The sialic acids are a family of 9-carbon carboxylated sugars containing nearly 50 members that are derivatives of N-acetylneuraminic acid (Neu5Ac), N-glycolylneuraminic acid (Neu5Gc) and deaminoneuraminc acid (Kdn; 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid). Diversity in these structures arises by acetylation, sulfation, methylation, lactylation, and lactonization. In most cases, the sialic acids are present as monosialyl residues at the non-reducing terminal positions of glycan chains on glycoproteins and glycolipids. By virtue of their net negative charge at physiological pH, they serve as mediators of ligand-receptor and cell-cell interactions. In rare cases, the sialic acids are linked to each other to form a polymerized structure on glycoprotein, disialic acid (diSia), oligosialic acid (oligoSia), and polysialic acid (polySia). Recently, several glycoproteins that contain di/oligo/polySia structures are reported with highly sensitive chemical detection methods. In this protocol, we introduce the basic chemical labeling of sialic acid and polymerized sialyl structures. These methods leads to deep understanding on the diversity in sialic acid, linkage, and degree of polymerization. |
Category | Isolation & structural analysis of glycans |
Protocol Name | Specific labeling and chemical analysis of sialic acid and sialyloligo/polymers |
Authors
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Sato, Chihiro
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Bioscience and Biotechnology Center, Nagoya University
Kitajima, Ken
Bioscience and Biotechnology Center, Nagoya University
*To whom correspondence should be addressed.
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KeyWords |
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Reagents
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For chemical labeling of sialic acid
0.2 N Trifluoroacetic acid (TFA)
0.01 N TFA
1,2-diamino-4,5-methylenedeoxybenzene (DMB)1) solution, freshly prepared
7 mM of DMB dihydrochloride was dissolved in 5 mM trifluoroacetic acid containing 1 M 2-mercaptoethanol and 18 mM sodium hydrosulfite.
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For fluorometric C7/C9 analysis2)
Solution A, 40 mM sodium acetate buffer (pH 5.5), stored at 4°C
Solution B, 0.25 M periodate, freshly prepared
Solution C, 3% ethyleneglycol, stored at 4°C
Solution D, 0.2 M sodium borohydride in 0.2 M sodium borate buffer (pH 8.0)
Solution E, 0.2 M trifluoroacetic acid, stored at 4°C
Solution F, 0.01 M trifluoroacetic acid, stored at 4°C
Solution G, DMB solution,
7 mM of DMB dihydrochloride was dissolved in 5 mM trifluoroacetic acid containing 1 M 2-mercaptoethanol and 18 mM sodium hydrosulfite.
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For mild hydrolysis and anon exchange chromatography3) 4)
0.01 M trifluoroacetic acid
DMB solution
7 mM of DMB dihydrochloride was dissolved in 5 mM trifluoroacetic acid containing 1 M 2-mercaptoethanol and 18 mM sodium hydrosulfite. |
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Instruments
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For chemical labeling of sialic acid
HPLC system equipped with one pump, one fluorescent detector, and a recorder
Column: TSK-gel ODS-120T (250×4.6 mm i.d.)
Solvent: methanol/acetonitrile/water (7:9:84, v/v/v)
Elution: isocratically at 1.0 mL/min
Detection: Fluorescent detector (Ex, 373 nm; Em, 448 nm) |
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For fluorometric C7/C9 analysis2)
HPLC system equipped with one pump, one fluorescent detector, and recorder
Column: TSK-gel ODS-120T (250×4.6 mm i.d.)
Solvent: methanol/acetonitrile/water (7:9:84, v/v/v)
Elution: isocratically at 1.0 mL/min
Detection: Fluorescent detector (Ex, 373 nm; Em, 448 nm) |
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For mild hydrolysis and anon exchange chromatography3) 4)
HPLC system equipped with gradient pump, one fluorescent detector, and recorder
Column: Mono or Mini Q HR5/5 (0.5 × 5 cm, GE Healthcare, Little Chalfont, UK),
Resource Q (1 mL, GE Healthcare), or CarbopacPA-100 (4 ×250 mm, Dionex) anion exchange column
Solvent: a linear gradient of NaCl (0–0.4M) after 15–30 min wash with 5 to 20 mM Tris-HCl (pH 8.0)
Elution: isocratically at 0.5 to 1.0 mL/min
Detection: Fluorescent detector (Ex, 373 nm; Em, 448 nm) |
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Methods |
1. |
Chemical labeling of sialic acid
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1) |
Hydrolyze sialoglycoconjugates with 0.2N TFA for 2 h to release sialic acid. |
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Incubate with equal volume of 0.1N TFA and DMB solution at 50°C for 2 h. |
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Inject into the HPLC system as described above. |
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2. |
Fluorometric C7/C9 analysis2)
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Samples (0.25–1000 ng as Sia) dissolved in 25 μL of Solution A. |
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Add 2 μL of Solution B and left at 0°C for 3 h in the dark. |
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Add 5 μL of Solution C and 32 μL of solution D and stood at 0°C overnight (more than 3 h). |
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Set volume to 100 μL with water and add 100 μL of Solution E to adjust to 0.1 M trifluoroacetic acid then perform same procedure of 1. |
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3. |
Mild hydrolysis and anon exchange chromatography3) 4)
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Samples (0.25–1000 ng as Sia) dissolved in 20 μL of 0.01N TFA |
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Add 20 μL of DMB solution and incubate at 50°C for 1 h or 4°C for over night |
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Analyze with HPLC as described above |
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Discussion | Chemical labeling of sialic acid with DMB (Fig. 1) is easy and highly sensitive way; however, we have to take other α-keto acid molecules abundant in cells into account. Therefore, it is very important to confirm the peak by co-injection with authentic sample or by mass spectrometry. In addition, due to highly sensitive methods, we have to take care of the contamination from the outside or from food the organism takes.
As for fluorometric C7/C9 analysis, the following limitations are noted in the application of this method. First, this method is applicable to only α2→8-linked oligo/polymer of N-acylneuramininc acid and N-glycolyneuraminic acid, and cannot be used for DP analyses of α2→9, α2→8/α2→9-mixed linkage polymers or α2→5Oglycolyl-linkage. Secondly, the detected C9-derivatives do not always arise from α2→8-linkages, because 8-O-substituted Neu5Acyl residues may also give the same C9-derivatives. Therefore, mild alkali-treatment of samples for saponification is usually carried out prior to periodate oxidation. Thirdly, the molar proportion of C9-derivatives to C7-derivatives does not directly represent the DP unless it is from a linear polySia chain. Thus, the method does not yield the DP for multiply sialylated chains present in the same sample.
In case of the detection of oligo/polySia, we have to take it into account that oligo/polySia easily degrades under mild acidic condition. Therefore, it is difficult to confirm the precise DP of the original oligo/polySia containing glycan. We can tell the largest DP we can observe. |
Figure & Legends |
Figure & Legends
Fig. 1. Structure of DMB-Sia |
Copyrights |
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Date of registration:2015-03-24 13:53:03 |
- Hara, S., Takemori, Y., Yamaguchi, M., Nakamura, M., and Ohlura, Y. (1987) Fluorometric high-performance liquid chromatography of N-acetyl- and N-glycolylneuraminic acids and its application to their microdetermination in human and animal sera, glycoproteins, and glycolipids Anal. Biochem. 164, 138–145 [PMID : 3674364]
- Sato, C., Inoue, S., Matsuda, T., and Kitajima, K. (1998) Development of a highly sensitive chemical method for detecting alpha2-->8-linked oligo/polysialic acid residues in glycoproteins blotted on the membrane., Anal Biochem 261, 191–197 [PMID : 9716421]
- Sato, C., Inoue, S., Matsuda, T., and Kitajima, K. (1999) Fluorescent-assisted detection of oligosialyl units in glycoconjugates., Anal Biochem 266, 102–109 [PMID : 9887218]
- Sato, C. and Kitajima, K. (2012) Advanced technologies in sialic acid and sialoglycoconjugate analysis. Topics Curr. Chem., in press
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