Method for enriching and detecting sialylated oligosaccharides

Abstract

The application discloses a method for enriching and detecting sialyl-oligosaccharide. The method for enriching sialyl-oligosaccharide comprises the following steps: performing solid phase extraction on a crude sialyl-oligosaccharide product through modified defatted cotton. The method for preparing the modified defatted cotton comprises the following steps: performing a hydrothermal reaction on defatted cotton and an ammonium-based cationizing agent containing an epoxy group under alkaline conditions, and then adjusting the pH to neutral to obtain the modified defatted cotton. The method for enriching sialyl-oligosaccharide can be used for enriching and identifying sialyl-oligosaccharide in a sample, the material is simple to prepare, the reaction condition is mild, and the method has good enrichment efficiency. The detection method using the method for enriching has high sensitivity, low detection limit and strong anti-interference ability.

Description

Technical Field

[0001] This invention relates to a method for enriching and detecting sialic acid oligosaccharides. Background Technology

[0002] Sialic acid glycans have complex compositions, diverse structures, and a wide abundance dynamic range; low-abundance glycans often fail to reach the detection limits of some analytical methods. Secondly, glycans have a polyhydroxy structure, making them highly hydrophilic. Sialic acid, located at the N-terminus of the glycan, carries a negative charge, resulting in low ionization efficiency and detection sensitivity in mass spectrometry. Furthermore, the enzymatic cleavage of glycans from glycoproteins inevitably introduces salts, causing coexistence of glycans with peptides and proteins, interfering with glycan detection in mass spectrometry.

[0003] To address the aforementioned challenges, glycans can be purified and enriched before mass spectrometry analysis. This reduces the interference of impurities on glycan mass spectrometry analysis and improves the detection sensitivity of mass spectrometry for glycans, thereby enabling accurate qualitative analysis of glycans.

[0004] Existing methods for enriching sialic acid oligosaccharides include fluorine chemical tagging, glucosinolate-Schiff base modified silica gel dynamic adsorption, and other materials based on hydrophilic interactions that can partially enrich sialic acid oligosaccharides when enriching N-glycans. However, these methods suffer from drawbacks such as complex operation and poor enrichment effect. Therefore, it is necessary to provide a method that is simple to prepare, has mild reaction conditions, and improves detection sensitivity. Summary of the Invention

[0005] The technical problem this invention aims to solve is to overcome the shortcomings of existing technologies in detecting sialic acid glycans, such as low detection sensitivity and interference from impurities, by providing a method for enriching and detecting sialic acid oligosaccharides. The enrichment method of this invention involves simple material preparation and can effectively enrich sialic acid oligosaccharides in samples.

[0006] The present invention solves the above-mentioned technical problems through the following technical solution:

[0007] This invention provides a method for enriching sialic acid oligosaccharides, comprising the following steps:

[0008] The crude sialic acid oligosaccharide was subjected to solid-phase extraction using modified defatted cotton.

[0009] The preparation method of the modified degreased cotton includes the following steps: degreased cotton and an ammonium cationizing agent containing epoxy groups are subjected to hydrothermal reaction under alkaline conditions, then the pH is adjusted to neutral, and the modified degreased cotton is obtained after drying.

[0010] In the present invention, in the preparation method of the modified degreased cotton, the degreased cotton can be selected from conventional degreased cotton in the art.

[0011] In this invention, preferably, the ammonium cationizing agent containing epoxy groups is 2,3-epoxypropyltrimethylammonium chloride.

[0012] In this invention, the hydroxyl groups of the defatted cotton react with the epoxy groups in the ammonium cationizing agent containing epoxy groups. Preferably, in the reaction, the amount of the ammonium cationizing agent containing epoxy groups is excessive to ensure that as many cations as possible are modified.

[0013] In this invention, preferably, the temperature of the hydrothermal reaction is 60-70°C, for example 62.5-67.5°C, and more preferably, the temperature of the hydrothermal reaction is 65°C.

[0014] In this invention, preferably, the hydrothermal reaction time is 7 to 9 hours, for example 7.5 to 8.5 hours, or even 8 hours.

[0015] In some embodiments of the present invention, the hydrothermal reaction is carried out by stirring at 65°C for 8 hours.

[0016] In this invention, the alkaline conditions can be adjusted using conventional alkaline substances in the art, such as sodium hydroxide. The amount of sodium hydroxide used relative to 2.5g of absorbent cotton can be 4-6g, or 4.5-5.5g; preferably, the amount of sodium hydroxide used is 5g.

[0017] In this invention, the preparation method of the modified degreased cotton further includes a drying step after adjusting the pH to neutral.

[0018] In this invention, the crude sialic acid oligosaccharide can be a sample that conventionally contains sialic acid oligosaccharides in the art. The sample containing sialic acid oligosaccharides can be obtained, for example, by a sugar-cutting experiment on a sample containing sialic acid oligosaccharides, or by a sugar-cutting experiment on a glycoprotein sample.

[0019] The glycoprotein sample may include sialic acid glycopeptide, fetal protein, or egg yolk.

[0020] The sugar-cutting experiment may include the following steps: enzymatically digesting the glycoprotein sample, precipitating the protein, centrifuging and collecting the supernatant.

[0021] The enzyme used in the enzymatic hydrolysis can be PNGase F glycosidase.

[0022] The amount of enzyme used in the enzymatic hydrolysis can be selected according to the sample, for example, it can be 1-3 μL or 2-4 μL. 1 μL of enzyme can be used to process 10-20 μg of protein.

[0023] The reaction temperature for the enzymatic hydrolysis can be 40–70°C, for example 45–55°C, and preferably 50°C.

[0024] The reaction time for the enzymatic hydrolysis can be 8 to 12 minutes, or 9 to 11 minutes, preferably 10 minutes.

[0025] The enzymatic hydrolysis can be carried out at 40–60°C for 8–12 minutes, or at 45–55°C for 9–11 minutes, and preferably at 50°C for 10 minutes.

[0026] In the protein precipitation step, protein precipitation can generally be carried out using an organic solvent, such as acetonitrile and icy ethanol.

[0027] In the centrifugation step, preferably, the centrifugation parameters are set to centrifugation at a speed of 5000-20000g for 1-15 minutes, for example, centrifugation at 15000g for 5 minutes.

[0028] Preferably, after the sugar-cutting experiment, the following steps are also included: drying the sialic acid oligosaccharide sample obtained from the sugar-cutting experiment, redissolving it in a solvent, and then obtaining crude sialic acid oligosaccharide.

[0029] The solvent may be acetonitrile, for example, an 80% aqueous solution of acetonitrile. In this invention, the solid-phase extraction can be carried out in a solid-phase extraction column as conventionally performed in the art, and the modified defatted cotton preferably includes the following steps: loading the crude sialic acid oligosaccharide sample into a solid-phase extraction column packed with the modified defatted cotton.

[0030] Preferably, the solid-phase extraction column is a pipette tip column. Specifically, for a 200 μL pipette tip column, the mass of modified degreased cotton can preferably be 4.5–5.5 mg or 4.8–5.2 mg. In this invention, a 200 μL pipette tip column is most suitable, and the amount of cotton can be adjusted according to the sample loading volume.

[0031] Preferably, the loading volume of the crude sialic acid oligosaccharide is 10 μL or more, for example, 15 or 20 μL.

[0032] Before loading the sample, the solid-phase extraction column can be cleaned according to conventional methods in the field. For example, it can be cleaned sequentially with ultrapure water, pure acetonitrile, and 80% ACN aqueous solution.

[0033] In this invention, the solid-phase extraction may include the following steps: loading the crude sialic acid oligosaccharide onto a solid-phase extraction column, centrifuging and enriching the solid-phase extraction system, washing, and eluting.

[0034] Preferably, the centrifugation gradient of the centrifugation enrichment step is 50g for 2 min, 550g for 2 min, and 2000g for 1 min, repeated 3 times. (Where 50g, 550g, and 2000g refer to the centrifugation speed settings.)

[0035] Preferably, the cleaning step includes the following operation: centrifuging at 2000g for 1 min with water and 80% acetonitrile.

[0036] The eluent used for elution can be 0.1% TFA (trifluoroacetic acid), 50 mM NH4HCO3, or 25 mM NH4HCO3. Preferably, it is 25 mM NH4HCO3.

[0037] The elution process includes the following steps: using a 25mM NH4HCO3 aqueous solution as the eluent, then centrifuging the solid-phase extraction column at 50g for 2 min, 550g for 2 min, and 2000g for 1 min, and then collecting the eluent.

[0038] The process after elution includes the following steps: collecting the eluent and vacuum drying it to obtain the enriched sample.

[0039] This invention also provides a method for detecting sialic acid oligosaccharides, comprising the following steps:

[0040] (1) Enrichment: The sample to be tested is enriched using the method described above to obtain enriched sample A;

[0041] (2) Derivatization treatment: The enriched sample A is subjected to a derivatization reaction with a derivatization reagent to obtain a derivatized product;

[0042] (3) Detection: The derivatives were detected by liquid chromatography-mass spectrometry (LC-MS). The LC-MS conditions were as follows: the mobile phase consisted of phase C and phase D, wherein phase C was an aqueous solution containing 50 mM ammonium formate at pH 4.4, and phase D was acetonitrile. The elution gradient was calculated as follows, with phases C and D accounting for 100% by volume:

[0043]

[0044]

[0045] Here, % refers to the volume percentage of phase D in the mobile phase.

[0046] In this invention, the sample to be tested may be a sample containing sialic acid oligosaccharides, such as crude sialic acid oligosaccharides or glycoprotein samples, and the glycoprotein samples may be, for example, sialic acid glycopeptides, fetal proteins or egg yolks.

[0047] In this invention, the derivatizing reagent may be selected from 2-AB, 2-AA, or other reagents in the art that can be used to enhance mass spectrometry response, preferably a 166.67 mg / mL 2-AB solution.

[0048] When the crude sialic acid oligosaccharide is selected from 15 μL of 1 mg / mL sialic acid glycopeptide standard solution, 50 μL of 1 mg / mL fetoglobulin, or one fresh raw egg, the derivatized sample is obtained after enrichment. The volume of the derivatization reagent can be 3 μL, 6 μL, or 8 μL. Preferably, the volume of the 2-AB solution is 6 μL.

[0049] In this invention, preferably, the temperature of the derivatization reaction is 50-80°C, for example 55-75°C, and more preferably 65°C.

[0050] In this invention, preferably, the derivatization reaction time can be 2.5 to 5 hours, or 3.5 to 4 hours, more preferably 4 hours.

[0051] In some embodiments of this application, the derivatization reaction includes the following steps: reacting enriched sialylated oligosaccharides with 20 μL of 166.67 mg / mL 2-AB solution at 65°C for 4 hours. Due to the solvent effect of DMSO causing abnormal peak shapes in the chromatogram, the volume of the added derivatization reagent was proportionally reduced to 6 μL, resulting in better peak shapes.

[0052] In this invention, the flow rate of the mobile phase in the liquid chromatography-mass spectrometry (LC-MS) detection is preferably 0.3 to 0.6 mL / min, for example, 0.56 mL / min.

[0053] In this invention, the "78%-45%" in the elution gradient can be understood in the art as the volume percentage of phase D in the mobile phase gradually decreasing from 78% to 45% during this time period. And so on.

[0054] In this invention, preferably, the chromatographic column used in the liquid chromatography-mass spectrometry detection is a Glycan BEHAmide Column (2.1×100mm, granulation of 1.7μm, Waters).

[0055] In this invention, the running time of chromatographic analysis in liquid chromatography-mass spectrometry (LC-MS) detection can be 20-40 minutes or 25-35 minutes, and preferably, the running time of chromatographic analysis parameters is 30 minutes.

[0056] In this invention, the injection volume in liquid chromatography-mass spectrometry (LC-MS) detection can be 40–60 μL or 45–55 μL, and preferably, the injection volume for chromatographic analysis is 50 μL.

[0057] In this invention, the temperature of the sample injection plate in the liquid chromatography-mass spectrometry (LC-MS) detection can be below 10°C, and preferably, the temperature of the sample injection plate is 4°C.

[0058] In this invention, the initial proportion of the aqueous mobile phase in the liquid chromatography-mass spectrometry (LC-MS) detection is no higher than 25%, and preferably, the initial proportion of the aqueous phase is 22%.

[0059] In this invention, after liquid chromatography-mass spectrometry (LC-MS) detection, qualitative analysis of the oligosaccharide structure may also be performed using software such as Xcalibur and Glycoworkbench.

[0060] The positive and progressive effects of this invention are as follows:

[0061] 1) Compared with other materials, the enrichment method of the present invention uses materials that are simple to prepare, have mild reaction conditions, and require low-cost and readily available raw materials. Furthermore, this material can effectively enrich sialylated oligosaccharides from samples, exhibiting good enrichment efficiency.

[0062] 2) The detection method of this invention has high sensitivity, low detection limit, and strong anti-interference ability. Utilizing high-resolution mass spectrometry for qualitative analysis of sialic acid oligosaccharides provides high resolution and can distinguish between two sugar chains with a molecular weight difference of 0.01 Da. Attached Figure Description

[0063] Figure 1 These are SEM images of degreased cotton (A, C) and cationic cotton (B, D).

[0064] Figure 2 This is a comparison of LC-MS images of the sample before and after enrichment in Example 1.

[0065] Figure 3 This is a comparison image of LC-MS before and after enrichment of the sample in Example 2.

[0066] Figure 4 This is the TIC image after enrichment of the sample in Example 3. Detailed Implementation

[0067] The present invention will be further illustrated by way of embodiments below, but the present invention is not limited to the scope of the embodiments described herein.

[0068] Example 1: Enrichment of sialic acid oligosaccharides from standard sialic acid glycopeptides

[0069] A method for enriching sialic acid oligosaccharides in standard sialic acid glycopeptides specifically includes the following steps:

[0070] 1) Weigh 2.5g of defatted cotton, 5g of sodium hydroxide, and 21g of 2,3-epoxypropyltrimethylammonium chloride (EPTMAC) into a volumetric flask, add 100mL of water, and stir for 12 hours.

[0071] 2) React at 65℃ for 8.5 hours. Then add an appropriate amount of dilute hydrochloric acid solution to adjust the pH to neutral, wash with deionized water, and freeze-dry to obtain cationic cotton material, i.e., modified degreased cotton material.

[0072] 3) Take 5mg of modified degreased cotton and fill it into a 200μL pipette tip column to prepare a solid phase extraction column.

[0073] 4) Prepare a sialic acid glycopeptide standard solution with a concentration of 1 mg / mL.

[0074] 5) Take 15 μL of sialic acid glycopeptide standard solution into a 1.5 mL centrifuge tube and add 4 μL of reaction buffer (non-reducing). Preheat the glycopeptide standard reaction solution at 75 °C for 5 min, cool to room temperature, add 1 μL of PNGase F glycosidase, mix gently, react in a 50 °C water bath for 10 min, and then vacuum dry.

[0075] 6) After reconstituted with 80% acetonitrile aqueous solution, the sample was loaded onto the column for enrichment experiment. The column tip was centrifuged with the following centrifugation gradient: 50g for 2 min, 550g for 2 min, and 2000g for 1 min, for 3 cycles. Then, 25mMNH4HCO3 was used as the eluent, and the eluent was dried under vacuum.

[0076] 7) Preparation of derivatization reagent: Weigh 10 mg of 2-AB (2-aminobenzamide), add 60 μL of 30% DMSO acetic acid solution, and then add 12 mg of NaBH3CN. Heat at 65 °C for a few minutes to dissolve.

[0077] 8) Take 6 μL of the 2-AB solution prepared in step 7) above and add it to the dry sample, and react at 65°C for 4 h.

[0078] 9) Dilute the reaction solution with 50 μL of 80% acetonitrile aqueous solution, and load 50 μL of the solution onto the liquid chromatography-mass spectrometry (LC-MS) sample. Analyze the derivatives using LC-MS to determine the types of sialic acid oligosaccharides in the sample.

[0079] Chromatographic conditions: flow rate 0.56 mL / min; run time 30 min; injection volume 50 μL. Injection plate temperature 4℃. Mobile phase: Phase C was an aqueous solution containing 50 mM ammonium formate (pH = 4.4), and Phase D was acetonitrile. Elution gradient: 0-1.5 min, 78% D; 1.5-24.8 min, Phase D gradually decreased to 45%; 24.8-25.8 min, Phase D gradually decreased to 40%; 25.8-25.9 min, Phase D gradually increased to 78%; 25.9-30 min, Phase D remained at 78%.

[0080] Figure 1 These are SEM images of absorbent cotton (A, C) and modified absorbent cotton (B, D), where... Figure 1 Parts A and C are SEM images of degreased cotton observed at different magnifications, while parts B and D are SEM images of modified degreased cotton observed at different magnifications.

[0081] Figure 2 This is a comparison of LC-MS chromatograms before and after enrichment of the sample in Example 1. As shown in the figure, the retention time of the peptide is approximately 8.32 min. After purification using the pipette tip column, the peptide peak has been removed, indicating that the pipette tip column has a poorer retention effect on peptides with weaker polarity than sialylated oligosaccharides, but a better retention effect on carbohydrate compounds. After enrichment and purification using the pipette tip column, a clear sialylated oligosaccharide peak can be observed in the chromatogram.

[0082] Example 2: Enrichment of sialic acid oligosaccharides in fetal proteins

[0083] A method for enriching sialic acid oligosaccharides from fetal globulins specifically includes the following steps:

[0084] 1) Weigh 2.5g of defatted cotton, 5g of sodium hydroxide, and 21g of 2,3-epoxypropyltrimethylammonium chloride (EPTMAC) into a volumetric flask, add 100mL of water, and stir for 12 hours.

[0085] 2) React at 65℃ for 8.5 hours. Then add an appropriate amount of dilute hydrochloric acid solution to adjust the pH to neutral, wash with deionized water, and freeze-dry to obtain cationic cotton material.

[0086] 3) Take 5 mg of cationic cotton material and pack it into a 200 μL pipette tip column to prepare a solid phase extraction column.

[0087] 4) Prepare a fetal protein aqueous solution (the fetal protein used for preparation is bovine fetal protein, analytical grade), with a concentration of 1 mg / mL.

[0088] 5) Take 50 μL of fetal protein solution into a 1.5 mL centrifuge tube and add 27 μL of reaction buffer (reduction). Preheat the fetal protein reaction solution at 75 °C for 5 min, cool to room temperature, add 2 μL of PNGase F glycosidase, mix gently, and react in a 50 °C water bath for 10 min.

[0089] 6) Add 400 μL of ice-cold ethanol to the system to precipitate the protein, and place it in a -80°C freezer to allow precipitation to complete. Centrifuge, collect the supernatant, and vacuum dry.

[0090] 7) After reconstituted with 80% acetonitrile aqueous solution, the sample was loaded onto the column for enrichment experiments. The enrichment steps were the same as in Example 1. The eluent was then dried under vacuum.

[0091] 8) Weigh 10 mg of 2-AB, add 60 μL of 30% DMSO acetic acid solution, then add 12 mg of NaBH3CN, and heat at 65°C for a few minutes to dissolve.

[0092] 9) Take 6 μL of the 2-AB solution prepared in step 8) above and add it to the dry sample, and react at 65°C for 4 h.

[0093] 10) Dilute the reaction solution with 50 μL of 80% acetonitrile, and load 50 μL of the solution onto the liquid chromatography-mass spectrometry (LC-MS) sample.

[0094] 11) Column: Flow rate 0.56 mL / min; run time 30 min; injection volume 50 μL. Injection plate temperature 4℃. Mobile phase: Phase C is an aqueous solution containing 50 mM ammonium formate (pH = 4.4), and Phase D is acetonitrile. The volume percentages of Phase C and Phase D are 100%. Elution gradient: 0-1.5 min, 78% D; 1.5-24.8 min, Phase D gradually decreases to 45%; 24.8-25.8 min, Phase D gradually decreases to 40%; 25.8-25.9 min, Phase D gradually increases to 78%; 25.9-30 min, Phase D is kept at 78%.

[0095] Figure 3 These are LC-MS comparison images of the sample before and after purification in Example 2. Figure 3 It can be seen that before and after purification by the pipette tip column, most of the non-sialylated oligosaccharides of fetal globulin are removed, and the chromatographic peaks with a retention time of more than 10 minutes are mainly sialic acid oligosaccharides. The number of chromatographic peaks before and after enrichment is basically the same. Therefore, the pipette tip column has a strong specific enrichment ability for sialic acid oligosaccharides.

[0096] Example 3: Enrichment of sialic acid oligosaccharides in egg yolks

[0097] A method for enriching sialic acid oligosaccharides in egg yolks specifically includes the following steps:

[0098] 1) Weigh 2.5g of defatted cotton, 5g of sodium hydroxide, and 21g of 2,3-epoxypropyltrimethylammonium chloride (EPTMAC) into a volumetric flask, add 100mL of water, and stir for 12 hours.

[0099] 2) React at 65℃ for 8.5 hours. Then add an appropriate amount of dilute hydrochloric acid solution to adjust the pH to neutral, wash with deionized water, and freeze-dry to obtain cationic cotton material.

[0100] 3) Take 5 mg of cationic cotton material and pack it into a 200 μL pipette tip column to prepare a solid phase extraction column.

[0101] 4) After removing the shells from fresh raw eggs, separate the yolks and place them in a beaker. Add about 15 mL of ultrapure water to dilute the yolks, stir well, and dispense into 100 μL tubes (about 50 μL of yolk solution). Add 1 mL of phenol aqueous solution (1:9, v / v) to each tube, and shake well at 20℃ and 450 rpm for 3.5 h. Concentrate and dry to obtain the crude product, and store at -20℃ for later use.

[0102] 5) Take the crude product from the egg yolk extraction, add 15 μL of water, shake to dissolve, and add 4 μL of reaction buffer (reduction). Preheat the reaction solution at 75℃ for 5 min, cool to room temperature, add 1 μL of PNGase F glycosidase, mix gently, react in a 50℃ water bath for 10 min, and then vacuum dry.

[0103] 6) After reconstituted with 80% acetonitrile aqueous solution, the sample was loaded onto the column for enrichment experiments. The enrichment steps were the same as in Example 1. The eluent was then vacuum dried.

[0104] 7) Weigh 10 mg of 2-AB, add 60 μL of 30% acetic acid DMSO solution, then add 12 mg of NaBH3CN, and heat at 65°C for a few minutes to dissolve.

[0105] 8) Take 6 μL of the 2-AB solution prepared in step 7) above and add it to the dry sample, and react at 65°C for 4 h.

[0106] 9) Dilute the reaction solution with 50 μL of 80% acetonitrile, and load 50 μL of the solution onto the liquid chromatography-mass spectrometry (LC-MS) sample.

[0107] 10) Column: Flow rate 0.56 mL / min; run time 30 min; injection volume 50 μL. Injection plate temperature 4℃. Mobile phase: Phase C is an aqueous solution containing 50 mM ammonium formate (pH = 4.4), and Phase D is acetonitrile. Elution gradient (based on 100% volume percentage of Phase C and Phase D): 0-1.5 min, 78% D; 1.5-24.8 min, Phase D gradually decreases to 45%; 24.8-25.8 min, Phase D gradually decreases to 40%; 25.8-25.9 min, Phase D gradually increases to 78%; 25.9-30 min, Phase D remains constant at 78%.

[0108] Figure 4 This is the TIC (Total Ion Chromatograph) chromatogram of the purified sample from Example 3. The types of sialic acid oligosaccharides detected in the egg yolk extract during the experiment are shown in Table 1 below. As shown in Table 1, this pipette tip column can effectively filter out non-sialic acid oligosaccharide components (such as peptides) in the system, effectively increasing the signal intensity of sialic acid oligosaccharides.

[0109] Table 1. Types of sialic acid oligosaccharides detected in egg yolk extracts in the experiment.

[0110]

[0111]

[0112] *2AB: This indicates that the oligosaccharide was detected after being labeled with 2-aminobenzamide. The absence of 2AB glycan structures in the table indicates that the detected glycan was not derived from 2-AB.

[0113] The experiment revealed that in step (8) of Example 1, during the derivatization step, when the volume of the added 2-AB mixed solution was changed from 6 μL to 20 μL (approximately 1 mg), although the peak area of ​​the product was the largest, the peak shape was poor due to the solvent effect. In contrast, the chromatographic peak shape was better with 6 μL.

[0114] Interference experiment:

[0115] Interference experiments were conducted by adding interfering substances to the standard sialic acid oligosaccharide in Example 1, with the rest remaining the same as in Example 1. In this experiment, bevacizumab glycans were used as interfering substances, with ratios to sialic acid glycopeptides of 10:1, 20:1, and 100:1, respectively. Both bevacizumab and sialic acid glycopeptide standards underwent glycosidase digestion experiments. After uniform mixing, this mixture was used as the loading solution for sialic acid oligosaccharide enrichment and purification experiments using a pipette tip column. As the concentration of the interfering substance gradually increased from 10, 20, and 100 times that of the standard sialic acid oligosaccharide, the peak area of ​​the sialic acid oligosaccharide derivatives showed minimal change, demonstrating the strong anti-interference ability of the pipette tip column, making it suitable for the enrichment and purification of sialic acid oligosaccharides in actual samples.

[0116] This invention can enrich sialic acid oligosaccharides in samples. The material preparation is simple and the enrichment efficiency is high, which has a good application prospect in the field of detection.

[0117] While specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples, and the scope of protection of the present invention is defined by the appended claims. Those skilled in the art can make various changes or modifications to these embodiments without departing from the principles and essence of the present invention, but all such changes and modifications fall within the scope of protection of the present invention.

Claims

1. A method for enriching sialic acid oligosaccharides, characterized in that, It includes the following steps: The crude sialic acid oligosaccharide was subjected to solid-phase extraction using modified defatted cotton. The solid-phase extraction includes the following steps: centrifuging, washing, and eluting the solid-phase extraction column after sample loading; the eluent used for elution is any one of 0.1% TFA, 50mM NH4HCO3, or 25mM NH4HCO3; The preparation method of the modified degreased cotton includes the following steps: degreased cotton and an ammonium cationizing agent containing epoxy groups are subjected to hydrothermal reaction under alkaline conditions, and then the pH is adjusted to neutral to obtain the modified degreased cotton. The ammonium cationizing agent containing an epoxy group is 2,3-epoxypropyltrimethylammonium chloride.

2. The method for enriching sialic acid oligosaccharides as described in claim 1, characterized in that, The temperature of the hydrothermal reaction is 60~70℃; And / or, the hydrothermal reaction time is 7-9 hours; And / or, the alkaline conditions are adjusted using an alkaline substance, wherein the alkaline substance is sodium hydroxide; And / or, the method for preparing the modified degreased cotton further includes a drying step after adjusting the pH to neutral.

3. The method for enriching sialic acid oligosaccharides as described in claim 2, characterized in that, The temperature of the hydrothermal reaction is 62.5~67.5℃; And / or, the hydrothermal reaction time is 7.5~8.5h; And / or, relative to 2.5g of absorbent cotton, the amount of sodium hydroxide used is 4-6g.

4. The method for enriching sialic acid oligosaccharides as described in claim 2, characterized in that, The temperature of the hydrothermal reaction is 65°C; And / or, the hydrothermal reaction time is 8 hours; And / or, relative to 2.5g of absorbent cotton, the amount of sodium hydroxide used is 4.5~5.5g.

5. The method for enriching sialic acid oligosaccharides as described in claim 2, characterized in that, The hydrothermal reaction was carried out by stirring at 65°C for 8 hours.

6. The method for enriching sialic acid oligosaccharides as described in claim 2, characterized in that, The amount of sodium hydroxide used is 5g relative to 2.5g of absorbent cotton.

7. The method for enriching sialic acid oligosaccharides as described in claim 1, characterized in that, The crude sialic acid oligosaccharide is a sample containing sialic acid oligosaccharides, which is obtained by glycoprotein samples through a sugar-cutting experiment.

8. The method for enriching sialic acid oligosaccharides as described in claim 7, characterized in that, The glycoprotein samples include sialic acid glycopeptides, fetal proteins, or egg yolks; And / or, the sugar-cutting experiment includes the following steps: enzymatically digesting the glycoprotein sample, precipitating the protein, centrifuging and collecting the supernatant.

9. The method for enriching sialic acid oligosaccharides as described in claim 8, characterized in that, The enzyme used in the enzymatic hydrolysis is PNGase F glycosidase; And / or, the reaction temperature of the enzymatic hydrolysis is 40~70℃; And / or, the reaction time for the enzymatic hydrolysis is 8-12 minutes; And / or, in the protein precipitation step, an organic solvent is used for protein precipitation, wherein the organic solvent is acetonitrile and icy ethanol; And / or, in the centrifugation step, the centrifugation parameters are set to centrifuge at a speed of 5000-20000g for 1-15 minutes; And / or, after the sugar-cutting experiment, the following steps are also included: drying the sialic acid oligosaccharide sample obtained from the sugar-cutting experiment, redissolving it in a solvent, and then obtaining crude sialic acid oligosaccharide.

10. The method for enriching sialic acid oligosaccharides as described in claim 9, characterized in that, The reaction temperature for the enzymatic hydrolysis is 45~55℃; And / or, the reaction time for the enzymatic hydrolysis is 9-11 minutes; And / or, the centrifugation parameters are set to 15000g for 5 minutes; And / or, the solvent is acetonitrile.

11. The method for enriching sialic acid oligosaccharides as described in claim 9, characterized in that, The reaction temperature for the enzymatic hydrolysis is 50℃; And / or, the reaction time for the enzymatic hydrolysis is 10 minutes; And / or, the solvent is an 80% aqueous solution of acetonitrile.

12. The method for enriching sialic acid oligosaccharides as described in claim 9, characterized in that, The enzymatic hydrolysis is carried out at 40-60°C for 8-12 minutes.

13. The method for enriching sialic acid oligosaccharides as described in claim 9, characterized in that, The enzymatic hydrolysis is carried out at 45-55°C for 9-11 minutes.

14. The method for enriching sialic acid oligosaccharides as described in claim 9, characterized in that, The enzymatic hydrolysis was carried out at 50°C for 10 minutes.

15. The method for enriching sialic acid oligosaccharides as described in claim 1, characterized in that, The modified defatted cotton includes the following steps: loading crude sialic acid oligosaccharide into a solid-phase extraction column packed with the modified defatted cotton.

16. The method for enriching sialic acid oligosaccharides as described in claim 15, characterized in that, The solid-phase extraction column is a pipette tip column; And / or, the loading volume of the crude sialic acid oligosaccharide is 10 μL or more; And / or, the solid-phase extraction column is also cleaned before loading the sample.

17. The method for enriching sialic acid oligosaccharides as described in claim 15, characterized in that, For a 200 μL pipette tip column, the mass of the modified degreased cotton filling is 4.5~5.5 mg; And / or, the loading volume of the crude sialic acid oligosaccharide is 15 or 20 μL; And / or, before loading the sample, the solid-phase extraction column is cleaned sequentially with ultrapure water, pure acetonitrile, and 80% ACN aqueous solution.

18. The method for enriching sialic acid oligosaccharides as described in claim 15, characterized in that, For a 200 μL pipette tip column, the mass of the modified degreased cotton filling is 4.8~5.2 mg.

19. The method for enriching sialic acid oligosaccharides as described in claim 1, characterized in that, In the centrifugation step, the centrifugation gradient is set to 50 g for 2 min, 550 g for 2 min, and 2000 g for 1 min, and the cycle is repeated 3 times. And / or, the cleaning step includes the following operation: centrifuging at 2000g for 1 min with water and 80% acetonitrile; And / or, the eluent used for elution is 25 mM NH4HCO3; The elution process includes the following steps: using a 25 mM NH4HCO3 aqueous solution as the eluent, then centrifuging the solid-phase extraction column at 50 g for 2 min, 550 g for 2 min, and 2000 g for 1 min, and then taking the eluent. The process after elution includes the following steps: collecting the eluent, vacuum drying it, and obtaining the enriched sample.

20. A method for detecting sialic acid oligosaccharides, characterized in that, It includes the following steps: (1) Enrichment: The sample to be tested is enriched by the enrichment method as described in any one of claims 1-19 to obtain enriched sample A; (2) Derivatization treatment: The enriched sample A is subjected to a derivatization reaction with a derivatization reagent to obtain a derivatized product; the derivatization reagent is 2-aminobenzamide; (3) Detection: The derivatives were analyzed by liquid chromatography-mass spectrometry (LC-MS). The LC-MS conditions were as follows: the mobile phase consisted of phase C and phase D, wherein phase C was an aqueous solution containing 50 mM ammonium formate at pH 4.4, and phase D was acetonitrile. The elution gradient was as follows, with the volume percentages of phases C and D being 100%: ， Where % refers to the volume percentage of phase D in the mobile phase; The chromatographic column used in the liquid chromatography-mass spectrometry (LC-MS) detection was a Glycan BEH Amide Column with dimensions of 2.1 × 100 mm and a diameter of 1.7 μm.

21. The method for detecting sialic acid oligosaccharides as described in claim 20, characterized in that, The sample to be tested is the crude sialic acid oligosaccharide or glycoprotein sample. And / or, the derivatizing reagent is a 166.67 mg / mL solution of 2-aminobenzamide; And / or, when the crude sialic acid oligosaccharide is selected from 15 μL of 1 mg / mL sialic acid glycopeptide standard solution, 50 μL of 1 mg / mL peptide globulin or 1 fresh raw egg, the derivatized sample is obtained after enrichment, and the volume of the derivatization reagent is 3-8 μL. And / or, the temperature of the derivatization reaction is 50–80°C; And / or, the derivatization reaction takes 2.5 to 5 hours.

22. The method for detecting sialic acid oligosaccharides as described in claim 21, characterized in that, The glycoprotein sample is sialic acid glycopeptide, fetal protein, or egg yolk; And / or, the volume of the derivatizing reagent is 3 μL, 6 μL or 8 μL; And / or, the temperature of the derivatization reaction is 55–75°C; And / or, the derivatization reaction takes 3.5 to 4 hours.

23. The method for detecting sialic acid oligosaccharides as described in claim 21, characterized in that, The volume of the 2-aminobenzamide solution is 6 μL; And / or, the temperature of the derivatization reaction is 65°C; And / or, the derivatization reaction takes 4 hours.

24. The method for detecting sialic acid oligosaccharides as described in claim 21, characterized in that, The derivatization reaction includes the following steps: reacting enriched sialylated oligosaccharides with 6 μL of 166.67 mg / mL 2-aminobenzamide solution at 65°C for 4 hours.

25. The method for detecting sialic acid oligosaccharides as described in claim 20, characterized in that, The flow rate of the mobile phase in the liquid chromatography-mass spectrometry (LC-MS) detection is 0.3–0.6 mL / min; And / or, the running time of the liquid chromatography-mass spectrometry (LC-MS) detection is 20-40 minutes; And / or, the injection volume for the liquid chromatography-mass spectrometry (LC-MS) detection is 40~60 μL; And / or, the temperature of the sample tray for the liquid chromatography-mass spectrometry (LC-MS) detection is below 10°C; And / or, after the liquid chromatography-mass spectrometry (LC-MS) detection, qualitative analysis of the oligosaccharide structure is also performed using software Xcalibur and Glycoworkbench.

26. The method for detecting sialic acid oligosaccharides as described in claim 25, characterized in that, The flow rate of the mobile phase in the liquid chromatography-mass spectrometry (LC-MS) assay is 0.56 mL / min; And / or, the running time of the liquid chromatography-mass spectrometry (LC-MS) detection is 25-35 minutes; And / or, the injection volume for the liquid chromatography-mass spectrometry (LC-MS) detection is 45~55 μL; And / or, the temperature of the sample tray for the liquid chromatography-mass spectrometry (LC-MS) detection is 4°C.

27. The method for detecting sialic acid oligosaccharides as described in claim 25, characterized in that, The running time for the liquid chromatography-mass spectrometry (LC-MS) assay is 30 minutes; And / or, the injection volume for the liquid chromatography-mass spectrometry (LC-MS) detection is 50 μL.

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