A Procedure for Alcian Blue Staining of Mucins on Polyvinylidene

Sep 5, 2012 - cannot be used to stain mucins with a low acidic glycan content. Meanwhile, periodic acid−Schiff staining can selectively visualize...
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A Procedure for Alcian Blue Staining of Mucins on Polyvinylidene Difluoride Membranes Weijie Dong, Yu-ki Matsuno, and Akihiko Kameyama* Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Open Space Laboratory C-2, 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, Japan S Supporting Information *

ABSTRACT: The isolation and characterization of mucins are critically important for obtaining insight into the molecular pathology of various diseases, including cancers and cystic fibrosis. Recently, we developed a novel membrane electrophoretic method, supported molecular matrix electrophoresis (SMME), which separates mucins on a polyvinylidene difluoride (PVDF) membrane impregnated with a hydrophilic polymer. Alcian blue staining is widely used to visualize mucopolysaccharides and acidic mucins on both blotted membranes and SMME membranes; however, this method cannot be used to stain mucins with a low acidic glycan content. Meanwhile, periodic acid−Schiff staining can selectively visualize glycoproteins, including mucins, but is incompatible with glycan analysis, which is indispensable for mucin characterizations. Here we describe a novel staining method, designated succinylation-Alcian blue staining, for visualizing mucins on a PVDF membrane. This method can visualize mucins regardless of the acidic residue content and shows a sensitivity 2-fold higher than that of Pro-Q Emerald 488, a fluorescent periodate Schiff-base stain. Furthermore, we demonstrate the compatibility of this novel staining procedure with glycan analysis using porcine gastric mucin as a model mucin.

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supported molecular matrix electrophoresis (SMME),10 which separates mucins on a PVDF membrane impregnated with a hydrophilic polymer. The analysis of the SMME-separated proteins does not require a transfer step to another PVDF membrane. Hence, this method can function as a highthroughput method that is not subject to protein loss due to low transfer efficiency. For these SMME− and SDS−AgPAGE-based methods, visualization of the mucins present on the PVDF membrane is of critical importance. In gel electrophoresis, silver staining and Coomassie blue staining are widely used for the visualization of proteins. Coomassie blue has also been used for staining proteins on PVDF membranes but is ineffective in mucin detection because of the hydrophilic nature of these proteins. Currently, mucins are stained with the Alcian blue or the periodic acid−Schiff (PAS) reagent. Alcian blue staining has been widely used to visualize mucopolysaccharides and acidic mucins in tissues, cells, and blotted membranes.8,9,11 However, this method cannot be used to stain mucins with a low acidic glycan content, such as porcine gastric mucin (PGM).10 Although PAS staining can selectively visualize glycoproteins, including mucins, regardless of the acidic residue content, this method is incompatible with glycan analysis since the periodic oxidation decomposes glycans. For the analysis of the glycans of

ucins are glycoproteins produced by a variety of epithelial cells for the protection and lubrication of the epithelial surfaces. These proteins have a high molecular mass and carry high-density clusters of O-linked glycans in the tandem repeat region of the molecule.1 Alterations in glycosylation and expression are closely associated with various disease states, including cancer and cystic fibrosis.2,3 In particular, most of the tumor-associated glycan antigens, such as CA19−9, are believed to be glycans on mucins.4,5 Thus, the isolation and characterization of mucins are critically important for gaining insight into the molecular pathology of diseases, including cancers, as well as the discovery of biomarkers for such diseases. Proteomic techniques are widely used to isolate and characterize proteins in the process of biomarker discovery. However, these techniques cannot be applied to mucin analysis because mucins are protease resistant and cannot enter sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS− PAGE) gels.6 For mucin analysis, agarose−polyacrylamide composite gels, which are prepared by mixing agarose with lowconcentration polyacrylamide gels, are generally used.6,7 Schulz et al. reported an analytical strategy for picomolar quantities of mucins.8 In this method, mucins are separated using SDS agarose−acrylamide composite gel electrophoresis (SDS− AgPAGE) and then electroblotted onto a polyvinylidene difluoride (PVDF) membrane; the O-linked glycans are subsequently released from the mucins for further analysis.8,9 As another approach for mucin characterization, we recently developed a novel membrane electrophoretic method, © 2012 American Chemical Society

Received: June 18, 2012 Accepted: September 5, 2012 Published: September 5, 2012 8461

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Figure 1. Staining of mucins on a PVDF membrane. (a) SMME-separated mucins from the crude PGM. (i) Conventional Alcian blue staining and (ii) succinylation-Alcian blue staining. Spot 1, proteoglycan; spot 2, hyaluronic acid; spot 3, acidic mucin; spot 4, PGM. (b) Succinylation-Alcian blue staining of the crude PGM using a variety of mixed solvents for succinylation. (i) Pyridine, (ii) 1:1 pyridine:acetonitrile, (iii) 1:4 pyridine:acetonitrile, and (iv) 1:11 pyridine:acetonitrile. The numbers on the right side of spots 3 and 4 indicate relative darkness, that is, “darkness of the spot” minus “darkness of the background”, as estimated by densitometry. (c) Staining of asialo-BSM on the SMME membrane. (i) Optimized succinylationAlcian blue staining and (ii) conventional Alcian blue staining. (d) Staining of electroblotted mucins on a PVDF membrane. (i) Pro-Q Emerald staining, (ii) conventional Alcian blue staining, and (iii) succinylation-Alcian blue staining. P is PGM, and B is BSM.

mucins with a low acidic residue content, the location of these mucins on an unstained membrane must be inferred from the staining pattern on a PAS-stained membrane. Thus, two PVDF membranes, one for Alcian blue staining and another for PAS staining, should be prepared when O-linked glycans of mucins are analyzed. However, this approach both is time-consuming and carries the risk of misassigning the location of the mucins. Because of these issues, a novel visualization method that is compatible with glycan analysis and mucin detection, irrespective of the acidic residue content, is desired. Here, we describe a novel procedure for the Alcian blue staining of mucins on a PVDF membrane. In this procedure, the glycans of the mucin molecules are modified with succinic anhydride, thereby enabling them to be stained with Alcian blue. This method can be applied to mucins on a blotted PVDF membrane and on a SMME membrane. Using PGM as a model mucin with a low acidic glycan content, we optimized the procedure for the method, evaluated the sensitivity of the staining, and demonstrated glycan analysis for the stained spots.

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(Tokyo, Japan). Other reagents and solvents were obtained from Wako Pure Chemical Industries Ltd. (Osaka, Japan) or Nacalai Tesque (Kyoto, Japan). All solvents used were of analytical reagent grade. SMME. A PVDF membrane (length, 6 cm; width, typically 6−10 cm) was quickly dipped into methanol and then immersed in a 0.1 M pyridine/formic acid buffer (pH 4.0) containing a 0.25% (w/v) hydrophilic polymer mixture [1:1 polyvinylalcohol (PVA)/polyethylene glycol (PEG)] for 30 min. The membrane was used for SMME. PGM or BSM (200 μg) was dissolved in 20 μL of a 0.1 M Tris-HCl buffer (pH 8.6) containing 20 mM dithiothreitol and 8 M urea. After incubation at room temperature (RT) for 3 h, 2 μL of an aqueous solution of iodoacetamide (250 mM) was then added, and the mixture was incubated at RT for 1 h in the dark. Desialylation of the BSM was performed by treating the reduced and alkylated BSM solution with 25 mM HCl (pH 1.6) at 80 °C for 1 h.12 Subsequently, the desialylated BSM was lyophilized and dissolved in distilled water. An aliquot (1 μL) of the prepared mucin solutions was directly spotted 1.5 cm from the bottom of the membrane. Electrophoresis was performed using an apparatus for cellulose acetate membrane electrophoresis (EPC105AA-type; Advantec, Tokyo, Japan) in a constant current mode at 1.0 mA/cm for 30 min. After the run, the membranes were stained by using the specified staining procedures. Electroblotting Mucins from AgPAGE. The agarose− polyacrylamide composite gels (0.5% agarose−2% polyacrylamide containing 0.1% SDS) were prepared as described previously.6 The solutions of PGM and BSM described above

EXPERIMENTAL SECTION

Materials and Reagents. Bovine submaxillary mucin (BSM) and PGM (type III, partially purified) were purchased from ICN Biomedicals Inc. (Aurora, OH) and Sigma-Aldrich (St. Louis, MO), respectively. Alcian blue 8GX was also obtained from Sigma-Aldrich. The Pro-Q Emerald 488 glycoprotein gel and blot stain kit was obtained from Molecular Probes (Eugene, OR). PVDF membranes (Immobilon-P) were purchased from Millipore (Bedford, MA). Succinic anhydride was obtained from Tokyo Chemical Industry Company Ltd. 8462

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were further denatured at 95 °C for 5 min by a sample loading buffer for a conventional SDS−PAGE. The denatured mucins were loaded onto the agarose−acrylamide composite gels and electrophoresed in 40 mM Tris/Tris-acetate buffer (pH 7.4) with 0.1% SDS and 2 mM ethylenediaminetetraacetic acid (EDTA) at 100 V for 1.5 h. After electrophoresis, the mucins were transferred to a PVDF membrane by the semidry method using the NuPAGE Transfer buffer (Life Technologies, Carlsbad, CA) for 30 min at 15 V. Afterward, the membranes were stained with the use of the specified staining procedures. Alcian Blue Staining. The SMME membranes, the succinylated SMME membranes, and the blotted membranes were gently shaken in 0.1% (w/v) Alcian blue 8GX dissolved in 0.1% acetic acid for 20 min and then washed with methanol for a few minutes to remove the background color. Succinylation-Alcian Blue Staining. Before succinylation, the membranes were dried by being heated at 80 °C for 20 min using a heating block. In the case of SMME, the membrane was immersed in acetone for 30 min prior to being heated. The reaction solution for the succinylation was prepared as follows: 500 mg of succinic anhydride was dissolved in 5 mL of acetonitrile containing pyridine in a 4:1 ratio (v:v). The dried membranes were incubated in the freshly prepared reaction solution in a sealed glass dish at RT for 2 h. Subsequently, the membranes were directly stained with Alcian blue. In order to acquire quantitative data, the stained membrane was made transparent by immersion in cis-4-nonene and densitometric analysis was performed with an image analyzer (LAS-1000 plus, Fujifilm, Tokyo, Japan). Glycan Release and Permethylation. Stained mucin spots were excised from the membrane and then transferred into a microtube. After the membrane pieces were wet with methanol, 30 μL of 0.5 M sodium borohydride (NaBH4) containing 50 mM NaOH was added to the microtubes, and the mixtures containing the membrane were incubated at 45 °C for 16 h. Next, the solution in the microtubes was neutralized with glacial acetic acid and subsequently applied onto a cationexchange solid-phase extraction cartridge (Oasis MCX, 1 mL; Waters, Milford, MA). After the samples had been washed with 500 μL of water two times, the flow-through fraction and washings were collected in a tube and lyophilized by a centrifugal evaporator. Next, 100 μL of 1% acetic acid in methanol was added to the tube, and the solution was evaporated again. This addition and evaporation procedure was repeated several times. The obtained residue was then permethylated, desalted, and dried, as previously reported.10,13 Mass Spectrometry. Spectra were acquired using a matrixassisted laser desorption ionization time-of-flight (MALDITOF) mass spectrometer (Reflex IV; Bruker Daltonik, Bremen, Germany). Ions were generated by a pulsed 337 nm nitrogen laser and were accelerated to 20 kV. All the spectra were obtained using the reflectron mode with a delayed extraction of 200 ns. For sample preparation, 0.5 μL of 2,5-dihydroxybenzoic acid (1 mg/mL) in 30% ethanol was spotted onto an Anchorchip plate (Bruker Daltonik) and dried. Permethylated glycans were dissolved in 50 μL of acetonitrile, and an aliquot (1 μL) of the solution was spotted onto the 2,5-dihydroxybenzoic acid crystal and dried.

Figure 2. Comparison of the sensitivity between Pro-Q Emerald and succinylation-Alcian blue staining. Indicated numerals are amounts (μg) of the crude PGM applied to the SMME. (a) Pro-Q Emerald staining and (b) succinylation-Alcian blue staining.

mucopolysaccharides) through electrostatic forces.14,15 Alcian blue is widely used for staining glycosaminoglycans and acidic mucins but cannot stain mucins with low acidic glycan contents.10 The presence of high-density clusters of O-linked glycans is one of the characteristic properties of mucin molecules. The introduction of carboxyl groups to the cluster of glycans represents one approach for transforming the neutral mucins into acidic mucin derivatives, which should possess the capacity to bind to Alcian blue. Succinylation is one of the most common methods for improving the physicochemical properties of polysaccharides, through the introduction of carboxyl groups.16,17 The hydroxyl groups of the glycans are modified with succinic anhydride to produce succinyl monoesters with an acidic residue. We evaluated the effectiveness of this modification for improving Alcian blue staining by using PGM as a model mucin. Partially purified PGM (crude PGM extract), which comprises proteoglycan, hyaluronic acid, an acidic mucin, and PGM, was electrophoresed by SMME, as described previously.10 The SMME membrane was dried and then immersed in a 10% (w/v) succinic anhydride solution in acetonitrile containing pyridine at a ratio of 4:1 (v/v). After incubation at RT for 2 h, the membrane was directly stained with Alcian blue according to the conventional method. As shown in Figure 1a, PGM (bottom spot) was clearly visualized after succinylation. Proteoglycan, hyaluronic acid, and the acidic mucin, which were also present in the crude PGM, were also stained. In contrast, the PGM completely failed to stain following conventional staining without succinylation. The SMME membrane was prepared by immersing a PVDF membrane in a 0.25% (w/v) aqueous solution of PVA and PEG in a 1:1 ratio.18 The hydroxy groups of PVA in the SMME membrane should also be modified by succinylation. Indeed, the entire membrane was colored using Alcian blue staining when the succinylation was performed in pyridine alone as the solvent (Figure 1b, lane i). The areas containing the mucins presented a high density of hydroxyl groups in contrast to other areas on the SMME membrane. In order to contrast the mucin areas by Alcian blue staining, the reaction conditions were adjusted to be more mild. We examined a variety of mixed ratios of pyridine and acetonitrile for the reaction solvent. Succinic anhydride was dissolved at a 10% (w/v) concentration that was nearly at saturation. As shown in Figure 1b, the relative darkness of the mucin spots increased upon the addition of acetonitrile. However, spot 3 was weakly stained after the reaction with the use of a mixed solvent containing pyridine and acetonitrile in a 1:11 ratio. On the basis of these results, we decided that the reaction conditions described above were best for the succinylation-Alcian blue staining of mucins. On the

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RESULTS AND DISCUSSION Succinylation-Alcian Blue Staining. Alcian blue is a cationic dye containing four isothiouronium residues and can sensitively bind negatively charged macromolecules (e.g., 8463

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Figure 3. MALDI-TOF MS spectra of O-linked glycans released from the SMME-separated mucins of the crude PGM. The glycans were permethylated. (a) An acidic mucin (spot 3) in the crude PGM, conventional Alcian blue staining (top), succinylation-Alcian blue staining (bottom). (b) The major mucin (spot 4) in the crude PGM, the corresponding area of the conventional Alcian blue staining membrane (top), succinylationAlcian blue staining (middle), Pro-Q Emerald staining (bottom). Indicated signals (m/z 1249.77 and 1380.87) with asterisks are derived from Alcian blue.

basis of the absorbance of the carbonyl groups in the Fourier transform infrared (FT-IR) spectra of the membranes, we roughly estimated that these conditions modified one-fourth of the hydroxyl groups on the membrane (see Figure S1 of the Supporting Information). We then examined whether other mucins, in addition to PGM, could be stained with good contrast under the optimized conditions. As shown in Figure 1c, the desialylated BSM, which could not be stained with the conventional Alcian blue, could be stained clearly under the optimized conditions. This result suggested that under optimized conditions, mucins carrying a high-density cluster of glycans could be stained, even if the degree of modification encompassed only one-fourth of the hydroxyl groups.

This method could be applied to mucins blotted on a PVDF membrane. The crude PGM was subjected to SDS−AgPAGE with BSM as a reference mucin, and the mucins were then electrically transferred onto a PVDF membrane. Three membranes were prepared by the same method and were stained with conventional Alcian blue staining, succinylationAlcian blue staining, or Pro-Q Emerald staining (Pro-Q is a fluorescent PAS reagent) (Figure 1d).19 With the use of conventional Alcian blue staining, the BSM spot was similarly visualized as it was with Pro-Q Emerald staining, while PGM could be seen only on the lower portion of the PGM spot. The lower portion in the PGM lane may have been proteoglycan and/or hyarulonic acid in the crude PGM. Notably, 8464

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succinylation-Alcian blue staining. This might be due to accurate excision, which became possible because of the visualization from the succinylation-Alcian blue staining.

succinylation-Alcian blue staining was able to stain BSM and PGM as successfully as Pro-Q Emerald staining was. Sensitivity. By using a dilution series of the crude PGM, we compared the sensitivity of the succinylation-Alcian blue staining with that of the Pro-Q Emerald staining, which is roughly 16-fold more sensitive than the standard colorimetric PAS-based method using acidic fuchsin dye.10,19 The diluted PGM solutions were applied to the SMME, and the membranes were stained with Pro-Q Emerald or succinylation-Alcian blue after electrophoretic separation. As shown in Figure 2, the succinylation-Alcian blue and Pro-Q Emerald allowed for visualization of the PGM at 0.6 and 1.25 μg, respectively. Thus, the sensitivity of succinylation-Alcian blue staining was ∼2-fold higher relative to that of Pro-Q Emerald staining. Densitometric analysis showed a linear correlation with the amount and the darkness of the PGM spots stained with 0.3−10 μg of succinylation-Alcian blue (see Figure S2 of the Supporting Information). Compared with Pro-Q Emerald, which is a fluorescent dye showing a linear dynamic range over 3 orders of magnitude,19 the linear dynamic range of the Alcian blue was small.20 The succinylation-Alcian blue also stained proteoglycan, hyarulonic acid, and acidic mucin. These acidic impurities in the crude PGM are not visualized in Pro-Q Emerald staining because these molecules are resistant to periodic oxidation.21,22 A low concentration may be another reason underlying their lack of visible staining. Thus, the succinylation-Alcian blue was able to stain such minor components in addition to the major mucin of the crude PGM. Compatibility with Glycan Analysis. In the characterization of mucins, glycan analysis is important for biomarker discovery and for the elucidation of the molecular pathology involved with these proteins.23 To analyze the glycans of mucins, the Alcian blue-stained spots were excised and the membrane pieces were subjected to a reductive β-elimination reaction to release the glycans.8−10 The glycans, which were obtained as alditols, were permethylated with methyl iodide in the presence of sodium hydroxide and subsequently analyzed by a mass spectrometer.8−10 During the succinylation-Alcian blue staining, the hydroxyl groups of the mucin glycans were partially and randomly modified to the corresponding succinyl esters. However, these esters would have been hydrolyzed during the reductive β-elimination and the permethylation, since esters are easily hydrolyzed under aqueous alkaline conditions. Next, we examined the glycan analysis of the mucins (spots 3 and 4 in Figure 1a) visualized using succinylation-Alcian blue staining or Pro-Q Emerald staining and compared their MS spectra with those of the conventional Alcian blue stained mucins. The mucin spots visualized with each staining method were treated using the identical procedure described in the Experimental Section. As shown in Figure 3, both staining procedures for Alcian blue and succinylation-Alcian blue resulted in almost identical MS spectra for the acidic mucin (spot 3) and PGM (spot 4). In contrast, PGM stained with Pro-Q Emerald gave unassignable signals, which can be attributed to glycans decomposed by periodic acid oxidation. Many of these signals were 2−8 units larger than the original glycan signals. All glycan signals in the MS spectra were assigned and are summarized in the Supporting Information. This result indicated that the succinylation-Alcian blue staining is compatible with glycan analysis via reductive β-elimination and permethylation. Furthermore, the signal-to-noise ratio of the MS spectra for spot 4 was significantly improved by the

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CONCLUSION In this study, we demonstrated that succinylation-Alcian blue staining could allow for the visualization of mucins on a PVDF membrane, irrespective of their acidic residue contents. The sensitivity was ∼2-fold higher than that of Pro-Q Emerald, while the staining procedure was quite simple. Furthermore, in contrast to PAS staining, this method is compatible with glycan analysis. Studies on biomarker and molecular pathology involving mucins will be accelerated by combining SMME and AgPAGE with succinylation-Alcian blue staining. This developed staining method can be applied to the detection of polysaccharides, including chitosan on a PVDF membrane (data not shown). Proteins other than mucins could be visualized with the succinylation-Alcian blue staining under the conditions used in this study (data not shown), since the succinylation should also modify the amino groups and hydroxyl groups of proteins. This would not pose a problem for mucin detection because mucins could selectively be migrated using SMME with a pyridine/formic acid buffer (pH 4.0).10 Pro-Q Emerald staining, which must be accompanied with glycan degradation, is widely used for the visualization of glycoproteins on PVDF membranes in glycoproteomic research. Thus, although not currently possible, the ability to selectively stain glycoproteins will be attractive to those performing glycoproteomic research. In this context, further studies to expand this method to other glycoproteins are underway.

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +81-29-861-3487. Fax: +81-29-861-3123. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Ms. N. Oomichi for technical advice on AgPAGE. FT-IR was conducted at the Nano-Processing Facility operated by the Innovation Center for Advanced Nanodevices (ICAN) and the National Institute of Advanced Industrial Science and Technology (AIST), Japan. This work was supported by a Grant-in-Aid for Scientific Research (KAKENHI 23310154) from the Japan Society for the Promotion of Science (JSPS).

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