Structural Characterization of Chemically Derivatized

Aug 7, 1999 - ... from the multiply charged precursor ions, were predominant and were used for the structural readout. Some oxonium ions that were obs...
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Anal. Chem. 1999, 71, 4100-4106

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Structural Characterization of Chemically Derivatized Oligosaccharides by Nanoflow Electrospray Ionization Mass Spectrometry Wenjun Mo,† Hiroko Sakamoto,† Atsushi Nishikawa,‡ Noriko Kagi,§ James I. Langridge,| Yasutsugu Shimonishi,† and Toshifumi Takao*,†

Institute for Protein Research, Osaka University, Yamadaoka 3-2, Suita, Osaka 565-0871, Japan, Department of Biochemistry, Okayama University of Science, 1-1 Ridai-cho, Okayama 700-0005, Japan, Jasco International Co., Ltd., Myojin-cho 1-chome 11-10, Hachioji, Tokyo 192-0046, Japan, and Micromass UK Ltd., Floats Road, Wythenshawe, Manchester M23 9LZ, United Kingdom

Oligosaccharides released from several glycoproteins were derivatized with either 4-aminobenzoic acid 2-(diethylamino)ethyl ester (ABDEAE) (Yoshino, K.; et al. Anal. Chem. 1995, 67, 4028-4031) or 2-aminopyridine. The resulting derivatives were analyzed on a nanoflow electrospray ionization (ESI) quadrupole-inlet time-of-flight mass spectrometer using the low-energy collision-induced dissociation technique. In the MS/MS spectra, the oxonium (b or internal series) and y series ions, which are derived from the multiply charged precursor ions, were predominant and were used for the structural readout. Some oxonium ions that were observed in the low-mass region, but that were not found in the PSD analyses (Mo, W.; et al. Anal. Chem. 1998, 70, 4520-4526), rendered a more detailed structural insight. The oxonium ions at m/z 512.2, which are derived from the fucosylated oligosaccharides of immunoglobulin Y and thyroglobulin, were observed, suggesting that fucosylation had occurred proximal to the outer nonreducing terminus. In addition, the data herein show that structural elucidation can be routinely achieved at a low sample concentration. For the case of ABDEAE derivatives, this can be achieved at the 50 fmol/µL level and with the actual sample consumption at the attomole level using nanoflow ESI MS/MS. It is clear that virtually all proteins that are processed and transported by/through cellular secretory apparatus (i.e., the endoplasmic reticulum and the golgi systems) are glycosylated.1 Glycoforms play a wide variety of roles in cell biology, for example, in the conformation and stability of proteins, control of the halflife of proteins and cells, and modulation of protein functions and * Corresponding author: (tel) +81-6-6879 8602; (fax) +81-6-6879 8603; (email) [email protected]. † Osaka University. ‡ Okayama University of Science. § Jasco International Co., Ltd. | Micromass UK Ltd. (1) Lewin, B. Genes VI; Oxford University Press: New York, 1997; pp 10301033.

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serving as ligands for specific binding events which mediate protein targeting and cell-matrix or cell-cell interactions.2 Because compositional and structural information of the carbohydrate moieties of glycoproteins is not encoded in DNA sequences, their structural characterization requires analytical techniques that are capable of direct sampling. Typically, glycoprotein-derived glycans are chemically derivatized and then profiled by high-performance separation techniques or/and mass spectrometry, a recently emerged alternative. Since there is no suitable chromophore in ordinary sugar chain moieties, small amounts of free oligosaccharides are difficult to analyze with a high degree of sensitivity. Prior to mass spectrometric analysis, excess salts and hydrophobic impurities, many of which may suppress the MALDI or ESI ionization process, could not be efficiently removed by commonly used techniques. MH-type quasi-molecular ions are difficult to observe in the positive-ion mode due to the low proton binding affinity of the residues as well. As a result, the detection of underivatized sugars by MS is usually done via monitoring of the salt adduct ions. The metal adducts can be detected with reasonable sensitivity,3 affording some cross-ring fragment ions in the MS/MS experiment, which are structurally informative in terms of resolving specific oligosaccharide linkages.4-6 Nonetheless, MS/MS experiments based on adduct ions generally provide fewer fragment ions and require higher collision energy7 (hence, inadvertently lowering the sensitivity). In addition, even for commercially available “standard” free oligosaccharides, the singly charged quasi-molecular ions are usually observed as clusters of salt adducts, i.e., (M + Na)+, (M + K)+, (M - H + 2Na)+, (M - H + K + Na)+ etc., which substantially complicate the spectra and lower the sensitivity by diversifying the total intensity to each of the signals. (2) (3) (4) (5) (6)

Varki, A. Glycobiology 1993, 3, 97-130. Bahr, U.; Pfenninger, A.; Karas, M. Anal. Chem. 1997, 69, 4530-4535. Konig, S.; Leary, J. A. J Am. Soc. Mass Spectrom. 1998, 9, 1125-1134. Gaucher, S. P.; Leary, J. A. Anal. Chem. 1998, 70, 3009-3014. Weiskoft, A. S.; Vouros, P.; Harvey, D. J. Anal. Chem. 1998, 70, 44414447. (7) Bru ¨ ll, L. P.; Kova´cik, V.; Thomas-Oates, J. E.; Heerma, W.; Haverkamp, J. Rapid Commun. Mass Spectrom. 1998, 12, 1520-1532. 10.1021/ac990247i CCC: $18.00

© 1999 American Chemical Society Published on Web 08/07/1999

Several chemical derivatization methods, which involve reductive amination of oligosaccharides, have been proposed and have been shown to permit 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),8 trimethyl(paminophenyl)ammonium chloride (TMAPA),9 or 2-aminoacridone (AMAC).10 It should be noted that the method based on permethylation and peracetylation 11-14 allows cross-ring fragmentation, which can provide extensive information on the branch sites in an oligosaccharide.15-17 Derivatization of oligosaccharides with ABDEAE, coupled with MS, was first proposed for the sensitive detection of oligosaccharides8,18 and was later extended to the application of de novo structural elucidation using PSD MALDI-TOF MS.19 In conjunction with the use of a normal-phase resin for rapid and simple purification,20 the ABDEAE derivatization approach was demonstrated to be applicable to 90 pmol of a glycoprotein as the starting material giving rise to a derivative with a 1000-fold enhancement in sensitivity with respect to MS measurement over techniques which used free oligosaccharides. In this study, we describe measurements of ABDEAE and PA derivatives on a nanoelectrospray3,21,22 quadrupole-inlet time-of-flight (Q-TOF) MS, which, together with MALDI-TOF MS, represents a well-established technique in current mass spectrometry. EXPERIMENTAL SECTION ABDEAE hydrochloride (ABDEAE-HCl) was purchased from Tokyo Chemical Industry (Tokyo, Japan). Sodium cyanoborohydride, ribonuclease B, transferrin, and thyroglobulin were obtained from Sigma Chemical (St. Louis, MO). Peptide-N-glycosidase F (PNGase F, which cleaves the β-aspartylglycosylamine linkage of Asn-linked carbohydrates) was acquired from New England Biolaboratory (Beverly, MA). Neuraminidase (sialidase) was purchased from Boehringer Mannheim (Mannheim, Germany). Standard PA-derivatized oligosaccharides, Gal2GlcNAc2Man3FucGlcNAc2-PA (>99%) and Man6GlcNAc2-PA (>95%) were purchased from Takara Biomedicals (Tokyo, Japan). All reagents were of analytical grade and were used without further purification. (8) Yoshino, K.; Takao, T.; Murata, H.; Shimonishi, Y. Anal. Chem. 1995, 67, 4028-4031. (9) Dell, A.; Carman, H.; Tiller, P. R.; Thomas-Oates, J. E. Biomed. Environ. Mass Spectrom. 1988, 16, 19-24. (10) Okafo, G. N.; Burrow, L. M.; Neville, W.; Truneh, A.; Smith, R. A. G.; Camilleri, P. Anal. Chem. 1996, 68, 4424-4430. (11) Egge, H.; Katalinic, J. P. Mass Spectrom. Rev. 1987, 8, 331-339. (12) Domon, B.; Mueller, D. R.; Richter, W. J. Org. Mass Spectrom. 1994, 29, 713-718. (13) Lemoine, J.; Chirat, F.; Domon, B. J. Mass Spectrom. 1996, 31, 908-912. (14) Perreault, H.; Costello, C. E. J. Mass Spectrom. 1999, 34, 184-197. (15) Sheeley, D. M.; Reinhold, V. Anal. Chem. 1998, 70, 3053-3059. (16) Viseux, N.; De Hoffmann, E.; Domon, B. Anal. Chem. 1997, 69, 31933198. (17) Viseux, N.; Costello, C. E.; Domon, B. J. Mass Spectrom. 1999, 34, 364376. (18) Takao, T.; Tambara, Y.; Nakamura, A.; Yoshino, K.; Fukuda, H.; Fukuda, M.; Shimonishi, Y. Rapid Commun. Mass Spectrom. 1996, 10, 637-640. (19) Mo, W.; Takao, T.; Sakamoto, H.; Shimonishi, Y. Anal. Chem. 1998, 70, 4520-4526. (20) Sakamoto, H.; Takao, T.; Mo, W.; Fukuda, H.; Tambara, Y.; Besada, V.; Shimonishi, Y. Proc. 46th ASMS Conf. Mass Spectrom. Allied Topics, Orlando, FL, 1998; p 1368. (21) Wilm, M.; Mann, M. Anal. Chem. 1996, 68, 1-8. (22) Okafo, G. N.; Langridge, J.; North, S.; Organ, A.; West, A.; Morris, M.; Camilleri, P. Anal. Chem. 1997, 69, 4985-4993.

N-Glycans derived from the enzymatic cleavage of sugar chains from ribonuclease B, thyroglobulin, and transferrin were subjected to ABDEAE derivatization, the detailed reaction conditions and purification protocols of which have been described in a previous report.19 PA derivatives were prepared from the N-glycans derived from the immunoglobulin Y (IgY)23 using methodology described elsewhere.24 The enzymatic removal of sialic acids from N-glycans with sialidase was performed in a 50 mM sodium acetate buffer (pH 5.1) with an approximate enzyme/substrate ratio of 1 unit/ 100 nmol by incubating at 37 °C for 12 h. Although the final products of the derivatization were purified/quantified by RPHPLC, the possibility that each resulting fraction could contain a mixture of isomers cannot be excluded. Mass spectrometric analyses of the ABDEAE-derivatized glycans were carried out on the Q-TOF mass spectrometer (Micromass, Manchester, U.K.), which is a hybrid quadrupole orthogonal acceleration tandem mass spectrometer, fitted with a Z-spray nanoflow electrospray ion source. Samples were dissolved in and diluted with 0.05% aqueous formic acid, with ∼2 µL of such a solution being loaded on a borosilicate nanoflow tip prior to analysis. The mass spectrometer was operated with a source temperature of 80 °C and a drying gas flow of 50 L/h. A potential of 1.0-1.5 kV was applied to the nanoflow tip in the ion source, which resulted in a flow rate of about 10-20 nL/min into the analyzer. Data were acquired in a continuum mode with an integration time of 2 s. For the acquisition of MS data, the quadrupole was used in the radio frequency mode allowing approximately a decade in mass to be transmitted to the TOF analyzer. For MS/MS studies, the quadrupole was used to select the parent ions, which were subsequently fragmented in the hexapole collision cell using argon as the collision gas at a pressure of ∼3 × 10-2 Pa and an appropriate collision energy (2035 eV). Data acquisition was performed on a Mass Lynx system based on Windows NT. MS/MS data were processed by a maximum entropy data enhancement program, MaxEnt 3 (Micromass), which is capable of deconvoluting a spectrum where peaks in a variety of charge states are present, thus producing a simplified spectrum consisting only of monoisotopic peaks in a single charge state. PSD MALDI-TOF MS data for comparison were acquired from a Voyager Elite XL time-of-flight mass spectrometer (PerSeptive Biosystems, Framingham, MA), whose parameters and experimental conditions have been described elsewhere.19 RESULTS AND DISCUSSION For all CID MS/MS experiments, protonated quasi-molecular ions (M + 2H)2+ or (M + 3H)3+, which, at conditions of lowenergy collision-induced dissociation (CID), undergo a much higher degree of decomposition than the salt adduct ions,7 were selected as the precursors. In addition, MS/MS of the protonated ion species afforded a simple spectrum, leading to a straightforward spectral interpretation by avoiding inconveniences, such as the mixing of salt-containing and nonsalt-containing fragment signals. The minimum sample concentration for MS/MS measurements reached 50 fmol/µL in the case of ABDEAE derivatives, (23) Hatta, H.; Kim, M.; Yamamoto, T. Agric. Biol. Chem. 1990, 54, 25322535. (24) Hase, S.; Ibuki, T.; Ikenaka, T. J. Biochem. 1984, 95, 197-203.

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Figure 1. Nanoelectrospray MS spectra of the molecular ion regions of derivatized oligosaccharides. (a) ABDEAE-derivatized Man5GlcNAc2; the singly charged ions m/z 725.9 and 730.9 possibly were the contaminants from the borosilicate nanoflow tip. (b) A commercially available PA-derivatized Man6GlcNAc2. (c) ABDEAE-derivatized NeuNAc2Gal2GlcNAc2Man3GlcNAc2 from transferrin. (d) PA-derivatized NeuNAcGal2GlcNAc3Man3FucGlcNAc2 from IgY. Samples used in (a), (c), and (d) were prepared in the laboratory and purified with RP-HPLC.

Scheme 1. ABDEAE-Derivatized Transferrin N-Glycana

a The mass numbers correlate to the monoisotopic ones calculated on the basis of the structures of fragments, and only one of the possible assignments is depicted for each observed m/z value.

giving rise to an actual sample consumption at the attomole level. PA derivatives were measured at a sample concentration of ∼1 pmol/µL. Even after RP-HPLC purification, PA-derivatized glycans showed a stronger propensity in forming salt adduct clusters during MS measurement than the ABDEAE counterparts (see Figure 1). Hence, the sensitivity of ABDEAE derivatives in term of minimum sample concentration required for MS/MS is ∼1 order of magnitude higher than that of PA derivatives. The ABDEAE-derivatized NeuNAc2Gal2GlcNAc2Man3GlcNAc2 (see Scheme 1) from transferrin was subjected to the low-energy CID MS/MS obtained from the triply charged precursor ion (M + 3H)3+ at m/z 815.27. Figure 2 shows the spectrum that has been processed with MaxEnt 3 based on the raw data (not shown). This result was also compared with the data on the same analyte 4102 Analytical Chemistry, Vol. 71, No. 18, September 15, 1999

acquired from the PSD MALDI-TOF MS experiment (data not shown).19 It is noteworthy that the oxonium ions (i.e., b series ions and internal series ions) and the y series ions (which result from the retention of a positive charge at the ABDEAE group) derived from the reducing terminus were the predominant ions observed. In addition, the loss of residues from the nonreducing terminus for the observed y series ions appeared as an overlapping pattern rather than as a fixed one (i.e., in the case of the PSD spectrum, the successive loss of NeuNAc, followed by Hex f HexNAc is a dominant feature19). The y series ions readily reveal the relative alignment of the constituent saccharides. In addition, information on the degree of branching in the nonreducing termini can be obtained through observing the array of signals (the yn-1 ions) formed by the truncation of any one of the reducing-terminal

Figure 2. Nanoelectrospray low-energy CID MS/MS spectrum of NeuNAc2Gal2GlcNAc2Man3GlcNAc2-ABDEAE derived from transferrin. Solid arrowhead and dashed arrowhead lines indicate the y and the oxonium (b or internal series) ions, respectively.

Figure 3. Low-energy CID MS/MS spectrum of PA derivatives of NeuNAcGal2GlcNAc3Man3FucGlcNAc2 obtained from IgY. The expanded region (the raw MS/MS spectrum) shows the fragment ion at m/z 512, which has a residual composition of a Hex, HexNAc, and dHex.

residues (see Figure 3). In contrast to the PSD mass spectra, the CID MS/MS spectra afforded the extra oxonium ions in the lowmass region, which were useful in elucidating structural details in the vicinity of the nonreducing termini. The difference between the PSD and the CID spectra may originate from differences in the fragmentation mechanisms for the derivatized oligosaccha-

rides. In the case of PSD MALDI-TOF MS, the precursor ions are singly charged and tend to follow a unimolecular decomposition during their flight in a field-free region. In contrast, for the case of nanoelectrospray MS/MS, the precursor ions are multiply charged (the charges are distributed on the derivatization group and the amide group of GlcNAc) and undergo a collision-induced Analytical Chemistry, Vol. 71, No. 18, September 15, 1999

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Scheme 2. PA-Derivatized N-Glycan of Immunoglobulin Ya

a The mass numbers correlate to the monoisotopic ones calculated on the basis of the structures of fragments, and only one of the possible assignments is depicted for each observed m/z value.

Figure 4. Low-mass region of MS/MS spectra of an ABDEAE-derivatized fucosylated N-glycan (Gal2GlcNAc2Man3FucGlcNAc2) from thyroglobulin (a) and the commercially available Gal2GlcNAc2Man3FucGlcNAc2-PA (b). The N-glycan from thyroglobulin was enzymatically treated with the sialidase prior to the ABDEAE derivatization. The insets show the presence (a) or the absence (b) of the dHex-containing oxonium ions at m/z 512.2.

dissociation process. As a result, pairs of fragment ions (i.e., y and b series ions), which are “complementary” in m/z over the molecular weight, are often generated as the result of the cleavage of a glycosidic bond. 4104 Analytical Chemistry, Vol. 71, No. 18, September 15, 1999

Figure 3 shows the nanoelectrospray MS/MS spectrum from (M + 3H)3+ at m/z 787.6 (see Figure 1d) of the PA-derivatized fucosylated N-glycan from IgY, which resulted in a fragmentation pattern similar to those of the ABDEAE derivatives. The observed

Scheme 3. ABDEAE-Derivatized, Sialidase Treated N-Glycan from Thyroglobulina

a The mass numbers correlate to the monoisotopic ones calculated on the basis of the structures of fragments, and only one of the possible assignments is depicted for each observed m/z value.

Scheme 4. A Standard PA-Sugar Chain (Takara, Tokyo)a

a The mass numbers correlate to the monoisotopic ones calculated on the basis of the structures of fragments, and only one of the possible assignments is depicted for each observed m/z value.

y series ions could be assigned according to one of the known oligosaccharides (see also Scheme 2) reported for IgY.25,26 From the yn-1 ions, all nonreducing terminal residues, i.e., GlcNAc, NeuNAc, Gal, and Fuc, were revealed, suggesting that the oligosaccharide is of the bisecting biantennary, fucosylated, monosialylated type. It is noteworthy, that in the low-mass region, the y series ion at m/z 446.3 could be assigned to the known (FucR1f6)GlcNAcfPA structure; whereas the oxonium ion at m/z 512.2 (inset, Figure 3), which has the residual composition of a hexose, a deoxyhexose, and a N-acetylhexosamine, was observed but could not be assigned to the proposed structure (see Scheme 2). Analogous fucose-containing ion has also been observed in the study of one of the ABDEAE-derivatized glycans from thyroglobulin (see Figure 4a and Scheme 3). The data suggest that the two oligosaccharides, prepared in the present

study, appear to contain the isoforms of fucosylation, i.e., fucosylation proximal to the outer nonreducing terminus as well as that at the reducing terminal GlcNAc, which cannot be resolved by RP-HPLC. To confirm the origin of the ion at m/z 512.2, a commercially available high-purity (>99%) PA derivative, the structure of which is shown in Scheme 4, was measured under the same MS/MS conditions (see Figure 4b). An ion at m/z 446.3 was observed, corresponding to the fucosylation at the reducing terminus, but an ion at m/z 512.2 corresponding to a composition of dHex, Hex, and HexNAc was not observed. Fucosylation proximal to the nonreducing terminus, which may occur at either the Gal (i.e., FucR1f2) or the GlcNAc (i.e., FucR1f3), was first observed in glycoproteins expressed by human promyelocytic leukemic cells (HL-60)27 and was later concluded to be an aberrant glycosylation.28 This does not, however, rule out the possibility

(25) Ohta, M.; Hamako, J.; Yamamoto, S.; Hatta, H.; Kim, M.; Yamamoto, T.; Oka, S.; Mizuochi, T.; Matsuura, F. Glycoconjugate J. 1991, 8, 400-413. (26) Matsuura, F.; Ohta, M.; Murakami, K.; Matsuki, Y. Glycoconjugate J. 1993, 10, 202-213.

(27) Mizoguchi, A.; Takasaki, S.; Maeda, S.; Kobata, A. J. Biol. Chem. 1984, 259, 11949-11957. (28) Yamashita, K.; Koide, N.; Endo, T.; Iwaki, Y.; Kobata, A. J. Biol. Chem. 1989, 264, 2415-2423.

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that it could occur frequently albeit at a low abundance.29 CONCLUSION ABDEAE and PA derivatives of oligosaccharides prepared from several glycoproteins have been successfully applied to nanoelectrospray Q-TOF MS for purposes of obtaining highly sensitive structural analytical data. The ABDEAE, attached to the reducing terminus not only permits high-sensitivity detection, with the actual sample consumption for an MS/MS study at the attomole level, but also provides useful “reading frames” for structural elucidation in MS/MS studies. Compared with PSD MALDI-TOF MS, nanoelectrospray low-energy CID MS/MS affords both y series and oxonium ions (b series or internal ions) via a divergent (29) Guile, G. R.; Harvey, D. J.; O’Donnell, N.; Powell, A. K.; Hunter, A. P.; Zamze, S.; Fernandes, D. L.; Dwek, R. A.; Wing, D. R. Eur. J. Biochem. 1998, 258, 623-656. (30) Tsuji, T.; Yamamoto, K.; Irimura, T.; Osawa, T. Biochem. J. 1981, 195, 691-699. (31) Yamamoto, K.; Tsuji, T.; Irimura, T.; Osawa, T. Biochem. J. 1981, 195, 701-713.

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pathway allowing a more detailed structural characterization. This procedure allowed the detection of the fucosylation proximal to the outer nonreducing terminus for the first time in the fucosylated glycoforms isolated from IgY25,26 and thyroglobulin.30,31 Given the effectiveness and the high sensitivity, the derivatization of oligosaccharides in conjunction with the nanoelectrospray MS/MS technique provides arguably the most powerful analytical method hitherto for the structural characterization of oligosaccharides. 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.).

Received for review March 3, 1999. Accepted June 9, 1999. AC990247I