New application and method of magnetic bead solution for enriching and purifying glucoside

Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to a novel application and method of a magnetic bead solution for enriching and purifying glucoside, and the magnetic bead solution in the application is used for enriching and purifying glucoside. The enrichment and purification method comprises the following steps: S1, providing a magnetic bead solution; s2, the magnetic bead solution is placed on a magnetic frame, magnetic beads are gathered, and supernate is removed; s3, adding an N-glucoside sample into the container containing the magnetic beads, and uniformly mixing; s4, adding an organic solvent into the mixed liquid, uniformly mixing and standing; s5, the container is placed on the magnetic frame again, the magnetic beads are gathered, and supernate is removed; s6, adding a washing solution into the container containing the magnetic beads, blowing, beating and uniformly mixing, placing the container on the magnetic frame again to gather the magnetic beads, and removing the supernatant; and S7, repeating the washing step to further purify the glucoside. The method has the technical advantages that enrichment and purification are simpler, more convenient and quicker, the treatment process is shortened, the loss of glycoside substances is avoided, and the consistency of detection results is ensured.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and in particular relates to a new use and method of a magnetic bead solution for enriching and purifying glycosides. Background Art

[0003] HILIC is the preferred method for separating complex carbohydrates, which has been widely recognized by many researchers (T.Ikegami, Hydrophilic interaction chromatography for the analysis of biopharmaceutical drugs and therapeutic peptides: a review based on theseparationcharacteristics of the hydrophilic interaction chromatographyphases.J. Sep. Sci. 42 (2019) 130e213.). In particular, the rapid development of HILIC materials in recent years can well achieve the identification of glycosidic bond isomers (Liu Z, Xu M, Zhang W, Miao X, Wang PG, Li S, Yang S. Recent development in hydrophilic interaction liquid chromatography stationary materials for glycopeptide analysis. Anal Methods. 2022 Nov 18;14(44):4437-4448.).

[0004] The current mainstream method is to enrich and purify glycosides using solid phase extraction (SPE) cleanup columns with HILIC fillers or 96-well plate cleanup modules. However, the SPE cleanup column operation steps are cumbersome, the elution volume is large, and in order to increase the injection concentration, additional concentration and re-dissolution operations are required, so the entire purification process is time-consuming; and the 96-well plate cleanup module is expensive and requires a special vacuum solid phase extraction device.

[0005] HILIC beads are a proprietary magnetic polymer microsphere. With mixed mode function, they can produce high affinity for biomolecules and then elute in a small volume, thereby achieving concentration during sample processing. HILIC beads are used to enrich glycosides in high-concentration organic phase systems and separate at low concentrations, thereby achieving the purpose of enriching and purifying glycosides. The samples obtained later can be analyzed or detected using high-performance liquid chromatography or ultra-high-performance liquid chromatography equipped with fluorescence detection, LC-MS mass spectrometry, matrix-assisted laser desorption / ionization mass spectrometry or capillary electrophoresis. Summary of the invention

[0007] In order to solve the problems of the prior art, a new use and method of a magnetic bead solution for enriching and purifying glycosides is provided. HILIC beads are used to enrich and purify glycosides removed from glycoproteins, thereby shortening the processing time while ensuring the enrichment quantity.

[0008] The specific technical solutions of the present invention are as follows: The invention provides a use of a magnetic bead solution, wherein the magnetic bead solution is used for enriching and purifying glycosides.

[0009] Preferably, the use of the magnetic bead solution provided by the present invention also includes other technical features, wherein the magnetic bead solution is a HILIC beads reagent.

[0010] The present invention also provides a method for glycoside enrichment and purification, comprising the following steps: S1. providing a magnetic bead solution; S2. placing the magnetic bead solution on a magnetic rack to aggregate the magnetic beads and remove the supernatant; S3. adding an N-glycoside sample to a container containing the magnetic beads and mixing them evenly; S4. adding an organic solvent to the mixed liquid, mixing them evenly and letting them stand; S5. placing the container on the magnetic rack again to aggregate the magnetic beads and remove the supernatant; S6. adding a washing solution to the container containing the magnetic beads, mixing them evenly by blowing, and then placing it on the magnetic rack again to aggregate the magnetic beads and remove the supernatant; S7. repeating the washing steps to further purify the glycoside.

[0011] Preferably, the method for glycoside enrichment and purification provided by the present invention also includes other technical features, wherein the type of the magnetic beads is magnetic beads with hydrophilic interactions.

[0012] Preferably, the method for glycoside enrichment and purification provided by the present invention also includes other technical features, wherein the fluorescent labeling mixture is used to label glycosides for subsequent detection and analysis.

[0013] Preferably, the method for glycoside enrichment and purification provided by the present invention also includes other technical features, wherein the organic solvent is acetonitrile or a mixture thereof.

[0014] Preferably, the method for glycoside enrichment and purification provided by the present invention also includes other technical features, wherein the N-glycoside sample added in S3. is labeled with a fluorescent labeling mixture or is not labeled with a fluorescent labeling mixture.

[0015] Compared with the prior art, the technical advantages of the present invention are as follows: Compared with traditional solid phase extraction (SPE) cartridge or plate purification methods, HILIC beads technology provides a simpler and faster means of enrichment and purification. Using this technology, the entire process can be significantly shortened and can usually be completed within 10 to 15 minutes. In addition, no loss of glycosides was found during the operation of this method, ensuring the consistency of the test results, and there was no significant difference compared with unpurified samples. This method not only meets the needs of high-throughput testing in the research and development and production processes, but is also cost-effective because it does not require the use of expensive commercial kits and is compatible with multiple detection platforms such as mass spectrometry, liquid chromatography and capillary electrophoresis. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the fluorescence image before N-glycoside enrichment and purification.

[0018] Figure 2 This is the fluorescence image after N-glycoside enrichment and purification. DETAILED DESCRIPTION

[0019] The terms used in the present invention, unless otherwise specified, generally have the meanings that are commonly understood by those of ordinary skill in the art.

[0020] In the following examples, various processes and methods not described in detail are conventional methods well known in the art.

[0021] The reagents used in the following examples were purchased from common commercial sources, and the experimental operations and experimental conditions not specified were referred to the conventional operations and conventional conditions in the art.

[0022] The specific implementation of the present invention is described below in conjunction with embodiments and drawings.

[0023] Embodiment 1: HILIC beads were used to enrich and purify the fusion protein N-glycoside (neutral glycoform and sialic acid glycoform), and UPLC was used for separation and detection; Take 200 μL of magnetic bead solution, place it on the magnetic stand for 1 min, and remove the supernatant.

[0024] The EP tube was taken out from the magnetic stand, and 10 μL of N-glycoside sample labeled with fluorescent labeling mixture was added to the EP tube containing magnetic beads, and mixed by pipetting, and 90 μL of acetonitrile was added, and mixed by pipetting 3-5 times, and allowed to stand for 2 min.

[0025] Place the EP tube on the magnetic stand for 1 min, remove the supernatant, take the EP tube out of the magnetic stand, add 400 μL of washing solution, pipet and mix 3-5 times, place the EP tube on the magnetic stand for 1 min, remove the supernatant, and repeat this step once.

[0026] Remove the EP tube from the magnetic rack, add 15uL of elution buffer, mix by pipetting 2-3 times, and incubate at room temperature for 2 minutes. Place the EP tube on the magnetic rack for 1 minute, pipette the supernatant into a new EP tube, and repeat this step once. Mix the two elution buffers together, and test 2uL on the instrument. For the final results, see Figure 1-2 .

[0027] It is worth noting that the reaction steps are specifically as follows: 1. Glycoside enrichment and purification Take 100 μL of magnetic bead solution, place it on the magnetic stand for 1 min, and remove the supernatant.

[0028] The EP tube was taken out from the magnetic stand, and 10 μL of N-glycoside sample (labeled with fluorescent labeling mixture) was added to the EP tube containing magnetic beads, and mixed by pipetting. 90 μL of acetonitrile was added, and mixed by pipetting for 3-5 times, and then allowed to stand for 2 min.

[0029] Place the EP tube on the magnetic stand for 1 min, remove the supernatant, take the EP tube out of the magnetic stand, add washing solution, mix by pipetting 3-5 times, place the EP tube on the magnetic stand for 1 min, remove the supernatant, and repeat this step once.

[0030] 2. Glycoside elution Remove the EP tube from the magnetic rack, add 15uL of elution buffer, mix by pipetting 2-3 times, and incubate at room temperature for 2 minutes. Place the EP tube on the magnetic rack for 1 minute, pipette the supernatant into a new EP tube, and repeat this step once. Mix the two elution buffers together for later use.

[0031] Embodiment 2: Based on Example 1, in the step of "adding 10 μL of N-glycoside sample labeled with fluorescent labeling mixture into the EP tube containing magnetic beads", the N-glycoside sample labeled with fluorescent labeling mixture can be replaced by an N-glycoside sample not labeled with fluorescent labeling mixture.

[0032] The above is a detailed description of the embodiments, which is convenient for those skilled in the art to correctly understand and use the present invention. Any improvements or modifications to the technical solutions obtained by those skilled in the art based on the present invention on the basis of the prior art, without innovative work, only through analysis, analogy or limited enumeration, etc., should be within the scope of protection determined by the claims.

Claims

1. A use of a magnetic bead solution, characterized in that The magnetic bead solution is used to enrich and purify glycosides.

2. The use of a magnetic bead solution as claimed in claim 1, characterized in that The magnetic bead solution is HILIC beads reagent.

3. A method for glycoside enrichment and purification, characterized in that The following steps are involved: S1. Provide magnetic bead solution; S2. Place the magnetic bead solution on a magnetic rack to aggregate the beads and remove the supernatant; S3. Add the N-glycoside sample to the container containing the magnetic beads and mix well; S4. Adding an organic solvent to the mixed liquid, mixing well and allowing to stand; S5. Place the container on the magnetic stand again to aggregate the beads and remove the supernatant; S6. Add washing solution to the container containing magnetic beads, mix well by pipetting, place on the magnetic rack again to aggregate the magnetic beads and remove the supernatant; S7. Repeat the washing steps to further purify the glycoside.

4. A method for glycoside enrichment and purification as claimed in claim 3, characterized in that The type of magnetic beads is magnetic beads with hydrophilic interaction.

5. A method for glycoside enrichment and purification as claimed in claim 3, characterized in that The fluorescent labeling mixture is used to label glycosides for subsequent detection and analysis.

6. A method for glycoside enrichment and purification as claimed in claim 3, characterized in that The organic solvent is acetonitrile or a mixture thereof.

7. A method for glycoside enrichment and purification as claimed in claim 3, characterized in that The N-glycoside sample added in S3. is labeled with a fluorescent labeling mixture or is not labeled with a fluorescent labeling mixture.

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