Comparison of methods to release mucin-type O-glycans for glycomic

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Comparison of methods to release mucintype O-glycans for glycomic analysis Yukinobu Goso, Tsukiko Sugaya, Kazuhiko Ishihara, and Makoto Kurihara Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b01346 • Publication Date (Web): 19 Jul 2017 Downloaded from http://pubs.acs.org on July 21, 2017

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Analytical Chemistry

Comparison of methods to release mucin-type O-glycans for glycomic analysis

Yukinobu Goso,1, * Tsukiko Sugaya,1 Kazuhiko Ishihara,2 and Makoto Kurihara3

1

Department of Biochemistry, Kitasato University School of Medicine, Sagamihara 252-

0374, Japan 2

Kitasato Junior College of Health and Hygienic Sciences, Minami-uonuma 949-7241,

Japan 3

Department of Applied Bioscience, Kanagawa Institute of Technology, Atsugi 243-0292,

Japan

ASSOCIATED CONTENT Supporting Information Additional information is noted in text.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]

ACS Paragon Plus Environment


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ABSTRACT Mucin-type O-glycans (O-glycans) are one of the most common glycans attached to proteins. To develop an optimized glycomic analysis protocol, O-glycans were released from glycoproteins using hydrazine, ammonia, or sodium hydroxide treatment, followed by hydrophilic interaction liquid chromatography to evaluate O-glycan release. We found that porcine gastric mucin or bovine fetuin treated at 60°C for 6 h with hydrazine gas in the presence of malonic acid yielded O-glycans with only a small amount of degraded, socalled “peeled” products. Ammonia treatment also yielded intact O-glycans, but with additional peeled products containing GlcNAc at the reducing end. In contrast, sodium hydroxide treatment yields mainly peeled glycans, including those containing GlcNAc at the reducing end. Importantly, O-glycans obtained from rat gastric mucin treated with hydrazine, and labeled with anthranilic acid, had a nearly identical profile following hydrophilic interaction liquid chromatography as permethylated O-glycan alditols analyzed by mass spectroscopy. Taken together, the data suggest that glycan release using hydrazine treatment, followed by high-performance liquid chromatography after fluorescent labeling, is a suitable method for glycomic analysis of mucin-type O-glycans.


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INTRODUCTION Many proteins are posttranslationally glycosylated, with the attached glycans essentially defining the role of such proteins1, 2. Mucin-type O-glycan (O-glycan) is one of the most common protein modifications, constitutes more than half the weight of mucin, and is critical for mucin function3. For instance, genetically engineered mice deficient in the key glycosyl transferase have gastric or intestinal mucosal imbalance and are prone to cancer4, 5. Therefore, glycomic analysis is necessary to fully elucidate the role of mucin, as well as other glycoproteins, in physiological and pathological processes6–8. Liquid chromatography of fluorescently-labeled glycans is an attractive method for glycomic analysis because of its high sensitivity and high throughput. However, this approach requires the release of O-glycans with free reducing-end aldehydes from glycoproteins, to which fluorescent tags can be added. Accordingly, many reagents have been proposed to obtain such glycans, including hydrazine9–11, ammonia12, or simple alkalis13, 14. Of these, we initially selected hydrazine, based on reports that O-glycans are released without significant degradation or so-called “peeling”15. However, we found that hydrazine causes significant degradation unless a weak acid, especially malonic acid, is added during treatment16. Nevertheless, significant degradation was still observed after hydrazine treatment at 65°C for 18 h, although intact, i.e., unpeeled, O-glycans were obtained in high yield. Therefore, the reaction conditions were further optimized in this study to reduce peeling, and ammonia and sodium hydroxide were evaluated as alternatives



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to hydrazine. In addition, we compared hydrazine treatment to reductive beta-elimination using alkaline borohydride, a commonly used method to obtain minimally degraded glycan alditols, which can then be permethylated and analyzed by mass spectroscopy.

EXPERIMENTAL SECTION Materials. Anthranilic acid, i.e., 2-aminobenzoic acid, was purchased from Wako Chemical (Osaka, Japan). Hydrazine and Dowex 50 (200–400 mesh) were obtained from Masuda Chemical (Takamatsu, Japan) and Bio-Rad (Hercules, CA), respectively. QAEToyopearl and a TSKgel Amide-80 column (4.6 mm internal diameter × 25 cm) were purchased from Tosoh (Tokyo, Japan), and PD MiniTrap G-10 and Oasis HLB 1 cc (1 mg) were obtained from GE Healthcare Bio-Sciences Japan (Tokyo, Japan) and Waters (Milford, MA), respectively. Porcine gastric mucin type I, bovine fetuin, and Discovery® DPA-6S SPE column were obtained from Sigma-Aldrich (St. Louis, MO), and rat gastric mucin was obtained from the corpus of rat stomachs as previously described17. Release of O-glycans by hydrazine treatment. Porcine gastric mucin partially purified as described18 and bovine fetuin were treated with hydrazine according to published methods19. Briefly, 1 mL samples containing 150 µg protein, with or without 10 µmol malonic acid, were placed in glass tubes (10 mm internal diameter × 75 mm) and lyophilized. Subsequently, the tubes were inserted into Waters Pico-Tag Reaction Vials and thoroughly dried over P2O5 in vacuo for more than 16 hours. Under dry nitrogen in a plastic


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glove bag, anhydrous hydrazine was then added into the reaction vials but outside the glass sample tubes. Vials were then evacuated to less than 200 Pa, sealed, and heated at 65°C for 18 h, at 60°C for 6 h, or at the indicated temperature and duration. Hydrazine was removed in vacuo with a cold trap and then over H2SO4. Finally, the released oligosaccharides were re-acetylated with acetic anhydride in saturated sodium bicarbonate, desalted, and fractionated into neutral and acidic glycans on Dowex 50 and QAE-Toyopearl columns as described19. O-glycans from rat gastric mucin were obtained in the same manner. Release of O-glycans by ammonia treatment. Porcine gastric mucin was reacted with ammonia according to Huang et al.12 Briefly, 1 mg protein/tube was treated at 60°C for 40 h with 1 mL concentrated ammonium hydroxide saturated with ammonium carbonate. Ammonia and ammonium carbonate were removed by repeated evaporation in a SpeedVac concentrator (Thermo Scientific, Waltham, MA) after the addition of water. The resulting glycosylamines were then treated at 37°C for 30 min with 10 µL 0.5 M boric acid. Subsequently, boric acid was removed by repeated evaporation with nitrogen gas after the addition of methanol. Finally, neutral glycans were obtained in the flowthrough from a sequential Dowex 50 and QAE-Toyopearl columns. Release of O-glycans by sodium hydroxide treatment. Porcine gastric mucin (1 mg/tube) was treated at 45°C for 15 h with 1 mL 0.05 M NaOH. After neutralizing with acetic acid, neutral O-glycans were obtained in the flowthrough from sequential Dowex 50 and QAE-Toyopearl columns.



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Release of O-glycans by alkaline borohydride treatment and subsequent methylation. Rat gastric mucin was treated with alkaline borohydride according to Carlson’s method20. Briefly, 150 µg protein/tube was treated at 45°C for 15 h with 50 µL 0.05 M sodium hydroxide containing 1 M sodium borohydride. After acidification and removal of borate, neutral glycans were obtained in the flowthrough from a QAE-Toyopearl column. The glycans were then permethylated as described by Ciucanu and Costello21. Fluorescent labeling. O-glycans were derivatized with anthranilic acid (AA) as described22, diluted to 95 % v/v acetonitrile, and applied to a DPA-6S column (150 mg in 3 mL) pre-equilibrated with acetonitrile. The column was then washed with 8 mL of 97 % v/v acetonitrile, and eluted with 2.5 mL water. After evaporation, AA-labeled glycans were dissolved in 0.1 mL 0.1 M pyridinium acetate pH 5.0 and applied to a PD MiniTrap G-10 column equilibrated with the same buffer. The column was then washed with 0.3 mL buffer, and eluted with 2 mL of the same solution. Glycans were also labeled with 2-aminopyridine (PA) according to Ohara et al.23, and purified on a DPA-6S column in the same manner. Prior to preparative high-performance liquid chromatography, mass spectrometry, and tandem mass spectrometry, fluorescently labeled glycans were further purified on an Oasis HLB column, with elution in 5 % v/v acetonitrile. Hydrophilic interaction liquid chromatography. Glycans were analyzed by hydrophilic interaction chromatography on a JASCO LC-2000 HPLC instrument with a fluorescence detector. In brief, samples were injected onto a TSKgel Amide-80 column


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equilibrated in a 13:87 v:v ratio of Buffer A (0.2 % acetic acid and 0.2 % triethylamine in water) to Buffer B (0.1 % acetic acid in acetonitrile), eluted at 30°C or 70°C and 0.7 mL/min over 87–57 % Buffer B in 60 min for analysis of fluorescently labeled glycans, over 87–62 % Buffer B in 150 min for preparative purposes, over 87–52 % Buffer B in 210 min for rat gastric mucin AA-labeled glycans, and over 85–75 % Buffer B in 10 min for analysis of PA-labeled monosaccharides. Fluorescence was detected at emission/excitation wavelengths 360 nm/425 nm, 320 nm/400 nm, and 310 nm/380 nm for AA-labeled glycans, PA-labeled glycans, and PA-labeled monosaccharides, respectively. During preparative chromatography, peak fractions were collected and then concentrated using a SpeedVac concentrator. Analysis of reducing-end monosaccharides. Reducing-end monosaccharides in PAlabeled glycans were analyzed according to Makino et al.24 except that hydrophilic interaction chromatography was used instead of ion-exchange chromatography. Briefly, labeled glycans were hydrolyzed at 100°C for 4 h with 4 M HCl. After removal of HCl, reducing-end monosaccharides were re-N-acetylated and analyzed. Water was used as a blank, while PA-labeled GalNAc, GlcNAc, and Gal were used as standards. AA-labeled glycans were hydrolyzed in the same manner, and analyzed according to Anumula25. Mass spectrometry and tandem mass spectrometry. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectra were acquired on a Bruker Autoflex III mass spectrometer (Bruker Daltonik, Bremen, Germany) operating in positive



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ion mode at an accelerating voltage 20 kV. 2,5-dihydrobenzoic acid was used as the matrix, and all spectra were measured in reflectron mode. Sugar composition was determined using GlycoMod26. Tandem mass analysis was performed in LIFT mode according to the manufacturer’s protocols, and data were analyzed using GlycoWorkbench27.

RESULTS AND DISCUSSION Optimization of O-glycan release using hydrazine gas. We previously demonstrated that a weak acid, especially malonic acid, inhibits peeling when O-glycans are released from porcine gastric mucin or fetuin using hydrazine16. Nevertheless, significant peeling was still observed following hydrazine treatment for 18 h at 65°C, although intact Oglycans were obtained at high yield. Therefore, in this study, we first optimized the reaction conditions to further minimize peeling, starting with hydrazine treatment at 60°C for 6 h, the most frequently used reaction scheme11, 15. As shown in Figure 1A, six major O-glycans were obtained from porcine gastric mucin under these conditions 16. Fractions 1 and 2 were identified as Fuc-Gal-AA and GlcNAc-Gal-AA, respectively, by sugar analysis19, mass spectrometry, and tandem mass spectrometry16. Both are peeled products. By similar analyses, fractions 3, 4, 5, and 6 were identified as intact GlcNAc-Gal-GalNAc-AA, GlcNAc-Gal-(GlcNAc-)GalNAc-AA, GlcNAc-Gal-(Fuc-Gal-GlcNAc-)GalNAc-AA, and GlcNAc-Gal-(GlcNAc-Gal-GlcNAc-)GalNAc-AA, respectively. The relative amount of peeled products (1 and 2) to intact products (3–6) was lower at 60°C for 6 h, than at 65°C


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for 18 h, both in the absence and presence of malonic acid. In particular, approximately 28 % and 12 % of O-glycans were peeled when released at 60°C for 6 h in the absence and presence of malonic acid, respectively. These values increased to 40 % and 20 % when Oglycans were released at 65°C for 18 h. However, the amount of intact O-glycans released at 60°C for 6 h in the presence of malonic acid was only approximately 43 % of the intact products released at 65°C for 18 h, although comparable amounts were produced in the absence of malonic acid. Similarly, lower amounts of peeled products were obtained from fetuin treated with hydrazine for 6 h at 60°C in the presence of malonic acid (Figure 1B), with the peeled product Neu5Ac-Gal-AA accounting for only 6.7 % of major products. In contrast, 15 % of major products were peeled when released at 65°C for 18 h16. The time-dependent release of O-glycans at 60°C is plotted in Figure 2A. Although the yield of major intact glycans at 6 h was 45 % of the yield at 18 h, the ratio of degraded products to all major products was lowest at 6 h. Strikingly, the ratio at 6 h was also lowest at 60°C than at all other tested temperatures (Figure 2B). These results indicate that hydrazine treatment for 6 h at 60°C yields an optimal amount of intact O-glycans. However, as O-glycan release is clearly incomplete under these conditions, it is possible that individual O-glycans were unevenly released. To test this possibility, the ratios among fractions 3–6 were compared between treatment at 60°C for 6 h and treatment at 65°C for 18 h. As listed in Table S-1, the ratios of fractions 3, 4, and 5 to fraction 6 was almost the same under both conditions, indicating uniform release of individual O-glycans.



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Collectively, the results suggest that hydrazine treatment at 60°C for 6 h in the presence of malonic acid is suitable for glycomic analysis. Kozak et al. have reported that the addition of ethylenediamine tetraacetic acid (EDTA) to the reaction mixture, to chelate calcium ions, suppressed peeling during hydrazine treatment of glycoproteins28, 29. Based on these data, these authors hypothesized that metal cations, including calcium, might enhance peeling. Our data from a previous study showed that addition of disodium malonate to the reaction tube did not reduce peeling, but instead enhanced it16. Taken together, this may indicate that sodium ions may facilitate peeling in a manner similar to the calcium ion. Another conclusion that could be reached from the Kozak et al. study is that EDTA acts as an acid similar to malonic acid. Contrary to this conclusion, the disodium salt of EDTA also suppressed peeling. In our study, we employed gas-phase hydrazine treatment whereas Kozak et al. employed liquid-phase treatment. Although malonic acid also suppressed peeling following liquid phase treatment, significantly more peeling occurred in the liquid phase than in the gas phase, because the water concentration in the reaction vessel might be lower in the gas phase than in the liquid phase as described16. In this regard, water might produce hydroxide ions which are known to cause peeling12. Further studies will be required to clarify this mechanism further. Evaluation of O-glycan release from porcine gastric mucin treated with ammonia. O-glycans can also be released using other alkaline reagents including ammonia. Indeed, ammonia is frequently used, because the glycosylamines obtained seem resistant to


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peeling12. The hydrophilic interaction chromatogram of products obtained from porcine gastric mucin treated with ammonia is shown in Figure 3A. The AA-labeled glycans obtained with hydrazine (1–6) were also obtained with ammonia, as identified by mass spectrometry and tandem mass spectrometry (Table 1). However, additional fractions (a, b, and c) were obtained, and these were identified to be dHex-Hex-HexNAc-AA, HexNAcHex-HexNAc-AA, and HexNAc-(dHex-)-Hex-HexNAc-AA, respectively (Table 1 and Figure S-1). As these three glycans are not produced by alkaline borohydride treatment19, they are likely to be degraded products. Of note, fractions 3 and b were found to have the same sequence, HexNAc-Hex-HexNAc-AA, and probably differed because of reducing-end HexNAc residues. To test this hypothesis, fractions 1–6 and a–c were hydrolyzed with HCl and monosaccharide moieties were analyzed according to Anumura22. Gal was identified as the reducing-end monosaccharide in fractions 1 and 2 (data not shown), but traces of hexosamine were observed in other fractions. We note that, for unknown reasons, hydrolysis of AA-labeled glycans with hexosamine as the reducing-end monosaccharide may result in loss of fluorescence. To analyze reducing-end monosaccharides, PA-labeled glycans were used instead of AA-labeled glycans, because a method for the former has already been established24. Hydrophilic interaction chromatography of PA-labeled glycans indicated that products corresponding to AA-labeled glycan fractions 1–6 and a–c were obtained, although fractions 1 and 2 were lower in quantity (Figure S-2). Some amount of fractions 1 and 2



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were probably lost during purification on the DPS-6A column, as these two fractions were detected in the wash (data not shown). Mass spectrometry and tandem mass spectrometry confirmed that PA-labeled fractions 1–7 and a–c are the same products as the AA-labeled glycans, except that the fluorescent tags were different (Table 1). The reducing-end monosaccharide in fractions 3–7 was GalNAc, but GlcNAc in fractions a–c (Figure S-3), implying that fractions a–c are likely degraded products derived from core 2 structures, as described later. Taken together, the data indicate that in comparison to hydrazine treatment, ammonia treatment produces late-stage degradation products. O-glycan release from porcine gastric mucin treated with NaOH. In contrast, NaOH treatment may cause complete peeling by producing free-end O-glycans. To test this hypothesis, O-glycans were released with 0.05 M NaOH for 15 h at 45°C, conditions that are typically used in alkaline borohydride treatment. Fractions 3–6 were lost using this treatment, indicating complete peeling (Figure 3B). Fractions 1 and 2 were present, but in smaller quantities, especially fraction 2. This result may indicate that Fuc-Gal and GlcNAcGal are further degraded by exposure to alkaline conditions long periods of time. In contrast, significant amounts of fractions a–c persisted. Interestingly, analysis of fractions x, y, and z in Figure 3B indicated that these glycans were 18 mass values lighter than Hex1HexNAc2dHex1AA, Hex1HexNAc3AA, and Hex1HexNAc3dHex1AA, respectively. Hence, these fractions are probably peeled products containing Morgan-Elson chromogens at the reducing end19 (Figure S-4), and are derived from non-derivatized forms of fractions


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5, 6, and 7, respectively, as described later. Evaluation of release methods. Based on all these data, the reaction mechanisms of O-glycan release by hydrazine, ammonia, and NaOH are proposed, and illustrated in Figure S-5 using fraction 6 as a model glycan. All treatments produce O-glycans by ß-elimination, probably via hydroxide ions12,

15

, which however also promotes degradation of some

products. In the presence of hydrazine, intact glycans are converted to hydrazones, which are resistant to peeling (Figure S-5A). Peeled products are also converted to hydrazones, and thus probably escape further degradation. During re-acetylation and subsequent steps, the hydrazones formed from intact glycans, as well those of peeled glycans containing Gal at the reducing end, are released with free ends. On the other hand, hydrazones of peeled products containing Morgan-Elson chromogens (shown in Figure S-5A near the top) at the reducing end, and which contain core 2 branch, do not seem to be converted to free glycans, and are therefore not labeled with AA (Figure S-5A), as previously noted19. The underlying cause for this phenomenon is unknown, although the unsaturated structure of dehydrated GalNAc may prevent further conversion. On the other hand, ammonia treatment produces glycosylamines, which are thought to be resistant to peeling12 (Figure S-5B). Moreover, glycosylamines from intact glycans, as well as from peeled products containing core 1 branch, are converted to free glycans. Glycosylamines containing core 2 branch are also converted to free glycans, unlike



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hydrazones. However, glycans containing Morgan-Elson chromogens may be unstable under acidic conditions, and are therefore degraded to glycans with GlcNAc at the reducing end (fractions a–c) as shown in Figure S-5B. We note that in similar studies, the composition of O-glycans was different when released by ammonia or alkaline borohydride30. If this is indeed the case, then careful attention is required during analysis, since some peeled products, including fractions a–c, have GlcNAc at the reducing end, and are not easily distinguishable by tandem mass spectrometry from intact O-glycans. Improvements to the ammonia treatment protocol, including the use of ammonium carbamate31, may mitigate this risk. Nevertheless, products may have to be specifically tested for the presence of GlcNAc. As hypothesized, simple alkaline treatment using NaOH did not produce intact glycans (Figure S-5C). However, significant amounts of fractions a–c were obtained, suggesting that glycans with Morgan-Elson chromogens may be stable under alkaline conditions, but are rapidly degraded after pH neutralization, as already described. Fractions a, b, and c are probably produced from non-derivatized forms of fractions 5, 6, and 7, via intermediates that elute as fractions x, y, and z, respectively. We note that release methods based on NaOH are rapid and are performed in line to suppress peeling13, 14. Nevertheless, products may have to be characterized in the same manner as products obtained with ammonia treatment. Peeling yields two forms of glycans that contain core 1 or core 2 branch. The former have Gal at the reducing end, and are frequently observed, whereas the latter are not


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commonly found. One possible reason for this phenomenon seems to be the popularity of bovine fetuin and bovine submaxillary mucin as model glycoproteins that are readily available and are well characterized. However, these glycoproteins do not contain large amounts of glycans containing core 2 structures. On the other hand, porcine gastric mucin may be more suitable for detailed characterization of peeling, although commercially available samples contain many impurities and require further purification. We note that the variability of results from a recent comparison of analytical methods to profile O-linked glycans32,

33

are probably due in part to the behavior of the peeled

products described in this study. Glycomic analysis of rat gastric mucin O-glycans. Of the many methods that have been used for glycomic analysis, one of the most popular is mass spectrometry of permethylated O-glycans obtained with alkaline borohydride, which are only minimally peeled. However, since hydrazine treatment seems to yield O-glycans suitable for fluorescent labeling and hydrophilic interaction chromatography, these methods were directly compared. As shown in Figure 4B, a few AA-labeled glycan fractions obtained from rat gastric mucin were peeled, as was observed in porcine gastric mucin. Importantly, almost all the major O-glycans obtained with hydrazine were also detected by mass spectrometry of permethylated O-glycans obtained with alkaline borohydride (Figures 4, S6 and S-7). Thus, both methods released O-glycans with comparable composition, highlighting the suitability of hydrazine treatment for glycomic analysis of mucin-type O-



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glycans. High-performance liquid chromatography of fluorescently labeled O-glycans has the following advantages. First, glycan isomers can be distinguished more easily. For instance, two isomers of Hex1HexNAc2 (r1 and r2) and Hex2HexNAc2dHex1 (r6 and r7) could be separated (Figure 4B). Although these isomers were also detected on mass spectrometry, mass spectrometry may not distinguish potential isomers in other fractions. Furthermore, glycans can be separated and analyzed in detail based on different properties using a combination of different forms of high-performance liquid chromatography, including reversed-phase,

normal-phase,

and

graphite

carbon

chromatography.

Finally,

chromatographic analysis is quantitative, as glycans are fluorescently labeled only at the reducing end.

CONCLUSION We have established a method to release mucin type O-glycans from glycoproteins with minimal peeling. The method enables glycomic analysis, and may help elucidate the physiological significance of these glycans.

ACKNOWLEDGMENTS The authors thank Yuichi Sato (Kitasato University School of Allied Health Sciences) for use of the mass spectrometer. This study was supported in part by Japan Society for the


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Promotion of Science KAKENHI grant 25460934.

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Figure legends Figure 1. Effect of reaction duration and temperature on release of O-glycans from porcine gastric mucin (A) and fetuin (B). Glycoproteins (150 µg/tube) were reacted with anhydrous hydrazine gas in the absence (control) and presence of malonic acid (malonic acid), and analyzed by hydrophilic interaction chromatography after products were fluorescently tagged with anthranilic acid. Peaks were normalized to the highest peak in each chromatogram. The proposed glycan structures are illustrated, with yellow circles, blue squares, yellow squares, red triangles, and purple diamonds denoting Gal, GlcNAc, GalNAc, Fuc, and Neu5Ac, respectively34. AA, anthranilic acid.

Figure 2. Release of O-glycans from porcine gastric mucin (150 µg/tube) treated with anhydrous hydrazine gas in the presence of 10 µmol malonic acid for the indicated time at 60°C (A) and for 6 h at the indicated temperature (B). Products were fluorescently tagged with anthranilic acid, and analyzed by hydrophilic interaction chromatography. Intact, i.e., not peeled, products (sum of the area of fractions 3 to 6, open circle) and peeled products (sum of the area of fractions 1 and 2, closed circle) were quantified (upper panels), and the degree of peeling was evaluated as the ratio of the latter to the sum of both (lower panels). Values are mean ± SD (n = 4).

Figure 3. Hydrophilic interaction chromatography of AA-labeled glycans obtained from



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porcine gastric mucin treated with ammonia and NaOH. In (A), porcine gastric mucin (1 mg/tube) was treated for 40 h at 60°C with ammonium hydroxide saturated with ammonium carbonate. After ammonia was removed, products were treated with boric acid. Neutral glycans were labeled with anthranilic acid and analyzed by hydrophilic interaction chromatography. (Inset) The relative fluorescence intensity after 21 min was arbitrarily tripled to improve clarity. In (B), porcine gastric mucin (1 mg/tube) was treated at 45°C for 15 h with 0.05 M NaOH, and neutralized with acetic acid. Products were then labeled and analyzed as in (A). Non-glycan peaks are marked with asterisks.

Figure 4. Mass spectrum of permethylated O-glycans obtained from rat gastric mucin treated with alkaline borohydride (A), and hydrophilic interaction chromatography of AAlabeled glycans obtained from rat gastric mucin treated with hydrazine gas (B). In (A), the glycoprotein was treated at 45°C for 15 h with 0.05 M NaOH and 1 M sodium borohydride. Products were then permethylated and analyzed by MALDI-TOF mass spectrometry. In (B), the glycoprotein was reacted at 60°C for 6 h with anhydrous hydrazine gas in the presence of malonic acid. Products were then fluorescently labeled with anthranilic acid, and analyzed by hydrophilic interaction chromatography. Proposed glycan structures, as determined by tandem mass spectrometry (Figures S-6 and S-7), are illustrated according to Figure 1. Closed dots in (B) indicate glycans identified to be degradation products by tandem mass spectrometry. Non-glycan peaks are marked with asterisks.


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for TOC only



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Table 1. Structures of O-glycans obtained from porcine gastric mucin treated with ammonia. Fraction

Proposed composition

a

m/z [M+Na] AA-glycan Calculated

+

b

Reducing end

c

Proposed structure

PA-glycan

Observed

Calculated

Observed

1

Hex1dHex1

470.2

470.0

427.2

426.9

Gal

2

Hex1HexNAc1

527.2

527.0

484.2

484.0

Gal

a

Hex1HexNAc1dHex1

673.2

673.1

630.2

630.1

GlcNAc

3

Hex1HexNAc2

730.3

730.2

687.3

687.1

GalNAc

b

Hex1HexNAc2

730.3

730.2

687.3

687.2

GlcNAc

c

Hex1HexNAc2dHex1

876.3

876.3

833.3

833.3

GlcNAc

4

Hex1HexNAc3

933.3

933.4

890.3

890.5

GalNAc

5

Hex2HexNAc3dHex1

1241.5

1241.4

1198.5

1198.5

GalNAc

6

Hex2HexNAc4

1298.5

1298.4

1255.5

1255.6

GalNAc

7

Hex2HexNAc4dHex1

1444.5

1444.5

1401.5

1401.7

GalNAc

a

Sugar composition was determined by MALDI-TOF-MS.

b

Reducing end was determined by hydrophilic interaction chromatography of PA-labeled glycans hydrolyzed in HCl.

c

Structures were estimated by MALDI-TOF-MS/MS. Yellow circle, Gal; yellow square, GalNAc; blue square, GlcNAc; red triangle, Fuc; AA, anthranilic acid.


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Figure 1


Figure 2

Figure 3



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Figure 4


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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 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment


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 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment

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84x47mm (300 x 300 DPI)


Comparison of methods to release mucin-type O-glycans for glycomic

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