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Specific labeling and chemical analysis of sialic acid and sialyloligo/polymers
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Specific labeling and chemical analysis of sialic acid and sialyloligo/polymers

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Introduction Protocol References Credit lines
Category
Isolation & structural analysis of glycans
Protocol Name

Specific labeling and chemical analysis of sialic acid and sialyloligo/polymers

Authors
Sato, Chihiro *
Bioscience and Biotechnology Center, Nagoya University

Kitajima, Ken
Bioscience and Biotechnology Center, Nagoya University
*To whom correspondence should be addressed.
KeyWords
Reagents

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.

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.


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.

Instruments

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)

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)

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)

Methods
1.

Chemical labeling of sialic acid

1) 

 Hydrolyze sialoglycoconjugates with 0.2N TFA for 2 h to release sialic acid.

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2) 

 Dry up with speed vaq.

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3) 

 Incubate with equal volume of 0.1N TFA and DMB solution at 50°C for 2 h.

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4) 

 Inject into the HPLC system as described above.

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2.

Fluorometric C7/C9 analysis2)

1) 

 Samples (0.25–1000 ng as Sia) dissolved in 25 μL of Solution A.

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2) 

 Add 2 μL of Solution B and left at 0°C for 3 h in the dark.

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3) 

 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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4) 

 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)

1) 

 Samples (0.25–1000 ng as Sia) dissolved in 20 μL of 0.01N TFA

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2) 

 Incubate at 50°C for 1 h.

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3) 

 Add 20 μL of DMB solution and incubate at 50°C for 1 h or 4°C for over night

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4) 

 Analyze with HPLC as described above

Comment 0
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

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