High-Sensitivity Detection and Postsource Decay of 2-Aminopyridine

Anal. Chem. 1997, 69, 2919-2926

High-Sensitivity Detection and Postsource Decay of 2-Aminopyridine-Derivatized Oligosaccharides with Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Masahiko Okamoto,* Ken-ichi Takahashi, Tadashi Doi, and Yoshiyuki Takimoto

Environmental Health Science Laboratory, Sumitomo Chemical Company, Ltd., 1-98, 3-Chome, Kasugade-naka, Konohana-ku, Osaka 554, Japan

The sensitivities of oligosaccharide derivatives in matrixassisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) were compared using two matrixes, 2,5-dihydroxybenzoic acid (DHBA) and r-cyano-4-hydroxycinnamic acid (CHCA). For this purpose, maltopentaose was tagged with 2-aminopyridine (PA), 4-aminobenzoic acid ethyl ester (ABEE), and trimethyl(p-aminophenyl)ammonium chloride (TMAPA). DHBA was more advantageous for enhancement than CHCA. Among the derivatives, the sensitivity with the PA-tagged maltopentaose showed a 100-fold improvement over the native one with DHBA as a matrix, while the oligosaccharide derivatized with ABEE and TMAPA gave 30- and 10fold increases in sensitivity over the underivatized one. To obtain structural information from these derivatized oligosaccharides, postsource decay (PSD) during flight in the field-free drift in MALDI-TOFMS was measured. Predictable and reproducible fragmentation patterns could be obtained in all cases. Furthermore, we found matrixdependence fragmentation with the PA-labeled oligosaccharide. With CHCA, a simple spectrum ascribable to Y series ions was obtained. On the other hand, both B and Y series ions were clearly observed in the DHBA case. The results demonstrate the usefulness of derivatives for sensitive analysis of oligosaccharides with MALDI. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) is known to have several advantages over other mass spectrometric methods in terms of sensitivity, efficiency, and simplicity.1-4 This technique is thought to be particularly suitable for analysis of oligosaccharides5 which play important roles in biochemistry and pharmacology.6 Diversity in oligosaccharide structures, combined with multiple glycosylation sites, invariably gives rise to an impressive array of glycoforms, each of which can be isolated pure using available (1) Beavis, R. C.; Chait, B. T. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 68736877. (2) Tanaka, K.; Waki, H.; Ido, Y.; Akita, S.; Yoshida, Y. Rapid Commun. Mass. Spectrom. 1988, 8, 151-153. (3) Karas, M.; Bahr, U.; Ingendoh, A.; Nordhoff, E.; Stahl, B.; Strupat, K.; Hillenkamp, F. Anal. Chim. Acta 1990, 241, 175-186. (4) Karas, M.; Ingendoh, A.; Bahr, U.; Hillenkamp, F. Biomed. Environ. Mass Spectrom. 1989, 18, 841-843. (5) Stahl, B.; Steup, M.; Karas, M.; Hillenkamp, F. Anal. Chem. 1991, 63, 14631466. (6) Dell, A.; Reason, A. J. Curr. Opin. Biotechnol. 1993, 4, 52-56. S0003-2700(96)00910-9 CCC: $14.00

© 1997 American Chemical Society

separation techniques. However, only limited quantities of glycoforms are generally obtainable with most separation protocols. Moreover, underivatized oligosaccharides are not amenable to picomole analysis even with MALDI-TOFMS, and a sensitive structural elucidation technique with a high reproducibility is urgently required. A number of workers have tried incorporating a derivatization step prior to mass spectrometric experiments,7-14 with the aim of increasing analytical performance and aiding detection. Reductive amination, involving reaction with a primary amine to form a Schiff base, which is then stabilized by reduction to secondary amines, has, for example, been applied. Such reactions provide chromophores and cationic (protonable) sites for detection by highperformance liquid chromatography (HPLC),8-12,14 and improvement of sensitivity in fast atom bombardment (FAB),7-10,14 electrospray (ESI),7,12,14 and MALDI studies.13 Some of the results represent between 1000 and 5000-fold enhancement of sensitivity over the native oligosaccharide.7,12,13 The recent development of postsource decay (PSD) mode MALDI-TOFMS has presented new possibilities in the structural analysis of biomolecules. Sensitive structural analysis of oligosaccharides has been demonstrated with the PSD-MALDI technique.15-20 However, only a few comparative studies have been conducted systematically among these derivatives. Under these circumstances, we compare the sensitivity of three derivatives [2-aminopyridine (PA), trimethyl(p-aminophenyl)ammonium chloride (TMAPA), and 4-aminobenzoic acid ethyl ester (ABEE)] in (7) Okamoto, M.; Takahashi, K.; Doi, T. Rapid Commun. Mass Spectrom. 1995, 9, 641-643. (8) Dell, A.; Carman, H.; Tiller, P. R.; Thomas-Oates, J. E. Biomed. Environ. Mass Spectrom. 1988, 16, 19-24. (9) Wang, W. T.; LeDonne, N. C.; Ackerman, B.; Sweeley, C. C. Anal. Biochem. 1984, 141, 366-381. (10) Hase, S. Methods Enzymol. 1994, 230, 225-237. (11) Poulter, L.; Burlingame, A. L. Methods Enzymol. 1990, 193, 661-689. (12) Yoshino, K.; Takao, T.; Murata, H.; Shimonishi, Y. Anal. Chem. 1995, 67, 4028-4031. (13) Takao, T.; Tambara, Y.; Nakamura, A.; Yoshino, K.; Fukuda, H.; Fukuda, M.; Shimonishi, Y. Rapid Commun. Mass Spectrom. 1996, 10, 637-640. (14) Suzuki, S.; Kakehi, K.; Honda, S. Anal. Chem. 1996, 68, 2073-2083. (15) Spengler, B.; Kirsch, D.; Kaufmann, R.; Lemoine, J. J. Mass Spectrom. 1995, 30, 782-787. (16) Lemoine, J.; Chirat, F.; Domon, B. J. Mass Spectrom. 1996, 31, 908-912. (17) Huberty, M. C.; Vath, J. E.; Yu, W.; Martin, S. A. Anal. Chem. 1993, 65, 2791-2800. (18) Spengler, B.; Dolce, J. W.; Cotter, R. J. Anal. Chem. 1990, 62, 1731-1737. (19) Ku ¨ ster, B.; Naven, T. J. P.; Harvey, D. J. Rapid Commun. Mass Spectrom. 1996, 10, 1645-1651. (20) Harvey, D. J. J. Chromatogr., A 1996, 720, 429-446.

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Figure 1. Comparison of the ionization efficiencies of derivatized maltopentaose in MALDI-TOFMS.

MALDI and PSD fragmentation with two matrixes, 2,5-dihydroxybenzoic acid (DHBA) and R-cyano-4-hydroxycinnamic acid (CHCA). The series of the PSD product ions was also compared in these derivatized oligosaccharides. In the present paper, we describe how when oligosaccharides are derivatized with PA they can be analyzed at high sensitivity in MALDI, which is comparable to the sensitivity achievable with HPLC-fluorescence. EXPERIMENTAL SECTION Derivatizing Reagents. TMAPA was bought from Sigma (St Louis, MO). ABEE and PA were obtained from Wako (Osaka, Japan). All other chemicals were of the highest grade commercially available and used without further purification. Derivatization and Purification of Oligosaccharides. The pentasaccharide maltopentaose, purchased from Wako, was used as a model oligosaccharide. The oligosaccharide derivatization by reductive amination with TMAPA, ABEE, and PA was performed following the method detailed in our earlier paper.7 HPLC purification was carried out according to the procedures described earlier.7 Each derivatized oligosaccharide collected was lyophilized prior to MS examination. These samples (∼10 mg each) were diluted with methanol to the concentration of 0.1-1 mg/ mL. MALDI-TOF Mass Spectrometry. The experiments were carried out using Reflex (Bruker-Franzen Analytik GmbH, Bremen, Germany) and Vision 2000 (Finnigan Mat GmbH, Bremen, Germany) TOF mass spectrometers. These machines were equipped with reflectors. Ions formed by a pulsed UV laser beam (nitrogen laser, λ ) 337 nm) were accelerated to a kinetic energy of 5 keV for Vision 2000 and 30 keV for Reflex. In the Vision 2000 case, ions were postaccelerated to 7 keV for detection with a secondary ion multiplier. An irradiance slightly above the threshold of ion detection was used. All the spectra were measured in the reflector mode without delayed extraction system. Analyte Preparation for Mass Spectrometric Investigation. DHBA and CHCA were used as matrixes. Both compounds were 2920 Analytical Chemistry, Vol. 69, No. 15, August 1, 1997

Figure 2. MALDI-TOFMS spectra of derivatized maltopentaose. Matrix, DHBA. These spectra were obtained with Vision 2000.

purchased from Aldrich (Milwaukee, WI). They were dissolved to a concentration of 10 g/L in a 10% (v/v) ethanol/water solution. Methanol solution of analytes were diluted with an appropriate volume of the matrix solution (typically from 1:4 to 1:10, v/v). Aliquots of the resulting mixtures (1 µL or less) were placed on silver plates. The solvent was removed in a gentle stream of air, and the solid sample/matrix mixture was then transferred into the vacuum chamber of the mass spectrometer. Mass Calibration. Peptides and oligosaccharides of known mass were used as standards. The measurements were calibrated with the following compounds: angiotensin I (human, Wako, 1296.5 Da), angiotensin II (human, Sigma, 1046.2 Da), substance P (Wako, 1347.6 Da), bombesin (frog, Sigma, 1619.9 Da), and γ-cyclodextrin (Wako, 1297.1 Da). Aliquots (0.5 µL) of 10-5 M aqueous solutions of the calibrants were added to the existing matrix. The base peak was used for calibration of MALDI-MS, and the fragment peaks of the peptides were utilized for calibration of PSD-MALDI. Comparative Studies. The ions were generated by irradiating the given target area with the output of N2 laser. To obtain a more accurate measurement of the signal strength, 50 shots were fired, and the resulting peak height was measured after the spectra were averaged. Means of three measurements were thus obtained for each oligosaccharide sample. PSD Experiments after Adding LiCl. A methanol solution (10 µL) of the analyte was diluted with 40 µL of matrix solution,

Figure 3. PSD-MALDI spectra of PA-tagged maltopentaose (MW 906) in CHCA (top) and DHBA (bottom). The spectra were measured by Reflex. [MNa]+ was selected as the parent ion.

and then 10 µL of 20 mM LiCl solution was added to this mixture. Aliquots of the resulting mixture (∼1 µL) were placed on the plates. The PSD-MALDI spectra were measured according to the procedure given below, after removing the solvent. PSD-MALDI Measurement. In Vision 2000, the spectra were measured at 5 keV acceleration voltage and reflection voltage was decreased in successive 20% steps. Depending on the mass range to be studied, 9-11 segments were acquired (30-50 laser shots for each window). The full spectrum was the result of the combination of different segments. In the case of Reflex, total

acceleration voltage was 28.5 keV, reflection voltage was decreased in successive 20% steps, 14 segments were obtained, and 50 singleshot spectra were summed up. RESULTS AND DISCUSSION We selected maltopentaose as a model oligosaccharide. Since in its native form it can not be reliably analysed at low levels in MALDI, at least 5 picomole was needed using DHBA as the matrix. Mohr et al.21 and Harvey22 compared sensitivities for underivatized oligosaccharides in many matrixes. There are few Analytical Chemistry, Vol. 69, No. 15, August 1, 1997

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Figure 4. PSD-MALDI spectra of PA-tagged maltopentaose in DHBA. The experiments were conducted with Vision 2000: (a) Li-free [MNa]+ as the parent ion. (b) With 20 mM LiCl, [MLi]+ was selected as the parent ion.

reports, however, on the comparison of the sensitivities among various oligosaccharide derivatives in DHBA and CHCA. We chose the reducing end reagents, PA, ABEE, and TMAPA, which become incorporated by reduction of the Schiff base, to enhance sensitivity. The comparison results of their ionization efficiencies in MALDI with the matrixes DHBA and CHCA are shown in Figure 1. DHBA was more advantageous for enhancement than CHCA. Among the derivatives, the sensitivity with the pyridyl(21) Mohr, M. D.; Bo¨rnsen, K. O.; Widmer, H. M. Rapid Commun. Mass Spectrom. 1995, 9, 809-814. (22) Harvey, D. J. Rapid Commun. Mass Spectrom. 1993, 7, 614-619.

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aminated maltopentaose showed a 100-fold improvement over the native maltopentaose with DHBA as a matrix. On the other hand, TMAPA-tagged maltopentaose, which was earlier found to exhibit extremely high sensitivity in positive ESIMS,14 demonstrated a low ionization efficiency in this experiment. This indicates that the sensitivity attained by the derivatization with TMAPA cannot be attributed to the constitutive positive charge of its quaternary ammonium group. The matrix may play key roles in the ionization efficiency produced by the MALDI process. Figure 2 shows three representative MALDI spectra of derivatized oligosaccharides obtained with DHBA, the mass

Table 1. Calculated and Observed Masses of PSD Product Ions of PA-Tagged Maltopentaosea

a

calcd fragment mass (av)

obsd fragment mass

∆m (Da)

mass accuracy (10-3)

assignment

791.7 767.7 671.6 605.5 509.4 443.4 422.4 420.4 363.4 347.3 288.3 281.3 260.3 259.3 185.1 163.2

791.6 767.5 671.6 605.7 508.9 443.5 423.2 420.5 361.5 347.5 289.5 281.4 261.2 259.2 185.4 165.4

-0.1 -0.2 0 +0.2 -0.5 +0.1 +0.8 +0.1 -1.9 +0.2 +1.2 +0.1 +0.9 -0.1 +0.3 +2.2

0.13 0.26 0 0.33 0.98 0.23 1.89 0.28 5.23 0.58 4.16 0.36 3.46 0.39 1.62 13.5

0,2A 5 Y′4 + Na B4 + Na Y′3 + Na B3 + Na Y′2 + Na 2,4A + K 3 Y′2 B2 + K B2 + Na 1,5X′′ 1 Y′1 + Na 2,4A + K 2 Y′′1 B1 + Na B1

Mass accuracy is given by ∆m/(calculated fragment mass), Matrix, DHBA.

Figure 5. PSD-MALDI spectrum of ABEE-tagged maltopentaose (MW 977) in DHBA. [MNa]+ was selected as the parent ion. This spectrum was obtained with Reflex.

accuracy being between 0.02 and 0.06%. Fragmentation of the analyte did not occur to any noticeable extent. The intensity of the protonated molecular ion was inconsiderable with the PA and ABEE derivatives. The analyte was detected mainly as a monosodium (m/z 929.6 for the PA derivative, m/z 1000.3 for the ABEE derivative) cation. The molecular ion was observed as m/z 963.5 with the TMAPA derivative, because it has a moiety containing a permanent positive charge at the reducing end. With CHCA, the spectra were quite similar to those obtained with DHBA in terms of mass accuracy and the molecular ion regions. Very recently, Takao et al. reported that ABEE derivatives show 4 times the sensitivity of PA derivatives in MALDI with CHCA.13 The

discrepancy may be ascribed in part to the influence of the sample preparation and loading, since Mohr et al. have already reported that the reproducibility in MALDI was greatly influenced by the sample preparation and loading.21 It is may be attributed to their use of instruments equipped with delayed extraction ion source (DE), because the signal-to-noise ratio is improved with DE.23 We tried the PSD-mode MALDI-TOFMS to obtain structural information from derivatized oligosaccharides, and peptide sequencing using this approach has been reported by several groups. The technique is particularly promising, because it is (23) Juhasz, P.; Roskey, M. T.; Smirnov, I. P.; Haff, L. A.; Vestal, M. L.; Martin, S. A. Anal. Chem. 1996, 68, 941-946.

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Figure 6. PSD-MALDI spectrum of TMAPA-tagged maltopentaose (MW 963) in DHBA. [M]+ was selected as the parent ion. The spectrum was taken by Reflex. Table 2. Sequence Ions for Different Derivatives of Maltopentaose in CID Experiments FAB-CIDb (high-energy collision)

PSD-MALDIa method PA ABEE TMAPA a

DHBA Bi, Yi Bi, Yi Yi, Zi

CHCA Yi Bi, Yi Yi, Zi

(+) 1,5Xi,

Yi, Zi Yi, Yi-EtOH Yi, Zi

1,5Xi,

ESI-MS/MSc (low-energy collision)

(-) -d 2,4Ai,

Yi

-

(+) Yi Yi Yi, Zi

(-) 2,4Ai,

Zi

-

This work. b Reference 29. c References 7 and 29. d -, sensitivity insufficient for spectra measurement.

applicable even for sequencing of peptides or proteins with molecular weights exceeding 5000.24,25 Application of PSD-MALDI for sequencing of oligosaccharides has been also reported.15-20 Despite this, there were very few reports where the series of sequence ions from each derivative were compared systematically. In the present study, a complete structural analysis with rather good mass resolution could be performed in all cases. Interestingly, among the derivatives, we found the fragmentation pattern with PA was strongly affected by the matrix compound used (Figure 3). This phenomenon is reproducible, since the fragmentation distributions were observed for both instruments, Reflex and Vision 2000, several times. This is the first report on the matrix-dependent fragmentation difference in PSD-MALDITOFMS, although the matrix dependence of metastable fragmentation of glycoproteins in MALDI-TOFMS has been already (24) Spengler, B.; Kirsch, D.; Kaufmann, R.; Jaeger, E. Rapid Commun. Mass Spectrom. 1992, 6, 105-108. (25) Kosaka, T.; Ichikawa, T.; Kinoshita, T. Rapid Commun. Mass Spectrum. 1995, 9, 1342-1344.

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reported.26 The explanation for this phenomenon, “matrixdependent fragmentation”, is not definitive, because the ion formation mechanism in MALDI has not been well elucidated yet. This would result from the difference of the parent ion, [M + Na]+ and [M + H]+, in PSD. Since DHBA tends to form the sodiated ion rather than the protonated ion in comparison with CHCA, the dominant intact ion ratio [M + Na]+/[M + H]+ in DHBA would be different from that in CHCA. This might be causing the fragmentation differences, because the ion selection device on the machines used cannot distinguish between [M + Na]+ and [M + H]+. The observed fragment ions were labeled according to the nomenclature of Domon and Costello.27 With the DHBA matrix, we could observe two classes of sequence ions: one with m/z 280.0, 442.5, 605.6, and 768.5, and the other m/z 346.2, 509.4, and 671.5. The former ions were easy to assign as [Yi + Na]+, but the latter could not be assigned definitively, because they were (26) Karas, M.; Bahr, U.; Strupat, K.; Hillenkamp, F.; Tsarbopoulos, A.; Pramanik, B. N. Anal. Chem. 1995, 67, 675-679. (27) Domon, B.; Costello, C. E. Glycoconjugate J. 1988, 5, 397-409.

Figure 7. PSD-MALDI spectrum of PA-tagged maltopentadectaose (DP ) 15, MW 2527) in DHBA. [MNa]+ was selected as the parent ion. The experiments was conducted with Vision 2000.

ascribable to both [Bi + Na]+ and 3,5Xi ions. To distinguish these sequence ions, we carried out a PSD-MALDI experiment on [M + Li]+ after adding lithium chloride to the analyte. If the ions were of [Bi + Na]+ series type, a mass difference shift of 16 Da would be observed. Figure 4 shows the effects of lithium chloride addition to PA-tagged maltopentaose. All the sequence ions observed in the spectrum were shifted by a mass difference of 16 Da. Therefore, we concluded that PA-maltopentaose gave two sequence ions, [Bi + Na]+ and [Yi + Na]+, with DHBA. On the other hand, with CHCA matrix, a simple spectrum was obtained (Figure 3). These sequences ions were ascribable to both [3,5Ai + Na]+ and Yi ions. The PSD experiments with lithium chloride showed that these ions were assigned to Yi ions. Table 1 summarizes the identified cleavages in the PSD fragment ion spectrum of pyridylaminated maltopentaose with DHBA. The PSD fragment ion masses had acceptable precision and mass accuracy. The average mass accuracy of fragment ion mass assignment was 2.1 × 10-3. The best mass accuracy of ∼0.2 × 10-3 was obtained for fragment ion masses above 400 Da, and the lowest of 13.5 × 10-3 for fragments below 200 Da. This reflects the performance that errors in digital control of the retarding reflection fields are currently limiting the mass accuracy.28 The fragments with rather low mass accuracy were potassium adduct ions, and these are due to the performance of parent mass selection of the machine. In the ABEE and TMAPA case, we could not observe such a matrix effect, while the spectra obtained with CHCA were identical to those obtained with DHBA. The PSD spectra of ABEE-maltopentaose (977 Da) and TMAPAmaltopentaose (963 Da) are summarized in Figures 5 and 6,

respectively. The PSD-MALDI sensitivities of these derivatives in DHBA and CHCA were between one-tenth and one-fiftieth times those of PA in DHBA. The characteristics of the sequence data from PSD-MALDI, in comparison with results of CID experiments in linked scan mode with a two-sector instrument and in triple quadrupole29 are shown in Table 2. The experiments performed on PA-labeled maltopentaose using PSD-MALDI with DHBA demonstrated that both B and Y series ions could be clearly observed from only one spectrum. Since this method is very sensitive, hundreds or even thousands of spectra, including PSD-MALDI examples, can be achieved with only one sample. Examination by PSD-MALDI with CHCA proved possible, and the data were complementary. Additionally, this method can be applied to the sequential analysis of oligosaccharides sized over 2500 Da, which are difficult to analyze with the usual CID measurements. As shown in Figure 7, sequential information from both ends of the chain were clearly observed. As a consequence of these features, structural elucidation of unknown compounds promises to be relatively easy. In our investigation, this advantage could not be obtained in other CID experiments. Although the ABEE-tagged oligosaccharide gave sequence ions from both termini by switching the ionization mode with FAB and ESI, or by measuring PSD-MALDI, such switching proved operationally difficult. In addition, the sensitivity in MALDI was about one-third lower than with the PA derivative, and matrix-dependent fragmentation could not be observed. The high potential of PA derivatization in PSD-MALDI was confirmed by its application to the naturally occurring oligosaccharides, lacto-N-fucopentaoses (LNFPs) I, II, III, and V.

(28) Kaufmann, R.; Spengler, B.; Luetzenkirchen, F. Rapid Commun. Mass Spectrom. 1993, 7, 902-910.

(29) Okamoto, M.; Takahashi, K.; Doi, T. Abstracts of 16th Japanese Carbohydrate Symposium, Kagoshima, Japan, 1994; pp 142-143.

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Figure 8. PSD-MALDI spectrum of PA-tagged LNFP III (MW 931) in DHBA. [MNa]+ was selected as the parent ion. This spectrum was recorded using Vision 2000.

These compounds are all present as sugars or their metabolites in human milk.30 The sequence ion features were coincident with those obtained for the model oligosaccharide maltopentaose, as shown in Figure 8. The PA-derivatized LNFP III having the branched structure gave the predictable fragmentation pattern, carrying information on sequence and branching in PSD-MALDI. This spectrum showed more structural information than those observed in benzylamino LNFP derivatives reported by Lemoine et al.16 CONCLUSION This study has demonstrated that PA-derivatized oligosaccharides can significantly enhance the ionization efficiency and carry clear information on sequence and branching from PSD-MALDI spectra. PA-tagged oligosaccharides have been widely used for sensitive quantitative and qualitative analysis by HPLC with fluorescent detection.10 Two-dimensional mapping with many standard PAoligosaccharides, together with the additivity rule and exoglycosidase digestion, has been applied for pattern analysis of their structures.32,33 From these results, the method described here can be concluded to be a powerful tool for neutral oligosaccharide analysis in general. The oligosaccharide structural analysis could (30) Yamashita, K.; Tachibana, Y.; Kobata, A. J. Biol. Chem. 1977, 252, 5408. (31) Talbo, G.; Mann, M. Rapid Commun. Mass Spectrom. 1996, 10, 100. (32) Hase, S.; Sugimoto, T.; Takemoto, H.; Ikenaka, T.; Schmid, K. J. Biochem. 1986, 99, 1725. (33) Tomiya, N.; Awaya, J.; Kurano, M.; Endo, S.; Arata, Y.; Takahashi, N. Anal. Biochem. 1988, 171, 73.

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become much more reliable and fast in the near future, using PSD-MALDI in combination with HPLC analysis of fluorescencelabeled oligosaccharides derivatized with PA. Preliminary studies indicate that this methodology was not practical for the analysis of acidic oligosaccharides. The sensitivity of sialooligosaccharides in MALDI was not so high in comparison with that of neutral oligosaccharides, even when they were derivatized with PA. In addition, prompt fragmentation in positiveion mode in MALDI could remove the sialic acid as Talbo and Mann have reported.31 Further experiments on acidic oligosaccharides are required. ACKNOWLEDGMENT A part of this work was presented at the 17th Japanese Carbohydrate Symposium (July 20, 1995, Kyoto, Japan). We thank Drs. Uwe Rapp of Bruker-Franzen Analytik GmbH, Arnd Ingendoh and Hidenori Ikezawa of Thermo Quest (formerly Finnigan Mat Instruments Inc.), Tokyo, Japan, Kiyonobu Fukunaga of Nissei Sangyo Co. Ltd., Tokyo, Japan, and Takashi Nirasawa of Bruker Japan Co. Ltd., Tsukuba-city, Ibaraki, Japan, for measuring the PSD-MALDI spectra. Our special gratitude is extended to Mr. Takao Nakagawa and Ms. Mayumi Toriu of our laboratory for their efficient technical contributions. Received for review September 10, 1996. Accepted May 14, 1997.X AC960910S X

Abstract published in Advance ACS Abstracts, July 1, 1997.