A method for analyzing sialylated sugar chains based on permanent charge reagent derivatization

Abstract

The application relates to an analysis method of sialylated sugar chains and their connecting isomers based on permanent charge reagent derivatization, and belongs to the field of biochemical detection. A kind of permanent charge reagent, 1-(4-aminobutyl) pyridinium bromide (ABP), is used to react with sialylated sugar chains under strong activation conditions, so that the sialylated sugar chains are derived into permanent charge sialylated sugar chain derivatives, the carboxyl groups are neutralized to protect unstable sialic acid residues, and the special structure effectively improves the problem that the ionization efficiency of sialylated sugar chains in mass spectrometry is low and is often inhibited by other samples. Secondly, by using ABP and its structural analogue 1-(3-aminopropyl) pyridinium bromide (APP) through two-step specific recognition reactions, alpha-2,3 and alpha-2,6 connecting sialic acid sugar chain isomers can be specifically converted into derivatives with a molecular weight difference of 14 Da, so that the connecting isomers of sialylated sugar chains can be distinguished through mass spectrometric analysis.

Description

Technical Field

[0001] This invention relates to the field of biochemical detection, specifically to a method for analyzing sialylated glycans based on permanent charge reagent derivatization. Background Technology

[0002] Sialic acid, a nonose sugar with a unique chemical structure, plays a crucial role in the physiological and pathological processes of living organisms by being linked to the non-reducing ends of sugar chains through various mechanisms (mainly α-2,3 or α-2,6 glycosidic bonds). Therefore, accurate analysis of sialylated sugar chains and their linked isomers is a key prerequisite for identifying disease-related characteristic sugar chains and understanding molecular mechanisms. Mass spectrometry, with its high sensitivity and specificity, and its ability to provide detailed molecular structure information, has become a primary tool for the analysis of carbohydrate compounds. However, sialic acid contains a carboxyl group with a negative charge, resulting in low ionization efficiency and instability of sialylated sugar chains in mass spectrometry, making them prone to decomposition or detachment from the sugar chain. Therefore, chemical modification is used to stabilize sialic acid before analysis. For example, patent document (JP2013068594A) discloses a method in which the reducing end of a free sugar chain is immobilized on a solid support, and PyAOP is used as a condensing agent and methylamine hydrochloride as a nucleophile to amidate the carboxyl group of the non-reducing end of the sugar chain. It also discloses an example of using mass spectrometry to quantitatively determine and structurally analyze the sugar chain in a sample modified by methylamidation. However, the disclosed method cannot identify the linkage type of sialic acid; isomers of sialylated sugar chains with different linkage methods have the same molecular weight and cannot be directly distinguished by mass spectrometry.

[0003] Chemical derivatization methods play a crucial role in both proteomics and glycomics research. For sialylated glycans, researchers have stabilized sialic acid and improved its ionization efficiency in mass spectrometry through carboxyl derivatization reactions, including amidation, acetylation, and p-methylaniline derivatization. Furthermore, based on the reactivity differences between α-2,3 and α-2,6-linked sialic acids, different chemical modifications are performed under appropriate reaction conditions to selectively convert α-2,6 and α-2,3-linked sialic acids into different molecules, resulting in mass differences between the linked isomers, which can then be identified and detected by mass spectrometry. Relevant derivatization reagents include alcohols (EtOH / MeOH) and amines (methylamine / isopropylamine). Patent document (JP2013076629A) discloses a method in which methyl esterification of sialic acid is performed using 1-methyl-3-p-tolyltriazene (MTT). Then, under acidic conditions, α-2,3-linked sialic acid is hydrolyzed to remove the methyl group, while α-2,6-linked sialic acid remains unhydrolyzed, resulting in a difference in quality. This method can distinguish between α-2,3-linked and α-2,6-linked sialic acid, but the disclosed method leads to differences in ionization between the two types, which is detrimental to quantitative analysis. Summary of the Invention

[0004] The purpose of this invention is to overcome the deficiencies of the prior art and provide a method for derivatizing sialylated sugars using 1-(4-aminobutyl)pyridine bromide (ABP) and 1-(3-aminopropyl)pyridine bromide (APP) reagents with permanent charges, including one-step derivatization of sialylated sugar chains and a two-step identification and derivatization method for sialic acid-linked isomers.

[0005] The technical solution to achieve the objective of this invention is to provide a method for analyzing sialylated glycans based on permanent charge reagent derivatization, comprising the following steps:

[0006] S1. Place the sialylated glycan sample to be analyzed in a centrifuge tube, add 1-(4-aminobutyl)pyridine bromide (ABP) solution and hexafluorophosphate (7-azabenzotriazole-1-oxy)tripyrrolidinephosphine (PyAOP) solution in sequence, shake well to mix thoroughly, and then place in a 37°C water bath for 2 hours;

[0007] S2. After the reaction is complete, add 85% acetonitrile (ACN) to quench the reaction and purify with a cotton-filled hydrophilic interaction solid phase extraction column (CottonHILICSPE);

[0008] S3. The purified sample was analyzed in positive ion mode using matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS) or electrospray mass spectrometry (ESI-MS).

[0009] Furthermore, the ABP solution in S1 has a concentration of 1M and uses dimethyl sulfoxide (DMSO) as the solvent.

[0010] Furthermore, the PyAOP solution in S1 has a concentration of 250 mM, and the solvent is N-methylmorpholine / DMSO with a volume ratio of v / v = 3:7.

[0011] An analytical method for distinguishing sialylated glycan-linked isomers based on permanent charge reagent derivatization includes the following steps:

[0012] S1. Place the sialylated sugar sample to be analyzed in a centrifuge tube, add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC)-1-hydroxybenzotriazole (HOBt) solution, and let stand at room temperature for 0.5 h;

[0013] S2. Add a solution of the derivatizing reagent 1-(3-aminopropyl)pyridinium bromide (APP), shake thoroughly, and then place in a water bath at 25°C for 2 hours.

[0014] S3. After the reaction is complete, add 85% ACN to quench the reaction, then perform Cotton-HILICSPE purification, and freeze-dry the purified solution;

[0015] S4. Add ABP solution to the freeze-dried sample obtained above, shake thoroughly, and then place in a 37°C water bath for 1 hour;

[0016] S5. After freeze-drying the sample obtained in S4 above, add PyAOP solution, shake thoroughly, and then place it in a 37°C water bath for 1 hour.

[0017] S6. After the reaction is complete, 85% ACN is added to quench the reaction, followed by Cotton-HILICSPE purification;

[0018] S7. The purified sample was analyzed by MALDI-MS or ESI-MS in positive ion mode.

[0019] Furthermore, the concentration of the EDC-HOBt solution in step S1 is 500 mM, and the solvent is DMSO.

[0020] Furthermore, in step S2, the concentration of the APP solution is 1M, and the solvent is DMSO.

[0021] Furthermore, the ABP solution in step S4 has a concentration of 1M and the solvent is 1M triethylammonium bicarbonate buffer.

[0022] Furthermore, the concentration of the PyAOP solution in step S5 is 250 mM, the solvent is N-methylmorpholine / DMSO, and the volume ratio is v / v = 3:7.

[0023] Furthermore, the CottonHILICSPE purification described in step S6 specifically includes the following steps: A. Take about 1 mg of medical absorbent cotton and load it evenly into a 100 μL pipette tip. Wash the cotton column with 100 μL of deionized water and 100 μL of 85% ACN, respectively, and repeat each three times.

[0024] B. Draw up the sample solution to be purified, and after 5 seconds, return the liquid to the pump. Repeat this step 20 times.

[0025] C. Take 100 μL of 85% ACN, discard the liquid after 5 seconds, and repeat this step three times;

[0026] D. Take 20 μL of water containing 0.1% trifluoroacetic acid, and after 5 seconds, return the liquid to the container. Repeat this step 20 times.

[0027] Furthermore, the ACN in step C contains 1% trifluoroacetic acid.

[0028] After adopting the above technical solution, the present invention has the following positive effects:

[0029] (1) The present invention uses a permanently charged reagent, 1-(4-aminobutyl)pyridinium bromide (ABP), to react with sialylated sugar chains under strong activation conditions to derivatize them into sialylated sugar chain derivatives with permanent charges. This derivative can neutralize carboxyl groups to protect unstable sialic acid residues. Since the derivatizing reagent itself is charged, there is no need for an ionization process. Therefore, it effectively improves the problem that sialylated sugar chains have low ionization efficiency in mass spectrometry and are often suppressed by other samples.

[0030] (2) The derivatization reagents used in this invention all carry permanent charges. One-step derivatization of sialylated sugars not only improves the stability of sialylated sugar chains, but also has good ionization efficiency. The ions formed in mass spectrometry are simple, which can reduce the complexity of the spectrum.

[0031] (3) The two-step specific identification derivatization method of the present invention can also distinguish α-2,3 or α-2,6 sialic acid linked isomers. The two-step specific identification derivatization uses a pair of structurally similar reagents, APP and ABP, which have similar ionization efficiencies. This helps to accurately quantify the content of isomers and can specifically convert α-2,3 and α-2,6 linked sialic acid glycan isomers into derivatives with a molecular weight difference of 14 Da. Thus, the linked isomers of sialylated glycans can be distinguished by mass spectrometry analysis.

[0032] (4) The present invention requires only one or two steps of reaction, the reaction conditions are easy to achieve, the reaction method is simple and easy to implement, and the reaction time is short. Attached Figure Description

[0033] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein...

[0034] Figure 1 This is a schematic diagram of the chemical structures of the derivatization reagents APP and ABP of this invention;

[0035] Figure 2 This is a schematic diagram illustrating the one-step derivatization of sialylated sugar chains according to the present invention;

[0036] Figure 3 The mass spectrum is the result of a one-step derivation of 3-SL or 6-SL according to the present invention;

[0037] Figure 4 This is a schematic diagram of the two-step specific recognition and derivatization of sialylated sugar chains according to the present invention;

[0038] Figure 5 This is the mass spectrum of the mixture of 3-SL and 6-SL after two-step specific derivatization according to the present invention; Detailed Implementation

[0039] Example 1: A method for analyzing sialylated glycans based on permanent charge reagent derivatization

[0040] Using α-2,3-linked sialyl lactose (3-SL) and α-2,6-linked sialyl lactose (6-SL) as models, one-step derivatization was performed on each. The derivatization reagent was ABP, and its structure is shown below. Figure 1 As shown, the reaction diagram is as follows: Figure 2 .

[0041] The specific steps are as follows:

[0042] S1. Weigh 100 μg of 3-SL and 6-SL standards into 0.6 mL centrifuge tubes, and add 10 μL of 1-(4-aminobutyl)pyridine bromide (ABP) solution (1 M; solvent: DMSO) and 10 μL of hexafluorophosphate (7-azabenzotriazol-1-oxy) tripyrrolidinephosphine (PyAOP) solution (250 mM; solvent: N-methylmorpholine / DMSO, v / v = 3:7) in turn. After thorough shaking and mixing, place the tubes in a 37 °C water bath for 2 h.

[0043] S2. After the reaction is complete, add 300 μL of 85% ACN to quench the reaction and then purify using a cotton-filled hydrophilic interaction solid-phase extraction column (CottonHILICSPE).

[0044] S3. The purified sample was analyzed by electrospray ionization mass spectrometry (ESI-MS) in positive ion mode.

[0045] The derivatized products were characterized by ESI-MS, and the results are as follows: Figure 3 As shown, since the ABP reagent itself carries a fixed charge, the derived 3-SL and 6-SL mainly react with [M] charge. + The (m / z 766.32) ion form was detected by mass spectrometry and had a high signal intensity.

[0046] Example 2: An analytical method for distinguishing sialylated glycan-linked isomers based on permanent charge reagent derivatization.

[0047] Equal volumes of 3-SL and 6-SL standards were mixed and subjected to a two-step specific recognition derivatization using APP and ABP as derivatization reagents, with structures as follows: Figure 1 As shown, the reaction diagram is as follows: Figure 4 As shown. The specific steps are as follows:

[0048] S1. Weigh 100 μg of 3-SL and 6-SL standards into a 0.6 mL centrifuge tube, add 10 μL of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC)-1-hydroxybenzotriazole (HOBt) solution (500 mM; solvent: DMSO), and let stand at room temperature for 0.5 h.

[0049] S2. Add a 1M solution of the derivatizing reagent 1-(3-aminopropyl)pyridine bromide (APP) (solvent: DMSO), shake thoroughly, and then react in a water bath at 25°C for 2 hours.

[0050] S3. After the reaction is complete, add 150 μL of 85% ACN to quench the reaction, then perform Cotton-HILIC SPE purification, and freeze-dry the purified solution.

[0051] S4. Add 10 μL of ABP solution (1M; solvent is 1M triethylammonium bicarbonate buffer) to the sample obtained above, shake thoroughly, and then place in a 37°C water bath for 1 hour.

[0052] S5. After freeze-drying the above sample, add 10 μL PyAOP solution (250 mM; solvent is N-methylmorpholine / DMSO, v / v = 3:7), shake thoroughly, and then place in a 37°C water bath for 1 h.

[0053] S6. After the reaction is complete, add 300 μL of 85% ACN to quench the reaction, followed by CottonHILIC SPE purification.

[0054] S7. The purified sample was analyzed by matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS) in positive ion mode.

[0055] The derivatized products were characterized by MALDI-MS, and the results are as follows: Figure 5 As shown, the derivatized 6-SL reacts with the first nucleophile APP, while 3-SL reacts with the second affinity reagent ABP. The mass-to-charge ratio of the two derivatized products differs by 14, making them easily distinguishable. Furthermore, equal amounts of 3-SL and 6-SL exhibit similar signal intensities after derivatization, indicating that this derivatization method does not lead to differences in ionization, thus aiding in quantitative analysis.

[0056] Example 3: Cotton HILICSPE Purification

[0057] Specifically, the following steps are included:

[0058] A. Take about 1 mg of medical absorbent cotton and load it evenly into the tip of a 100 μL pipette. Wash the cotton column with 100 μL of deionized water and 100 μL of 85% ACN, respectively, and repeat each three times.

[0059] B. Draw up the sample solution to be purified, and after 5 seconds, return the liquid to the pump. Repeat this step 20 times.

[0060] C. Take 100 μL of 85% ACN (containing 1% trifluoroacetic acid), discard the liquid after 5 seconds, and repeat this step three times;

[0061] D. Take 20 μL of water containing 0.1% trifluoroacetic acid, and after 5 seconds, return the liquid to the container. Repeat this step 20 times.

[0062] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for analyzing sialylated glycans based on permanent charge reagent derivatization, characterized in that, Includes the following steps: S1. Place the sialylated glycan sample to be analyzed in a centrifuge tube, add 1-(4-aminobutyl)pyridine bromide solution and hexafluorophosphate (7-azabenzotriazole-1-oxy)tripyrrolidine phosphorus solution in sequence, shake well to mix thoroughly, and then place in a 37°C water bath for 2 hours. S2. After the reaction is complete, add 85% acetonitrile to quench the reaction, and purify with a hydrophilic interaction solid-phase extraction column filled with cotton. S3. The purified sample was analyzed by matrix-assisted laser desorption / ionization mass spectrometry or electrospray mass spectrometry in positive ion mode.

2. The method for analyzing sialylated glycans based on permanent charge reagent derivatization according to claim 1, characterized in that, The 1-(4-aminobutyl)bromopyridine solution in S1 has a concentration of 1M and uses dimethyl sulfoxide as the solvent.

3. The method for analyzing sialylated glycans based on permanent charge reagent derivatization according to claim 1, characterized in that, The concentration of the hexafluorophosphate (7-azabenzotriazol-1-oxy) tripyrrolidinephosphine solution in S1 is 250 mM, and the solvent is N-methylmorpholine / dimethyl sulfoxide with a volume ratio of v / v=3:

7.

4. An analytical method for distinguishing sialylated glycan-linked isomers based on permanent charge reagent derivatization, characterized in that, Includes the following steps: S1. Place the sialylated sugar sample to be analyzed in a centrifuge tube, add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide-1-hydroxybenzotriazole solution, and let stand at room temperature for 0.5 h; S2. Add the derivatizing reagent 1-(3-aminopropyl)pyridine bromide solution, shake thoroughly, and then place in a 25°C water bath for 2 hours; S3. After the reaction is complete, add 85% acetonitrile to quench the reaction, then purify with a hydrophilic interaction solid-phase extraction column filled with cotton, and freeze-dry the purified solution. S4. Add 1-(4-aminobutyl)pyridine bromide solution to the freeze-dried sample obtained above, shake thoroughly, and then place in a 37°C water bath for 1 hour; S5. After freeze-drying the sample obtained in S4 above, add hexafluorophosphate (7-azabenzotriazole-1-oxy) tripyrrolidinephosphine solution, shake thoroughly, and then place in a water bath at 37°C for 1 hour. S6. After the reaction was completed, 85% acetonitrile was added to quench the reaction, followed by purification using a hydrophilic interaction solid-phase extraction column filled with cotton. S7. The purified sample was analyzed by matrix-assisted laser desorption / ionization mass spectrometry or electrospray mass spectrometry in positive ion mode.

5. The analytical method for distinguishing sialylated glycan-linked isomers based on permanent charge reagent derivatization according to claim 4, characterized in that, The concentration of the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide-1-hydroxybenzotriazole solution in step S1 is 500 mM, and the solvent is dimethyl sulfoxide.

6. The analytical method for distinguishing sialylated glycan-linked isomers based on permanent charge reagent derivatization according to claim 4, characterized in that, The concentration of the 1-(3-aminopropyl)pyridine bromide solution in step S2 is 1M, and the solvent is dimethyl sulfoxide.

7. The analytical method for distinguishing sialylated glycan-linked isomers based on permanent charge reagent derivatization according to claim 4, characterized in that, The 1-(4-aminobutyl)pyridine bromide solution in step S4 has a concentration of 1M and the solvent is 1M triethylammonium bicarbonate buffer.

8. The analytical method for distinguishing sialylated glycan-linked isomers based on permanent charge reagent derivatization according to claim 4, characterized in that, The hexafluorophosphate (7-azabenzotriazol-1-oxy) tripyrrolidinephosphine solution in step S5 has a concentration of 250 mM and uses N-methylmorpholine / dimethyl sulfoxide as the solvent, with a volume ratio of v / v = 3:

7.

9. The analytical method for distinguishing sialylated glycan-linked isomers based on permanent charge reagent derivatization according to claim 4, characterized in that, The purification process using a cotton-filled hydrophilic interaction solid-phase extraction column in step S6 specifically includes the following steps: A. Take about 1 mg of medical absorbent cotton and load it evenly into the tip of a 100 μL pipette. Wash the cotton column with 100 μL of deionized water and 100 μL of 85% acetonitrile, respectively, and repeat each three times. B. Draw up the sample solution to be purified, and after 5 seconds, return the liquid to the pump. Repeat this step 20 times. C. Take 100 μL of 85% acetonitrile, discard the liquid after 5 seconds, and repeat this step three times; D. Take 20 μL of water containing 0.1% trifluoroacetic acid, and after 5 seconds, return the liquid to the container. Repeat this step 20 times.

10. The analytical method for distinguishing sialylated glycan-linked isomers based on permanent charge reagent derivatization according to claim 9, characterized in that, The acetonitrile in step C contains 1% trifluoroacetic acid.

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