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Solid-phase chemical modification for sialic acid linkage analysis: Application to glycoproteins of host cells used in influenza virus propagation Shuang Yang, Ewa Jankowska, Martina Kosikova, Hang Xie, and John F. Cipollo Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b02514 • Publication Date (Web): 09 Aug 2017 Downloaded from http://pubs.acs.org on August 13, 2017

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Analytical Chemistry

Solid-phase chemical modification for sialic acid linkage analysis: Application to glycoproteins of host cells used in influenza virus propagation Shuang Yang1, Ewa Jankowska1, Martina Kosikova2, Hang Xie2, and John Cipollo1* 1

Laboratory of Bacterial Polysaccharides, Division of Bacterial, Parasitic and Allergenic

Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, MD 20993 2

Division of Viral Products, Office of Vaccines Research and Review, Center for Biologics

Evaluation and Research, Food and Drug Administration, Silver Spring, MD 20993 *Corresponding to Dr. John Cipollo, G637, Bldg 52/72, 10903 New Hampshire Ave, Silver Spring 20993, Email: [email protected]

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Abstract Differentiation between the sialyl linkages is often critical to understanding biological consequence. Here we present a facile method for determining these linkages in glycans. Analysis of sialic acids is challenging due to their labile nature during sample preparation and ionization. Derivatization is often required via chemical reaction. Amidation derivatizes all sialic acids regardless of linkage, while esterification enables differentiation between α2,3-linked and α2,6-linked sialic acids. Reactions have been primarily performed on free glycans in solution but have been recently adapted to solid-phase providing unique advantages such as simplified sample preparation, improved yield, and high throughput applications. Here, we immobilized glycoproteins on resin via reductive amination, modified α2,6-linked sialic acids through ethyl esterification and α2,3-linked sialic acids via amidation. N-glycans and O-glycans were released via enzyme and chemical reactions. The method was applied for analysis of three different MDCK cell lines used for influenza propagation and where distributions of α2,3 and α2,6 sialic acids are critical for cell performance. Linkage specific distribution of these sialic acids was quantitatively determined and unique for each cell line. Our study demonstrates that protein sialylation can be reliably and quantitatively characterized in terms of sialic acid linkage of each glycan using the solid-phase esterification/amidation strategy.


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Introduction All eukaryotic cell surfaces are essentially populated with diverse glycans. It is often the case that terminal monosaccharide residues in these oligosaccharides are directly involved in recognition events in a range of biological processes 1. The common residues on those oligosaccharides contain a variety of sialic acids. The residues richly substitute glycoproteins of the cell surface and secreted proteins in vertebrates 1. Sialylated glycoproteins (sialylglycoproteins) are involved in a broad range of biological processes such as intercellular adhesion, signaling, microbial attachment 2,3 and coagulation 4. Often the biological activity can require the presence of sialic acid substitution in a specific configuration such as a α2,3 or α2,6 linkage. The particular sialic acid monosaccharide can also be of consequence. The capping by N-Acetylneuraminic acid (Neu5Ac) or N-Glycolylmeuraminic acid (Neu5Gc) further complicates the interaction of sialic acids with influenza virus, affecting the binding efficiency of receptor on avian flu 5,6. Clearly the ability to differentiate between these configurations is of critical importance to the understanding of the underlying biological process 7. For instance, influenza virus hemagglutinin (HA) receptor site recognition is modulated by sialic acid structural context within the target tissue 8,9. Sialic acids have a range of chemical properties that are significant in biological processes. They are negatively charged and likely inhibit intermolecular and intercellular interactions through charge repulsion 10. They also can act as critical ligand components leading to recognition by sialic acid binding lectins and other proteins 11. As described previously, they are present in alternative linkage configurations. Thus the altered expression of particular types of sialic acids or linkages can dramatically change their recognition function. For instance, the ratio of sialic acid linkage in the terminal disaccharide pair, i.e. Neu5Acα2,3Gal vs Neu5Acα2,6Gal, has been associated with disease symptoms and progression 12.The level of α2,6-linked sialic acids have been shown to be increased in breast cancer and the altered ratios



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of α2,3- versus α2,6-linked sialic acids may be useful for disease diagnosis 12. Analysis by the SNA lectins and mRNA expression confirmed that the expression of β-galactoside: α2,6sialyltransferease (ST6Gal-1) has been enhanced in tumor tissues as well as cell lines over the cancer-free or benign specimens 13,14; whereas a down-regulation of ST3Gal-IV mRNA was associated with human renal carcinoma malignant progression 15. Thus the overexpression of the Golgi enzyme ST6Gal-1 can also enhance α2,6-linked sialylation on N-glycans. Because of this, ST6Gal-1 can be used as a mediator of tumor progression since the ST6Gal-1 null phenotypes showed selectively altered expression of genes associated with focal adhesion signaling and decreased phosphorylation 16. Transmission of avian virus to human can require a shift in HA receptor specificity centered on terminal sialic acid linkages. Avian influenza virus preferentially bind to glycoprotein receptors with α2,3-linked sialic acids; in contrast human influenza virus strains favorably bind to those with α2,6 linkage 17. However, a virus variant of avian origin with an altered receptor specificity for α2,6 capped glycan patterns may gain transmitability in humans increasing the chance of pandemic 17,18. Interestingly, the epithelial cells of the pig trachea produce both α2,3 and α2,6-linked sialic acids, which make them a fortuitous intermediate host for viral transmission between avian and human influenza virus strains, serving as a “mixing vessel” for the emergence of new pandemic viruses 19. It is thus essential to study sialic acid linkages in the human epithelial cells to better understand how, where, and what ratios the sialyl linkages are presented in the target tissues as well as in model cells and animal systems. Sialic acid linkages can be studied with lectin affinity, enzymes, or mass spectrometry (MS). Lectins have been routinely used for recognizing or enriching specific moiety of sialic acids. Sambucus nigra L (SNA-I) can recognize α2,6-linked sialic acids 20, while maackia amurensis (MAL) targets α2,3-linked sialic acids 21. However, lectin affinity may not be as specific as required for some applications. Glycoproteins with low abundance sialyl moieties 4 ACS Paragon Plus Environment

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may be under represented in some cases. Thus, the efficiency and specificity of lectin enrichment and/or linkage identification can be of concern 22. Use of neuraminidase is another option to study sialic acid linkage. In general, neuraminidase α2,3 can be applied for removal of α2,3-linked sialic acids; yet, imperfect specificity and activity across the range of sialyl oligosaccharides remains a concern with these enzyme driven techniques and no neuraminidase is readily available for removal of α2,6 exclusively. Identification of sialic acid linkages and glycan isomers has been facilitated by advancements in liquid chromatography (LC) and MS. The linkages and isomers can be determined by tandem mass spectrometry (MS/MS) from permethylated glycans 23,24,25. Moreover, it is possible to determine isomers of sialic acids by further fragmenting the MS/MS ions although these methods are non-trivial 26. The sialic acid is lost at relatively low energy leading to very low abundances of diagnostic Aions, the presence of which are key for linkage configuration determination 27,28. To overcome these obstacles, chemical derivatization has been pursued for differential analysis of sialic acids. There are two approaches for sialic acids derivatization: performing the reaction either in solution or on solid-phase 29,30,31. Amidation or esterification has been widely employed for modification of sialic acids in solution 32,33,34,35. Amidation can effectively stabilize sialic acids, but both α2,3 and α2,6 linkages are concurrently converted to amide, and are thus indistinguishable from one another. Esterification can react with α2,3 or α2,6 differently, forming lactone and ester respectively 35,36,37. Even though derivatization of sialic acids in solution is efficient, it often requires multiple chromatographic purification steps prior to MS analysis. Excess amounts of reagent chemicals may be required to achieve complete reaction, leading to the need for extensive clean-up protocols. On the contrary, a chemical reaction performed on solid-phase can mitigate these challenges since reagents are removed by extensive washing steps without sample loss. Sialic acids have been reliably modified on solid-phase by amidation via carbodiimide coupling 30,38,39 or by esterification 40. The linkage can be differentially modified



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by esterification, formation of lactone and ester. However, lactone stabilities differ for α2,8 and α2,3 as the latter is quickly hydrolyzed in water 41,42,43 and this situation can lead to ambiguities in some settings where both linkages may be present. In this study, we developed a novel method for the sequential derivatization of α2,6- and α2,3-linked sialic acids via chemical modification. The method consists of glycoprotein immobilization, esterification of α2,6-linked sialic acid, amidation of α2,3-linked sialic acid, enzymatic release of N-glycans, and chemical release of O-glycans (Figure 1). Glycoproteins are conjugated to the functionalized aldehydes on the resin via reductive amination Figure 1a); the immobilized glycoproteins are derivatized via esterification using hydroxybenzotriazole hydrate (HBot)/ethanol/ N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), formation of ethyl ester on α2,6-linked sialic acids (Figure 1b); glycoproteins are further reacted with p-Toluidine (pT) in the presence of EDC at pH 4-6 (Figure 1c), stabilization of α2,3-linked sialic acids; N-glycans are then released from the solid support (resin) and analyzed by MS (Figure 1e); O-glycans are cleaved by ammonia (β-elimination) in the presence of 1-phenyl-3methyl-5-pyrazolone (PMP) (Figure 1f). We used this novel method to study sialic acid linkages of sialylation on Madin-Darby Cannie Kidney (MDCK) cells commonly used for influenza propagation. Experimental Methods Protein immobilization for sialic acid derivatization. We used sialylglycopeptide (α2,3-linked sialic acid: SGP2,3; α2,6-linked sialic acid: SGP2,6; Fushimi Pharmaceutical Co. Ltd., Japan) (10 µg, 20 µg, 30 µg, and 40 µg), fetuin from bovine serum (20 µg), human serum (2 µL) (Sigma), and proteins from MDCK cells (0.5 mg). Except for peptide samples, proteins were denatured using 10 µL 10× denaturing buffer (New England BioLabs) (100 µL solution; 90 µL distilled water (DI)) at 100°C for 10 min. Samples were conjugated to Aminolink resin as following: 1) resin condition: wash resin using 500 µL 1× binding buffer (pH10, 50 mM sodium carbonate and 6 ACS Paragon Plus Environment

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100 mM sodium citrate) in a snap-cap spin-column (SCSC) (Fisher Scientific), vortexing and centrifugation at 2000× g for 30 sec, repeat once; 2) immobilization: add 100 µL to 325 µL DI, then add 50 µL 10× binding buffer. Samples were incubated 3 h before adding 25 µL of 1 M NaCNBH3 for another 3 h. The resin was washed with 500 µL 1× PBS twice, followed by adding 475 µL 1× PBS and 25 µL of 1 M NaCNBH3. The reaction was proceeding for 3 h. After washing the resin with 1 M Tris-HCl (500 µL, twice), the resin active sites were blocked with 1 M Tris-HCl in presence of 50 mM NaCNBH3. The resin was finally washed with 1 M NaCl (500 µL, twice), DI water (500 µL, twice) and ethanol (500 µL, twice); 3) α2,6-linked sialic acid ethyl esterification: prepare 250 µL 500 mM HBot and equal volume of 500 mM EDC.HCl in ethanol (anhydrous). These solutions were added to the samples in SCSC. The samples were incubated in a ThermoMixer C (Eppendorf) at 37°C for 2 h, followed by resin cleanup using ethanol (500 µL, twice), 1 M NaCl (500 µL, twice), and DI water (500 µL, twice); 4) α2,3-linked sialic acid pToluidine amidation: prepare 460 µL of 1 M p-Toluidine solution (400 µL 1 M HCl, 42.84 mg pT, 40 µL EDC, and 25 µL HCl (36-38%). Reaction was performed at room temperature (20~25°C) for 3 h; 5) N-glycan release: after washing resins with 10% formic acid (500 µL, thrice), 10% acetonitrile (ACN) (500 µL, thrice), 1 M NaCl (500 µL, thrice), and DI water (500 µL, thrice), PNGase F (1 µL) was added to the resins together with 30 µL of 10× GlycoBuffer (New England BioLabs), and 270 µL DI water. Digestion was conducted at 37°C for overnight. N-glycans were collected in the flow-through and purified by graphitized carbon column; 6) O-glycan βelimination: the resins were further washed with 1 M NaCl and DI water (500 µL, thrice). Oglycans were cleaved using 200 µL NH4HCO3 and 250 µL of 0.5 M PMP in methanol, incubated at 55°C for 24 h. The released O-glycans were extracted with chloroform and purified using C18 SPE column. Some of the details have been described in our previous works 39,44. Analysis of sialic acid linkages using neuraminidase. To compare the performance of chemical derivatization on sialic acids for linkage analysis, we use neuraminidase α2,3 (Neu3)



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and neuraminidase α2,3,6,8 (Neu3,6,8) to selectively remove sialic acids. The design of experiment was given in Supporting Information Table S1. Proteins from each cell line were aliquoted to three subsets (0.5 mg per subset). Proteins were immobilized to the Aminolink resin, as described previously. Further proteins were subjected to the treatment of neuraminidase. For example, London-MDCK includes L (no neuraminidase), L3 (neuraminidase α2,3), and L3,6,8 (neuraminidase 3,6,8). N-glycans were then released with PNGase F and purified with graphitized carbon column. Chemicals and reagents were purchased from Sigma unless specified otherwise. Results Proof of concept on differential linkages of sialic acids. To demonstrate whether sialic acid linkages can be differentially modified on the solid phase, we used standard sialylglycopeptide (SGP) SGP2,3 and SGP2,6 (Supporting Information Figure S1). The peptide sequence is H2NKVANKT-COOH SGP2,3 has α2,3 linkage between galactose and terminal sialic acid, whereas SGP2,6 has α2,6 linkage between them. Three samples were tested as shown in Figure 2: (a) 10 µg SGP2,3, (b) 10 µg SGP2,6, and (c) mixture of 10 µg SGP2,3 and 10 µg SGP2,6. Sample (a) and (b) were treated by HBot-EDC in ethanol, while (c) was sequentially treated by HBot-EDC in ethanol and pT-EDC following the procedures described in Figure 1. The released N-glycans were detected in MALDI-MS. The native sialic acid generates three peaks at 1663.6, 1976.7, and 2289.8 Da due to loss one and two sialic acid residues. Results showed that α2,3-linked sialic acid remains in its original form without reaction activity (Figure 2a). Several peaks were observed in MALDI due to loss of one and two sialic acids. In contrast, α2,6-linked sialic acid forms a stable ethyl ester, with a mass shift of 28.2 Da per sialic acid. After sample (c) was treated by both ethyl esterification and amidation, both α2,3-linked and α2,6-linked sialic acids were successfully modified. Two peaks are formed: one at 2302.1 Da with ethyl ester (α2,6) (28.2 Da), and another at 2423.9 Da with pT (α2,3) (89.0 Da) (Figure 2c). The result shows that


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a combination of amidation/esterification can be used to discriminate between sialic acid linkages. Linearity of the modified sialic acids. To characterize ionization of α2,3 and α2,6 sialic acids after chemical modification, we designed a study with known ratios between SGP2,6 and SGP2,3. The nominal ratio (SGP2,6/ SGP2,3) is 0.25, 0.67, 1.50, and 4.00. The SGP was conjugated to the resin and treated with reagents as described in Figure 1. The N-glycans were measured using MALDI-MS with an internal control (Maltoheptaose, DP7; 1 µM). The intensity of SGP2,6 (Int(α2,6)) was plotted against SGP2,3 (Int(α2,3)). As shown in Figure 2d, glycans modified by ethyl ester have slightly better ionization than pT (p-Toluidine) modified counterparts. Based on these data, we generated a polynomial fitting equation to correct the intensity of α2,3-linked sialic acids as shown in the Figure 2. The relative abundance of each sialic acid thus can be estimated. Quantitative analysis of sialic acids. Both bovine fetuin and human serum (Sigma) were evaluated using pT-only and ethanol-pT modification. Five sialylated N-glycans of high abundance have been reported in bovine fetuin via permethylation 45. To compare performance, one set of fetuin (20 µg) was immobilized to the resin for chemical modification using pT only, while the second set (20 µg) was modified using ethanol-pT. By pT modification only, the profile of N-glycans from fetuin is similar to that by permethylation, which stabilizes but does not differentiate between sialyl linkages. Peak compositions included S3H6N5, S2H5N4, S4H6N5, S2H6N5, and SH5N4 in a descending order (Figure 3a) (where N = HexNAc, H = Hexose, and S = NeuAc). However, fetuin N-glycans exhibit more complex pattern after chemical modification using our linkage specific procedure (Figure 3a&b; Supporting Information Table S2). Several observations were made: (1) all five sialylated N-glycans were identified using the ethanol-pT method; (2) several N-glycans contain more than one structure, e.g., S2H5N4 consisting of S22,6H5N4 and S2,6S2,3H5N4, or S3H6N5 consisting of S32,6H6N5, S22,6S2,3H6N4,



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and S2,6S22,3H6N5. (3) The relative abundance of N-glycans is similar, e.g., the most abundant N-glycan is the sum of S32,6H6N5, S22,6S2,3H6N5, and S2,6S22,3H6N5. The novel method can distinguish these linkages and provide relative quantitation of the sialyl species. We further applied the method for analysis of sialylated N-glycans in human serum. The profile of N-glycan composition is similar to that generated by permethylation, in which S2H5N4 is the highest abundant sialyl-N-glycans in serum 46. By combination of ethyl esterification with pT amidation, the linkages of sialic acids were readily determined (Supporting Information Figure S2). The non-sialylated N-glycans, or asialyl-N-glycans, have same profiles in pT or ethanol-pT modified method. Similar to the fetuin, some of sialyl-N-glycans have multiple structures due to different sialic acid linkages, e.g., S32,6H6N5 and S2,3S22,6H6N5. This cannot be easily elucidated by analyzing glycans through permethylation or amidation. However, the ratios and relative abundances of the different sialyl forms are readily discerned using the current method. Overexpressed sialylation on MDCK cells. Three different MDCK cell lines, MDCK derived by CBER (CBER-MDCK) (Xie et al. J Virol 2013) 47, MDCK of London lineage (London-MDCK) 48, and MDCK stably transfected with human 2,6-sialyltransferease (SIAT1-MDCK) 49, were cultured and used for analysis of sialylation. CBER-MDCK is a cloned subline that shows homogeneous morphology with cell borders hardly distinguishable in the monolayer (Figure 4a). In contrast, both London-MDCK and SIAT1-MDCK cells show heterogeneity in cell size and shape with cell borders clearly visible (Figure 4b&c). Using fluorescence-labeled lectins as probes, it was revealed that all three MDCK cell lines expressed both α2,3-linked and α2,6linked sialic acids as determined by flow cytometry (Figure 4). Both CBER-MDCK and SIAT1MDCK showed comparable expression of α2,3-linked sialic acids as indicated by mean fluorescence intensity (MFI) (Figure 4a&c), while London-MDCK cells had 50% reduction in MFI indicative of significantly less expression of α2,3-linked sialic acids (Figure 4b). Also compared


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to SIAT1-MDCK that had overexpression of α2,6-linked sialic acids (MFI= 64690, Figure 4), CBER-MDCK and London-MDCK showed approximately 50% and 70% reduction in MFI specific for α2,6-linked lectin, respectively (Figure 4a&b). These results suggested that overall London-MDCK cells had less expression of both α2,3-linked and α2,6-linked sialic acids than SIAT1-MDCK and CBER-MDCK cells. However, the flow cytometry analysis could not show the type of glycans expressed on these cell lines neither was able to reveal the kind of sialyl linkages involved. Thus, we applied our novel method that sequentially derivatizes α2,6-, α2,3-, and α2,6-linked sialic acids via chemical modification to analyze N-glycans and O-glycans expressing on MDCK cells. MALDI-MS profile of glycans in MDCK cell lines. Proteins from three cell lines were immobilized, chemically modified, and digested by enzymes as described in Method for differential analysis of sialic acid linkages. The N-glycans from three cell lines are listed in Supporting Information Table S3 and structures for each N-glycans are given in Supporting Information Table S4. A total of 86 N-glycans were identified, including asialyl-N-glycans (29), α2,6-linked sialic acids (39), α2,3-linked sialic acids (7), hybrid of α2,6- and α2,3-linked sialic acids (11). The MALDI-MS profile is given in Figure 5. Several observations were made from these data: (1) all cells are highly glycosylated by high-mannose, including Man3-9GlcNAc2. These N-glycans are the most abundant species in MDCK cells; (2) even though most sialic acids are identified in three cell lines, many of them are present in low abundance with negligible amount detected in London-MDCK and CBER-MDCK; (3) overall, sialyl-N-glycans are highly expressed in SIAT1-MDCK cells; (4) α2,6-linked sialic acids are the dominant sialyl-Nglycans (39), compared to α2,3-linked sialic acids (7) in SIAT1-MDCK cells; (5) fucosylated Nglycans are also upregulated in the overexpressed MDCK cells, whereas these N-glycans are barely detectable in wild-type cell lines.



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We further compared changes on different types of N-glycans in these cell lines. The twenty-nine asialyl-N-glycans were plotted as shown in Figure 6a, in which the fold-change is based on the ratio of median of all asialyl-N-glycans between SIAT1 versus London or CBER cells. The same strategy is employed to plot others for glycosylation comparison. If over 1.5-fold change is defined as biological significance, asialyl-N-glycans are essentially at the same level across SIAT1 (1.39-fold, p = 0.01), London, and CBER cells. This observation is agreement with the expectation. On the other hand, sialic acids have been upregulated regardless of sialic acid linkages. For instance, the expression of 57 sialyl-N-glycans are increased in SIAT1 (3.36-fold, p = 0.001) over London or CBER (Figure 6b). Among 57 N-glycans, 38 have α2,6-linked sialic acids, whose abundance in SIAT1 is 3.15-fold of those in London cells (Figure 6c). Similarly, the median intensity of six α2,3-linked sialic acids increased by 1.96 fold in SIAT1 (Figure 6d). In addition, the hybrid linkages of sialic acids are also significantly increased in SIAT1 cell lines (Figure 6e). As an example, we plotted the three sialylated N-glycans in three cell lines, illustrating their upregulation in the overexpressed SIAT1 cells (Figure 6f). We also characterized the O-glycans in the MDCK cell lines. As shown in Supporting Information Figure S3, there are three dominant O-glycans that are overexpressed in LondonMDCK and CBER-MDCK cells. London-MDCK has S2,6N, S22,6HFN, and S2,3HN; CBER-MDCK contains S22,6HFN and S2,3HN; SIAT1-MDCK only has S2,3HN. Although the SIAT1-MDCK shows overexpression of sialyl on its N-glycosylation, O-glycosylation was not overexpressed in these cells and was actually reduced for S2,6N and S22,6HFN. It should be noted that Oglycosylation can be impacted by alteration of glycosyl machinery since glycosylation reflects the coordinated effort of a complex array of enzymes and other factors 50. Our recent work discovered that inhibition of O-glycosylation regulated N-glycosylation in ovarian cancer cells 51. Our method is thus very useful for studying N-glycans and O-glycans through the same platform.


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Analysis of sialic acid linkages by neuraminidase. To demonstrate whether our method has advantage over other approaches for the analysis of linkages, we used neuraminidases to treat sialic acids. The experimental design can be found in Supporting Information Table S1, which includes proteins from three MDCK cells and treats proteins by neuraminidase- α2,3 (Neu2,3) or neuraminidase-α2,3,6,8 (Neu2,3,6,8). The N-glycans are profiled by MALDI-MS (Supporting Information Table S5). The N-glycans are classified as asialyl, α2,6-linked, and α2,3-linked. By neuraminidase digestion, we identified a total of 90 N-glycans from three MDCK cell lines, including 42 asialyl and 48 sialyl N-glycans. In contrast, the chemoenzymatic solidphase) identified 86 N-glycans including 29 asialyl, 39 α2,6-linked, 7 α2,3-linked, and 11 hybrid of α2,6 and α2,3-linked (Table 1). Enzymatic digestion enables the determination of asialyl oligosaccharides; however, it cannot determination of all glycosylic linkages for sialic acids. For example, Neu2,3 can determine α2,3, but no enzyme can determine α2,6. As shown in Table 1, eighteen sialyl Nglycans are determined with α2,6 linkage and nine with α2,3 linkage; eight N-glycans could be either α2,3 or α2,6 linkages; there are 13 N-glycans that cannot be determined with enzymatic digestion, we are able to determine all sialyl N-glycans and their sialyl linkages. Thus sialic acid linkages can be reliably investigated by the sequential chemical modifications. Discussion We have developed a novel mass spectrometry-based method for analysis of sialic acid linkages based on chemical modification. Because samples are carried out on a solid support, they can be easily processed by a variety of chemical modifications and enzymatic treatments. It is otherwise impractical to perform these chemistries without using the solid support. When solid-phase ethyl esterification is performed, it only modifies α2,6-linked sialic acids through formation of ethyl ester. To ensure complete reaction, an excess amount of chemicals can be added, which may be impractical for reaction in solution since the removal of excess chemicals 13 ACS Paragon Plus Environment


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is challenging using chromatographic cleanup 35. Any remaining reagent can be completely removed by extensive washing steps, inherently beneficial on sample preparation without loss of sample using this solid phase method. Next, α2,3-linked sialic acids are modified by use of amidation such as pT in the presence of EDC at pH 4-6. With these steps, we can easily distinguish α2,3- and α2,6-linked sialic acids. The method may be also possible to detect α2,8-linked oligo- or poly-sialic acids. It has been reported that α2,8-linked oligosialic acids or polysialic acids can form stable lactone via acid-catalyzed lactonization 52,53,54. The α2,3-linked sialic acid forms lactone intermediate but quickly hydrolyzes to its original form (Supporting Information Figure S4), whereas α2,8linked oligosialic acid may produce a stable lactone. Therefore, it is theoretically possible to distinguish α2,3 by amidation, α2,6 by esterification, and α2,8 by lactonization. We detected two peaks from human serum that may contain α2,8-linked sialic acids. The MS/MS fragmentation supports this hypothesis (Supporting Information Figure S5). Our on-going work is to verify this hypothesis by combining with enzymatic and lectin methods. Distinguishing between sialic acids linkages may provide an effective mean for studying sialylation in diseases. Our results show that fetuin N-glycans are much more complex than might be expected when the sialyl linkages are taken into account. Similarly, it is impossible to identify α2,3 versus α2,6 dependent disease biomarkers in human serum without considering their linkages, since most those circulating proteins are highly sialylated glycoproteins 55,56. Importantly, it has been shown that avian influenza viruses such as H5N1 could infect and spread in humans because they seem to target different regions of a patient’s respiratory tract, in which α2,3- and/or α2,6-linked sialic acids are present in different ratios and contexts along the airway passage 6. Likewise, overexpression of sialyltransferease should significantly enhance the synthesis of specific linkages of sialic acids in target cells, thus enhancing their susceptibility to influenza virus infection. For example, SIAT1-MDCK cells with α2,614 ACS Paragon Plus Environment

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sialyltransferease overexpression not only have substantially increased expression of α2,6-Galterminated glycans, but also have higher expression of α2,3-Gal-terminated glycans than London-MDCK cells. Enhanced expression of α2,6-linked sialic acids on SIAT1-MDCK promoted binding of human influenza virus resulting in increased sensitivity to neuraminidase inhibitors 49. Altered expression of α2,6-linked or α2,3-linked terminal sialic acids could dramatically change human susceptibility to avian influenza viruses. Additionally, MDCK cell lines are commonly used in influenza-related research and regulatory activities including basic virology and vaccine production. Thus, the ability to monitor the expression patterns of different linkages of sialic acids on MDCK cells can help to better inform choice of a cell substrate for individual viruses of interest. Given the importance of sialic acid expression in host cell susceptibility to influenza infections, we believe that our mass spectrometry-based chemical modification method will also be useful to assist in investigation of host range and adaptation for influenza and other viruses that target sialyl ligands. Elucidation of sialic acids on virus target tissues can be impacted by various factors, such as stabilization approach for sialic acids 34,35,57, chemical diversity of the sialic acids 58, ability to differential linkages of sialic acids 59, release of N- and O-linked glycans 51

, reproducibility of the method used 60, and purification methods used 45,61,62. This

chemoenzymatic method allows for permanent immobilization of glycoproteins or glycopeptides on solid-phase by formation of covalent bond between lysine or N-terminal with aldehyde 39,44. Stabilization of sialic acid linkages is achieved by sequential chemical reactions, while excess amount of chemicals can be used to ensure complete modification of sialic acids. More importantly, it allows for analysis of N-glycans and O-glycans by sequential release via enzymatic digestion and chemical treatment. It generates highly reproducible data and purification can be done using graphite column “clean-up” or direct analysis of samples can be performed when a volatile buffer is used for N-glycan cleavage.



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The chemoenzymatic solid-phase method can be easily scaled up for use as a highthroughput platform. It can also be adapted to microfluidic or pipette-tip based platform 29,63. A large number of samples can be performed with improved reproducibility. Acknowledgements We thank Dr. Yuan C. Lee and Dr. Ronald L. Schnaar from Johns Hopkins University for useful discussion on stability of lactonization of sialic acids. We thank Dr. Karli R. Reiding and Dr. Manfred Wuhrer for help on esterification experiments. Dr. Lisa Parsons helps on mass spectrometry instrumentation. Supporting Information The Supporting information is available free of charge on the ACS Publications website http.acs.analchem.com. Experimental methods, quantitative analysis of sialic acid linkages, MDCK o-glycans, MS/MS fragmentation in Supplementary Information. N-glycans and Oglycans from MDCK in Supporting Information Tables. Competing interests: The authors declare no competing financial interests. References 1.

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Figure 1. Schematic workflow of sequential derivatization of sialic acids via an enzymatic solid-phase method. (a) Immobilization: glycoproteins are conjugated to the Aminolink resin via reductive amination. The active aldehyde sites are blocked to prevent further reaction with chemicals and enzymes; (b) Esterification: α2,6-linked sialic acids react with ethanol (100%) in the presence of EDC; (c) Carbodiimide coupling: α2,3-linked sialic acids are modified with pToluidine in the presence EDC at pH 4-6; (d) Enzymatic release: N-glycans are digested with PNGases, whereas O-glycans remain on the solid support; (e) Flow-through: N-glycans are collected in the supernatant for LC-MS; (f) β-elimination: O-glycans are cleaved using ammonia in the presence of PMP.



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Figure 2. MALDI-MS analysis of α(2,6)-linked and α(2,3)-linked sialic acids after chemical modification by ethanol and p-Toluidine. (a) No modification on α(2,3)-linked sialylglycopeptide (SGP2,3) after ethanol esterification. Several peaks are observed in MALDIToF MS due to loss of one or two sialic acids; (b) Formation of ester on α(2,6)-linked sialylglycopeptide (SGP2,6); (c) Modification of SGP2,6 by esterification and SGP2,3 by amidation on the solid support sequentially; (d) Quantification of ionization of α(2,6)-linked versus α(2,3)linked SGP. The fitting curve is used for estimation of abundance of both sialic acid linkages. The error bars in (d) are calculated from standard deviation.


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Figure 3. Identification of different sialic acid linkages from bovine fetuin using chemical modification on the solid support. (a) Profiling of N-glycans from bovine fetuin with carbodiimide coupling by p-Toluidine-EDC. Five sialylated N-glycans are SH5N4, S2H5N4, SH6N5, S2H6N5, S3H6N5, and S4H6N5, where N = HexNAc, H = Hexose, and S = NeuAc. (b) Linkages of sialic acids present in bovine fetuin using esterification and amidation. Nine different sialylated N-glycans are present in bovine fetuin, including S2,6H5N4, S22,6H5N4/ S2,3S2,6H5N4, S22,6H6N5/ S2,6S2,3H6N5, S32,6H6N5, S32,6H6N5/ S22,6S2,3H6N5/ S2,6S22,3H6N5, and S32,6S2,3H6N6.



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Figure 4. The morphology of MDCK cell lines and their sialic acid expression determined by flow cytometry. Representative images (20X) of MDCK monolayers are shown. The percentage and mean fluorescence intensity (MFI) of MDCK cells stained positive for α2,3linked lectin (red peaks) or positive for α2,6-linked lectin (blue peaks) are displayed in representative flow cytometry histograms. Shaded peaks represent unstained cells. (a) CBERMDCK cells; (b) London-MDCK cells;(c) SIAT1-MDCK cells.


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Figure 5. MALDI-ToF MS profiles of N-glycans in MDCK cell lines. (a) N-glycans identified from London-MDCK cells. High mannose oligosaccharides are dominantly present, while sialic acids glycans are low in abundance; (b) High abundance sialic acids glycans are identified in sialyltransferease overexpressing SIAT1 cells; (c) Low abundance of sialic acids are detected in CBER cells.



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Figure 6. Comparison of asialyl- and sialyl-glycans in MDCK cell lines. (a) Relative abundance of asialyl oligosaccharides present in MDCK cell lines are shown. The same amount of asialyl-N-glycans (29) are detected in London, SIAT1, and CBER MDCK cells (p < 0.01); (b) Sialylated N-glycans across 57 structures are upregulated in sialyltransferease overexpressing MDCK cells (SIAT1) (3.36 fold, p = 0.001). London and CBER cells have similar levels of sialyl glycans; (c) α2,3-linked sialic acids in SIAT1 cells are increased by 1.96 fold over London cells, while CBER cells remain at same level; (d) α2,3-linked sialic acids in SIAT1 cells are upregulated by 3.15 fold in comparison to London or CBER cells; (e) Hybrid sialic acid with both α2,6 and α2,3 are also highly upregulated in overexpressed SIAT1 cells (2.93 fold); (f) Three most abundant sialic acids in three cell lines are shown.


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Table 1. Comparison of Glycans identified from MDCK cells using enzymatic (neuraminidase 2,3 and neuraminidase 2,6) or chemoenzymatic method (esterification and amidation). Proteins were immobilized on the resin for enzymatic digestion. α2,3 + α2,6 stands for hybrid sialic acid terminals consisting of both linkages; while α2,6 and/or α2,3 are sialic acids that can be either linkage. N/D = not detected. Identification (N-glycans or Linkages) Item

Chemoenzymatic solid-phase Enzyme (Our method)

Total N-glycans

90

86

Non-sialyl

42

29

α2,6

18

39

α2,3

9

7

α2,3 + α2,6

0

11

α2,6 and/or α2,3

8

0

N/D

13

0



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ToC

The universal platform for analysis of linkage of sialylated N-linked and O-linked glycans. Derivatization of sialic acis can distinguish α2,3, α2,6 and α2,8 via esterification and carbodiimide reaction. N-linked and O-linked glycans are sequentially released by enzymes and chemical reaction.


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Solid-phase chemical modification for sialic acid ... - ACS Publications

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