments has also been shown on ESI-tandem-MS systems using linear ion trap technology.4 In this context, the amount of X-/A-ions is shown to be dependent on parameters of fragmentation such as excitation and collision energy applied within the linear ion trap. In general, characteristics of low- and high-energy CID have been investigated by many groups in the past5-8 and been the (1) Mechref, Y.; Novotny, M. V.; Krishnan, C. Anal. Chem. 2003, 75, 48954903. (2) Domon, B.; Costello, C. Glycoconjugate J. 1988, 4, 397-409. (3) Stephens, E.; Maslen, S. L.; Green, L. G.; Williams, D. H. Anal. Chem. 2004, 76, 2343-2354. (4) Sandra, K.; Devreese, B.; Van Beeumen, J.; Stals, I.; Claeyssens, M. J. Am. Soc. Mass Spectrom. 2004, 15, 413-423. (5) Gillece-Castro, B.; Burlingame, A. L. Methods Enzymol. 1990, 193, 689. (6) Kovacik, V.; Hirsch, J.; Kovac, P.; Heerma, W.; Thomas-Oates, J. E. J. Mass Spectrom. 1995, 30, 949. 10.1021/ac048399n CCC: $30.25

© 2005 American Chemical Society Published on Web 04/14/2005

subject or part of several reviews9-11 as well. Properties such as influence of cationization, energy applied to induce fragmentation, and kinds of ions produced show consistent results in terms of present ion series. However, newly introduced generations of mass spectrometers seem to improve the results to some extent with regard to quality of spectra as well as the sample amount necessary for analysis.9 By altering machine designs, e.g., by combination of sector field instruments with orthogonal acceleration time-of-flight analyzers12 or recent MALDI-TOF/TOF designs,1,13 it was gradually possible to increase, for example, the yield of cross-ring fragmentations and thus provide information about sequence branching and linkage positions. Spectra may now exhibit high numbers of cross-ring cleavages instead of predominating series of glycosidic cleavages as obtained under low-energy CID conditions occurring in postsource decay reactions.14,15 The information content of fragment ions may vary to a substantial degree between glycosidic and cross-ring cleavage ions. While the first ones provide evidence of the sequence of glycans, they contain little or no information regarding linkages or branching of sugar compounds. This information on the other hand is provided by detailed cross-ring fragmentations with complementary ions determining the linkage of certain residues. In combination with a detailed knowledge of the given oligosaccharide synthesis pathways in different cell types, this information may be sufficient for an initial analysis of protein carbohydrate contents and structure. Further MS-based methods, e.g., in combination with sequential glycosidase treatment,16 can accomplish a comprehensive picture of the carbohydrate of interest. Beneath the problem of mere production of valid MS/MS data, the interpretation of the experimental spectra is still performed manually in the first place. Up to now several tools for processing of oligosaccharide-derived tandem-MS data sets have been published. GlycosuiteDB17 represents a curated database with structures derived from literature research. Recently a tool termed GlycosidIQ18 was published equipping the database with the option of searching an experimental MS/MS data set against theoretical fragments derived from the database entries. GlycoSuiteDB entries are also retrievable via the GlycoMod19 tool (www.expasy.org), which calculates possible oligosaccharide compositions to given molecular ions. In contrast to GlycosuiteDB, (7) Lemoine, J.; Fournet, B.; Despeyroux, D.; Jennings, R.; Rosenberg, R.; de Hoffmann, E. J. Am. Soc. Mass Spectrom. 1993, 4, 197-203. (8) Brull, L. P.; Kovacik, V.; Thomas-Oates, J. E.; Heerma, W.; Haverkamp, J. Rapid Commun. Mass Spectrom. 1998, 12, 1520-1532. (9) Zaia, J. Mass Spectrom. Rev. 2004, 23, 161-227. (10) Reinhold, V. N.; Reinhold, B. B.; Costello, C. E. Anal. Chem. 1995, 67, 1772-1784. (11) Harvey, D. J. Mass Spectrom. Rev. 1999, 18, 349-450. (12) Harvey, D. J.; Bateman, R. H.; Green, M. R. J. Mass Spectrom. 1997, 32, 167-187. (13) Suckau, D.; Resemann, A.; Schuerenberg, M.; Hufnagel, P.; Franzen, J.; Holle, A. Anal. Bioanal. Chem. 2003, 376, 952-965. (14) Huberty, M. C.; Vath, J. E.; Yu, W.; Martin, S. A. Anal. Chem. 1993, 65, 2791-2800. (15) Spengler, B.; Kirsch, D.; Kaufmann, R.; Lemoine, J. Org. Mass Spectrom. 1994, 29, 782. (16) Ku ¨ ster, B.; Naven, T. J.; Harvey, D. J. J. Mass Spectrom. 1996, 31, 11311140. (17) Cooper, C. A.; Harrison, M. J.; Wilkins, M. R.; Packer, N. H. Nucleic Acids Res. 2001, 29, 332-335. (18) Joshi, H. J.; Harrison, M. J.; Schulz, B. L.; Cooper, C. A.; Packer, N. H.; Karlsson, N. G. Proteomics 2004, 4, 1650-1664. (19) Cooper, C. A.; Gasteiger, E.; Packer, N. H. Proteomics 2001, 1, 340-349.

another tool called GlycosciencesDB is based on the SweetDB,20 comprising different sources for glycan structures that can be compared to experimental mass spectrometric data by GlycoFragment and GlycoSearchMS.21 Further tools such as SweetSubstitute22 or individual macros such as Dona23 are offered for annotation of ion series to individual MS/MS spectra. These algorithms and database comparisons will not completely avoid the need for further detailed experimental work such as the characterization by reagent array analysis,24 but due to the easy use, they may be among the first choice for a fast sweep of carbohydrate complexity. In this report, a range of experimental conditions is revisioned that influence the quality of MALDI-TOF/TOF-derived mass spectra of oligosaccharides. Beside the cationization state and sample complexity, instrument-specific settings have been compared in regard to obtain the highest information content possible by comparing LID and heCID spectra. While LID spectra are generated by increased laser intensity for sample acquisition, heCID spectra containing analytically valuable A-/X-fragmentations are obtained by using different collision gases. The latter ones provide the basis for a successful comparison with existing databases such as GlycoSuiteDB and Sweet. It is demonstrated that heCID spectra of individual compounds receive superior and more confident identification scorings by GlycoSuiteDB than the corresponding LID spectra. EXPERIMENTAL SECTION Materials. Maltoheptaose, ribonuclease B (bovine), ovalbumin (chicken egg), R1-antitrypsin (human), and dextran 9000 were purchased from Sigma (Steinheim, Germany). Standard sialylated oligosaccharides were obtained from Dextra Laboratories (Reading, U.K.). Matrixes such as R-cyano-4-hydroxycinnamic acid (HCCA), 2,4,6-trihydroxyacetophenone (THAP), 2,5-dihydroxybenzoic acid (DHB), sinapinic acid, and 6-aza-2-thiothymine (6ATT) were obtained from Sigma and recrystallized twice as needed. Arabinosazone was synthesized according to Chen et al.25 All chemicals used were of analytical grade or higher. Preparation of Protein-Derived Oligosaccharides. Deglycosylation of ovalbumin and ribonuclease B was performed using peptide-N-glycosidase F (PNGaseF). Briefly, 1 mg of protein was dissolved in 1 mL of 100 mM sodium phosphate buffer, pH 7.0. Upon addition of 1% β-mercaptoethanol, the sample was denatured at 95 °C for 5 min and 3 units of PNGaseF (Roche, Basel, Switzerland) was added to the sample after cooling to room temperature. Digestion was performed at 37 °C for 16 h. Reduction of Oligosaccharides. Reduction of oligosaccharides has been facilitated according to Schulz et al.26 Briefly, the (20) Loss, A.; Bunsmann, P.; Bohne, A.; Schwarzer, E.; Lang, E.; von der Lieth, C. W. Nucleic Acids Res. 2002, 30, 405-408. (21) Lohmann, K. K.; von der Lieth, C. W. Nucleic Acids Res. 2004, 32, W261266. (22) Clerens, S.; van den Ende, W.; Verhaert, P.; Geenen, L.; Arckens, L. Proteomics 2004, 4, 629-632. (23) Spina, E.; Sturiale, L.; Romeo, D.; Impallomeni, G.; Garozzo, D.; Waidelich, D.; Glueckmann, M. Rapid Commun. Mass Spectrom. 2004, 18, 392-398. (24) Edge, C. J.; Rademacher, T. W.; Wormald, M. R.; Parekh, R. B.; Butters, T. D.; Wing, D. R.; Dwek, R. A. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 63386342. (25) Chen, P.; Baker, A. G.; Novotny, M. V. Anal. Biochem. 1997, 244, 144151. (26) Schulz, B. L.; Packer, N. H.; Karlsson, N. G. Anal. Chem. 2002, 74, 60886097.

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sugars were mixed with 50 mM NaOH in 0.5 M NaBH4 and incubated for 1 h at 50 °C. Reaction was terminated by adding acetic acid, and the sugars were purified via cation exchange chromatography and repeated evaporation of boric acid with 1% acetic acid in methanol. Samples were reconstituted in water for further analysis. Purification of Glycans. Purification of protein-derived glycans was performed using self-packed carbon microcolumns by packing graphitic carbon solid-phase extraction material (Restek, Bellefonte, PA) into µC18-ZipTips (Millipore, Schwalbach, Germany).27 The columns were precleaned with acetonitrile and equilibrated with 0.1% TFA. After trapping of an aliquot of diluted sample, the resin was washed with 0.1% TFA and the sample was eluted in minimal volumes of 25% acetonitrile, 0.075% TFA. Preparation of Matrixes. DHB and 6ATT were prepared as a saturated solution in 80% acetonitrile, 0.02% TFA equally mixed and diluted 1:5 in the same solvent. TFA was added to maintain a constant acidic pH during crystal formation. Dried droplet preparation was employed for all of the analysis with a sample to matrix volume of 1:1. Samples were prepared in concentrations ranging from 200 fmol to 10 pmol per spot while most measurements were performed using 1-2 pmol of sample. To study the effect of different cationization states, LiCl, NaCl, KCl, and CsCl were added at concentrations of 1 mM within the matrix solution yielding 500 µM within the final sample volume. LC Separation of Glycans. Glycans were rapidly separated by RP-chromatography on an Agilent 1100 HPLC system (Agilent Technologies, Waldbronn, Germany) employing a 2 mm i.d. × 150 mm Nucleosil 100-5-C18 column (Bischoff Chromatography, Leonberg, Germany) with a linear gradient for 30 min to 40% B. Solvents consisted of 0.1% formic acid (A) and 0.1% formic acid, 84% acetonitrile (B), respectively. Sampling directly after the injection peak yielded fractions of isolated dextran 9000 isomers. Mass Spectrometric Conditions. Mass spectrometric analysis was performed using a MALDI-TOF/TOF with LIFT capability (Ultraflex TOF/TOF, Bruker Daltonik GmbH, Bremen, Germany). All spectra shown have been acquired with a basic MALDI-TOF/ TOF (or -LIFT) method comprising the following voltage parameters: ion source I 8 kV, ion source II 7 kV, lens 3.8 kV, reflector 29 kV, reflector II 14.5 kV, LIFT1 19 kV, LIFT2 2.3 kV. The 337nm nitrogen laser was operated at a frequency of 50 Hz. The precursor ion selector was set to 1% of the molecular ion mass during all experiments. For heCID pressure within the high vacuum source was set to 2 × 10-6-1 × 10-5 mbar. Under LID conditions, the pressure was ∼4 × 10-7 mbar. The reflector gain was set to 12× with an increment of 75% for fragment ions. Laser intensity was adapted to an individual threshold for each compound. In general, LID laser intensity was well above (50-75%) the threshold for generation of molecular ions in order to enhance the intensity of LID ions. On the contrary, for heCID fragmentation, the laser intensity was set only slightly (5%) above the threshold for acquisition of molecular ions. RESULTS AND DISCUSSION MALDI Matrixes. Different MALDI matrixes (DHB, HCCA; THAP, 6ATT, arabinosazone) have been evaluated for their compatibility with heCID of neutral oligosaccharides. Under the (27) Packer, N. H.; Lawson, M. A.; Jardine, D. R.; Redmond, J. W. Glycoconjugate J. 1998, 15, 737-747.


Figure 1. Images of sample preparation using (A) DHB and (B) DHB/ATT as matrix. Samples were mixed in equal volumes with matrix and directly spotted onto a polished steel target. The DHB/ ATT matrix shows smaller needles and a minor dependency on sweet spots as compared to DHB.

chosen experimental conditions, DHB was the most useful matrix so far regarding the abundance of cross-ring ions in general. In addition to more conventional matrixes, a mixture of DHB and 6ATT was applied. It resulted in a more homogeneous sample surface than DHB without further need for recrystallization ontarget as shown in Figure 1. Spectra collected from random points of a single preparation showed close resemblance of spectra quality. Moving to the outer rim of the preparation slightly increased laser intensity was necessary to obtain a constant number of fragment ions as observed for spectra taken from the middle of the preparation. However, this increase of laser energy showed no adverse effects on fragmentation types observed as discussed later in the text or as may be seen in Figure 4. The abundance of cross-ring fragmentation was assessed with maltoheptaose as model substance and resulted in a slight overall increase of cross-ring ions of up to 25% in contrast to acquisition performed with DHB as matrix. The DHB/6ATT matrix also offers an alternative to DHB when fragmentation of acidic sugars is intended. DHB has been known to be less suitable for the ionization of acidic sugars.11 However, in our hands, 6ATT and THAP alone showed no enhanced fragmentation of acidic sugar compounds either. Therefore, spectra of acidic sugars have been acquired using DHB/6ATT as matrix as shown in Figures 6 and 7. Similar to maltoheptaose, a slight increase in cross-ring fragmentation was observed for DHB/ 6ATT in contrast to DHB. As an example, the ratios of 3,5A3R and 1,5X ions to the Y ion can be used. With DHB, an already high 2R 2R degree of cross-ring fragmentation (1.35 3,5A3R/Y2R and 0.48 1,5X2R/ Y2R) could be observed. It is slightly increased in the DHB/6ATT preparation (1.44 3,5A3R/Y2R and 0.57 1,5X2R/Y2R for the outer rim region and 1.21 3,5A3R/Y2R and 0.66 1,5X2R/Y2R for the inner ring). However, no additional fragment ions could be observed when comparing DHB- and DHB/6ATT-derived spectra. HCCA and arabinosazone results were inferior compared to DHB and DHB/6ATT. In particular, heCID spectra with HCCA as matrix resembled the common LID spectra obtained from DHB, suggesting a readily decay of molecular ions due to laser induced dissociation. heCID Gas Pressure Conditions. With energy for the heCID process originating from the collision of sample ions with gaseous molecules, the pressure applied to the collision cell as well as some collision gases was evaluated. Covering a range from 2 × 10-6 to 1 × 10-5 mbar, the fragmentation pattern remained almost

Figure 2. MALDI-heCID-TOF/TOF-MS/MS spectra of maltoheptaose ionized with different alkali adducts. The spectra show lithiated (upper spectrum) and sodiated maltoheptaose (middle spectrum) as well as a fragmentation spectrum of maltoheptaose as potassium adduct (lower spectrum). Lithiated species resulted in a higher intensity of low m/z ions while potassium adducts exhibit a preference for high m/z fragmentation products. The sodiated species ranks between those extremes. Insets show a particular excerpt from the spectra showing in detail the assigned ions. Notably the abundance of the 1,5X-ion differs between the different molecular ion species as well as the relative intensity of the C-2-ion series.

the same. Differences between spectra could be observed in the low m/z region showing an increase of ion abundance and intensity with higher pressure settings of the collision cell. Further below 2 × 10-6 mbar, the energy was not sufficient for CID and the spectra resembled those of the LID process. In general, most spectra shown have been acquired with a pressure setting of 5 × 10-6 mbar, which offers balance between sufficient fragmentation and overall high intensity of fragment ions throughout the spectra. The nature of collision gases employed seems to have minor effects on type of ions produced giving credit to measurements with He, Ar, and N2. Therefore, Ar remained the preferred collision gas during analyses. This statement is only in partial agreement with former findings7 that the change from He to Ar results in greatly enhanced fragmentations derived from more than one bond cleavages. Influence of Cationization State. To study the influence of different parameters potentially influencing the MALDI-heCID process, we chose maltoheptaose and dextran as model systems. Since both matrixessDHB and DHB/6ATTsresult preferably in single metal coordinated ions of oligosaccharides, the effect of different cations (Li+, Na+, K+, Cs+) in combination with DHB/ 6ATT matrix using maltoheptaose as model was reviewed. The generation of alkali adducts in MALDI-mass spectrometry of oligosaccharides is known to affect fragmentation products and sensitivity.28 Furthermore, the comparison between protonated and

sodiated ions during collision-induced dissociation shows marked differences.29 The influence on MALDI-heCID-derived spectra of three alkali-coordinated ions is shown exemplary by spectra for maltoheptaose in Figure 2. heCID spectra display an intensive series of B-/Y-ions as well as X- and A-ion series. While Li+ supports increase of low m/z ions, quite the opposite was observable using K+ as additive. Presumably the [M + K+] precursor ions are more stable under those conditions. Using Na+, the generated ions cover the whole range of the acquired spectrum with similar intensity. Furthermore, Cs+ led to complete abolishment of observable fragmentation under the employed conditions yielding only the signal of the molecular ion (data not shown). These data are in accordance with observations made previously by Penn et al.30 The addition of Li+ to large oligosaccharides was found to increase the amount of fragmentation for heCID in combination with a Fourier transform mass spectrometer. The fragmentation yields for protonated and alkali cationized oligosaccharides were found to be inversely related to cation size (H+ > Li+ > K+ > Rb+ > Cs+). Cs+ was determined to minimize fragmentation due to enhanced stabilization of the generated (28) Hofmeister, G. E.; Zhou, Z.; Leary, J. A. J. Am. Chem. Soc. 1991, 113, 59645970. (29) Orlando, R.; Bush, C. A.; Fenselau, C. Biomed. Environ. Mass Spectrom. 1990, 19, 747-754. (30) Penn, S. G.; Cancilla, M. T.; Lebrilla, C. B. Anal. Chem. 1996, 68, 23312339.


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Figure 3. MALDI-heCID-TOF/TOF-MS/MS spectra of a dextran 9000 hexasaccharide. The insets show the complete spectra while the main view shows excerpts. The left spectrum was acquired from the LC-purified compound while the right spectrum is derived from the dextran 9000 mixture. The spectra exhibit a dependency of the fragmentation pattern on the complexity of the analyte. The LC purified analyte shows a higher abundance of cross-ring cleavage ions, e.g. 1,5X-, 0,2X-, and 3,5A-ions as compared to the unpurified compound.

molecular ions, which is in turn attributed to its increased number of coordination sites. These can interact with up to five sugar rings stabilizing the molecular ion via Cs+-O interactions. While this effect may be useful to minimize undesired fragmentation, e.g., of fucosylated glycans,31 it prevents also the successful fragmentation by MALDI-heCID-TOF/TOF in the given case of maltoheptaose. Abundance and intensity of cross-ring fragmentation also differs between the different cationization states. The ratio between 1,5X and Y ions increases when changing from sodium to 4 4 potassium ions. This observation may be explained by the activation energy necessary for glycosidic bond cleavages, which has been determined to be lowest for Li+ coordinated molecular ions.32 Moving on to larger ions, this energy barrier is increased and allows for higher abundance of cross-ring fragmentations. It is also notable that the overall intensity of the C-2 ion series12 being present throughout the spectrum is increased by the use of KCl. This indicates a susceptibility of formation of this specific ion series in the presence of different cations. Due to their isobaric nature, no differentiation between 2,4A-ion series and 0,2X-ions can be made on the basis of single spectrum information alone. Therefore, reduction of maltoheptaose in the presence of NaBH4 causes a +2 Da mass shift of fragment ions originating from the reducing terminus. Following MALDI-heCID-TOF/TOF analysis of the reduced compound clearly indicates the presence of a 0,2Xion series and the absence of the 2,4A-ion series. Furthermore, it is evident that the Y-ion signals comprise isobaric C-ion series as well by shifting the Y-ions by 2 Da. C- and C-2-ion are generated in equal amounts under the conditions employed judging by the intensity of their corresponding fragments. Thus, the reduction of oligosaccharides and subsequent comparison of heCID of reducing and reduced oligosaccharide species proves to be a valuable tool in order to differentiate between individual ion series and confirming the nature of individual A- and X-ions. Complexity of Carbohydrate Mixtures. Regarding the fragmentation of oligosaccharides out of complex mixtures, a decrease (31) Tseng, K.; Lindsay, L. L.; Penn, S.; Hedrick, J. L.; Lebrilla, C. B. Anal. Biochem. 1997, 250, 18-28. (32) Cancilla, M. T.; Wong, A. W.; Voss, L. R.; Lebrilla, C. B. Anal. Chem. 1999, 71, 3206-3218.


of the ratio between glycosidic and cross-ring fragmentation was observed in comparison to isolated compounds. The model system for this purpose comprises a hexasaccharide (m/z 1013.3 Da) of a dextran 9000 mixture. It was analyzed once out of the complete mixture and again from a reversed-phase LC fraction containing the isolated hexasaccharide. As can be deduced from Figure 3 MALDI-heCID-TOF/TOF analysis of the isolated glycan resulted in a strong series of 1,5X-ions along with 0,2X- and 3,5A-ion series (refer to Chart 1 for annotation). Although the same ions were detectable using the dextran 9000 mixture, the intensity ratio of glycosidic cleavages compared to cross-ring fragmentation was increased (Figure 3). The pronounced B-/Y-ion series could result from slightly increased laser energy necessary for producing sufficient amounts of ions from the more complex sample. Due to suppression effects during the MALDI process, the ionization efficiency of the individual component presumably decreases along with the intensity of observable low abundant ions. The same effect however was not encountered when using mixtures of moderate complexity as, for example, the glycan mixture derived of an enzymatic digest of chicken egg ovalbumin. The spectra acquired from individual oligosaccharides resembled those of the much less complex ribonuclease B glycans. LID/heCID Comparison. The used mass spectrometer offers three different modes of fragmentationslaser-induced dissociation, high-energy collision-induced dissociation, and in-source decay.13 The latter one is only of minor use for the analysis of small MW compounds. heCID and LID mainly differ in the pressure of inert gas in the collision cell adjacent to the ion source as well as in employed laser energies. Fragmentation of maltoheptaose and other model compounds of high mannose and acidic sugars with either LID or heCID may be seen in figures 4-6. Figure 4 depicts the fragmentation of maltoheptaose with heCID and LID as well as a mixture of both modesstermed LID/CID. Basically LID results in a complete series of B-/Y-ion pairs throughout the entire mass range while no cross-ring fragmentation could be observed. heCID on the contrary produced series of 1,5X-, 0,2X-, and 3,5A-ions. By gradually increasing the laser energy above the threshold for the generation of molecular ions, the glycosidic B-/Y-ions again dominated the spectrum while suppressing formation of A-/X-ions.

Chart 1. Fragmentation Patterns and Annotation of Observed Ions from Selected Oligosaccharides During heCID and LID

a Depicted are selected fragments of (A) NeuAc Gal GlcNAc (Figure 6), (B) dextran hexasaccharide (Figure 3) and (C) Man GlcNAc 2 3 1 5 2 (Figure 5) derived from LID and heCID data.

Figure 4. MALDI-TOF/TOF-MS/MS spectra excerpts of maltoheptaose using heCID (left), LID (middle), and a combination of heCID/ LID as primary fragmentation method. heCID produces the highest intensity of 1,5X-, 0,2X-, and 3,5A-ion series, which are not detectable in the LID mode at all. Using heCID pressure conditions but an increased laser energy setting, the intensity of the cross-ring cleavages is diminished in favor of the B-/Y-ions.

In general, this behavior is also seen with the employed highmannose glycans and the acidic oligosaccharides. LID and LID/ CID spectra produced less analytical valuable cross-ring cleavages as well as an overall decreased number of fragments. This emphasizes the need for accurate and for the most part manual adjustment of laser intensity during the analysis. Similar differences between high- and low-energy CID have been reported, e.g.,

for the fragmentation of glycoalkaloids33 as well as N-linked carbohydrates.12,34 Although the modes of ionization and detection differ from the system employed in this study, the results indicate similar trends for fragmentation of compounds as observed with LID and heCID by MALDI-TOF/TOF analysis. heCID Fragmentation of High-Mannose Glycans. As further example for heCID fragmentation of biological relevant neutral N-glycans, ribonuclease B glycans were used. In Figure 5, the MALDI-heCID-TOF/TOF spectrum of underivatized Man5GlcNAc2 is shown with minor insets depicting excerpts from the heCID and the corresponding LID spectrum. Briefly, heCID resulted in a rich series of A- and X-ions. Their intensities are among the ions dominating this spectrum (refer to Chart 1 for annotation). Of particular interest are ions resulting from fragmentation of the core mannose, giving evidence about the branching structure. Corresponding to m/z 671.2 Da we identified the so-called D-ion. This ion species proposed by Harvey et al.17 comprises the central mannose as well as the attached 6′-antenna. In this case, it consists of four mannose residues (as sodium adduct). Further fragmentation by the 0,4A3 and the 3,5A3 ion indicates the attachment of a Man3 residue to the 6′-atom of the (33) Claeys, M.; Van den Heuvel, H.; Chen, S.; Derrick, P. J.; Mellon, F. A.; Price, K. R. J. Am. Soc. Mass Spectrom. 1996, 7, 173-181. (34) Harvey, D. J. J. Mass Spectrom. 2000, 35, 1178-1190.


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Figure 5. MALDI-TOF/TOF-MS/MS spectra of Man5GlcNAc2 derived of ribonuclease B. The upper main spectrum depicts a heCID-MALDITOF/TOF-MS/MS experiment with an intense series of X- and A-ions demonstrating the usefulness of heCID for the structural elucidation of oligosaccharides. In addition to the common ion series, a protonated series of glycosidic cleavages occurred as indicated within the annotation. The insets in the lower parts show the same region of the upper spectrum acquired with heCID (left) and LID (right). The CID spectrum exhibits far more cross-ring cleavages in addition to more intense glycosidic cleavages including 2-fold fragmentations such as the D-ion at m/z 671.2 Da.

Figure 6. MALDI-TOF/TOF-MS/MS spectra of NeuAc2Gal3GlcNAc1. The upper spectrum shows the heCID fragmentation of the molecule while the lower left inset depicts the corresponding LID spectrum. The other two insets show representative excerpts from the LID (middle) and heCID spectrum (lower right). The use of heCID results in an increased number of glycosidic and cross-ring cleavage ions including the D-ion (m/z 516.8 Da). Being most prominent peaks corresponding to the loss of sialic acid have been adjusted in intensity. Signals marked with asterisks correspond to multiple possible fragment ions while rhombi mark ions of metastable decay. 3280


core mannose as well. A particular high mass resolution is needed when differentiating between nearly isobaric ions, e.g., the C2R (m/z 527.1 Da), C2R-2 (m/z 525.1 Da), and the protonated HexNAc1Hex2 ion (m/z 528.1 Da). The MALDI-heCID spectrum of Man5GlcNAc2 shows an intense series of protonated ions despite the fact that the molecular ion is a sodiated species. Protonated ions are present for a selection of glycosidic cleavages, e.g., m/z 204.0 Da (GlcNAc1 + H+), m/z 366.0 Da (Man1GlcNAc1 + H+), m/z 528.1 Da (Man2GlcNAc1 + H+), m/z 690.1 Da (Man3GlcNAc1 + H+), and m/z 893.3 Da (Man3GlcNAc2 + H+). During the generation of molecular ions as lithiated species as well as potassium adducts, this ion series did not shift in its components’ m/z values during fragmentation, indicating a formation mechanism independent of the cationic adducts. Furthermore, a mass inaccuracy of roughly 1 Da seems implausible, since adjacent ions are well within the calibration range (e.g., Y1R, B1R with ∆m < 0.1 Da). The only theoretical possible sodiated fragment for the m/z 204 Da peak would account for a C1-ion. However, such an ion was not observed within the MALDI-heCID-TOF/TOF-MS/MS spectra of maltoheptaose, which should contain the C1-ion of a hexose residue as well. Those observations suggest indeed the existence of a protonated ion series within the MALDI-heCIDTOF/TOF-MS/MS spectra although the mechanism for its formation is still unclear. The presence of protonated ion series within MALDI-TOF/TOF-MS/MS spectra has also been reported in a recent work by Kurogochi and Nishimura35 as well. In their study, pyridylaminated derivatives of neutral glycans were analyzed although without the use of heCID. The use of HCCA instead of DHB as matrix was recommended to suppress the protonated ion series. However, this matrix seems to be unsuitable for the presented heCID approach since the abundance of cross-ring fragmentations is reduced. Stephens et al.3 demonstrated this drawback with neutral glycans showing the decrease of X- and A-ions and the predominant formation of glycosidic cleavages analyzing neutral glycans. Curiously, neither Stephens et al. nor Mechref et al.1 reported the formation of a protonated ion series employing DHB as matrix but a mass spectrometer of slightly different design (Applied Biosystems 4700 proteomics analyzer). The high mass accuracy achieved by heCID-MALDI-TOF/TOF analysis represents a further advantage when searching with MS/MS data against already existing curated databases such as GlycosuiteDB (www.glycosuite.com) or GlycosciencesDB (www.glycosciences.de). By using the data of the heCID analysis of Man5GlcNAc2, a corresponding structure is readily identified. Although isomers, for example, with Man(1-2) linkage in the 6′antenna, are present within the hit list, false positives can be ruled out by providing additional data like the origin of the glycansin this particular case bovine. It is of interest, that the LID and CID spectra differ remarkably in their scoring. GlycosidIQsthe fragmentation algorithm for GlycoSuiteDBsperforms its scoring by two different parameters: Beneath the biological index, a mixture of segmentation score and correspondence score are used. Comparing LID and heCID spectra, the biological index judging the monosaccharide composition gives a first indication if the result is reasonable in a biological way of thinking. Both spectra obtain the same biological index since their basic sugar composition (Hex5HexNAc2) is identical. The segmentation score however characterizes how well the structure matched based upon

the fragments that were assigned. The lower the score the better the result is judged. While heCID information results in a score of 1, the LID spectrum only receives a score of 16. This indicates a better suitability of heCID data due to the increased number of ions in total and of A-/X-ions in detail. This assessment is supported by the overall higher correspondence score of the heCID data (32.7% for glycosidic scoring and 33.0% for scoring of nonglycosidic ions matched) in contrast to the LID data set (14.3% glycosidic/7.9% nonglycosidic). Similar experiences could be made with the GlycoscienceDB, which is based on structural data sets such as NMR or X-ray crystallography. heCID data of Man5GlcNAc2 was among the top 10 hits while the LID data returned no reasonable database search result at all. heCID Fragmentation of Acidic Oligosaccharides. Acidic oligosaccharides proved to be a challenge for analysis by mass spectrometry. Due to the labile nature of the sialic acid attachment, a major loss of this residue is readily observed in MALDI-MS spectra as well as with ESI-MS methods. To minimize this loss, which occurs especially employing reflector mode, a range of socalled “cold matrixes” has been used in order to prevent the unintended fragmentation of the molecule. 6ATT has been shown to be suitable for this class of compound36,37 although not so far in combination with DHB. Anyway, recent years have seen a series of mixed matrixes that have proven useful for the analysis of distinct compoundsse.g., super DHB,38 a mixture of 2,5-dihydroxybenzoic acid and 2-hydroxy-5-methoxybenzoic acid for oligosaccharidessas well as HCCA/DHB mixtures for enhanced peptide analysis.39 Using DHB/6ATT, we were able to acquire spectra in good quality of acidic oligosaccharides with increased sample homogeneity on target compared to nonrecrystallized DHB. Figure 6 shows the MALDI-heCID-TOF/TOF-MS/MS spectrum of a NeuAc2Gal3GlcNAc1 oligosaccharide from human breast milk (refer to Chart 1 for annotation). Carrying two N-acetylneuraminic acid groups, this molecule would be expected to undergo fragmentation in the reflectron mode. Indeed the heCID (upper part Figure 5) and the LID spectrum (lower left part Figure 5) exhibited a prominent loss of two sialic acid residues. Apart from this loss, the LID spectrum provides only few further ions for the analysis of linkage or glycosidic sequence of the molecule. Only the Y3R-/Y3β-ion at m/z 567.8 Da delivers further information. In contrast to the rather poor results of the LID spectrum, the heCID fragmentation provides a range of ions showing either glycosidic ors to a lesser degreescross-ring cleavages. Again, the D-ion at m/z 516.8 Da provides evidence for the 6′-antenna by consisting of NeuAc1GlcNAc1. The linkage of the second antenna to the central GlcNAc residue may be determined by the m/z 285.9 Da 1,3A3R-/Y4R-ion although this particular ion may also account for C4R/0,2X2R. Nevertheless, the 3,5A3β-ion supports this first assumption by placing the second NeuAc residue on the 6-position of the central GlcNAc with a free hydroxyl group on the 3-position. Furthermore, the spectrum shows a complete series of YR-ions and several B-ions giving information about residue (35) Kurogochi, M.; Nishimura, S. Anal. Chem. 2004, 76, 6097-6101. (36) Papac, D. I.; Wong, A.; Jones, A. J. Anal. Chem. 1996, 68, 3215-3223. (37) Juhasz, P.; Costello, C. E. J. Am. Soc. Mass Spectrom. 1992, 3, 785-796. (38) Karas, M.; Ehring, H.; Nordhoff, E.; Stahl, B.; Strupat, K.; Hillenkamp, F.; Grehl, M.; Krebs, B. Org. Mass Spectrom. 1993, 28, 1476-1481. (39) Laugesen, S.; Roepstorff, P. J. Am. Soc. Mass Spectrom. 2003, 14, 9921002.


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Figure 7. Comparison of GlycoSuiteDB search results for NeuAc2Gal2Man3GlcNAc2. In (A), the search result for LID data is shown while (B) depicts the scoring of heCID fragmentation data of the same compound. (C) and (D) show cross-ring fragmentation ions derived of the central mannose residue by use of heCID-MALDI-TOF/TOF-MS/MS.

sequence. It is also notable that a series of internal glycosidic fragments could be annotated. However, those were often ambivalent regarding the possible residue combinations. Some signals marked with asterisks account for up to six different possible combinations of internal ions. Again a database search against GlycosuiteDB was conducted with both the LID and heCID data sets for the NeuAc1Gal3GlcNAc1 molecule. The results back the ones obtained from the search of neutral compounds. The heCID spectrum receives a segment score of 1 while the LID spectrum receives a lower score of 2. The difference in the segmentation score is less tremendous as seen with the Man5GlcNAc2 example but may be contributed to the less complicated structure and the reduced amount of peak data obtained by the mass spectrometric analysis. The scoring of both spectra differs by ∼5% for the glycosidic and the general scoring, indicating again a better sequence characterization by the MALDI-heCID spectrum. Comparison to the database systems yields the exact glycan structure as a component of human breast milk. Furthermore, the biantennary disialated glycan NeuAc2Gal2Man3GlcNAc4 (R-1-antitrypsin) was used as an example for a protein-derived acidic carbohydrate. The monosodiated molecular ion at m/z 2245.7 Da was again fragmented using LID and heCID and searched against the GlycoSuiteDB. Spectra quality resembled the one shown in Figure 6. Therefore, Figure 7 shows only the comparison of two searches conducted once with the heCID and with the LID data set, respectively. With both fragmentation methods, the identification of the glycan was possible, mainly because of the simple structure and the prominent loss of sialic acid in case of using LID. The LID data resulted in a worse overall scoring (12 000) than the heCID data set, which received an overall score of 2 with a good coverage of both glycosidic and cross-ring fragmentations. Clearly, heCID allows for a more 3282 Analytical Chemistry, Vol. 77, No. 10, May 15, 2005

reliable scoring of the results in this case. Regarding individual fragmentations, Panels C and D in Figure 7 show some complementary A-/X-ions of the core mannose obtained by heCID. Although 3,5X2R, 1,5X2R, and 3,5A5R do not unambiguously define the branching structure they show the potential of this method for the mass spectrometric-based linkage analysis. Further, crossring ions give hints but not definite proof of structural details, e.g., to the linkage of the nonreducing sialic acid residues. The 1,5A -ion does not determine the linkage between NeuAc and Gal 2 but a (2-6)-linkage may be supported by the presence of a 3,5A2ion. However, since 3,5A2 and 2,4X1 are isobaric and due to a supporting 2,4X0-ion in the heCID spectrum, the linkage of the nonreducing sialic acid residues still remains uncertain by means of mass spectrometric analysis alone. Remarks on Database Searches. Regarding computer-based evaluation of oligosaccharide fragmentation data, fundamental differences are to be acknowledged in comparison to the peptide analysis sector. While peptide sequencing for sequenced organisms is accessible via well-established database algorithms,40,41 even peptide structures of nonsequenced sources may be elucidated by de novo sequencing approaches.42 These approaches are not easily applicable for glycans for several reasons. First, no supporting database of all biological possible glycan structures is present. Existing collections of glycan structural data are most often based on confirmed structures within the literature.17,18 Theoretical spectra are generated from these structures for alignment with the experimental data. Further approaches, e.g., for the evaluation of glycan mass fingerprinting data, are based (40) Perkins, D. N.; Pappin, D. J.; Creasy, D. M.; Cottrell, J. S. Electrophoresis 1999, 20, 3551-3567. (41) Eng, J. K.; McCormack, A. L.; Yates, J. R. J. Am. Soc. Mass Spectrom. 1994, 4, 976-989. (42) Reinders, J.; Lewandrowski, U.; Moebius, J.; Wagner, Y.; Sickmann, A. Proteomics 2004, 4, 3686-3703.

on theoretical libraries of biosynthetically plausible structures.43 Present or envisioned collections of direct mass spectrometric data sets for comparison with new data are rare.44 Since amino acids differ (with the exception of leucine/isoleucine) in their mass, a second major drawback for the analysis of glycans is evident due to the isobaric nature of many monosaccharides. Upon an initial classification/identification of the carbohydrate, additional experiments are therefore necessary to confirm structural details. In general, a careful evaluation of database-derived identification results is certainly recommendable. It should be noted that obtained results may be subject to wrong assignments or that a truly new structure is not recognized or even worse, identified as an already known structure. Database searching with oligosaccharide fragmentation data sets should therefore be regarded as a first round of analysis followed by a second round of manual editing and follow-up experiments. In the growing field of glycoanalytics and glycomics, this preevaluation may indeed prove a valuable help and reference point. CONCLUSION In this report, the closer conditions of MALDI-heCID-TOF/ TOF-MS/MS analysis of acidic and neutral oligosaccharides have been reviewed. Therefore, a dependency of the fragmentation pattern has been shown for parameters such as cationization state, complexity of the sample, and the employed matrix. In this (43) Goldberg, D.; Sutton-Smith, M.; Paulson, J.; Dell, A. Proteomics 2005, 5, 865-875. (44) Tseng, K.; Hedrick, J. L.; Lebrilla, C. B. Anal. Chem. 1999, 71, 3747-3754.

context, the ionization of neutral and acidic compounds as sodium adducts has proven to be the method of choice while the complexity of the sample was in the low to moderate range. Parameters such as pressure variation within the collision cell in the range of roughly 1 order of magnitude as well as the nature of the collision gas had no substantial effect on the fragmentation pattern. Furthermore, the use of a mixture of DHB and 6ATT has been useful for the analysis of acidic and neutral compounds leading to MS/MS data sufficient for the analysis of employed model oligosaccharides by database algorithms. The employed methods of LID and heCID both provide data that can lead readily to the identification of the compound by existing databases. heCID is so far the preferred fragmentation mode, being especially suitable to produce cross-ring cleavages in addition to the more common glycosidic cleavages. Thereby, a more detailed analysis of the oligosaccharides under research is possible. This analysis can provide the basis for further detailed analysis, e.g,. by enzymatic digests, and should be seen as initial step for classification of oligosaccharides. ACKNOWLEDGMENT This work was supported by the Deutsche Forschungsgemeinschaft (FZT 82).

Received for review October 29, 2004. Accepted March 15, 2005. AC048399N


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High-Energy Collision-Induced

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