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Alcian blue staining can visualize BSM even at 78 ng. ... A faintly stained Alcian blue region in the area of spots 1 and 2 was present after the chon...
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Anal. Chem. 2009, 81, 3816–3823

Supported Molecular Matrix Electrophoresis: A New Tool for Characterization of Glycoproteins Yu-ki Matsuno,† Takuro Saito,‡ Mitsukazu Gotoh,‡ Hisashi Narimatsu,† and Akihiko Kameyama*,† Research Center for Medical Glycoscience, National Institute of Advanced Industrial Science and Technology (AIST), Open Space Laboratory C-2, 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, Japan, and Department of Surgery I, Fukushima Medical University, Fukushima 960-1295, Japan A new concept of separation technology, supported molecular matrix electrophoresis (SMME), is described. In SMME, analytes migrate in a molecular matrix supported by backbone materials. Here we introduce a novel strategy for the separation and characterization of mucins using SMME. Mucin, a highly tumor-associated glycoprotein, has great potential as clinical biomarker for diagnosis of various malignant tumors. However, due to their large size, polymeric nature, and heterogeneous glycosylation, analysis of mucins has been left behind by modern techniques. For mucin analysis, we employed a poly(vinylidene difluoride) (PVDF) membrane and poly(vinyl alcohol) (PVA) as the backbone material and the matrix molecule, respectively. Combining SMME with mass spectrometry and capillary electrophoresis, we demonstrate that a crude porcine stomach mucin consists of a neutral and a sulfated mucin and is contaminated by chondroitin sulfate-containing proteoglycan and hyaluronic acid. Furthermore, to demonstrate the feasibility of the strategy for biomarker discovery, we analyzed mucins in human pancreatic juice, which is an important source for clinical biomarkers of pancreatic tumors. This work revealed the presence of three types of mucin with distinct glycan profiles in human pancreatic juice. Glycosylation is the most common posttranslational modification of proteins. The attached glycans provide a number of critical functions to proteins and play a key role in various biological aspects.1,2 Furthermore, glycans are considered to have great potential as therapeutic targets or clinical biomarkers for diagnosis of various malignant diseases. However, characterization of glycoproteins is still not easy, and there is no universal method for easy and rapid analysis of glycans and glycoproteins. In particular, mucin-type glycoproteins (mucins) have been left behind by modern techniques in terms of isolation and characterization due to their large size, polymeric nature, and heterogeneous glycosylation. Mucins are the major constituents of epithelial mucus and are typically characterized by large molecular mass (∼2 MDa) and * To whom correspondence should be addressed. Phone: +81-29-861-3123. Fax: +81-29-861-3123. E-mail: [email protected]. † National Institute of Advanced Industrial Science and Technology. ‡ Fukushima Medical University. (1) Varki, A. Nature (London) 2007, 446, 1023–1029. (2) Dove, A. Nat. Biotechnol. 2001, 19, 913–917.

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high carbohydrate content (50% to ∼90% by weight) reflecting heavy glycosylation with a large number of O-linked glycans.3 Mucins have long been implicated in health and disease.4-6 In tumor states, regulation of mucin expression is disrupted,7 and tumor-associated structural alterations of O-linked glycans in mucins have also been reported.8 For example, in breast cancer, O-linked glycans in MUC1, which is expressed by more than 90% of breast cancers, are often truncated and highly sialylated (i.e., sialyl-T or sialyl-Tn antigens), and expression of sialyl-Tn antigen correlates with poor prognosis.9-11 Alteration of sulfated glycans has also been reported to be associated with some disorders. Ectopic expression of GlcNAc 6-O-sulfotransferase-2 (GlcNAc6ST2) in colonic or ovarian mucinous adenocarcinomas implies that specific sulfated glycans in mucins may correlate with malignancy.12,13 Sulfated mucins are also associated with cystic fibrosis and intestinal metaplasia.14,15 In contrast, a significant loss of mucin sulfation in several cancer cell lines has also been reported.16 Immunohistochemistry and in situ hybridization studies of pancreatic tumors have revealed that the expression profiles of mucins are closely related to a poor outcome in the patients,17 and that the sialyl-Tn antigen was markedly expressed along with malignant transformation.18 Thus, mucins and their O-linked (3) Rose, M. C.; Voynow, J. A. Physiol. Rev. 2006, 86, 245–278. (4) Corfield, A. P.; Myerscough, N.; Longman, R.; Sylvester, P.; Arul, S.; Pignatelli, M. Gut 2000, 47, 589–594. (5) Voynow, J. A. Paediatr. Respir. Rev. 2002, 3, 98–103. (6) Hollingsworth, M. A.; Swanson, B. J. Nat. Rev. Cancer 2004, 4, 45–60. (7) Andrianifahanana, M.; Moniaux, N.; Batra, S. K. Biochim. Biophys. Acta 2006, 1765, 189–222. (8) Brockhausen, I. EMBO Rep. 2006, 7, 599–604. (9) Burchell, J. M.; Mungul, A.; Taylor-Papadimitriou, J. J. Mammary Gland Biol. Neoplasia 2001, 6, 355–364. (10) Sewell, R.; Ba¨ckstro ¨m, M.; Dalziel, M.; Gschmeissner, S.; Karlsson, H.; Noll, T.; Ga¨tgens, J.; Clausen, H.; Hansson, G. C.; Burchell, J.; Taylor-Papadimitriou, J. J. Biol. Chem. 2006, 281, 3586–3594. (11) Leivonen, M.; Nordling, S.; Lundin, J.; von Boguslawski, K.; Haglund, C. Oncology 2001, 61, 299–305. (12) Seko, A.; Nagata, K.; Yonezawa, S.; Yamashita, K. Glycobiology 2002, 12, 379–388. (13) Kanoh, A.; Seko, A.; Ideo, H.; Yoshida, M.; Nomoto, M.; Yonezawa, S.; Sakamoto, M.; Kannagi, R.; Yamashita, K. Glycoconjugate J. 2006, 23, 453– 460. (14) Xia, B.; Royall, J. A.; Damera, G.; Sachdev, G. P.; Cummings, R. D. Glycobiology 2005, 15, 747–775. (15) Bodger, K.; Campbell, F.; Rhodes, J. M. J. Clin. Pathol. 2003, 56, 703– 708. (16) Brockhausen, I. Biochem. Soc. Trans. 2003, 31, 318–325. (17) Yonezawa, S.; Goto, M.; Yamada, N.; Higashi, M.; Nomoto, M. Proteomics 2008, 8, 3329–3341. (18) Itzkowitz, S.; Kjeldsen, T.; Friera, A.; Hakomori, S.; Yang, U. S.; Kim, Y. S. Gastroenterology 1991, 100, 1691–1700. 10.1021/ac900157c CCC: $40.75  2009 American Chemical Society Published on Web 04/15/2009

glycans are considered to be a potential diagnostic marker for the early detection of various cancers and a means of specific discrimination between cancers and benign diseases. Combinations of lectin affinity and proteomics techniques such as two-dimensional liquid chromatography-tandem mass spectrometry (2D LC-MS/MS) or 2D polyacrylamide gel electrophoresis (2D-PAGE), which revealed the potential of glycoproteins as disease biomarkers,19,20 contribute little to characterization of mucins in spite of the biological significance of these molecules. Difficulties are encountered in their analysis because they are generally protease-resistant and do not migrate into the resolving gel of SDS (sodium dodecyl sulfate)-PAGE gels due to their large molecular mass. Agarose gel electrophoresis and agarose/ polyacrylamide composite gel electrophoresis (AgPAGE) have been used for the separation and characterization of mucins in various biological samples.21-23 In these cases, characterization of mucins requires their transfer to a membrane such as poly(vinylidene difluoride) (PVDF). However, the recovery of the mucins in the transfer procedure is problematic for quantitative evaluation in biomarker discovery. Furthermore, agarose/polyacrylamide composite gels must be prepared to an appropriate concentration by users depending on the target mucins. Consequently, these methods are not suitable for large-scale analysis such as biomarker discovery in terms of throughput, ease of use, and reproducibility. Here we describe a novel strategy for the separation and characterization of mucins using a new technique, named “supported molecular matrix electrophoresis (SMME)”. We also show that the strategy can be applied to analysis of proteoglycan-type glycoproteins. Furthermore, to demonstrate the feasibility of the strategy for biomarker discovery, we analyzed mucins in human pancreatic juice which is an important source for clinical biomarkers of pancreatic tumors.24 This method has great potential to facilitate a breakthrough in glycan biomarker discovery. EXPERIMENTAL SECTION Materials and Reagents. Bovine submaxillary mucin (BSM) was purchased from ICN Biomedicals Inc. Porcine stomach mucin (PSM, type III, partially purified), Alcian blue 8GX, and direct blue71 (DB-71) were purchased from Sigma-Aldrich. Pro-Q emerald 488 glycoprotein gel and blot stain kit and 2-aminoacridone (AMAC) were purchased from Molecular Probes. PVDF membrane (Immobilon-P, pore size 0.45 µm) was purchased from Millipore. Poly(vinyl alcohol) (PVA, MW 22 000) and 2,5-dihydroxybenzoic acid (DHB) were purchased from Wako pure chemical corporation. Chondroitinase ABC, hyaluronidase from (19) Abbott, K. L.; Aoki, K.; Lim, J. M.; Porterfield, M.; Johnson, R.; O’Regan, R. M.; Wells, L.; Tiemeyer, M.; Pierce, M. J. Proteome Res. 2008, 7, 1470– 1480. (20) Block, T. M.; Comunale, M. A.; Lowman, M.; Steel, L. F.; Romano, P. R.; Fimmel, C.; Tennant, B. C.; London, W. T.; Evans, A. A.; Blumberg, B. S.; Dwek, R. A.; Mattu, T. S.; Mehta, A. S. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 779–784. (21) Spurr-Michaud, S.; Argu ¨ eso, P.; Gipson, I. Exp. Eye Res. 2007, 84, 939– 950. (22) Schulz, B. L.; Packer, N. H.; Karlsson, N. G. Anal. Chem. 2002, 74, 6088– 6097. (23) Andersch-Bjo¨rkman, Y.; Thomsson, K. A.; Holme´n Larsson, J. M.; Ekerhovd, E.; Hansson, G. C. Mol. Cell. Proteomics 2007, 6, 708–716. (24) Liang, J. J.; Kimchi, E. T.; Staveley-O’Carroll, K. F.; Tan, D. Int. J. Clin. Exp. Pathol. 2009, 2, 1–10.

Streptomyces hyalurolyticus, and standard samples of unsaturated disaccharides were purchased from Seikagaku Kogyo. Normal human plasma was from laboratory stock. SMME of Mucins. Mucin samples were dissolved in distilled water, and the solutions were centrifuged at 10 000g for 5 min to remove undissolved materials. The supernatant was then lyophilized by centrifugal evaporator and used for the analysis. Prior to electrophoresis, mucins were reduced and alkylated. BSM (100 µg) and PSM (200 µg) were dissolved in 0.1 M Tris-HCl buffer (pH 8.6)/20 mM DTT/8 M urea (20 µL), and the mixtures were incubated at room temperature for 3 h. An aqueous solution of iodoacetamide (250 mM, 2 µL) was then added, and the mixture was incubated at room temperature for 1 h in the dark. A portion of the mixture (1 µL) was directly used for SMME analysis. Human plasma was desalted by centrifugal filter device (10 kDa cutoff) and lyophilized by centrifugal evaporator. The lyophilized material (100 µg) was used as plasma proteins and reduced and alkylated in a similar manner to that already described. A PVDF membrane (length, 6 cm; width, typically 6-10 cm) was immersed in methanol and transferred into a solution of 0.25% (w/v) PVA dissolved in running buffer (0.1 M pyridine-formic acid buffer (pH 4.0)). After incubation for 30 min with gentle shaking, the membrane was used for electrophoresis. The prepared samples were spotted at a position 1.5 cm from the bottom of the membrane with intervals (1 cm) between each spot. Electrophoresis was performed using apparatus for cellulose acetate membrane electrophoresis (EPC105AA-type, Advantec) and was performed in constant current mode at 1.0 mA/cm for 30 min. After the run, the membrane was incubated for 10 min in a solution of 0.1% (w/v) Alcian blue in 0.1% (v/v) acetic acid and then washed with methanol for a few minutes to remove the background color. For protein staining, the membrane was incubated for 10 min in a solution of 0.008% (w/v) DB-71 in 10% (v/v) acetic acid/40% (v/v) ethanol and then washed with methanol to remove the background color. For Pro-Q emerald staining, Immobilon-FL (Millipore) was used as the PVDF membrane and staining was performed according to the manufacturer’s instructions. Glycosidase Treatments of PSM Sample. PSM (200 µg) was dissolved in 50 mM Tris-HCl buffer (pH 8.0, 20 µL), and an aqueous solution of chondroitinase ABC (100 mU/2 µL) was added to the solution. Digestion was performed at 37 °C for 16 h, and then the reaction mixture was dried in a centrifugal evaporator. The dried material was then reduced and alkylated in a similar manner to that already described, and the mixture was analyzed by SMME. In hyaluronidase digestion, PSM (200 µg) was dissolved in 20 mM acetate buffer (pH 6.0, 20 µL), and an aqueous solution of hyaluronidase (100 mU/2 µL) was added to the solution. Digestion was performed at 60 °C for 16 h, and the reaction mixture was treated as described for the chondroitinase ABC digestion. Glycan Analysis of Mucins. O-Linked glycans were released from mucins by on-membrane reductive β-elimination reaction. Mucin spots were excised from the membrane and cut into smallsize pieces (ca. 2 mm square). The membrane pieces were rewetted with methanol (2-5 µL) in a microtube. Then an aqueous solution of 50 mM NaOH/0.5 M NaBH4 (20 µL) was added to the membrane pieces in the tube, and the mixture was Analytical Chemistry, Vol. 81, No. 10, May 15, 2009

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incubated at 45 °C for 16 h. After the reaction, the mixture was neutralized with glacial acetic acid and then desalted by passing through an Oasis MCX cartridge (1 cc, Waters). The flow through fraction and washes were collected (ca. 500 µL) and lyophilized by centrifugal evaporator. A solution of 1% (v/ v) acetic acid/methanol (100 µL) was added to the lyophilized material, and the mixture was evaporated to remove boric acid as its methyl ester, and the procedure was repeated several times. The dried material (O-linked glycan alditols) was then permethylated. Permethylated glycans were prepared according to the method described previously25 with some minor changes. Dimethyl sulfoxide (DMSO) containing 1% (v/v) distilled water (10 µL) was added to the dried glycan sample under alkaline conditions with powdered sodium hydroxide. Methyl iodide (10 µL) was then added to the mixture, and the reaction was performed at room temperature for 15 min with vigorous shaking. Distilled water (1 mL) was slowly added to the reaction mixture, and then the mixture was applied to an Oasis HLB solid-phase extraction cartridge (1 cc, Waters) to purify the permethylated glycans. After washing with water (1 mL) several times, the permethylated glycans were eluted with 50% (v/v) acetonitrile (500 µL) and evaporated to dryness by centrifugal evaporator. The dried material was dissolved in acetonitrile (5 µL), and a portion (0.5 µL) of the solution was used for matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis. Mass spectra of the permethylated glycans were acquired in reflectron positive ion mode with a Reflex IV MALDI-TOF instrument (Bruker-Daltonics). DHB was used as the matrix throughout the work. Disaccharide Composition Analysis of Glycosaminoglycans in Proteoglycan. Glycosaminoglycans (GAGs) in proteoglycan were released from core proteins by a similar method as used for mucins. The released glycans were dissolved in 50 mM Tris-HCl buffer (pH 8.0, 20 µL), and an aqueous solution of chondroitinase ABC (100 mU/2 µL) was added to the glycan solution. Digestion was performed at 37 °C for 16 h, and then the reaction mixture was desalted by Oasis MCX cartridge as described above. The released unsaturated disaccharides were then derivatized with AMAC according to the method described previously26,27 with some minor changes. The unsaturated disaccharides were dissolved in 100 mM AMAC in a mixture (10 µL) of DMSO-acetic acid (17:3 v/v) and 1 M sodium cyanoborohydride (10 µL) in the same solvent. After incubating the mixture at 45 °C for 3 h, water (100 µL) and chloroform (500 µL) were added to the reaction mixture and mixed vigorously by a vortex mixer. After removing the chloroform layer, the aqueous solution was washed again with chloroform (500 µL), and the procedure was repeated three times. A portion of the aqueous solution was used for analysis by laser-induced fluorescence capillary electrophoresis (LIF-CE). AMAC-labeled unsaturated disaccharides were analyzed using a Beckman P/ACE MDQ glycoprotein system equipped with an (25) Ciucanu, I.; Costello, C. E. J. Am. Chem. Soc. 2003, 125, 16213–16219. (26) Militsopoulou, M.; Lamari, F. N.; Hjerpe, A.; Karamanos, N. K. Electrophoresis 2002, 23, 1104–1109. (27) Zinellu, A.; Pisanu, S.; Zinellu, E.; Lepedda, A. J.; Cherchi, G. M.; Sotgia, S.; Carru, C.; Deiana, L.; Formato, M. Electrophoresis 2007, 28, 2439–2447.

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argon laser-induced fluorescence detector (ex 488 nm, em 520 nm). Electrophoresis was performed using a fused-silica capillary (50 µm i.d., 20 cm effective length). Phosphate buffer (50 mM, pH 3.5) was used as the running buffer. Sample solutions were introduced into the capillary by pressure injection for 10 s (1 psi). Separation was performed by applying a potential of 20 kV (reverse polarity) at 25 °C. Enrichment of Mucin-Like Glycoproteins from Human Pancreatic Juice. Human pancreatic juice (5 mL) was collected by pancreatic duct drainage after pancreatectomy for pancreatic cancer. The juice was collected into a polypropylene tube and stored at -80 °C. A portion of the juice (100 µL) was used for the analysis of mucins in this study. A 3-fold volume of ethanol (300 µL) was added to the juice, and the mixture was kept in an ice bath for 2 h to precipitate proteins as well as mucins. After centrifugation of the mixture at 12 000g for 10 min, the precipitate was collected and then washed with ethanol. The precipitated proteins (ca. 320 µg) were dissolved into 0.1 M Tris-HCl buffer containing 2 M urea (pH 8.6, 100 µL), and an aqueous solution of trypsin (1 mg/mL, 6.5 µL) was added to the mixture. Digestion was performed at 37 °C for 24 h, and then the reaction mixture was centrifuged at 12 000g for 10 min to remove undissolved materials. The supernatant was passed through a centrifugal filter device (100 kDa cutoff) to remove digested peptides. After washing with the same buffer (500 µL × 2), the solution was concentrated to 10 µL and was then reduced and alkylated in a similar manner to that already described. A portion of the solution (1 µL) was used for SMME analysis. RESULTS Strategy for Separation and Characterization of Glycoproteins Using SMME. SMME is a new method of electrophoresis in which analytes migrate in a molecular matrix which is supported by backbone materials (Figure 1a). The matrix molecules and the backbone materials can be arranged depending on the nature of the analyte and the purpose of the experiment. For separation and characterization of mucins, we used a PVDF membrane and PVA as the backbone material and the matrix molecule, respectively. The supported PVA matrix can be easily prepared by immersing the PVDF membrane in a solution of PVA dissolved in the electrophoresis running buffer. The polyvinyl residue in PVA may contribute to hydrophobic binding with the PVDF backbone. The hydroxyl groups in PVA, therefore, should be oriented such that they form a contact surface with analytes. Mucins migrate through the PVA matrix to an electrode depending on their mass-to-charge ratio with interaction with the PVA molecule. A general scheme for characterization of glycoproteins using SMME is shown in Figure 1b. After SMME, mucins and proteoglycans can be visualized by Alcian blue staining without degrading glycan moieties. For sensitive detection of glycoproteins, periodic acid-Schiff base staining can be also applied. The Alcian blue stained spots are excised for characterization of glycans. O-Linked glycans of the stained glycoproteins in the PVA matrix can be released from the excised membrane by reductive β-elimination. The released glycans are permethylated and then analyzed by mass spectrometry. In the case of proteoglycans, the released GAGs are treated with specific enzymes such as chondroitinase ABC to digest to unsaturated disaccharides.

Figure 1. Novel strategy for characterization of glycoproteins using supported molecular matrix electrophoresis (SMME): (a) conceptual overview of SMME; (b) schematic diagram for characterization of glycoproteins using SMME.

Figure 2. SMME analysis of BSM and plasma proteins: (a and b) electrophoresis using a PVDF membrane as a separating support; (c-e) SMME. Staining with Alcian blue (a and c) and DB-71 (b, d, e).

After labeling of the disaccharides with AMAC, disaccharide compositions of GAGs are determined by LIF-CE.26,27 Separation of Mucins by SMME. We show results of SMME analysis of BSM and plasma proteins (Figure 2). In the case of electrophoresis using a PVDF membrane as a separating support, neither BSM nor plasma proteins migrated from the origin (Figure 2, parts a and b). Thus, the PVDF membrane could not be used as a support for electrophoresis of proteins, perhaps due to its hydrophobic properties. In SMME, BSM successfully migrated and is seen as a clear single spot when stained with Alcian blue (Figure 2c). Visualization of proteins could be performed with Alcian blue or DB-71 by similar PVDF membrane staining methods. In contrast, plasma proteins did not

migrate from the origin (Figure 2e). Staining of BSM with DB-71 revealed that the BSM sample contained contaminating proteins which were observed at the origin after SMME (Figure 2d). We have confirmed by SDS-PAGE and Coomassie brilliant blue staining that several protein bands contaminate the BSM sample used in this study (data not shown). In contrast, Alcian blue staining of plasma proteins after SMME did not show any visible spots. Thus, SMME using PVA matrix could selectively separate BSM from non-mucin-like proteins. We applied SMME to the analysis of a crude mucin sample, namely, partially purified PSM which is commercially available (Figure 3). SMME separated the crude PSM into four distinct spots when stained with Alcian blue (Figure 3a). Although spot 4 was only faintly stained with Alcian blue, Pro-Q emerald staining, which selectively stains glycans, revealed that spot 4 is a major glycoprotein of crude PSM (Figure 3b). There were no visible spots upon staining with DB-71 (data not shown). By digestion of the crude PSM with chondroitinase ABC prior to SMME, two faster migrating spots (1 and 2) almost disappeared (Figure 3c). Furthermore, spot 2 completely disappeared after digestion of the PSM with hyaluronidase from S. hyalurolyticus (Figure 3d). These results indicate that the crude PSM sample contains two clearly different mucins: one a neutral mucin (spot 4, major mucin) and the other an acidic mucin (spot 3, minor mucin, Alcian bluepositive), as well as chondroitin sulfate-containing proteoglycan (spot 1) and hyaluronic acid (spot 2). Glycan Analysis of SMME-Separated Glycoproteins. We analyzed O-linked glycans from the mucins (spots 3 and 4 in Figure 3a) of PSM separated by SMME. The Alcian blue stained spots 3 and 4 were excised, and the membrane pieces were Analytical Chemistry, Vol. 81, No. 10, May 15, 2009

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Figure 3. SMME analysis of crude PSM. The crude PSM was electrophoresed by SMME and stained with Alcian blue (a) and Pro-Q emerald (b). After chondroitinase ABC digestion (c) and hyaluronidase digestion (d), the PSM sample was electrophoresed by SMME and stained with Alcian blue.

subjected to a reductive β-elimination reaction to release glycans. The released glycans were permethylated and analyzed by MALDI-TOF MS (Figure 4a). The two mucins gave markedly different mass spectra. Neutral glycans were predominantly observed in the major mucin (spot 4), whereas various sulfated glycans were found in the minor acidic mucin (spot 3). The observed signal intensities in the mass spectra are presented as a histogram (Figure 4b), with the estimated monosaccharide compositions (Supporting Information Table S1). The estimated compositions for both the neutral and the sulfated glycans in PSM are consistent with previously reported data.28,29 Some unreported compositions for PSM were also found in this experiment. Furthermore, we showed that the glycans in the minor mucin mainly consist of fucosylated glycans and that sulfation occurs only on the fucosylated glycans (Supporting Information Table S1). We also characterized the proteoglycan in the crude PSM sample. Stained spot 1 in Figure 3a was excised and treated in a similar manner to the glycan released from the mucins. The released GAGs from proteoglycan were digested with chondroitinase ABC to produce unsaturated disaccharides. The obtained disaccharides were labeled with AMAC for analysis by LIF-CE (Figure 5). In capillary electrophoresis, spot 1 gave two peaks which were identified as monosulfated disaccharides, ∆di-6S (1) and ∆di-4S (2) (Figure 5). Peak identification was performed by coinjection of authentic unsaturated disaccharide standards. The relative amounts of ∆di-6S and ∆di-4S estimated by peak areas were 35% and 65%, respectively. Thus, spot 1 contains condroitin sulfate consisting of ∆di-6S and ∆di-4S. In addition, mucin-type O-linked glycans were not detected in the proteoglycan spot by MALDITOF MS analysis (data not shown). Analysis of Mucins in Human Pancreatic Juice. Pancreatic juice is an excellent source for the identification of diagnostic (28) Yamada, K.; Hyodo, S.; Matsuno, Y. K.; Kinoshita, M.; Maruyama, S. Z.; Osaka, Y. S.; Casal, E.; Lee, Y. C.; Kakehi, K. Anal. Biochem. 2007, 371, 52–61. (29) Thomsson, K. A.; Karlsson, H.; Hansson, G. C. Anal. Chem. 2000, 72, 4543–4549.

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Figure 4. MALDI-TOF MS analysis of O-glycans in mucin-glycoforms separated by SMME. (a) Mass spectra of permethylated glycans derived from spots 3 and 4 in Figure 3a. Capital letters “N” and “S” indicate neutral glycans and sulfated glycans, respectively. (b) Histograms of relative intensities of the glycan signals observed. The intensities of neutral glycans and sulfated glycans are indicated by black bars and red bars, respectively (the histograms represents only signal intensities but not the quantities). The signal numbers correspond to those described in Supporting Information Table S1.

biomarkers of pancreatic disorders. Depending on types of diseases, mucins in pancreatic juice might change in quantity and/ or their glycosylation. Ohuchida et al. suggested that quantitative assessment of MUC1 and MUC5AC mRNA in pancreatic juice has high potential for preoperative diagnosis of pancreatic cancer.30 To demonstrate the feasibility of SMME for analysis of mucin from biological fluid, we applied the present method to characterization of mucin in human pancreatic juice. After pretreatment as described in the Experimental Section, pancreatic juice was subjected to SMME (Figure 6). Three major spots (spots 1-3) and one minor spot were visualized by Alcian blue staining. The minor spot could be assigned as hyaluronic acid based on the similarity to the migrating position of the hyaluronic acid in the PSM sample (Figure 6b). Pro-Q emerald staining visualized an additional spot (spot 4) which could also be stained by DB-71. We analyzed the O-linked glycans of each spot (spots 1-4) in a similar manner to that described for glycan analysis of PSM. As shown in Figure 7a, O-linked glycans were detected from spots 1, 2, and 3 by MALDITOF MS, but not from spot 4. This result implies the presence of (30) Ohuchida, K.; Mizumoto, K.; Yamada, D.; Fujii, K.; Ishikawa, N.; Konomi, H.; Nagai, E.; Yamaguchi, K.; Tsuneyoshi, M.; Tanaka, M. Int. J. Cancer 2006, 118, 405–411.

Figure 5. LIF-CE analysis of AMAC-labeled unsaturated disaccharides derived from proteoglycan in the crude PSM sample. Electropherograms of unsaturated disaccharides derived from spot 1 in Figure 3a (a), commercially available standard of ∆di-6S (b), and ∆di4S (c) are shown. The disaccharide structures of the peaks were identified as shown. AMAC: 2-aminoacridone.

Figure 6. SMME analysis of mucins in human pancreatic juice. PSM (a) and the mucin sample prepared from pancreatic juice (b) were electrophoresed by SMME and stained with Alcian blue. The electrophoresed pancreatic juice on SMME membrane was also stained with Pro-Q emerald (c) and DB-71 (d).

glycoprotein with another type of glycan (N-linked glycan) or another type of glycoconjugate such as a glycolipid in spot 4. The observed signal intensities in the mass spectra are presented as a histogram (Figure 7b), with the estimated monosaccharide compositions summarized in Table 1. These data suggest that spot 1 contains sialyl-T and disialyl-T antigens as major glycan components. In contrast, spot 2 has a variety of O-linked glycans including fucosylated neutral glycans and sialylated glycans. Monosaccharide composition of sialylated glycan 4 in spot 2 corresponds with the sialyl-Lea epitope (CA19-9). O-Linked glycans from spot 3 consist only of neutral glycans which may correspond with core-2-type glycans. Although we have shown the monosaccharide compositions here, detailed structural determination of these putative structures should be

Figure 7. MALDI-TOF MS analysis of O-glycans in pancreatic juice mucins separated by SMME. (a) Mass spectra of permethylated glycans derived from spots 1-4 in Figure 6b. Capital letters “N” and “SA” in the figure indicate neutral glycans and sialoglycans, respectively. Capital letter “K” indicates glycan signals observed as [M + K]+ ions which correspond to potassium adduct ions. Small letter “i” indicates the signals from [M - 14 + Na]+ which correspond to incompletely permethylated glycans. Many signals observed between m/z 1100 and m/z 1400 in the spectrum of spot 3 were a contaminating polymeric compound which has intervals of 44 u. (b) Histograms of relative intensities of the glycan signals observed. The intensities of neutral glycans and sialoglycans are indicated by black bars and red bars, respectively (the histograms represents only signal intensities but not the quantities). The signal numbers correspond to those described in Table 1.

performed by tandem MS techniques in studies containing glycan biomarker assessment. DISCUSSION We have developed a new electrophoresis technique, SMME, which can be applied to characterization of glycoproteins. Using SMME employing PVA, we can selectively separate large-size Analytical Chemistry, Vol. 81, No. 10, May 15, 2009

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Table 1. O-Linked Glycans in Pancreatic Juice Mucins Observed in MALDI-TOF MS no.

obsd m/za

calcd m/z

compositionb,c

1 2 3 4 5 6 7 8 9 10 11 12 13

895.56 1256.79 1426.83 1519.00 1693.07 708.48 779.54 983.64 1157.77 1331.88 1506.01 1536.08 1710.23

895.46 1256.64 1426.74 1518.78 1692.87 708.38 779.42 983.52 1157.60 1331.69 1505.78 1535.79 1709.88

(NeuAc)(Hex)(HexNAc) (NeuAc)2(Hex)(HexNAc) (NeuAc)(HexNAc)4d (NeuAc)(Fuc)(Hex)2(HexNAc)2 (NeuAc)(Fuc)2(Hex)2(HexNAc)2 (Fuc)(Hex)(HexNAc) (Hex)(HexNAc)2 (Hex)2(HexNAc)2 (Fuc)(Hex)2(HexNAc)2 (Fuc)2(Hex)2(HexNAc)2 (Fuc)3(Hex)2(HexNAc)2 (Fuc)2(Hex)3(HexNAc)2 (Fuc)3(Hex)3(HexNAc)2d

a The glycans were observed as [M + Na]+. b Monosaccharide compositions were determined by database searching using GlycoMod (http://www.expasy.ch/tools/glycomod/). c NeuAc, N-acetylneuraminic acid; Fuc, fucose; Hex, hexose; HexNAc, N-acetylhexosamine. d These signals were not found in GlycoSuiteDB by the GlycoMod search. This suggests that the monosaccharide compositions have not been reported.

glycoproteins such as mucins and proteoglycans from regularsize proteins. The mechanism of the selective separation can be explained as follows: (1) mucins and proteoglycans can efficiently migrate to the anode side even in acidic buffer (pH 4.0) due to their acidic properties (which are a result of many intrinsic carboxylic acids and/or sulfonic acids in their glycans), (2) mucins and proteoglycans poorly adhere to the supported molecular matrix under aqueous conditions due to their hydrophilic properties which are a result of being highly glycosylated. In SDS gel electrophoresis, proteins are separated based on differences in their molecular masses. In contrast, SMME separates glycoproteins mainly based on differences in their charge density. The pore size (0.45 µm) of the PVDF membrane used in this study is significantly larger than that of SDS-PAGE gels. This allows the discrimination of mucin-glycoforms which have different amounts of acidic residues such as sialic acid and sulfonic acid. Furthermore, we have also shown that the proposed SMME is compatible with glycoprotein staining by the periodic acid-Schiff base method (Pro-Q emerald, Figures 3b and 6c). In addition, we also obtained preliminary results which demonstrate that immunostaining of mucins separated by SMME is also applicable, although fixation of mucin spots is required (data not shown). Thus, this method is maybe applicable in techniques using specific detection of glycoproteins by staining with lectins or antibodies. Furthermore, identification of the separated glycoproteins may also be possible by employing peptide mass fingerprinting (PMF) or immunostaining approaches. In comparison with a gel-based strategy such as AgPAGE, the proposed strategy is simple, rapid, and quantitative, because gel preparation and blotting processes are not required. Furthermore, membrane format electrophoresis is suitable for larger sample handling compared to CE (ca. 10100-fold larger), which allows a wide variety of applications such as preparative analysis or extensive analysis for mass spectrometry. Thus, SMME should become a versatile tool for evaluation of glycan alterations on mucins associated with diseases. We have shown separation of the glycoforms of PSM by SMME. The presence of glycoforms with different degrees of sulfation in PSM has already been revealed using cellulose acetate 3822

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membrane electrophoresis.31 Cellulose acetate membrane electrophoresis which was developed by Joachim Kohn in 1957 has been used for separation of serum proteins for a long time32 and is used for diagnosis even now as a simple and robust method. Although the separation mode of SMME performed in this study is similar to that of conventional cellulose acetate membrane electrophoresis, SMME has an advantage for characterization of glycoproteins. Cellulose acetate membrane cannot be used in onmembrane β-elimination because cellulose fragments which are released from the membrane during the alkaline β-elimination reaction interfere with MALDI-TOF MS analysis of glycans released from the mucin spot (data not shown). PVDF membrane and PVA which are employed in SMME do not interfere with MALDI-TOF MS analysis of the glycans at all. Using the present strategy, we examined the lower detection limit of O-linked glycans in BSM by a serial dilution (Supporting Information Figure S1 and Table S1). Alcian blue staining can visualize BSM even at 78 ng. This sensitive detection is based on the fact that BSM is a highly sialylated mucin. Therefore, in the case of detection of both acidic and neutral mucins, the use of a combination of Alcian blue and Pro-Q emerald staining is important as shown above. The reported four sialoglycans28 could be clearly observed even from 156 ng of BSM (actually, a portion of the sample (15.6 ng as BSM) was transferred onto target plate for MS analysis). SMME is also applicable to the analysis of proteoglycans as well as mucins (Figure 3a). Separation of mucins and the “mucin-like” proteoglycans, which are highly glycosylated anionic macromolecules (e.g., aggrecan), is generally difficult due to the similarity of their physicochemical properties. However, SMME can separate proteoglycans from PSM and gave a distinct proteoglycan spot (spot 1) and hyaluronic acid spot (spot 2). A faintly stained Alcian blue region in the area of spots 1 and 2 was present after the chondroitinase ABC digestion (Figure 3c). This implies the presence of heparin/heparan sulfate chain(s) in the proteoglycan (spot 1). In fact, we could detect a trace amount of unsaturated disaccharides derived from heparin/heparan sulfate by LIF-CE after heparin/heparan sulfate lyase digestion of the crude PSM solution (data not shown) but not from the spot on the membrane. SMME will be applicable to the analysis of GAGs as well as proteoglycans. It will provide a simple method for routine quality control in pharmaceutical heparin preparation, which recently attracted much attention in terms of the relationship between contaminated oversulfated chondroitin sulfate and an acute onset of serious side effects.33 Furthermore, we applied our method to mucin analysis of human pancreatic juice. Over the past several years, mucinproducing pancreatic tumors have been widely recognized.34 With increasing awareness has also come an evolution of classification which can make the distinction between malignant and benign. We demonstrated that three types of mucin having distinct glycan (31) Stanley, R. A.; Lee, S. P.; Roberton, A. M. Biochim. Biophys. Acta 1983, 760, 262–269. (32) Rocco, R. M. Clin. Chem. 2005, 51, 1896–1901. (33) Guerrini, M.; Beccati, D.; Shriver, Z.; Naggi, A.; Viswanathan, K.; Bisio, A.; Capila, I.; Lansing, J. C.; Guglieri, S.; Fraser, B.; Al-Hakim, A.; Gunay, N. S.; Zhang, Z.; Robinson, L.; Buhse, L.; Nasr, M.; Woodcock, J.; Langer, R.; Venkataraman, G.; Linhardt, R. J.; Casu, B.; Torri, G.; Sasisekharan, R. Nat. Biotechnol. 2008, 26, 669–675. (34) Grogan, J. R.; Saeian, K.; Taylor, A. J.; Quiroz, F.; Demeure, M. J.; Komorowski, R. A. AJR, Am. J. Roentgenol. 2001, 176, 921–929.

profiles exist in human pancreatic juice. These data were easily obtained from 100 µL of the juice. This amount of pancreatic juice can be collected endoscopically at the time of endoscopic retrograde cholangiopancreatography (ERCP). By analyzing many samples, useful markers for distinction between cancer and benign diseases maybe discovered. CONCLUSIONS SMME, a new method for the characterization of large-size glycoproteins described here, is a simple and rapid tool for the exploration of glycan alterations associated with various diseases such as cancer. In addition, the ease and rapidness of the method is suitable for practical use in a clinical setting. Therefore, SMME has the potential to be directly used as a diagnostic method at the bedside in the future. Furthermore, the concept of SMME has much potential for the development of various separation modes by selection of different primary supports and matrix molecules. The design of the format of SMME will enable the

separation/characterization of a wide variety of analytes as well as glycoproteins. ACKNOWLEDGMENT We thank Professor Y. Hashimoto, Fukushima Medical University, for his valuable advice on the analysis of human pancreatic juice mucins. We also thank Dr. W. Dong and Ms. K. Ogasawara for technical assistance. This work was performed as a part of the R&D project of the Industrial Science and Technology Frontier Program supported by the New Energy and Industrial Technology Development Organization (NEDO). SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review January 22, 2009. Accepted March 23, 2009. AC900157C

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