Glycoblotting-Assisted O-Glycomics: Ammonium ... - ACS Publications

Nov 15, 2010 - Kita-ku, Sapporo, Japan, and Division of Quantification of Health State (Feel Fine Corporation), Graduate School of. Life Science, Hokk...
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Anal. Chem. 2010, 82, 10021–10029

Glycoblotting-Assisted O-Glycomics: Ammonium Carbamate Allows for Highly Efficient O-Glycan Release from Glycoproteins Yoshiaki Miura,† Kentaro Kato,‡ Yasuhiro Takegawa,‡ Masaki Kurogochi,‡ Jun-ichi Furukawa,‡ Yasuro Shinohara,‡ Noriko Nagahori,‡ Maho Amano,‡,§ Hiroshi Hinou,‡ and Shin-Ichiro Nishimura*,†,‡ Ezose Sciences, Inc., 25 Riverside Drive Pine Brook, New Jersey 07058, United States, Graduate School of Life Science, and Frontier Research Center for Post-Genomic Science and Technology, Hokkaido University, N21, W11, Kita-ku, Sapporo, Japan, and Division of Quantification of Health State (Feel Fine Corporation), Graduate School of Life Science, Hokkaido University, N21, W11, Kita-ku, Sapporo, Japan Glycoblotting, high throughput method for N-glycan enrichment analysis based on the specific chemical ligation between aminooxy/hydrazide-polymers/solids and reducing N-glycans released from whole serum and cellular glycoproteins, was proved to be feasible for selective enrichment analysis of O-glycans of common (mucin) glycoproteins. We established a standard protocol of glycoblotting-based O-glycomics in combination with nonenzymatic chemical treatment to release reducing Oglycans predominantly from various glycoprotein samples. It was demonstrated that the nonreductive condition employing a simple ammonium salt, ammonium carbamate, made glycoblotting-based enrichment analysis of O-glycans possible without significant loss or unfavorable side reactions. A general workflow of glycoblotting using a hydrazide bead (BlotGlyco H), on-bead chemical manipulations, and subsequent mass spectrometry allowed for rapid O-glycomics of human milk osteopontin (OPN) and urinary MUC1 glycoproteins purified from healthy donors in a quantitative manner. It was revealed that structures of O-glycans in human milk OPN were varied with habitual fucosylation and N-acetyllactosamine units. It was also suggested that purified human urinary MUC1 was modified preferentially by sialylated O-glycans (94% of total) with 7:3 ratio of core 1 to core 2 type O-glycans. Versatility of the present strategy is evident because this method was proved to be suited for the enrichment analysis of general biological and clinical samples such as human serum and urine, cultured human cancer cells, and formalin-fixed paraffin-embedded tissue sections. It is our belief that the present protocols would greatly accelerate discovery of disease-relevant O-glycans as potential biomarkers. Glycosylation is one of the most important posttranslational modifications of proteins in eukaryotes and this step is essential * To whom correspondence should be addressed. Phone: +81 11 706 9043. Fax: +81 11 706 9042. E-mail: [email protected]. † Ezose Sciences, Inc. ‡ Frontier Research Center for Post-Genomic Science and Technology. § Division of Quantification of Health State (Feel Fine Corporation). 10.1021/ac101599p  2010 American Chemical Society Published on Web 11/15/2010

to modulate a wide range of protein and lipid functions both on the cellular surfaces and within the cells.1-3 Therefore, it is clear that glycomics, a term defining the sequence analysis or profiling of glycome, must be integrated with the results revealed by proteomics, the analysis of genomic complements of proteins. However, it should be emphasized that glycome (glycan structures) of the glycoproteins of interest are impossible to predict based on any genomic database because the biosynthetic process of the glycan moieties of glycoproteins is not template-driven and is subject to multiple sequential and competitive enzymatic pathways.4,5 In other words, there is no PCR-like glycan amplification technology for glycomics, while proteomics can use satisfactory quality and quantity of the any proteins of interest by means of PCR-based recombination technology.6 As a result, the therapeutic/diagnostic potential of complex glycans has not been well exploited with a few notable exceptions despite the growing importance of drastic structural alteration of glycome in carcinogenesis and cancer metastasis.7,8 Regarding glycomics for N-glycans, a major class of glycan chains attached to glycoproteins through the N-glycosidic linkage with asparagines residues,4 we have demonstrated that glycoblotting method,9 a PCR-like key technology for N-glycan enrichment analysis, allows for discovery research of novel diseaserelevant biomarkers,10,11 and rapid monitoring of dynamic (1) Zachara, N. E.; Hart, G. W. Chem. Rev. 2002, 102, 431–438. (2) Molinari, M. Nat. Chem. Biol. 2007, 3, 313–320. (3) Arnold, J. N.; Wormald, M. R.; Sim, R. B.; Rudd, P. M.; Dwek, R. A. Annu. Rev. Immunol. 2007, 25, 21–50. (4) Kornfeld, R.; Kornfeld, S. Annu. Rev. Biochem. 1985, 54, 631–664. (5) Brockhausen, I. Biochim. Biophys. Acta 1999, 1473, 67–95. (6) Pilobello, K. T.; Mahal, L. K. Curr. Opin. Chem. Biol. 2007, 11, 300–305. (7) Ludwig, J. A.; Weinstein, J. N. Nat. Rev. Cancer 2005, 5, 845–856. (8) Dube, D. H.; Bertozzi, C. R. Nat. Rev. Drug Discovery 2005, 4, 477–488. (9) Nishimura, S.-I.; Niikura, K.; Kurogochi, M.; Matsushita, T.; Fumoto, M.; Hinou, H.; Kamitani, R.; Nakagawa, H.; Deguchi, K.; Miura, N.; Monde, K.; Kondo, H. Angew. Chem., Int. Ed. 2004, 44, 91–96. (10) Miura, Y.; Hato, M.; Shinohara, Y.; Kuramoto, H.; Furukawa, J.; Kurogochi, M.; Shimaoka, H.; Tada, M.; Nakanishi, K.; Ozaki, M.; Todo, S.; Nishimura, S.-I. Mol. Cell. Proteomics 2008, 7, 370–377. (11) Furukawa, J.; Shinohara, Y.; Kuramoto, H.; Miura, Y.; Shimaoka, H.; Kurogochi, M.; Nakano, M.; Nishimura, S.-I. Anal. Chem. 2008, 80, 1094– 1101.

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N-glycoform alteration during differentiation of mouse embryonic carcinoma cells and mouse embryonic stem cells.12 While the N-glycans present widely in mammalian glycoproteins can be released by using specific enzymes, for example, peptide N-glycosidase F (PNGase F) and PNGase A in case for the glycoblotting of N-glycans from other species such as plants and insects glycoprteins,13 O-linked type glycans (O-glycans) attached to serine/threonine residues of glycoproteins have been mostly subjected to chemical treatments under some conditions for alkaline β-elimination, that is, Carlson degradation, alkylamineassisted alkaline solution, or conventional hydrazinolysis.14-23 However, these methods are usually accompanied with significant loss of the intact O-glycans due to serious peeling reactions caused by hydroxide anions and the needs of hazardous/toxic reagents, respectively. Even though the resulting alditols, stable and inactive reduced derivatives from hemiacetals, could be identified, timeconsuming and tedious procedures for the chromatographic separation appear to make rapid and large-scale analysis difficult. On the other hand, ammonia-based release of O-glycans from glycoconjugates may be an attractive option to produce O-glycans in the form with reducing end that is suited for the subsequent labeling of the hemiacetal with such as fluorescent and stable isotope reagents. Actually, concentrated ammonium hydroxide with saturated ammonium carbonate has often been employed instead of strong alkaline β-elimination or hydrazinolysis, although quantitative aspect has not been confirmed yet.24,25 However, it is clear that use of concentrated ammonia solution does not meet the criteria required for the large-scale glycomics at robotic glycoblotting systems.10-12 Our attention has been paid toward the effect of the hydroxide anions liberated by β-elimination in the presence of concentrated aqueous ammonia/ammonium carbonate on the peeling reaction to give rise to undesired degradation products. We thought that advent of much more facile and efficient protocols/reagents for generating reducing O-glycans from various biological sources would accelerate high throughput and quantitative O-glycomics of glycoproteins and glycosphingolipids on the basis of general platform of the glycoblotting technology. Here we communicate a standard method for the (12) Amano, M.; Yamaguchi, M.; Takegawa, Y.; Yamashita, T.; Terashima, M.; Furukawa, J.-i.; Miura, Y.; Shinohara, Y.; Iwasaki, N.; Minami, A.; Nishimura, S.-I. Mol. Cell. Proteomics 2010, 9, 523–537. (13) Kimura, A.; Tandang, M. -R.; Fukuda, T.; Cabanos, C.; Takegawa, Y.; Amano, M.; Nishimura, S. -I.; Matsumura, Y.; Maruyama, N.; Ustumi, S. J. Agric. Food Chem. 2010, 58, 2923–2903. (14) Carlson, D. M. J. Biol. Chem. 1968, 243, 616–626. (15) Patel, T.; Bruce, J.; Merry, A.; Bigge, C.; Wormald, M.; Jaques, A.; Parekh, R. Biochemistry 1993, 32, 679–693. (16) Royle, L.; Mattu, T. S.; Hart, E.; Langridge, J. I.; Merry, A. H.; Murphy, N.; Harvey, D. J.; Dwek, R. A.; Rudd, P. M. Anal. Biochem. 2002, 304, 70–90. (17) Yamada, K.; Hyodo, S.; Matsumoto, Y.; Kinoshita, M.; Maruyama, S.; Osaka, Y.; Casal, E.; Lee, Y. C.; Kakehi, K. Anal. Biochem. 2007, 371, 52–61. (18) Cooper, C. A.; Packer, N. H.; Redmond, J. W. Glycoconjugate J. 1994, 11, 163–167. (19) Chai, W.; Feizi, T.; Yuen, C. T.; Lawson, A. M. Glycobiology 1997, 7, 861– 872. (20) Hanisch, F. G.; Jovanovic, M.; Peter-Ktalinic, J. Anal. Biochem. 2001, 290, 47–59. (21) Czeszak, X.; Ricart, G.; Tetaert, D.; Michalski, J. C.; Lemoine, J. Rapid Commun. Mass Spectrom. 2002, 16, 27–34. (22) Zheng, Y.; Guo, Z.; Cai, Z. Talanta 2009, 78, 358–363. (23) Maniatis, S.; Zhou, H.; Reinhold, V. Anal. Chem. 2010, 82, 2421–2425. (24) Huang, Y.; Mechref, Y.; Novotny, M. V. Anal. Chem. 2001, 73, 6063–6069. (25) Huang, Y.; Konse, T.; Mechref, Y.; Novotny, M. V. Rapid Commun. Mass Spectrom. 2002, 16, 1199–1204.

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enrichment analysis of common O-linked type oligosaccharides of glycoproteins without significant loss of the intact O-glycans caused by the possible peeling reactions. MATERIALS AND METHODS Materials and Reagents. Peptide N-glycosidase F (PNGase F) was purchased from Roche (Mannheim, Germany). Ammonium carbamate (99%), 3-methyl-1-p-tolyltriazene (MTT), human normal serum, bovine submaxillary mucin (BSM), human milk osteopontin (OPN), O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine hydrochloride (BOA(F)), and dithiothreitol (DTT) were from SigmaAldrich, Inc., (St. Louis, MO). Anti-MUC1 mouse monoclonal antibody (clone C595) was purchased from Abcam Inc. (Cambridge, MA). Rat kidney FFPE sections were kind gift from Drug Safety Evaluation, Developmental Research Laboratories, Shionogi & Co., Ltd., Japan. BlotGlyco H bead (BlotGlyco) was purchased from Sumitomo Bakelite, Co., Tokyo, Japan. O-Benzylhydroxylamine hydrochloride (BOA) was from Tokyo Chemical Industry Co., Ltd., Japan. Other reagents and solvents were obtained from Wako Pure Chemicals Co., Tokyo, Japan, unless otherwise stated. General Protocol for Glycoblotting-Based O-Glycomics of Purified Glycoproteins. To the solution of glycoprotein (1-400 µg of BSM or 1-30 µg of OPN in 20 µL of H2O) was added 30 mg of dry powder of ammonium carbamate in a 0.2 mL PCR tube. The mixture was incubated at 60 °C for 20 h followed by addition of 500 µL water and evaporated at 60 °C. The residual materials were reconstituted in 500 µL of 150 mM aqueous acetic acid and evaporated at 60 °C. To the residue was added 20 µL of H2O and an internal standard (500 pmole of chitotetraose, GlcNAcβ1,4GlcNAcβ1,4GlcNAcβ1,4GlcNAc). An aliquot of the solution was subjected to the glycoblotting and subsequent methylation of sialic acids, and tagging by transiminization with BOA or BOA(F) in a similar protocol reported previously.11,12 In brief, BlotGlyco H beads (500 µL) (10 mg/mL suspension, Sumitomo Bakelite Co., Tokyo, Japan) were aliquoted onto a well of a MultiScreen Solvinert filter plate (Millipore, Billerica, MA). The sample solution was applied to a well followed by the addition of 180 µL of 2% acetic acid in acetonitrile (MeCN). The plate was incubated at 80 °C for 45 min to capture total glycans in sample mixtures specifically onto beads via stable hydrazone bonds. The plate was washed by 200 µL of 2 M guanidine-HCl in ammonium bicarbonate followed by washing with the same volume of water and 1% triethyl amine in methanol (MeOH). Each washing step was performed twice respectively. Unreacted hydrazide functional groups on beads were capped by incubation with 10% acetic anhydride in MeOH for 30 min at room temperature. Then the solution was removed by vacuum and then the bead was serially washed by 2 × 200 µL of 10 mM HCl, MeOH and dioxane, respectively. On-bead methyl esterification of carboxyl groups in sialic acids was carried out by incubation with 150 mM 3-methyl-1-p-tolyltriazene (MTT) in dioxane at 60 °C to dryness. It usually took 90 min in a conventional oven. Then the bead was serially washed by 200 µL of dioxane, water, MeOH, and water. The O-glycans on bead were subjected to the transiminization by treating with a mixture of 180 µL of 2% acetic acid in acetonitrile and 20 µL of 50 mM BOA or BOA(F) for 45-60 min at 80 °C. BOA/BOA(F)-tagged O-glycans were eluted by adding 100 µL of water, and an aliquot of the recovered O-glycans was directly mixed with a matrix solution (DHB:DHB(Na) ) 9:1,

10 mg/mL in 30% MeCN) and dried under vacuum to afford crystals. Samples were subjected to MALDI-TOFMS analysis on an Ultraflex III TOF/TOF mass spectrometer (Bruker Daltonics) equipped with a reflector, and controlled by the Flexcontrol 3.0 software package according to the general protocols reported in the previous paper.26 The peaks were detected generally as a formula of [M + Na]+ ions. In MALDI-TOFMS reflector mode, ions generated by a Smartbeam (pulsed UV solid laser, λ ) 355 nm, 50 Hz) were accelerated to a kinetic energy of 23.5 kV. Metastable ions generated by laser-induced decomposition of the selected precursor ions were analyzed without any additional collision gas. In MALDI-TOF/TOF mode, precursor ions were accelerated to 8 kV and selected in a timed ion gate. The fragments were further accelerated by 19 kV in the LIFT cell (LIFT means “lifting” the potential energy for the second acceleration of ion source), and their masses were analyzed after the ion reflector passage. Masses were automatically annotated by using FlexAnalysis 3.0 software package. External calibration of MALDI mass spectra was carried out using singly charged monoisotopic peaks of a mixture of human angiotensin II (m/z 1046.542), bombesin (m/z 1619.823), ACTH (18-39) (m/z 2465.199), and somatostatin 28 (m/z 3147.472). Structural identification of glycans was performed by mass spectrometric analysis as well as the use of database for glycan structures (http://glycosuitedb.expasy.org/glycosuite/glycodb). Purification and Glycoblotting-Based O-Glycomics of Urinary MUC1. The study using human urinary samples was approved by the local ethics committee of Hokkaido University and informed consent was obtained from the donors. MUC1 was isolated from urine of a healthy male individual using an antiMUC1 mouse monoclonal antibody-conjugated affinity bead, according to the previous method for the isolation of serum MUC127 with minor changes as follows: Urine (50 mL) collected by using 50 mL-Falcon tube from a healthy individual at first morning was stored overnight at 4 °C to cryoprecipitate in the presence of 0.05% NaN3, and the supernatant was concentrated by a centrifugal UF unit at 4000 g (30 kDa, Millipore). The concentrate was partially purified by gel filtration column on Superdex 200 (GE Healthcare Bio-Sciences), using 50 mM TrisHCl (pH 8.0) containing 200 mM NaCl. The MUC1-containing fractions that could be registered by Western blot were collected and concentrated at 4 °C to 500 µL by a centrifugal UF unit at 4000g (30 kDa, Millipore). For immunoaffinity purification, anti-MUC1 antibody C595 (200 µg) (Abcam, Cambridge, UK) was bound to protein G Sepharose bead (125 µL, GE Healthcare), and then cross-linked to the bead via a coupling reagent, dimethyl pimelimidate (DMP). The partially purified sample (250 µL) was mixed with C595 affinity beads overnight at 4 °C. After washing with 20 volumes of 50 mM Tris-HCl, pH 7.4, containing 150 mM NaCl and 0.05% Tween 20, MUC1 glycoprotein was eluted with 10 volumes of 0.1 M Gly-HCl, pH 3.0. The purity of isolated MUC1 was analyzed by LC/ESI-MS analysis of the clostripain digested sample, and confirmed the spectrum dominated by MUC1-derived glycopeptides. Western blot was performed with the anti-MUC1 antibody and antimouse alkaline phosphatase (AP) conjugates (26) Kurogochi, M.; Nishimura, S. -I. Anal. Chem. 2004, 76, 6097–6101. (27) Storr, S. J.; Royle, L.; Chapman, C. J.; Hamid, U. M. A.; Robertson, J. F.; Murray, A.; Dwek, R. A.; Rudd, P. M. Glycobiology 2008, 18, 456–462.

(Promega Corp, Madison, WI) using NBP/BCIP (Pierce Biotechnology, Rockford, IL) detection system. The obtained MUC1 fractions were concentrated to 150 µL by a centrifugal unit at 4 °C and 10 000g (30 kDa, Millipore) and the residue was once dried up by SpeedVac at 50 °C. Purification of MUC1 from urine samples was carried out at 4 °C and total procedures needed approximately 5 days. The purified MUC1 was dissolved in water (20 µL) and the solution was subjected to O-glycan release by means of ammonium carbamate and analyzed by modified condition of glycoblotting technique using a small scale of BlotGlyco bead (0.5 mg) and imine-exchange reagent (5 mM BOA(F)) in order to achieve high-sensitive MALDI-TOFMS analysis. Protocol for O-Glycomics of Human Serum, Cancer Cells, FFPE Sections Glycoproteins. Protocols for O-glycomics of glycoproteins from these biological samples were also established by suitable modification and detail conditions were described as follows. (a) Serum Glycoproteins. Commercially available human serum (20 µL) was treated beforehand with proteinase K (Hoffman La Roche Chemicals, Penzberg, Germany) to prevent filter clogging and directly employed for the treatment with 30 mg of ammonium carbamate to release whole O-glycans. The reaction mixture was subjected to the same procedure as described above in the glycoblotting analysis of purified glycoproteins. (b) Cellular Glycoproteins. In general, cells confluently grown in a 10 cm-φ dish were washed with ice-cold PBS and scraped in PBS containing 10 mM EDTA followed by washing with PBS. Cell pellets were dissolved with 100 µL of 20 mM Tris-HCl (pH 9) containing 2% SDS and denatured for 20 min at 100 °C. To lyse glycoproteins completely the mixtures were further incubated for 2 h at 60 °C and then centrifuged at 20 400g for 15 min at 4 °C. The obtained supernatants were aliquoted and a part of them was applied to protein quantification. The other part of them (∼50 µL of lysate) was applied to the subsequent reaction with 50 mg of dry powder of ammonium carbamate. The mixture was incubated for 20 h at 60 °C followed by addition of 300 µL of H2O to be applied to SpeedVac at 60 °C. The residual materials were dissolved with aqueous acetic acid and adjusted at pH 4-5. Glycoblotting was performed as described above, except the amount of BlotGlyco H beads (1.25 mg) and diluted BOA reagent (10 µL of 20 mM BOA(F)) to suppress ghost peaks. In case of human breast cancer cells (MCF-7), 400 µg of crude protein fraction obtained from a 10 cm-φ dish (1-3 × 106 cells) was used for further glycoblotting analysis, but the minimal amount of protein fraction required for the glycoblotting-based O-glycomics seems to be dependent on the cell size and contents of whole mucin glycoproteins. On the other hand, at least 200 µg of whole proteins, significantly, cells confluently grown in a 6 cm-φ or 10 cm-φ dish, depending on each cell size, eg, 1 × 106 cells for mouse ES cells12 while 1 × 107 for mouse T cells (data not shown), are required for the general protocol of N-glycomics of cellular glycoproteins. (c) Glycoproteins Extracted From FFPE Specimens. Four slices of 10 µm-thickness rat kidney FFPE sample were placed in a 1.5 mL tube, then deparafinized with heptane and methyl alcohol followed by air-dry. The proteinous pellet was incubated with 100 µL of 2% SDS in 20 mM Tris-HCl, pH 9, at 100 °C for 20 min and Analytical Chemistry, Vol. 82, No. 24, December 15, 2010

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Scheme 1. β-Elimination in Common O-Glycoside Linkages With Ser or Thr Residues in Alkaline Conditions and Plausible Mechanism of Subsequent Peeling Reaction

the extraction was continued by shaking at 60 °C for 2 h. The mixture was centrifuged at 20 000g for 30 min at 4 °C. The supernatant was transferred to a new 1.5 mL tube, and was added 50 mg of dry powder of ammonium carbamate. The mixture was subjected to the general protocol for glycoblotting and MS-based analysis as described above. RESULTS AND DISCUSSION Ammonium Carbamate As an Efficient Reagent to Release O-Glycans. Since the glycoblotting-based enrichment glycomics has been developed basically for the purpose of high throughput robotic protocol,10-12 conventional protocols for chemical O-glycan release are not suited for this system because the reagents are hazardous or highly toxic and induce unfavorable peeling reactions as illustrated in Scheme 1. We considered that one of the stable ammonium salts, ammonium carbamate, should be a promising alternate reagent because this reagent does not generate unfavorable hydroxide ion. Although it seems likely that ammonium carbonate may also become another potential ammonium salt, possible generation of hydroxide ion under the same condition must influence significantly yields and profiles of intact O-glycans. First, the efficiency of ammonium carbamate was evaluated preliminarily in comparison with a common conventional procedure (saturated ammonium carbonate/aqueous ammonia) by glycoblotting-based quantitative analysis of four major O-glycans released from BSM (Figure 1A and B). As anticipated, merit of an ideal mechanism in the β-elimination induced by ammonia derived from ammonium carbamate was evident because this reagent did not exhibit significant loss of GlcNAcβ1,3(Neu5AcR2,6)GalNAc or GlcNAcβ1,3 (Neu5GcR2,6)GalNAc and the profile of major O-glycans was quite similar to that estimated by highly sensitive capillary electrophoresis of the reducing O-glycans released from BSM in the presence of alkali borohydride.20 When the mixture obtained by treating BSM with ammonium carbonate/28% aqueous ammonia was subjected to the same glycoblotting procedure for subsequent O-glycomics, MALDI-TOF mass spectra showed the significant increase of disaccharide components, Neu5AcR2,6GalNAc and Neu5GcR2,6GalNAc, suggesting the loss of various complicated glycans branching at C-3 position of GalNAc residue due to the peeling reaction. It was also suggested that the present protocol greatly facilitated quantitative O-glycomics of BSM involving various minor O-glycans by a simple BOA tagging as shown in 10024

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Figure 1C. Totally, 14 O-glycans structures were estimated by MS/ MS analysis and/or database searching of known O-glycans structures and listed in Table S-1 (see also Supporting Information (SI) Figure S-1). Therefore, we concluded that the characteristics of ammonium carbamate appear to be beneficial to depress the peeling reaction and stabilize desired hemiacetal forms to be selectively enriched by general glycoblotting protocol including subsequent esterification of carboxyl groups of sialic acids and tagging the captured O-glycans by trans-iminization.10-12 Versatility of this simple procedure for direct O-glycan release using ammonium carbamate and subsequent glycoblotting was demonstrated by characterization of total O-glycan profile of human milk osteopontin (Figure 2). Osteopontin (OPN) is highly phosphorylated human glycoprotein having integrin-binding sequence (Arg-Gly-Asp, RGD) and widely distributed in many tissues and body fluids. It seems likely that posttranslational modification, such phosphorylation, sulfation, and glycosylation might be crucial as regulatory switches in multiple biological functions of OPN such as bone mineralization, cancer metastasis, cell-mediated immune response, inflammation, and cell survival.28 OPN has been known to have five potential O-glycosylation sites near the integrinbinding region. To the best of our knowledge, there is no report for precise structural characterization of O-glycans attached to this protein.29,30 Our present glycoblotting-assisted protocol revealed that human milk OPN bears highly complicated O-glycans, at least 44 kinds of glycoforms with habitual fucosylation, sialylation, and N-acetyllactosamine units as summarized in Table 1, although precise structural characterization by MS/MS analysis has not been performed yet. It has also been documented that human milk OPN has no N-glycosylation site while other OPN commonly involve two potential sites for N-glycosylation,29 indicating that all glycoforms detected herein were due to mucin type O-glycans released and enriched from the targeted sequence. When a glycoprotein of interest is limited in the amount in biological fluids, immunoprecipitation is a choice of method for enrichment the target. We selected MUC1 as a potential glycoprotein target because it has been well documented that O-glycans (28) Bellahcene, A.; Castronovo, V.; Ogbureke, K. U. E.; Fisher, L. W.; Fedarko, N. S. Nat. Rev. 2008, 8, 212–226. (29) Christensen, B.; Nielsen, M. S.; Haselmann, K. F.; Petersen, T. E.; Sørensen, E. S. Biochem. J. 2005, 390, 285–292. (30) Christensen, B.; Petersen, T. E.; Sørensen, E. S. Biochem. J. 2008, 411, 53–61.

Figure 1. Generation of reducing O-glycans from BSM. (A) Major O-glycans released by ammonium carbamate (top), and saturated ammonium carbonate in 28% aqueous ammonia (bottom). First, O-Glycans enriched by glycoblotting were tagged with O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine hydrochloride (BOA(F)) to prevent overlapping of ion peaks and MALDI-TOFMS was carried out in the presence of chitotetraose (m/z 1048.61) spiked as an internal standard. (B) Relative mass intensity of four major glycoforms estimated from the internal standard spiked. (C) 14 O-glycans of BSM identified by an optimized O-glycoblotting protocol using a BOA tagging in the presence of N-acetyllactosamine as an alternative internal standard. The symbols for representation of glycan structure recommended by nomenclature committee of the consortium for functional glycomics (http://www.functionalglycomics.org/static/consortium/Nomenclature.shtml).

of MUC1 tandem repeats is altered drastically in cancer progression or metastasis.31-34 Since some MUC1 O-glycopeptides are proved to be disease-relevant epitopes, extensive attention has been paid to structural elucidation of O-glycans of MUC1 from cancer patients.5,35-38 The present protocol elicited that human (31) Hanisch, F.-G.; Mu ¨ ller, S. Glycobiology 2000, 10, 439–449. (32) Taylor-Papadimitriou, J.; Burchell, J.; Miles, D. W.; Dalziel, M. Biochim. Biophys. Acta 1999, 1455, 301–313. (33) Hollingsworth, M. A.; Swanson, B. J. Nat. Rev. Cancer 2004, 4, 45–60. (34) Andrianifahanana, M.; Moniaux, N.; Batra, S. K. Biochem. Biophys. Acta 2006, 1765, 189–222. (35) Springer, G. F. Science 1984, 224, 1198–1206. (36) Karsten, U.; Diotel, C.; Klich, G.; Paulsen, H.; Goletz, S.; Mu ¨ ller, S.; Hanisch, F.-G. Cancer Res. 1998, 58, 2541–2549. (37) Karsten, U.; Serttas, N.; Paulsen, H.; Danielczyk, A.; Goletz, S. Glycobiology 2004, 14, 681–692.

urinary MUC1 samples purified by anti-MUC1 mAb from two normal human donors involve eightmajor O-glycans and they exhibited quite similar O-glycan profiles (Figure 3). It was suggested that normal human urinary MUC1 appears to express sialylated O-glycans (94% of total) with 7:3 ratio of core 1 to core 2 type structures, in which characteristic cross-ring1,5 A/Y1a-type cleavages39,40 were used for the differentiation between core 1 and core 2 with isomeric oligosaccharide structures (SI Figure S-2). Among sialylated O-glycans, our results showed the occurrence of two unusual glycoforms, Neu5AcR2,3Galβ1,3GlcNAcβ1, (38) Tarp, M. A.; Clausen, H. Biochim. Biophys. Acta 2008, 1780, 546–563. (39) Domon, B.; Costello, C. Glycoconjugate J. 1988, 5, 397–409. (40) Naruchi, K.; Hamamoto, T.; Kurogochi, M.; Hinou, H.; Shimizu, H.; Matsushita, T.; Fujitani, N.; Kondo, H.; Nishimura, S.-I. J. Org. Chem. 2006, 71, 9609–9621.

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Figure 2. MALDI-TOFMS of O-glycans enriched by a standard protocol using ammonium carbamate from human milk OPN. Numbers represent the peaks due to O-glycans and asterisk indicates internal standard. The compositions listed in Table 1 were estimated from measured masses of the glycans by the use of GlycoSuite database as described in the experimental section.

6GalNAc (core 6-type) and Galβ1,3 [Neu5AcR2,3(GalNAc?)Galβ1, 3GlcNAcβ1,6]GalNAc (core 2-type), in which the linkage between GalNAc and Gal in the core 2-type structure remains unclear.41 It was demonstrated that array displaying synthetic MUC1 glycopeptides involving cancer-related major O-glycans becomes a nice tool to determine highly disease-specific antigen/epitope.42,43 Therefore, these results should greatly encourage us to challenge further discovery study of diseasespecific new biomarkers from human urinary or serum MUC1 O-glycans by means of the present approach as well as other glycoprotein biomarker candidates that can be isolated using some antibodies. However, it seems likely that MALDI-TOFMSbased profiling could not cover smaller monosaccharides such as GalNAc (Tn antigen) and GlcNAc residues from glycoproteins even though they can be labeled by BOA and/or other labeling reagents, because molecular masses appear to be overlapped with signals due to matrix. Application to Direct Profiling of O-Glycans from Some Biological Materials. Next our interest was focused on the feasibility of the present protocol in various biological materials such as serum, culture cells, and formalin-fixed paraffin-embedded (FFPE) tissue samples. At present, we have no feasible data set of entire O-glycan expression level and the structural variant identified in a quantitative manner for human whole serum, urine, and any other biological samples even human cancer cell lines. Comparing with N-glycans,10-12 generally 60-80 major glycans widely distributing both in tissue/cellular and human serum glycoproteins, the expression level of O-glycans might be strongly dependent on that of membrane-associated several mucin glycoproteins and commonly very low. As anticipated, we could identify (41) Bhavanandan, V. P.; Zhu, Q.; Yamakami, K.; Dilulio, N. A.; Nair, S.; Capon, C.; Lemoine, J.; Fournet, B. Glycoconjugate J. 1998, 15, 37–49. (42) Ohyabu, N.; Hinou, H.; Matsushita, T.; Izumi, R.; Shimizu, H.; Kawamoto, K.; Numata, Y.; Togame, H.; Takemoto, H.; Kondo, H.; Nishimura, S.-I. J. Am. Chem. Soc. 2009, 131, 17102–17109. (43) Wandall, H. H.; Blixt, O.; Tarp, M. A.; Pedersen, J. W.; Bennett, E. P.; Mandel, U.; Ragupathi, G.; Livingston, P. O.; Hollingsworth, M. A.; Papadimitriou, J. T.; Burchell, J.; Clausen, H. Cancer Res. 2010, 70, 1306– 1313.

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only two major O-glycans from human whole serum, notably Neu5AcR2,3Galβ1,3GalNAc (sialyl-T) and Neu5AcR2,3Galβ1,3 (Neu5AcR2,6)GalNAc (disialyl-T) when 20 µL of serum was employed directly for the treatment with ammonium carbamate (30 mg) and followed by glycoblotting-based analysis (Figure 4(A)). It should be noted that considerable N-glycans were also released concurrently by treatment with ammonium carbamate from whole serum glycoproteins and entrapped by glycoblotting as indicated at a range from m/z 1200-3500, though they can be identified and differentiated from O-glycans by use of the protocol/ database for N-glycan profiling independently12 (SI Table S-2). Next, cell lysate of MCF-7 cells was subjected directly to the cellular O-glycomics protocol described in the experimental procedure. The results indicated that MCF-7 cells appear to express various core 2 type O-glycans in addition to the simple sialylated core 1 type O-glycans detected in human serum (Figure 4(B)). Eight O-glycoforms were estimated as listed in SI Table S-3, whereas six O-glycans could be identified by MS/MS analysis, in which three O-glycans have sialyl Lewis type motif. It was also revealed that N-glycans captured from MCF-7 cellular whole glycoproteins mostly differ from those of human serum (SI Table S-2). It is likely that MCF-7 cells express predominantly high mannose-type N-glycans such as M5 (m/z 1452.981), M6 (m/z 1615.08), M7 (m/z 1777.184), M8 (m/z 11939.264), and M9 (m/z 2101.351) rather than complex-type N-glycans widely distributed in popular serum glycoproteins. This N-glycan profile is quite similar to the results obtained in cases for some culture cell lines such as mouse ES cells and P19 cells previously reported.12 Glycomics of FFPE tissue samples was also tested by employing essentially the same protocol except the use of solubilization kit for the FFPE slices to remove glycogen (Figure 4(C)). It was shown that rat kidney FFPE sample can be subjected to the general protocol for O-glycomics and 7 O-glycans were detected in this tissue section in addition to 17 identified N-glycans (SI Table S-4). It is noteworthy that ammonium carbamate treatment and glycoblotting of common biological samples such as serum, culture cells, and FFPE tissue section allows for rapid profiling

Table 1. Signals and Estimated Composition of BOA-labeled O-Glycans Released from Human Milk Osteopontin as Shown in Figure 2. Hex; Hexose, HexNAc; N-Acetylhexosamine, Neu5Ac; N-Acetylneuraminic Acid, dHex; Deoxyhexose peak no.

deduced composition

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44

(Hex)1(HexNAc)1(Neu5Ac)1 (Hex)2(HexNAc)1(dHex)1 (Hex)2(HexNAc)1(dHex)2 (Hex)2(HexNAc)2(dHex)1 (Hex)2(HexNAc)2(dHex)1 (Hex)3(HexNAc)2 (Hex)1(HexNAc)1(Neu5Ac)2 (Hex)2(HexNAc)2(dHex)2 (Hex)2(HexNAc)2(Neu5Ac)1 (Hex)3(HexNAc)2(dHex)1 (Hex)2(HexNAc)2(dHex)1(Neu5Ac)1 (Hex)3(HexNAc)2(dHex)2 (Hex)1(HexNAc)4(dHex)2 (Hex)3(HexNAc)3(dHex)2 (Hex)3(HexNAc)3(dHex)1(Neu5Ac)1 (Hex)4(HexNAc)3(dHex)2 (Hex)4(HexNAc)4(dHex)1 (Hex)2(HexNAc)5(dHex)2 (Hex)3(HexNAc)3(dHex)2(Neu5Ac)1 (Hex)4(HexNAc)4(dHex)2 (Hex)3(HexNAc)3(dHex)3(Neu5Ac)1 (Hex)3(HexNAc)3(dHex)1(Neu5Ac)2 (Hex)4(HexNAc)4(dHex)3 (Hex)4(HexNAc)4(dHex)1(Neu5Ac)1 (Hex)4(HexNAc)3(dHex)3(Neu5Ac)1 (Hex)4(HexNAc)4(dHex)2(Neu5Ac)1 (Hex)5(HexNAc)5(dHex) (Hex)3(HexNAc)6(dHex)3 (Hex)4(HexNAc)4(dHex)3(Neu5Ac)1 (Hex)5(HexNAc)5(dHex)3 (Hex)4(HexNAc)4(dHex)2(Neu5Ac)2 (Hex)5(HexNAc)5(dHex)4 (Hex)5(HexNAc)5(dHex)3(Neu5Ac)1 (Hex)6(HexNAc)6(dHex)2 (Hex)5(HexNAc)5(dHex)3(Neu5Ac)1 (Hex)6(HexNAc)6(dHex)3 (Hex)5(HexNAc)5(dHex)4(Neu5Ac)1 (Hex)5(HexNAc)5(dHex)2(Neu5Ac)2 (Hex)6(HexNAc)6(dHex)4 (Hex)6(HexNAc)6(dHex)2(Neu5Ac)1 (Hex)1(HexNAc)8(dHex)1(Neu5Ac)3 (Hex)6(HexNAc)6(dHex)3(Neu5Ac)1 (Hex)7(HexNAc)7(dHex)3 (Hex)6(HexNAc)6(dHex)4(Neu5Ac)1

of major O-glycans derived from whole glycoproteins. Although global O-glycan profiling of serum and cells/tissues may indicate significant changes of a few major O-glycans expression level during cell differentiation, proliferation, or malignant alteration,31,32 generally known lower abundance of total O-glycans than Nglycans makes precise structural characterization and quantification difficult. CONCLUSION We established herein a standard protocol of glycoblottingbased O-glycomics in combination with nonenzymatic chemical treatment to release intact O-glycans predominantly from various glycoprotein samples. The nonreductive condition employing a simple ammonium salt, ammonium carbamate, made glycoblotting-based enrichment analysis of O-glycans possible without significant loss or unfavorable peeling reactions. A general workflow of glycoblotting using a hydrazide bead, on-bead

chemical manipulations, and subsequent mass spectrometry allowed for high throughput and quantitative O-glycomics of human milk OPN and urinary MUC1 glycoproteins. Versatility of the present strategy is evident because this method was proved to be suited for the enrichment analysis of a wide range of biological materials such as whole serum, cultured cancer cells, and rat kidney FFPE tissue sections. The method described herein to liberate O-glycans safely by simple chemical treatments in combination with glycoblotting technique and on-bead chemical modifications would greatly contribute to the broad range of study concerning biological relevance of O-glycans, especially in clinical and diagnostic benefits of glycoconjugates containing O-linked type oligosaccharides. The advantage of the present strategy by combined use of ammonium carbamate and a streamlined chemical manipulation on the glycoblotting platform9-12 is to eliminate both of the possible peeling reactions during O-glycan release by nonreductive β-elimination and the significant deletion of acidlabile sialic acid residues. However, it should be noted that further glycoproteomics study including O-glycomics at individual Oglycosylation site needs other optional approaches such as “reverse glycoblotting technique”, namely a protocol for the enrichment analysis of glycopeptides carrying sialic acid residue,44,45 and new fragmentation method by means of electron-capture dissociation(ECD)46-49 andelectrontransferdissociation(ETD)50-52 device/platforms giving highly informative c and z · ions without any significant degradation in the glycan moiety. In addition, it is clear that the progress of synthetic potentials of complex Oglycopeptides will greatly contribute to much more precise structural elucidation and functional analysis of mucin glycoproteins than the investigation using only isolated biological materials,53,54 because even though the glycoproteins of interest could be purified, they usually carry heterogeneous O-glycans at multiple O-glycosylation sites as demonstrated in the O-glycomics of human milk OPN and human urinary MUC1 (Figure 2 and Figure 3). Actually, we have revealed the essential epitope structure of KL-6/MUC1 glycoprotein, a diagnostic biomarker of interstitial pneumonia, by using an array displaying synthetic MUC1 glycopeptides library.39 (44) Kurogochi, M.; Amano, M.; Fumoto, M.; Takimoto, A.; Kondo, H.; Nishimura, S. -I. Angew. Chem. Int. Ed 2007, 46, 8808–8813. (45) Kurogochi, M.; Matsushita, T.; Amano, M.; Furukawa, J. -i.; Shinohara, Y.; Aoshima, M.; Nishimura, S. -I. Mol. Cell. Proteomics 2010, 9, . in press. (46) Zubarev, R. A.; Kelleher, N. L.; McLafferty, F. W. J. Am. Chem. Soc. 1998, 120, 3265–3266. (47) Satake, H.; Hasegawa, H.; Hirabayashi, A.; Hashimoto, Y.; Baba, T.; Masuda, K. Anal. Chem. 2007, 79, 8755–8761. (48) Matsushita, T.; Sadamoto, R.; Ohyabu, N.; Nakata, H.; Fumoto, M.; Fujitani, N.; Takegawa, Y.; Sakamoto, T.; Kurogochi, M.; Hinou, H.; Shimizu, H.; Ito, T.; Naruchi, K.; Togame, H.; Takemoto, H.; Kondo, H.; Nishimura, S.I. Biochemistry 2009, 48, 11117–11133. (49) Yoshimura, Y.; Matsushita, T.; Fujitani, N.; Takegawa, Y.; Fujihira, H.; Naruchi, K.; Gao, X. -G.; Manri, N.; Sakamoto, T.; Kato, K.; Hinou, H.; Nishimura, S. -I. Biochemistry 2010, 49, 5229–5941. (50) Syka, J. E. P.; Coon, J. J.; Schroender, M. J.; Shabanowitz, J.; Hunt, D. F. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 9528–9533. (51) Coon, J. J.; Ueberheide, B.; Syka, J.; Dryhust, D. D.; Ausio, J.; Shabanowitz, J.; Hunt, D. F. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 9463–9468. (52) Darula, Z.; Medzihradszky, K. F. Mol. Cell. Proteomics 2009, 8, 2515–2526. (53) Gamblin, D. P.; Scanlan, E. M.; Davis, B. G. Chem. Rev. 2009, 109, 131– 163. (54) Fumoto, M.; Hinou, H.; Ohta, T.; Ito, T.; Yamada, K.; Takimoto, A.; Kondo, H.; Shimizu, H.; Inazu, T.; Nakahara, Y.; Nishimura, S.-I. J. Am. Chem. Soc. 2005, 127, 11804–11818.

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Figure 3. MALDI-TOFMS of O-glycans enriched by a standard protocol using ammonium carbamate from human urinary MUC1 glycoprotein purified by immunoprecipitation. Asterisk indicates internal standard. See also, SI Figure S-2 for MS/MS spectra of four major O-glycans.

Figure 4. O-Glycomics of some biological samples. (A) Two abundant O-glycans, sialyl-T and disialyl-T antigens, enriched from whole human serum. (B) Four major O-glycans enriched from human breast cancer cells (MCF-7 cells). (C) Six major O-glycans enriched from FFPE tissue section of rat kidney. Asterisk means an internal standard and the red stars represent the signals due to O-glycans. “N” indicates the signals due to N-glycans. For further information on the deduced composition of minor O-glycans as well as major N-glycans, see SI Tables S-2-S-4. 10028

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ACKNOWLEDGMENT We thank Ms. K. Okada and Ms. H. Okamoto, Hokkaido University, for their technical assistances in O-glycan preparations. We also gratefully acknowledge Dr. T. Murai and Ms. T. Miyoshi, Drug Safety Evaluation, Developmental Research Laboratories, Shionogi & Co., Ltd., for providing FFPE specimens. This work was supported partly by a grant for “Development of Systems and Technology for Advanced Measurement and Analysis (SENTAN)” and “The Matching Program for Innovations in Future Drug Discovery and Medical Care” from the Japan Science and Technology Agency (JST) and the Ministry of Education, Culture, Science, and Technology, Japan. ABBREVIATIONS BSM: bovine submaxillary mucin, OPN: osteopontin, MUC1: human polymorphic epithelial mucin, PNGase F: protein Nglycanase F, MTT: 3-methyl-1-p-tolyltriazene, BOA: O-benzylhydroxylamine hydrochloride, BOA(F): O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine hydrochloride, DTT: dithiotheitol, PCR: polymerase chain reaction, MeCN: acetonitrile, I. S.: internal

standard, WR: tryptophanylarginine, FFPE: formalin-fixed paraffinembedded, GSLs: glycosphingolipids, HPLC: high performance liquid chromatography, MALDI-TOF: matrix-assisted laser desorption/ionization time-of-flight, MS: mass spectrometry, ECD: electron-captured dissociation, ETD: electron-transfer dissociation. NOTE ADDED AFTER ASAP PUBLICATION This paper was published on November 15, 2010 with errors in the laser used in for the MALDI-TOFMS analysis and the version of the FlexAnalysis software package. The corrected version was reposted on November 22, 2010. SUPPORTING INFORMATION AVAILABLE Tables S1-S4 and Figures S1-S3. This material is available free of charge via the Internet at http://pubs.acs.org.

Received for review June 26, 2010. Accepted October 29, 2010. AC101599P

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