JP2009162530A - Chemical modification method of biocomponent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a quantitative amidation method of a sialo-sugar chain usable for marker search. <P>SOLUTION: In the amidation method of a carboxyl group of a sialic acid residue executed by mixing the sialo-sugar chain with a nucleophilic agent and by adding a condensation agent thereto, which is characterized by generating a condensation reaction at pH3 or lower, a compound having a hydrazide group and carbodiimide are used as a preferable nucleophilic agent and a preferable condensation agent, respectively. By chemical modification of a biocomponent, quantitative and highly-sensitive detection of the sialo-sugar chain by a mass spectrometry which is difficult hitherto, and comparative quantitative analysis of a biomarker having the sialo-sugar chain become possible. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a technique for improving analytical sensitivity in mass spectrometry of biological components, and more particularly to a technique for improving analytical sensitivity by chemically modifying a sialo-glycan that is a biological component.

A protein having a sialoglycochain has attracted attention as a new diagnostic marker that has been suggested to be associated with diseases such as cancer.
For the purpose of marker search, mass spectrometry is an effective means to detect the sialoglycan sensitively, but sialic acid is unstable and easily decomposed. Analytical methods are used.

Examples of such chemical modification methods include complete methylation, esterification, and amidation.
Since complete methylation is performed under strongly alkaline conditions, sugar chains having an O-acetyl group are decomposed. In addition, since the hydroxyl group is methylated in addition to the carboxylic acid, in the case of a sugar chain originally having an O-methyl group as a part, it is impossible to specify the methylated site.
In addition, ester derivatives are more easily hydrolyzed than amide derivatives, and easily form lactones with nearby hydroxyl groups.

From these points, amidation is a promising technique for the purpose of marker search (see Patent Document 1 and Non-Patent Document 1), but the amidation efficiency differs depending on the binding position of sialic acid, and the reactivity In the case of a low bonding position, there is a problem that amidation cannot be performed.
That is, amidation using carbodiimide is generally performed at a pH of around 3.5 to 4.5 where carbodiimide itself has high reactivity. It has been reported that when the pH is higher than this, the reactivity becomes low, and when it is lower than this pH, the carbodiimide becomes unstable and decomposes (Non-patent Document 3). However, under these conditions, amidation is not possible in the case of a bonding position having low reactivity.
As described above, there is no quantitative sialo-glycan modification method that can be used for marker search.
JP 2005-148054 A Sekiya S, Wada Y, Tanaka K. Derivatization for stabilizing sialic acids in MALDI-MS. Anal Chem. 2005 Aug 1; 77 (15): 4962-8. Nakajima N, Ikada Y. Mechanism of amide formation by carbodiimide for bioconjugation in aqueous media. Bioconugate Chem. 1995 6. 123-30.

It is known that sialicosaccharides have different chemical reactivity depending on the linkage. However, if a sialic acid linkage with low reactivity can be completely modified in the search for biomarkers with sialo-glycan, comparative quantitative analysis is possible. It is considered to be.
The present invention has been made in view of the circumstances as described above, and an object of the present invention is to provide a quantitative sialo-glycan modification method that can be used for marker search.

The linkage of sialic acid that can be amidated by a known method is α2-6 sialic acid, and the sugar chain having the sialic acid is mixed with ammonium chloride, which is a compound having an amino group as a nucleophile, A method of adding 4- (4,6-dimethoxy-1,3,5-triazin-2-yl) -4-methylmorpholinium chloride as a condensing agent is employed.
As a result of intensive studies on a method that can completely modify a low-reactivity sialic acid linkage, the present inventors dissolved a biological sample in water, added a nucleophile, and then adjusted the pH to an appropriate value on the acidic side. It was found that the sialic acid moiety having low reactivity can be chemically modified by adjusting, adding an excessive amount of a condensing agent, and stirring or standing for a certain period of time.

The present invention has been completed based on these findings, and is as follows.
(1) A sialo-sugar chain characterized by mixing a sialo-sugar chain and a nucleophile, adjusting the pH to 3 or less, and adding a condensing agent to amidate the carboxyl group of the sialo-sugar chain. Method of amidation of
(2) The method for amidation of a sialo-sugar chain according to claim 1, wherein the nucleophile is a compound having a hydrazide group.
(3) The method for amidating a sialo-sugar chain according to claim 1, wherein the condensing agent is carbodiimide.
(4) The method according to any one of claims 1 to 3, wherein the sialo-sugar chain contains a sugar chain having α2-3 sialic acid.

  The method of the present invention enables highly sensitive and quantitative detection of sialo-glycans by mass spectrometry, which has been difficult in the past, by chemically modifying biological components, and comparative quantitative analysis of biomarkers having sialo-glycans It becomes.

Hereinafter, the amidation method of the sialo-sugar chain of the present invention will be described.
The method for amidating a carboxyl group of a sialic acid residue in the present invention is performed by mixing a sialo-sugar chain and a nucleophile and adding a condensing agent thereto.
The present invention is characterized in that the condensation reaction is carried out at a pH of 3 or less, preferably at a pH of 2 to 3.
In addition, when a sialic acid linkage with low reactivity such as α2-6 and a sialic acid linkage with low reactivity such as α2-3 are mixed in the sample, the modification reaction is performed in advance under normal conditions. After carrying out, amidation may be subsequently carried out by the method of the present invention.

  In the present invention, a compound having a hydrazide group is used as the nucleophile, and an acetohydrazide is preferably used as the compound having the hydrazide group. If the compound used as the nucleophilic agent becomes bulky, the yield in the condensation reaction decreases, which is not preferable. Therefore, it is preferable to use a small hydrazide, particularly acetohydrazide.

  In the present invention, carbodiimides are used as the condensing agent. Since the molecule to be modified is a sialo-sugar chain rich in hydrophilicity, the reaction is more preferably aqueous. Therefore, it is preferable to use water-soluble carbodiimide, particularly 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (hereinafter referred to as “EDAC”) as the condensing agent.

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.
(Reference example: amidation of sialo-glycans with different linkages by a known method)
First, the amidation of sialic acid by a known amidation method is shown. Amidation by a known method was performed on sialo-sugar chains with different linkages.
In this amidation method, ammonium chloride, which is a compound having an amino group, is mixed with a sugar chain having sialic acid, and 4- (4,6-dimethoxy-1,3,5-triazine-2yl which is a condensing agent is mixed. ) -4-Methylmorpholinium chloride is added, and α2-6 sialic acid has been reported as the linkage of sialic acid that can be amidated by this method.

Therefore, a fluorescently labeled N-type double-chain sugar chain (the following formula (A)) having two α2-6 sialic acids at the ends was used as a control for the modification reaction.
A comparison of sialicated glycans with different linkages is as follows: a fluorescently labeled N-type double-stranded glycan chain (formula (B) below) having one α2-6 sialic acid at the end, and α2-3 sialic acid at the end. One fluorescently labeled N-type double-stranded sugar chain (the following formula (C)) was used. Sugar chains B and C are positional isomers that differ only in the linkage of sialic acid.

First, a mixture of sugar chain A (4 pmol) and sugar chain B (4 pmol) was amidated by a known method and subjected to reverse phase high performance liquid chromatography (hereinafter referred to as “HPLC”). As shown in the upper part, two peaks (peaks 1 and 2) were observed.
When these two peaks 1 and 2 were subjected to mass spectrometry (hereinafter referred to as “MS”), the mass-to-charge ratio (hereinafter referred to as “m / z value”) was 2031.95 and 232.21, respectively. In order, the sugar chains A and B were amidated, and the mass numbers (2031.75, 2321.86) were added with Na ions.
Therefore, it was confirmed that the α2-6 sialic acid-containing sugar chain was easily amidated. This is consistent with past findings.

Next, amidation was similarly performed using a mixture of sugar chain A (4 pmol) and sugar chain C (4 pmol), and the mixture was subjected to reverse phase HPLC. As shown in the lower part of FIG. 4, 5) It was seen.
When these peaks 3, 4, and 5 were subjected to MS, the m / z values were 2031.96, 2332.94, and 2054.71, respectively, and the mass number (2031.75) and amidation in which Na ions were added to the amidated sugar C in order. This corresponds to the mass number (2321.86) in which Na ions were added to the sugar chain A, and the mass number (2054.71) in which two Na ions were added to the unreacted sugar chain C. It is known that a sugar chain in which sialic acid is not modified adds an extra number of Na ions by the same number as the number of sialic acids.
From the fact that unreacted sugar chain C was observed and that only amidated sugar chain C was observed, it was confirmed that the amidation of α2-3 sialic acid-containing sugar chains was inefficient. .
In this reaction, since the sugar chain A is almost completely amidated, it is judged that there is no possibility that the amidation reaction itself is insufficient.
Therefore, these results show that the known amidation method has different reactivity depending on the linkage of sialic acid.

Hereinafter, an example in which an α2-3 sialic acid-containing sugar chain is amidated using a new amidation method according to the present invention will be described.
Example 1
Sugar chain D having α2-3 sialic acid (formula (D), 4 pmol) is dissolved in 30 μl of 1M acetohydrazide solution, adjusted to pH 3.5 with HCl, and 4 μl of 1M EDAC solution is added. The reaction was performed at room temperature for 2 hours. Similarly, reactions with pH adjusted to 3.1, 2.5, 2.1, 1.5, and 1.2 were performed.
When the reaction product was subjected to reverse phase HPLC, as shown on the left side of FIG. 2, it was shown that the peak 6 was remarkably increased at pH 2.1, 2.5, and 3.1.
When the peak 6 and peak 7 in the figure were separated and subjected to MS, the m / z values were 790.35 and 712.43, respectively, and the mass number in which Na ions were added to the amidated sugar chain D in order (790.30) This corresponds to the mass number (712.28) in which one proton was added to the unreacted sugar chain D.
After the reaction of sugar chain D at pH 2.5, 4 μl of 1M EDAC solution was further added and the reaction was repeated for 2 hours twice. When the obtained reaction product was similarly subjected to reverse phase HPLC, peak 6 corresponding to the amidation product of sugar chain D became the main product as shown on the right side of FIG.

(Example 2)
Next, for comparison with known amidation methods, a new amidation method was examined using N-type double-chain sugar chains A, B, and C.
A fluorescently labeled N-type double-chain sugar chain having the two α2-6 sialic acids at the ends (the above formula (A)) was used as a control for the modification reaction.
A comparison of sialicated glycans with different linkages is as follows: a fluorescently labeled N-type double-chain glycan chain having one α2-6 sialic acid at the end (the above formula (B)), and α2-3 sialic acid at the end One fluorescently labeled N-type double-stranded sugar chain (the above formula (C)) was used. Sugar chains B and C are positional isomers that differ only in the linkage of sialic acid.
First, the above mixture of sugar chains A (4 pmol) and B (4 pmol) is dissolved in 50 μl of 1M acetohydrazide solution, adjusted to pH 2.5 using HCl, 4 μl of 2M EDAC solution is added, 2 The reaction was performed at room temperature for an hour. Thereafter, 4 μl of 2M EDAC solution was added again and reacted at room temperature for 2 hours. After that, 4 μl of 2M EDAC solution was added again and reacted at room temperature for 2 hours. When amidated over 6 hours in total was subjected to reversed phase HPLC, two peaks (peaks 8 and 9) were observed as shown in FIG.
When these two peaks were subjected to MS, the m / z values were 2088.77 and 2436.14, respectively, the mass number (2088.77) in which Na ions were added to the amidated sugar chain B, and the amidated sugar chain A This coincided with the mass number (2435.91) in which Na ions were added.
As described above, it was confirmed that the α2-6 sialic acid-containing sugar chain was amidated under these conditions.

Next, amidation was performed in the same manner using the mixture of sugar chains A and C, and the mixture was subjected to reverse phase HPLC. As shown in FIG. 3, two peaks (peaks 10 and 11) were observed.
When these two peaks were subjected to MS, the m / z values were 2089.02 and 2435.84, respectively, the mass number (2088.77) in which Na ions were added to the amidated sugar chain C, and the amidated sugar chain A This coincided with the mass number (2435.91) in which Na ions were added.
Therefore, it was confirmed that the α2-3 sialic acid-containing sugar chain was easily amidated by this method.
From these results, it can be seen that the new amidation method shown above can easily modify low-reactivity sialic acid which has been difficult to amidate until now.

(Example 3)
Furthermore, sugar chain E (formula (E) below) having a different position of α2-3 sialic acid, sugar chain F (position (I) below) that is a positional isomer of sugar chain E and linkage is α2-6 sialic acid )), And the sugar chain G to which a large number of sialic acids are bonded (the following formula (G)) was amidated in the same manner.

A mixture of sugar chains E and A, sugar chains F and A, and sugar chains G and A was modified by the same method as in Example 2 and subjected to reverse phase HPLC. As shown in the lower part, peaks 12 and 13, peaks 14 and 15, and peaks 16 and 17 were obtained.
The m / z values (2435.78, 2436.00, 2435.88) of these peaks 13, 15, and 16 coincided with the mass number (2435.91) in which Na ions were added to the amidated sugar chain A. The peaks 12, 14, 17 (m / z: 2088.88, 2089.01, 3496.04) corresponded to the mass numbers (2088.77, 2088.77, 3495.30) in which Na ions were added to the amidated sugar chains E, F, G, respectively.
From these results, it can be seen that the amidation method according to the present invention can amidate sialic acid efficiently even when the position of sialic acid is different or when the number of sialic acids is large.

Example 4
The sugar chain of Fetuin, which is a glycoprotein containing an α2-3 sialic acid-containing sugar chain, was analyzed using the amidation method of the present invention. Fetuin has the highest content of three sialic acids bonded to an N-type three-chain sugar chain, and 90% or more contains α2-3 sialic acid.
Fetuin was treated with PNGase F, and the resulting N-type sugar chain was labeled with pyridylamine, modified in the same manner as in Example 2, and analyzed by MS.
In the obtained MS spectrum, as shown in FIG. 5, the signal with the m / z value of 3148.28 is the highest, which corresponds to the three-chain sugar chain having the highest content with three sialic acids bound thereto. It corresponds to the mass number (theoretical value: 3148.17).
Thus, a result reflecting that α2-3 sialic acid was modified was obtained.

  According to the present invention, it has become possible to perform highly sensitive and quantitative detection of sialo-glycan by mass spectrometry, which has been difficult in the past, and comparative quantitative analysis of biomarkers having sialo-glycan. A new technique can be provided for disease diagnosis.

The figure which shows the result of the reverse phase HPLC of the reaction material obtained by known amidation reaction The figure which shows the result of the reverse phase HPLC of the reaction material obtained in Example 1 The figure which shows the result of the reverse phase HPLC of the reaction material obtained in Example 2 The figure which shows the result of the reverse phase HPLC of the reaction material obtained in Example 3 The figure which shows the m / z value of the reactant obtained in Example 4

Claims (4)

  After mixing the sialo-sugar chain and the nucleophilic agent, the pH is adjusted to 3 or less, and a condensing agent is added thereto to amidate the carboxyl group of the sialo-sugar chain. Method.   The method for amidating a sialoglycosaccharide according to claim 1, wherein a compound having a hydrazide group is used as the nucleophile.   The method for amidating a sialo-sugar chain according to claim 1, wherein the condensing agent is carbodiimide.   The method according to any one of claims 1 to 3, wherein the sialosugar chain contains a sugar chain having α2-3 sialic acid.

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