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Pure and Applied Chemistry

The Scientific Journal of IUPAC

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Volume 89, Issue 7

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1,3-Di(2-dipyridyl)propan-1,3-dione – a new fluorogenic labeling reagent for milk oligosaccharides

Zhi Peng Cai
  • Glycomics and Glycan Bioengineering Research Center (GGBRC), College of Food Science and Technology, Nanjing Agricultural University, Nanjing, People’s Republic of China
  • Other articles by this author:
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/ Wen Li Wang
  • Glycomics and Glycan Bioengineering Research Center (GGBRC), College of Food Science and Technology, Nanjing Agricultural University, Nanjing, People’s Republic of China
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/ Louis Conway
  • Glycomics and Glycan Bioengineering Research Center (GGBRC), College of Food Science and Technology, Nanjing Agricultural University, Nanjing, People’s Republic of China
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/ Kun Huang
  • Glycomics and Glycan Bioengineering Research Center (GGBRC), College of Food Science and Technology, Nanjing Agricultural University, Nanjing, People’s Republic of China
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/ Faisal Nureldin Awad
  • Glycomics and Glycan Bioengineering Research Center (GGBRC), College of Food Science and Technology, Nanjing Agricultural University, Nanjing, People’s Republic of China
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/ Li Liu
  • Corresponding author
  • Glycomics and Glycan Bioengineering Research Center (GGBRC), College of Food Science and Technology, Nanjing Agricultural University, Nanjing, People’s Republic of China
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/ Josef Voglmeir
  • Corresponding author
  • Glycomics and Glycan Bioengineering Research Center (GGBRC), College of Food Science and Technology, Nanjing Agricultural University, Nanjing, People’s Republic of China
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  • Other articles by this author:
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Published Online: 2017-03-18 | DOI: https://doi.org/10.1515/pac-2016-0914

Abstract:

We herein demonstrate the use of 1,3-di(2-dipyridyl)propan-1,3-dione (DPPD) as a fluorogenic label for oligosaccharides. A number of milk-derived oligosaccharide standards were successfully labeled with this reagent, with the advantage of greatly simplified sample preparation compared to other commonly used fluorescent tags. DPPD shows a selectivity for oligosaccharides which do not possess a 2-acetamido-2-deoxy-hexose moiety at the reducing terminus, potentially aiding in the identification of complex mixtures of carbohydrates. The use of DPPD for the structural determination of oligosaccharides through exoglycosidase treatment, quantitative analysis of reactions, and in the synthesis of labeled oligosaccharides was also explored. This reagent has, in addition to the analysis of individual and mixed oligosaccharides, potential applications in the study of glycosidases and glycosyltransferases and as such represents a valuable addition to the tools available to the glycoscientist.

Keywords: DPPD; fluorescence; free milk oligosaccharides; high-performance liquid chromatography (HPLC); ICS-28; oligosaccharide labeling; oligosaccharides

Article note:

A collection of invited papers based on presentations at the XXVIII International Carbohydrate Symposium (ICS-28), New Orleans, July 17–21, 2016.

Introduction

The remarkable biological roles played by naturally occurring oligosaccharides have become increasingly apparent in recent years. Some particularly notable oligosaccharides are those derived from human milk, which, among other biological functions, have been reported to prevent infection by pathogens in neonates, and act as prebiotics by stimulating the growth of beneficial bacteria such as bifidobacteria [1], [2], [3]. Furthermore, these oligosaccharides have been demonstrated to be beneficial for brain development and the maturation of the immune system in newborns [4], [5]. Due to the fact that the function of these compounds is highly dependent on their structure and that the oligosaccharides, especially milk oligosaccharides, extracted from natural sources tend to be mixtures with high structural diversity, it is essential to isolate and elucidate the structures of individual oligosaccharides for functional investigation. While HPLC is a powerful technique for the identification of oligosaccharides, an initial derivatization step is required to facilitate analysis since these biomolecules do not, in general, possess chromophores.

Fluorescent labeling has been reported to permit more sensitive analysis of carbohydrates in comparison to the use of UV-active tags [6], and so a number of these techniques have been developed [7]. Two of the most commonly used fluorescent derivatization agents are 2-aminobenzamide (2-AB) [8] and 2-aminopyridine (2-AP) [9], which are affixed to the reducing end of the oligosaccharide through a reductive amination using dimethylaminoborane, sodium cyanoborohydride or pyridine-borane as the reducing agents [7]. A major drawback of these techniques is that cleanup procedures are normally required to remove the excess derivatization reagent, which can cause significant interference with the analysis. Excess 2-AB is normally removed through paper chromatography [10], or by extraction with ethyl acetate [11]. 2-AP-derivatized oligosaccharides are generally purified by gel filtration [12], ion-exchange chromatography [13] or extraction with benzene [14]. However, all of these procedures are time-consuming and result in sample loss to some extent [15].

We have previously reported the development of a method for fluorescent derivatization of monosaccharide using a novel fluorogenic labeling reagent, 1,3-di(2-dipyridyl)propan-1,3-dione (DPPD) [16]. The non-fluorescent DPPD reacts with the monosaccharide aldehyde to create a fluorophore which can be detected by HPLC with high sensitivity. No removal of excess DPPD is required due to the non-fluorescent nature of the unreacted reagent. Here, we seek to demonstrate the validity of this labeling technique when applied to structurally diverse mixtures of oligosaccharides. We also seek to apply fluorescent DPPD labeling to the structural analysis of oligosaccharides, and to monitoring the progress of enzymatic reactions.

Materials and methods

Chemicals and enzymes

Oligosaccharide standards (see Fig. 1) α-l-Fuc-(1→2)-β-d-Gal-(1→4)-d-Glc (2′-FL), β-d-Gal-(1→3)-β-d-GlcNAc-(1→3)-β-d-Gal-(1→4)-d-Glc (LNT), β-d-Gal-(1→4)-β-d-GlcNAc-(1→3)-β-d-Gal-(1→4)-d-Glc (LNnT), α-l-Fuc(1→2)-β-d-Gal-(1→3)-β-d-GlcNAc-(1→3)-β-d-Gal-(1→4)-d-Glc (LNFP I), β-d-Gal-(1→3)-(α-l-Fuc-[1→4])-β-d-GlcNAc-(1→3)-β-d-Gal-(1→4)-d-Glc (LNFP II), β-d-Gal-(1→4)-[α-l-Fuc-(1→3)]-β-d-GlcNAc-(1→3)-β-d-Gal-(1→4)-d-Glc (LNFP III), α-l-Fuc-(1→2)-β-d-Gal-(1→3)[α-l-Fuc-(1→4)]-β-d-GlcNAc-(1→3)-β-d-Gal-(1→4)-d-Glc (LNDFH I) and β-d-GlcNAc-(1→2)-α-d-Man-(1→3)[β-d-GlcNAc-(1→2)-α-d-Man-(1→6)]-β-d-Man-(1→4)-β-d-GlcNAc-(1→4)-d-GlcNAc (NGA2) were purchased from Prozyme (Shanghai, China). α-Neu5NAc-(2→3)-β-d-Gal-(1→4)-d-Glc (3′-SL) was supplied by J&K (Beijing, China). Lactose monohydrate was obtained from Aladdin (Shanghai, China). All exoglycosidases were obtained from Prozyme (Shanghai, China). 1,3-di(2-dipyridyl)propan-1,3-dione (DPPD) was obtained from JK (Shanghai, China). Acetonitrile used for HPLC was purchased from Merck (Nanjing, China). All other chemicals used in this work were of the highest grade commercially available.

The free human milk oligosaccharides standards used in this study.
Fig. 1:

The free human milk oligosaccharides standards used in this study.

Derivatization of oligosaccharides with DPPD

Oligosaccharides were labeled with DPPD according to a previously described method [16]. Briefly, 5 μL of 0.16 M NaHCO3 solution and 30 μL of DPPD dissolved in methanol (0.1 M) were combined with 5 μL of 1 mM oligosaccharide solution. The mixture was maintained at 110°C overnight in a heat- and pressure-resistant resealable screw-capped glass tube. Samples were diluted in water or 80% acetonitrile prior to HPLC analysis.

HPLC detection conditions

Chromatographic separation of oligosaccharides was carried out using a Shimadzu LCMS 2020 system (Shimadzu Corporation, Kyoto, Japan), with a DGU-20A5R degasser, LC-30AD pump, SIL-30AC autosampler, RF-20Axs fluorescence detector (excitation 330 nm, emission 390 nm), and an ESI mass spectrometer. The analysis were performed using either a reversed phase column (Phenomenex Hyperclone 5 μm ODS 120 Å, 250×4.60 mm) or an Acquity BEH Glycan column (Waters 1.7 μm, 150×2.1 mm). The mobile phases were NH4COOH (pH 4.5, 50 mM) in water and acetonitrile for solvents A and B, respectively.

For the reversed phase column, a flow rate of 1.5 mL/min and injection volume of 10 μL were used. A linear gradient of 5–8% B was applied from 0–20 min; B was then increased to 95% over 1 min and held at 95% for 2 min. B was then decreased to 5% in 1 min, and the column was equilibrated with 5% B for 6 min. The elution profile for the column was as follows: a gradient of 95–78% B was applied from 0 to 6 min at a flow rate of 0.5 mL/min; 78–70% B from 6 to 20 min at 0.5 mL/min; 70–0% B in 1 min at 0.25 mL/min; held for 2 min at 0.25 mL/min; 0–95% B in 2 min at 0.25 mL/min; held for 1.5 min at 0.25 mL/min; the flow rate increased from 0.25 to 0.5 mL/min from 26.5–29.5 min; and finally the column was equilibrated with 95% B for 3.5 min at 0.5 mL/min before the next sample injection.

Structural analysis of a DPPD labeled oligosaccharide standard by exoglycosidase digestion and HPLC

The DPPD-labeled LNFP II standard was selected for sequence mapping through HPLC profiling. The oligosaccharide was digested for 16 h at 37°C by a β-galactosidase (Streptococcus pneumoniae) with a specificity for β(1-4)-linked galactose, and an α-fucosidase (Almond meal) with a specificity for non-reducing terminal α(1-3, 4)-linked fucose residues. The digestion was carried out according to the manufacturer’s instruction in a total volume of 10 μL. The reaction mixtures were diluted in 80% acetonitrile prior to HPLC analysis.

Sialyltransferase activity assay

The synthesis of sialylated DPPD-lactose was performed according to the procedures previously described by Huang et al. with minormodifications [17], [18]. Lactose (5 μL, 0.02 M solution in water) was labeled with DPPD and the solvents were removed under vacuum. To the residue was added Neu5Ac or Neu5Gc solution (5 μL, 0.02 M), CTP solution (5 μL, 0.02 M), MgCl2 solution (5 μL, 0.02 M), MES buffer (8 μL, 0.02 M, pH 6.5), α2,6-sialyltransferase (PdST6, 4 μL, ≥7mU), CMP-sialic acid synthase (NmCSS, 4 μL, ≥7 mU) and de-ionized water (5 μL). The mixture was incubated at 37°C for 16 h. The proteins were removed through extraction with chloroform and the aqueous remainder was analyzed by HPLC.

Results and discussion

HPLC detection of DPPD-labeled oligosaccharides

The oligosaccharide standards from milk employed in this study are shown in Fig. 1. The products were subjected to both reverse-phase (RP) chromatography and hydrophilic-interaction liquid chromatography (HILIC), which are the two most widely used methods for sugar separation [17], [18], [19], [20]. All eight oligosaccharide standards were successfully detected by the two methods when run separately, which indicated the success of the DPPD labeling procedure. Since the retention of the analyte on the HILIC column is based on hydrophilic interactions [19], the elution order of the oligosaccharides standards is, with some exceptions, the inverse of that on RP-HPLC. For some closely structurally related pairs of isomers, such as LNT and LNnT, and LNFP III and LNFP II, no good separation could be achieved with either column. The peaks in RP-HPLC profile (Fig. 2b) were noticeably broader and more poorly separated than the corresponding peaks generated using the Acquity BEH glycan column (Fig. 2c), and for this reason the Acquity BEH Glycan column was chosen for further experiments. It is worth noting that when this column is used, the acetonitrile content of the sample solvent must be similar to the initial solvent gradient composition, since excess water may affect the hydration layer of the stationary phase and result in a poor chromatogram [20].

HPLC chromatograms of DPPD-labeled human milk oligosaccharide standards were acquired using different columns. a) The labeling procedure. b) Chromatograms acquired using a reverse phase column (Phenomenex Hyperclone 5 μm ODS 120 Å, 250×4.60 mm); c) Chromatograms acquired using an Acquity BEH Glycan column (Waters 1.7 μm, 2.1×150 mm).
Fig. 2:

HPLC chromatograms of DPPD-labeled human milk oligosaccharide standards were acquired using different columns. a) The labeling procedure. b) Chromatograms acquired using a reverse phase column (Phenomenex Hyperclone 5 μm ODS 120 Å, 250×4.60 mm); c) Chromatograms acquired using an Acquity BEH Glycan column (Waters 1.7 μm, 2.1×150 mm).

Comparison of DPPD with 2-AB

To mimic a natural oligosaccharide sample which normally contains more than one structure, the standards were combined and derivatized with both DPPD and 2-AB. Both samples were analyzed by HPLC using the Acquity BEH Glycan column and identical elution profiles. As shown in Fig. 3a, the order of elution is the same for oligosaccharides labeled with both DPPD and 2-AB. However, the DPPD-labeled sugars were eluted faster than those labeled with 2-AB, indicating that 2-AB-labeled oligosaccharides are more hydrophobic than their DPPD-labeled counterparts. In both cases, the labeling efficiency was clearly structure-dependent as the HPLC peak areas varied despite the equal concentrations of each standard in the sample. Moreover, differences between the peak areas in the HPLC profiles of the DPPD-labeled and 2-AB-labeled samples indicate that the labeling efficiency was also reagent-dependent. The two pairs of isomers (peak c and d and peak f and g), which were co-eluted when labeled with DPPD, could also not be separated when labeled with 2-AB. The complete chromatographic resolution of all oligosaccharides in a sample, especially isomers, is a perennial problem; one way of circumventing this issue is to use a different column, such as porous graphitic carbon (PGC) which has been reported to excel in the resolution of isomeric oligosaccharides [19], used in combination with a HILIC or RP column in a multidimensional HPLC system for improved separation. Alternatively, digestion with highly structurally specific exoglycosidases in combination with HPLC analysis is another potential solution.

HPLC chromatograms of a): 2-AB and b): DPPD-labeled human milk oligosaccharides standards.
Fig. 3:

HPLC chromatograms of a): 2-AB and b): DPPD-labeled human milk oligosaccharides standards.

The successful use of DPPD for labeling mono- and oligo-sugars led us attempt to extend this methodology to N-glycan labeling using NGA2 as a test substrate. However, after analysis by HPLC, no peak corresponding to the DPPD-labeled NGA2 was observed, while the peak for 2-AB labeled NGA2 was clearly present. The inability of DPPD to label this substrate is likely due to the presence of GlcNAc at the reducing end of the oligosaccharide. Since GlcNAc does not possess the C2 hydroxy group required for the formation of the furan moiety which makes up part of the fluorophore, the labeled oligosaccharide exhibits no fluorescence. In contrast, the milk-derived oligosaccharides from this study, and the monosaccharides tested in our previous study [16] all possessed C2 hydroxy groups on the root saccharide and were all successfully labeled. The reaction of a carbohydrate with 2-AB requires only a carbonyl group, [7] and so GlcNAc can be successfully labeled using this reagent. The ability of DPPD to thus discriminate between N-glycans and other oligosaccharides will be of great use for the analysis of oligosaccharides from natural sources.

Despite the dependence of labeling efficiency on both sugar structure and choice of labeling reagent which we have noted above, the use of 2-AB in conjunction with HPLC for the relative quantification of carbohydrates after exoglycosidase digestion has been reported [21], [22]. We wished to determine whether DPPD could be used for similar applications, and to this end we investigated the relationship between labeling efficiency and sample concentration. Lactose, 3′-fucosylactose, and 3′-sialyllactose in concentrations ranging from 0.04 to 0.4 mM were labeled with DPPD as well as 2-AB (Fig. 4). Despite the substrate- and reagent-dependent differences in labeling efficiency, strong linear relationships between peak area and sample quantity were observed for all three samples using both labeling reagents, indicating that the labeling efficiency does not vary significantly with concentration and that DPPD can thus be used to monitor the relative concentration of a carbohydrate.

(a) Calibration curves of lactose, 3′-fucosylactose and 3′-sialyllactose labeled with DPPD; (b) calibration curves of lactose, 3′-fucosylactose and 3′-sialyllactose labeled with 2-AB. ×, 3′-fucosylactose; ▲, lactose; ■, 3′-sialyllactose.
Fig. 4:

(a) Calibration curves of lactose, 3′-fucosylactose and 3′-sialyllactose labeled with DPPD; (b) calibration curves of lactose, 3′-fucosylactose and 3′-sialyllactose labeled with 2-AB. ×, 3′-fucosylactose; ▲, lactose; ■, 3′-sialyllactose.

Application to an exoglycosidase assay

Exoglycosidase digestion is a powerful tool for determining the sequence and structure of glycan chains [23]. A mixture of glycans can be separated into individual oligosaccharides and each of these oligosaccharides can be digested sequentially with various linkage-specific exoglycosidases. In order to verify the applicability of DPPD to the exoglycosidase treatment of oligosaccharides, LNFP II containing terminal fucose and galactose was employed as a standard. This oligosaccharide was labeled with DPPD, and subjected to digestion by β-galactosidase and α-fucosidase separately and in combination, and the products of digestion were analyzed by HPLC. As shown in Fig. 5, no change in retention time was observed after β-galactosidase digestion. The peak shifted from a to c after the treatment with α-fucosidase, and peak c further shifted to peak d after the treatment with β-galactosidase (Fig. 5). The α-fucosidase used in this report can release non-reducing terminal α(1-3, 4)-linked fucose, while β-galactosidase can release β(1-4)-linked galactose from the non-reducing terminus of an unbranched oligosaccharide. The results of this study clearly show that DPPD labeling can facilitate sugar structure analysis or assays of exoglycosidase activity.

The DPPD labeled LNFP II was digested with various exoglycosidases. a) Before digestion. b) Digestion with β(1-3,4)-galactosidase. c) Digestion with β(1-3,4)-fucosidase. d) Digestion with both β(1-3,4)-galactosidase and β(1-3,4)-fucosidase.
Fig. 5:

The DPPD labeled LNFP II was digested with various exoglycosidases. a) Before digestion. b) Digestion with β(1-3,4)-galactosidase. c) Digestion with β(1-3,4)-fucosidase. d) Digestion with both β(1-3,4)-galactosidase and β(1-3,4)-fucosidase.

Application to a sialyltransferase assay

The use of glycosyltransferases to synthesize oligosaccharides is a valuable alternative to chemical approaches [24]. Methods for monitoring the progress of reactions catalyzed by these enzymes are therefore of great utility. In order to investigate the suitability of DPPD for this application, the synthesis of two lactose-based sialosides (one containing Neu5Ac and the other containing Neu5Gc) was carried out. The synthesis of the sialic acid-containing structures required CMP-sialic acid synthetase and α2,6-sialyltransferase enzymes, which were cloned from Neisseria meningitidis and Photobacterium damsela, respectively. Initially the sialic acid (Neu5Ac or Neu5Gc) is converted to the corresponding CMP-sialic acid by CMP-sialic acid synthase. The α2,6-sialyltransferase then catalyzes the transfer of a sialyl group from CMP- sialic acid to a glycosyl acceptor which in these reactions was DPPD-Lac (Fig. 6a). As shown in Fig. 6b, two chemo-enzymatically synthesized DPPD-Lac sialosides were obtained by these means. The peaks corresponding to DPPD-Lac-Neu5Ac and DPPD-Lac-Neu5Gc, were clearly differentiated, despite the difference in their structures of only a single oxygen atom. This one-pot multiple-enzyme reaction system is an efficient means of producing sialic acid-containing structures, avoiding the separation and purification of intermediate CMP- sialic acid conjugates. Yu et al. have performed these reactions using 3-azidopropyl lactoside or 4-methylumbelliferyl α-d-lactoside as glycosyl acceptors, indicating that while a terminal galactose residue is required, the transferase is otherwise tolerant of structural diversity in the oligosaccharide acceptor [25], [26]. DPPD-labeled lactose provides a convenient foundation for the construction of sialic acid-containing structures which may find application as substrates for neuraminidases and study of other biological processes.

(A) The enzymatic sialylation of DPPD-labeled lactose. (B) HPLC analysis of sialyltransferase activity. a: A reaction mixture containing CMP-sialic acid synthetase, α2,6-sialyltransferase and Neu5Ac. b: A reaction mixture containing CMP-sialic acid synthetase, α2,6-sialyltransferase and Neu5Gc. c: A reaction mixture containing only lactose.
Fig. 6:

(A) The enzymatic sialylation of DPPD-labeled lactose. (B) HPLC analysis of sialyltransferase activity. a: A reaction mixture containing CMP-sialic acid synthetase, α2,6-sialyltransferase and Neu5Ac. b: A reaction mixture containing CMP-sialic acid synthetase, α2,6-sialyltransferase and Neu5Gc. c: A reaction mixture containing only lactose.

Conclusion

In this report we described a new fluorogenic tag for oligosaccharides, DPPD. Several oligosaccharides standards from human milk were labeled with this reagent and were analyzed by RP-HPLC or HILIC-HPLC with fluorescence detection. The resolution of the resulting chromatograms was found to be comparable to those generated with commonly used fluorescence tags such as 2-AB, with the added advantage that there is no requirement for purification prior to HPLC, which may be both time-consuming and laborious, and result in sample loss. The specificity of DPPD for oligosaccharides with 2-hydroxy groups at the reducing monosaccharides enables DPPD to act as a selective labeling reagent for mixed samples, for example distinguishing between hexoses and N-acetyl hexoses. While the labeling efficiency of DPPD did appear to be structure dependent, it was shown not to depend on the concentration of the substrate, which should allow this reagent to be used for the quantitative monitoring of reactions. The use of DPPD-labeled lactose as a sialyltransferase acceptor in a one-pot, multiple enzyme reaction system was also demonstrated, and this approach may have future applications in glycosyltransferase and glycosidase screening experiments.

Abbreviations

2-AB

2-aminobenzamide

2-AP

2-aminopyridine

CMP

cytidine 5′-monophosphate

DPPD

1,3-di(2-dipyridyl)propan-1,3-dione

2′-FL

2′-fucosyllactose

HILIC

hydrophilic-interaction liquid chromatography

HPLC

high-performance liquid chromatography

Lac

lactose

LNT

lacto-N-teraose

LNnT

lacto-N-neotetraose

LNFP I

lacto-N-fucopentaose I

LNFP II

lacto-N-fucopentaose II

LNFP III

lacto-N-fucopentaose III

LNDFH I

lacto-N-difucohexaose I

Neu5Ac

N-acetylneuraminic acid

Neu5Gc

N-glycolylneuraminic acid

NmCSS

Neisseria meningitidis CMP-sialic acid synthase

PdST6

Photobacterium damselae α-2,6-sialyltransferase

2-PF

2-pyridylfuran

RP

reversed-phase

Acknowledgments

The authors would like to thank the Natural Science Foundation of China (Projects 31471703, A0201300537 and 31671854 to J.V. and L.L.), the Natural Science Foundation of Jiangsu Province (Projects Q0201500580 to L.L. and BK20160731 to Z.P.C.) and the 100 Foreign Talents Plan (grant number JSB2014012 to J.V.).

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About the article

Zhi Peng Cai, Wen Li Wang, and Louis Conway: These authors contributed equally to this work.


Published Online: 2017-03-18

Published in Print: 2017-07-26


Citation Information: Pure and Applied Chemistry, Volume 89, Issue 7, Pages 921–929, ISSN (Online) 1365-3075, ISSN (Print) 0033-4545, DOI: https://doi.org/10.1515/pac-2016-0914.

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