Structural Analysis of Oligosaccharides Derivatized with 4

protocol for the preparation of ABDEAE-derivatized oligosaccha- rides. The procedure .... satellite to the Y series ions (Figure 5), indicating that a...
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Anal. Chem. 1998, 70, 4520-4526

Structural Analysis of Oligosaccharides Derivatized with 4-Aminobenzoic Acid 2-(Diethylamino)ethyl Ester by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Wenjun Mo, Toshifumi Takao,* Hiroko Sakamoto, and Yasutsugu Shimonishi

Institute for Protein Research, Osaka University, Yamadaoka 3-2, Suita, Osaka 565-0871, Japan

Oligosaccharides derivatized with 4-aminobenzoic acid 2-(diethylamino) ethyl ester (ABDEAE) can be analyzed by ESI (Yoshino, K.; et al. Anal. Chem. 1995, 67, 40284031) and MALDI (Takao, T.; et al. Rapid Commun. Mass Spectrom. 1996, 10, 637-640) mass spectrometry. In this study, oligosaccharides derived from the enzymatic cleavage of the sugar chains of glycoproteins ribonuclease B, erythropoietin, and transferrin were subjected to ABDEAE derivatization, prior to analysis on a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF MS) for high-resolution mass measurement and a postsource decay (PSD) experiment. In the mass measurement of ABDEAE derivatives, quasi-molecular ion species have been observed in monoisotopic resolution using 2,5-dihydroxybenzoic acid as the matrix from spots that contain 50-200 fmol of sample; in the PSD analyses from the spots contained 500 fmol-1 pmol of sample, the predominant backbone ion series which covers the entire mass range for all the derivatives, the internal ion series which reflect the branched trimannosyl core structure of N-glycans, and the low m/z fingerprint ion of ABDEAE were consecutively observed, permitting structure elucidation of the oligosaccharides. Given the effectiveness of this derivatization in terms of its high sensitivity and resolution with respect to MALDI-TOF MS, current methodology is clearly applicable to the sensitive detection and accurate structural analysis of N-glycans. The carbohydrate moieties of glycoproteins and their chemical constitution have attracted considerable interest, because of their role in a variety of biological processes. For the case of hormonal glycoproteins, antibodies, secretin family, etc., it is clear that sugar moieties play an important role in the biological activities and biological functions, such as cell-cell interactions, in particular for processes involving recognition, targeting, and adhesion.1 Hence, the development of new, simple, and highly sensitive methods for the structural analysis of carbohydrates would be useful and desirable. Since there is no suitable chromophore in ordinary sugar chain moieties, small amounts of free oligosac* Corresponding author: (tel) +81-6-879 8602; (fax) +81-6-879 8603; (e-mail) [email protected]. (1) Cumming, D. A. Glycobiology 1991, 1, 115-130.

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charides are difficult to analyze with high sensitivity. With the advent of innovative instrumentation, coupled with new ionization methods, e.g., electrospray ionization (ESI),2,3 and matrix-assisted laser desorption/ionization (MALDI),4,5 mass spectrometry has become an increasingly important method for the accurate and sensitive detection and structural elucidation of carbohydrates. However, in contrast to the case of peptides and proteins, the detection of free oligosaccharides at the low-picomole level remains a difficult and strenuous task. Several chemical derivatization methods for oligosaccharides have been proposed and have been proven to be effective for sensitive detection by MS. These methods involve attaching a high-proton-affinity or positively charged site to the reducing terminus with 2-aminopyridine (PA), 4-aminobenzoic acid 2-(diethylamino) ethyl ester (ABDEAE),6 trimethyl(p-aminophenyl)ammonium chloride (TMAPA),7 or 2-aminoacridone (AMAC)8 as a derivatization agent. Derivatization of oligosaccharides with ABDEAE was first proposed for use in sensitive detection using ESI-MS6 and later was extended to MALDI MS.9 In both cases, the resulting ABDEAE derivatives have shown an increase in sensitivity up to the femtomole level, which corresponds to a ∼1000-fold enhancement over techniques using free oligosaccharides. In addition, ABDEAE derivatives are usually well separated by means of reversed-phase high-performance liquid chromatography (RPHPLC). In this study, we describe the application of this methodology to several oligosaccharides obtained by the enzymatic cleavage of N-glycans from glycoproteins and demonstrate the effectiveness of the technique to the structural characterization of oligosaccharides by postsource decay (PSD) experiments using MALDI-TOF MS. (2) Whitehouse, C. M.; Dreyer, R. N.; Yamashita, M.; Fenn, J. B. Anal. Chem. 1985, 57, 675-679. (3) Fenn, J. B. J. Am. Soc. Mass Spectrom. 1993, 4, 524-535. (4) Karas, M.; Bachmann, D.; Bahr, U.; Hillenkamp, F. Int. J. Mass Spectrom. Ion Processes 1987, 78, 53-68. (5) Hillenkamp, F.; Karas, M. Methods Enzymol. 1990, 193, 280-295. (6) Yoshino, K.; Takao, T.; Murata, H.; Shimonishi, Y. Anal. Chem. 1995, 67, 4028-4031. (7) Dell, A.; Carman, H.; Tiller, P. R.; Thomas-Oates, J. E. Biomed. Environ. Mass Spectrom. 1988, 16, 19-24. (8) Okafo, G. N.; Burrow, L. M.; Neville, W.; Truneh, A.; Smith, R. A. G.; Camilleri, P. Anal. Chem. 1996, 68, 4424-4430. (9) Takao, T.; Tambara, Y.; Nakamura, A.; Yoshino, K.; Fukada, H.; Fukada, M.; Shimonishi, Y. Rapid Commun. Mass Spectrom. 1996, 10, 637-640. 10.1021/ac9803838 CCC: $15.00

© 1998 American Chemical Society Published on Web 09/12/1998

EXPERIMENTAL SECTION Chemicals. ABDEAE hydrochloride (ABDEAE-HCl) was purchased from Tokyo Chemical Industry (Tokyo, Japan). MALDI matrixes, 2,5-dihydroxybenzoic acid (DHBA) and R-cyano-4hydroxycinnamic acid (CHCA), sodium cyanoborohydride, and glycoprotein ribonuclease B and transferrin, which were used as substrates to obtain targeting oligosaccharides by enzymatic cleavage, were obtained from Sigma Chemical (St. Louis, MO). Recombinant erythropoietin was kindly provided by Chugai Pharmaceutical Co., Ltd. (Tokyo, Japan). Peptide-N-glycosidase F (PNGase F, which cleaves the β-aspartylglycosylamine linkage of Asn-linked carbohydrates) was purchased from New England Bio-laboratory (Beverly, MA). Maltohexaose, 2-mercaptoethanol, and heptafluoro-n-butyric acid (HFBA) were from Nacalai Tesque (Kyoto, Japan). All reagents were of analytical grade and used without further purification. Derivatization and Purification of Oligosaccharides. Oligosaccharide mixtures of MannGlcNAc2 (with 5 e n e 9), the oligosaccharide complex containing Gal3GlcNAc3Man3FucGlcNAc2 (see Scheme 2), and oligosaccharide NeuNAc2Gal2GlcNAc2Man3GlcNAc2 (see Scheme 3) were prepared through the enzymatic cleavage of the sugar chain from ribonuclease B, erythropoietin, and transferrin by PNGase F, respectively. The protocols used for the enzymatic cleavage of oligosaccharides from glycoproteins were as follows: 1.5 µL of 2-mercaptoethanol was added into 25 µL of an aqueous solution containing 5 nmol of glycoprotein in a 1.5-mL polypropylene Assist tube (Assist Trading Co., Ltd., Tokyo, Japan). The resulted reaction mixture was then heated at 100 °C for 15 min; after cooling, the solution was diluted by the addition of 161 µL of 1 mM NH4HCO3 prior to the addition of 2 µL of PNGase F solution (containing 1000 units). The final solution was then incubated at 37 °C for 12 h. After adding acetic acid (2 µL) and acetone (200 µL) to the resulting mixture, the precipitated deglycosylated protein was removed by centrifugation and the supernatant solution (which contained the carbohydrates) was lyophilized. The ABDEAE-derivatized oligosaccharides were prepared with minor modifications of the method described previously.6,9 ABDEAE-HCl (0.5 µmol) and glacial acetic acid (0.5 µL) were added to 0.5 µL of an aqueous solution containing 1 nmol of oligosaccharide. The reaction tube was then vortexed and maintained at 90 °C for 10 min. Sodium cyanoborohydride (500 nmol) in 5 µL of water was added to the reaction mixture, and the reaction tube was maintained at this temperature for an additional 1 h. The resulting derivatives from ribonuclease B, erythropoietin, and transferrin were immediately separated from the reagent by RP-HPLC using a Cosmosil 5C18-AR column, 10 mm i.d. × 250 mm (Nacalai, Kyoto, Japan) on a Waters model 600E multisolvent delivery system (Milford, MA). The gradient formed between solvent A (0.05% HFBA in water) and solvent B (0.05% HFBA in acetonitrile) was used for the separation. The reaction products were eluted using a linear gradient of solvent B (5-25% in 40 min, 25-80% in 20 min) at a flow rate of 2.0 mL/min. The ABDEAEderivatized oligosaccharides, which exhibit maximum absorption at 309.8 nm, were quantified by UV absorbance at 310 nm using a Waters model 486 tunable absorbance detector. Mass Spectrometry. DHBA and CHCA were used as matrixes for the MALDI-TOF MS experiments. DHBA was

dissolved to a concentration of 12 mg/mL in a 10% (v/v) solution of ethanol/water; CHCA was saturated in a solution of a 50% aqueous acetonitrile solution containing 0.1% trifluoroacetic acid (TFA). For the sample preparation, 50-200 fmol or 0.5-1 pmol of an ABDEAE-derivatized oligosaccharide dissolved in 0.5 µL of aqueous 0.1% TFA was mixed with 0.5 µL of DHBA matrix solution on a sample plate, for use in high-resolution mass measurement, or with 0.5 µL of the CHCA matrix, for PSD analysis, before drying in ambient air. Positive-ion PSD MALDI-TOF MS was performed on a Voyager Elite XL time-of-flight mass spectrometer equipped with a delayedextraction system (PerSeptive Biosystems, Framingham, MA) with flight paths of 4.2 and 6.5 m for the linear mode and reflectron mode, respectively. The sample was evaporated and ionized by irradiation with an N2 laser with a pulse width of 3 ns at a wavelength of 337 nm and accelerated at 20 (for linear mode) or 25 kV (for reflectron mode) potential in the ion source with a delay ranging from 50 to 200 ns. Ions of interest can be separated from undesired ones by the rapid switching of an electrical deflector (timed ion selector), which is situated immediately in front of the reflectron and which has a resolution of ∼100 in terms of mass separation. After passing through the timed ion selector into a reflectron, the ions could be mass selectively focused on the detector, which was operated in the reflectron and PSD mode, by altering the potential of the reflectron in a stepwise manner. The segments of the product ion spectra, measured successively at each potential on the reflectron, were stitched together to create a complete product ion spectrum. RESULTS AND DISCUSSION ABDEAE Derivatization and the Purification of the Oligosaccharides from Glycoproteins. Figure 1 shows the general protocol for the preparation of ABDEAE-derivatized oligosaccharides. The procedure used for the reduction of disulfide bonds in a glycoprotein was also found to be efficient for denaturation, which was necessary for effective cleavage with a glycanase such as PNGase F. Following PNGase F treatment, the deglycosylated protein was precipitated and removed by centrifugation, and the resulting supernatant was then subjected to derivatization. Although it was difficult to completely remove the deglycosylated protein as a precipitate when small amounts of glycoproteins were used, the presence of the protein precipitate had no effect on the subsequent derivatization. Finally, the reaction mixture of the derivatives was subjected to RP-HPLC using the HFBA solvent system, in which the excess ABDEAE reagent and some hydrophobic impurities are removed. For the case of ribonuclease B (see Figure 2), the derivatives corresponding to the known high-mannose-type oligosaccharides (Man5-9GlcNAc2-ABDEAE) were eluted from 30 to 32 min and were well-separated, on the basis of the difference in the number of mannose residues except for Man9GlcNAc2-ABDEAE, which coeluted with Man8GlcNAc2-ABDEAE. It should be noted that each fraction may constitute a mixture of isomers. Derivatives obtained from transferrin, which contain complex-type oligosaccharides with NeuNAc residues at the nonreducing termini, were separated on the basis of the difference in the number of sialic acids (data not shown). All fractionated derivatives were identified by MALDI-TOF MS, and their observed molecular masses are summarized in Table 1. The yield for the case of reductive Analytical Chemistry, Vol. 70, No. 21, November 1, 1998

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Table 1. Theoretical Values and Experimental Data (Observed in DHBA Matrix) for the MH+ of the ABDEAE Derivatives m/z ABDEAE derivatives

theor

obsd

maltohexaose Man5GlcNAc2 Man6GlcNAc2 Man7GlcNAc2 Man8GlcNAc2 Man9GlcNAc2 Gal3GlcNAc3Man3FucGlcNAc2 NeuNAc2Gal2GlcNAc2Man3GlcNAc2

1211.49 1455.60 1617.64 1779.70 1941.76 2103.81 2372.95 2443.95

1211.51 1455.68 1617.53 1779.64 1941.80 2103.96 2373.27 2444.23

Figure 1. Scheme for preparation of ABDEAE-derivatized oligosaccharides from a glycoprotein containing N-glycans.

Figure 2. RP-HPLC profile of the reaction mixture of ABDEAE derivatives obtained from ribonuclease B.

amination is in excess of 95% using the free oligosaccharide maltohexaose, while the total yield calculated by summing the peak areas obtained in the chromatograms for the derivatives is 40-50% for ribonuclease B and 30-40% for transferrin and erythropoietin using 1-5 nmol each as the starting materials. Sensitive and Accurate Mass Measurement of ABDEAEDerivatized Oligosaccharides. In previous mass spectrometric studies of derivatized sugars, [MH - H2O]+, MH+, [M + Na]+, and [M + K]+ ions have been separately or collectively observed. The presence of these ions in a specific mass spectrometric detection is dependent on the derivatizing group, matrix, and laser intensity. Mass measurements on the ABDEAE derivatives were obtained using DHBA and CHCA as matrixes. For the measurements involving the DHBA matrix, MH+ ions, [M + Na]+, and [M + K]+ adducts were consistently observed with high resolution. The upper trace of Figure 3 shows the mass spectrum of 4522 Analytical Chemistry, Vol. 70, No. 21, November 1, 1998

Figure 3. Spectra of the molecular ion region of ABDEAE derivatives of the high-mannose-type N-glycans. The upper trace shows the spectrum obtained from the spot containing 200 fmol of Man6GlcNAc2-ABDEAE using the DHBA matrix with delayed extraction in the reflectron mode of MALDI-TOF MS. The lower trace shows the spectrum obtained from the spot containing 200 fmol of Man6GlcNAc2ABDEAE using the CHCA matrix with delayed extraction in linear mode, in which the mass resolution of MH+ ion was much lower than that of the salt adducts, indicating that the MH+ ion has undergone extensive decomposition in the field-free drift region. The inset of the lower trace shows the spectrum obtained from the spot containing 50 fmol of Man5GlcNAc2-ABDEAE using the CHCA matrix with delayed extraction in linear mode on a Voyager Linear instrument (with a flight path of 1.2 m), and the average mass values were shown.

Man6GlcNAc2-ABDEAE acquired from the DHBA matrix with delayed extraction in the reflectron mode. The observed values of MH+ on all ABDEAE derivatives were in good agreement with the theoretical values (see Table 1) and in a mass resolution between 5000 and 8000. In practice, high resolution allows the

Figure 4. PSD spectrum from the MH+ ion of Man8GlcNAc2-ABDEAE using the CHCA matrix from the spot containing 1 pmol of sample. Dashed arrowhead lines and solid arrowheads indicate the internal ion series and the Y′ backbone ion series, respectively. For each observed m/z value, only one of the possible assignments to the oligosaccharide structure is depicted in Scheme 1.

unequivocal determination of the spacing between various molecular ion species (e.g., MH+, [M + Na]+, and [M + K]+), which is particularly helpful for the determination of the molecular weight of unknown samples. It should be noted that the sensitivity in terms of sample amount per spot was less than that reported previously,9 in which the ABDEAE-derivatized oligosaccharides were measured using a MALDI-TOF MS linear mode with a minimum amount of 10 fmol of sample. This decrease might well be explained by the more intensive ion dispersion in the MALDITOF MS used in this experiment, Elite XL, which has a much longer flight path (see Experimental Section) than that of Elite RP (1.3 m) used in the previous experiment. The lower trace in Figure 3 is the spectrum of Man6GlcNAc2-ABDEAE from the CHCA matrix, which was acquired with the linear TOF mode. The resolution on the Man6GlcNAc2-ABDEAE precursor ions was ∼2300, while the resolution of the salt adducts was ∼4300. In other measurements using the CHCA matrix, the MH+ was poorly resolved in the linear TOF mode and, at times, could not be observed at all in the reflectron mode. These results suggest that, during MALDI-TOF MS experiments using the CHCA matrix, MH+ ion species of the derivatives in the postsource flight tube undergo extensive dissociation, which is a disadvantage for the high-resolution mass measurement but favorable for the subsequent PSD analysis owing to the high decomposition rate of the molecular ions (see below). This conclusion is in agreement with previous reports10 that CHCA can be classified as a “hot matrix” which can transfer energy more effectively from matrix to samples

and, as a result, is suitable for sensitive mass detection, while DHBA can be considered as a “cool matrix” appropriate for the observation of intact molecular ions and high-resolution mass measurements. Thus, for the case of an unknown sample, the m/z value of MH+ is first determined using the DHBA matrix, and in the subsequent PSD experiment, the derivative, mixed with CHCA matrix, which can cause the analytes to undergo a higher degree of decomposition in PSD process,10 can be measured by setting the timed ion selector to this observed value, regardless of the low intensity or even the absence of molecular ions in the PSD spectrum (see Figures 5 and 6). It is noteworthy that a similar matrix effect was observed for the PA-derivatized sugars (data not shown). PSD Analysis for the Structural Elucidation of ABDEAEDerivatized Sugar Chains. MALDI-TOF PSD analyses of ABDEAE-derivatized sugars were made in the CHCA matrix. In all PSD experiments, MH+ ions were selected as the precursors, thus allowing a more direct structural investigation, in contrast to some previous reports11-13 in which salt adducts were chosen as precursors for MS/MS. Figure 4 is the PSD spectrum of Man8(10) Kussmann, M.; Nordhoff, E.; Rahbek-Nielsen, H.; Haebe, S.; Rossel-Larsen, M.; Jakobsen, L.; Gobom, J.; Mirgorodskaya, E.; Kroll-Kristensen, A.; Palm, L.; Roepstorff, P. J. Mass Spectrom. 1997, 32, 593-601. (11) Lemonie, J.; Chirat, F.; Domon, B. J. Mass Spectrom. 1996, 31, 908-912. (12) Okamoto, M.; Takahashi, K.; Doi, T.; Takimoto, Y. Anal. Chem. 1997, 69, 2919-2926. (13) Rouse, J. C.; Strange, A. M.; Yu, W.; Vath, J. E. Anal. Biochem. 1998, 256, 33-46.

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Figure 5. PSD spectrum from the MH+ of Gal3GlcNAc3Man3FucGlcNAc2-ABDEAE using the CHCA matrix from the spot containing 1 pmol of sample. The inset shows the molecular ion region acquired in the reflectron mode using the DHBA matrix. For each observed m/z value, only one of the possible assignments to the oligosaccharide structure is depicted in Scheme 2.

Scheme 1. N-Glycan of Ribonuclease Ba

a The mass values correlate with the monoisotopic ones calculated on the basis of the structures of fragments.

GlcNAc2-ABDEAE, showing the capability of the current experimental methodology in revealing the structure of the analyte (see Scheme 1; only one of the possible assignments is depicted for each observed m/z value of the fragment ions). The PSD spectrum of Man8GlcNAc2-ABDEAE (and also other ABDEAE derivatives) contained the distinctive Y series ions14 (which are formed by cleavage at the glycosidic bond and contain reducing termini), which arise from the remote charge fragmentation mechanism, and the fragmented ions of the ABDEAE moiety at (14) Domon, B.; Costello, C. E. Glycoconjugate J. 1988, 5, 397-409.

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99.9 Da. Internal series ions, which were caused by the chargeinduced fragmentation from the ABDEAE-GlcNAc group, have also been observed with good reproducibility for all the ABDEAEGlcNAc-containing derivatives (see also below). The branching structures of the peripheral portion of the high-mannose-type ABDEAE derivatives could not be “de novo” resolved in this study; however, it has recently been reported that some of the isomeric structures can be differentiated using “knowledge-based” strategy,13 i.e., by comparing the peak intensities of the fragment ions with those in the “spectral library” (which consists of MS/MS spectra measured on isomerically pure samples). In this study, however, more information about the branching structures for complex-type N-glycans was acquired from the PSD spectra (see below), which is largely due to the fact that each residue in a peripheral branch has a mass different from that of the neighboring one. In general, the accurate de novo characterizations of branching structures of oligosaccharides constitute a problem that remains to be solved. One asialo form (Gal3GlcNAc3Man3FucGlcNAc2)15 of the derivatized oligosaccharides derived from erythropoietin was subjected to a PSD experiment, using the CHCA matrix (Figure 5). The inset of Figure 5 shows the molecular ion region observed (15) Sasaki, H.; Bothner, B.; Dell, A.; Fukuda, M. J. Biol. Chem. 1987, 262, 12059-12076.

Figure 6. PSD spectrum from the MH+ of NeuNAc2Gal2GlcNAc2Man3GlcNAc2-ABDEAE using the CHCA matrix from the spot containing 1 pmol of sample. The inset shows the molecular ion region acquired in the reflectron mode using the DHBA matrix. For each observed m/z value, only one of the possible assignments to the oligosaccharide structure is depicted in Scheme 3.

with the DHBA matrix and the MH+ ion, which was used as the precursor for the PSD experiment. It should be noted that the molecular ions could not be detected in the CHCA matrix, indicating that the precursor ions had undergone nearly a 100% decomposition in the flight tube and provided strong fragment ion signals even at low sample concentrations. In this measurement, the ions corresponding to the molecular species with truncation of a deoxyhexose (dHex) were successively observed satellite to the Y series ions (Figure 5), indicating that a dHex residue is linked to the HexNAc at the reducing terminus in a branching structure (Scheme 2). The intact molecular ions of sialic acid containing oligosaccharides are observed only with difficulty, due to the facile decomposition of NeuNAc residues.16,17 ABDEAE derivatization has also proven to be effective for the measurement of sialic acidcontaining oligosaccharides. The inset of Figure 6 demonstrates the stability of MH+ ions in the DHBA matrix of the ABDEAE derivative of NeuNAc2Gal2GlcNAc2Man3GlcNAc2 (derived from the enzymatic cleavage of the glycoform from transferrin). The PSD spectrum from the MH+ obtained using the CHCA matrix gave a complete sequence of sugar residues for this glycan. In addition, two series of internal ions were observed, both of which constitute the three Hex and one HexNAc (see also Figure 5); whereas, these series were not observed for ABDEAE-maltohexaose (data not shown). The observation of these internal ions, (16) Harvey, D. J. J. Mass Spectrom. 1995, 30, 1311-1324. (17) Powell, A. K.; Harvey, D. J. Rapid Commun. Mass Spectrom. 1996, 10, 10271032.

Scheme 2. Triantennary N-Glycan from Erythropoietina

a The mass values marked by asterisks correlate with the average ones calculated on the basis of the structures of fragments.

which also had been detected in the PSD from underivatized highmannose-type N-glycans,13 reflects the branched core mannose structure (see Schemes 1-3). Furthermore, the fragmentation upon PSD appears to be in a fixed, rather than a random pattern; e.g., in the case of NeuNAc2Gal2GlcNAc2Man3GlcNAc2-ABDEAE, the successive loss of NeuNAc and then Hex f HexNAc is a dominant feature (Figure 6). The observation of the Y ion series allows a quick determination of the relative alignment of the constituent saccharides (see Schemes 1-3); more significantly, the repetition of the “beat” of Hex and HexNAc allows the identification of the antennary structure of complex-type N-glycans (i.e., two beats were observed for the biantennary structure of Analytical Chemistry, Vol. 70, No. 21, November 1, 1998

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Scheme 3. N-Glycan of Transferrina

a The mass values marked by asterisks correlate with the average ones calculated on the basis of the structures of fragments.

the oligosaccharide (Figure 6), three beats for the triantennary structure (Figure 5)). Since ions usually undergo unimolecular decomposition during PSD, such a specific pattern of fragmentation is probably energetically favorable or a function of the ion structure in the gaseous phase. CONCLUSIONS The ABDEAE derivatization, which is suitable for structural analysis by PSD MALDI-TOF MS, has been effectively applied to N-glycans cleaved from glycoproteins. The overall yield of the derivatives, calculated for ribonuclease B and transferrin, was 3050%, based on the starting glycoproteins (1-5 nmol), although it was consistently higher than 95%, based on the amount of free oligosaccharides. When this method was applied to 90 pmol of (18) Sakamoto, H.; Takao, T.; Mo, W.; Fukuda, H.; Tambara, Y.; Besada, V. and Shimonishi, Y. Proc. 46th ASMS Conf. Mass Spectrom. Allied Topics, Orlando, FL, 1998. (19) Mo, W.; Takao, T.; Shimonishi, Y. Rapid Commun. Mass Spectrom. 1997, 11, 1829-1834.

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ribonuclease B, it was possible to perform both the mass profiling of N-glycans and structural analyses by MALDI-TOF PSD, using the resulting ABDEAE derivatives.18 A “cool” matrix, such as DHBA was used for the highresolution mass measurement, from which the molecular weight of the derivatized sugar can be accurately determined. A “hot” matrix, such as CHCA, which provides for a high rate of decomposition of the molecular ion of an ABDEAE derivative, was employed for the sensitive PSD analysis. The overall sensitivity achieved in these experiments is not only much higher than those in the previous mass spectrometric studies on oligosaccharides but is also similar to or even higher than that in previous PSD studies on peptides (under the same experimental conditions).19 In addition, the interpretation of PSD spectra for a number of ABDEAE derivatives has turned out to be simple, structurally informative, and highly reproducible. With the effectiveness of the derivatization on PSD analysis, this methodology would be applicable to the sensitive detection and accurate structural analysis of unknown glycoforms derived from glycoproteins/ glycopeptides. ACKNOWLEDGMENT This work was, in part, supported by Grants-in-Aid for Scientific Research (No. 10558099) from the Ministry of Education, Science and Culture of Japan, the Suntory Institute for Bioorganic Research (to T.T.), and the Mitsubishi Foundation (to Y.S.). The authors are grateful to Mr. M. Fukuda from Nihon PerSeptive Ltd. for confirming the sensitivity on the derivatives in an independent set of PSD MALDI-TOF MS experiments. Received for review April 7, 1998. Accepted August 7, 1998. AC9803838