CN115007119A - Zwitterion-containing hydrophilic microsphere and preparation and application thereof - Google Patents
Zwitterion-containing hydrophilic microsphere and preparation and application thereof Download PDFInfo
- Publication number
- CN115007119A CN115007119A CN202110234609.5A CN202110234609A CN115007119A CN 115007119 A CN115007119 A CN 115007119A CN 202110234609 A CN202110234609 A CN 202110234609A CN 115007119 A CN115007119 A CN 115007119A
- Authority
- CN
- China
- Prior art keywords
- hydrophilic
- zwitterions
- microsphere
- preparation
- propane
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
- B01J20/26—Synthetic macromolecular compounds
- B01J20/261—Synthetic macromolecular compounds obtained by reactions only involving carbon to carbon unsaturated bonds
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
- B01D15/265—Adsorption chromatography
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
- B01D15/30—Partition chromatography
- B01D15/305—Hydrophilic interaction chromatography [HILIC]
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
- B01J20/28014—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their form
- B01J20/28016—Particle form
- B01J20/28021—Hollow particles, e.g. hollow spheres, microspheres or cenospheres
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
- B01J20/282—Porous sorbents
- B01J20/285—Porous sorbents based on polymers
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/20—Partition-, reverse-phase or hydrophobic interaction chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/40—Concentrating samples
- G01N1/405—Concentrating samples by adsorption or absorption
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2220/00—Aspects relating to sorbent materials
- B01J2220/40—Aspects relating to the composition of sorbent or filter aid materials
- B01J2220/48—Sorbents characterised by the starting material used for their preparation
- B01J2220/4812—Sorbents characterised by the starting material used for their preparation the starting material being of organic character
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2220/00—Aspects relating to sorbent materials
- B01J2220/50—Aspects relating to the use of sorbent or filter aid materials
- B01J2220/52—Sorbents specially adapted for preparative chromatography
Landscapes
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Organic Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Health & Medical Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Physics & Mathematics (AREA)
- Solid-Sorbent Or Filter-Aiding Compositions (AREA)
Abstract
The invention relates to a hydrophilic microsphere containing zwitterions and preparation and application thereof. Specifically, a 'one-pot method' strategy is adopted, in a dispersion medium consisting of water and N-heptane, a functional monomer 3- [ N, N-dimethyl- [2- (2-methylpropane-2-enoyloxy) ethyl ] ammonium ] propane-1-sulfonic acid inner salt and a cross-linking agent N, N-methylene bisacrylamide are subjected to reversed-phase suspension polymerization under the heating initiation effect of an initiator sodium persulfate to obtain the hydrophilic microsphere material containing zwitterions. The preparation of the hydrophilic material can be completed only by one step, the production process is simple, the reaction condition is mild, and the hydrophilic material can be directly used as an adsorbent of hydrophilic interaction chromatography (HILIC) without late grafting or derivation and is used for separating and enriching glycopeptides in biological samples (body fluid, tissue, cell enzymolysis solution and the like of human and/or animals).
Description
Technical Field
The invention relates to a hydrophilic microsphere containing zwitterions, a preparation method thereof and application thereof in glycopeptide separation and enrichment, and particularly relates to a hydrophilic microsphere material containing zwitterions, which is obtained by performing reversed-phase suspension polymerization on a functional monomer 3- [ N, N-dimethyl- [2- (2-methylpropane-2-enoyloxy) ethyl ] ammonium ] propane-1-sulfonic acid inner salt and a cross-linking agent N, N-methylene bisacrylamide in a dispersion medium consisting of water and N-heptane under the initiation action of an initiator sodium persulfate. Due to the existence of zwitterions, the material has good hydrophilic performance, can be directly used as an adsorbent of hydrophilic interaction chromatography and is used for separating and enriching glycopeptides in complex biological samples.
Background
Glycosylation of proteins is considered one of the most important post-translational modifications in protein modification, which is involved in most life processes, such as: abnormal glycosylation of proteins, such as cellular communication, signal transduction, and cellular metabolism, often leads to certain diseases, such as Alzheimer's disease, nervous system diseases, and cancer, etc., and thus, studies on glycosylation of proteins have been of great importance in the field of life sciences (1 Alvarez-Manillea, et al, "diabetes for glycobiological analysis: Size expression pathology identification of therapeutic glycosylation sites with N-linked glycosylation sites." Journal of protein research.2006,5, 701-. The "shotgun method" is currently the most widely used strategy for studying protein glycosylation, and mass spectrometry is the most important analytical tool. However, the relative abundance of glycopeptides in the protein enzymatic hydrolysate is only 2-5%, interference of non-glycopeptides is very serious, and mass spectrometry of glycopeptides in the enzymatic hydrolysate is very difficult to directly perform. Therefore, efficient enrichment of glycopeptides prior to mass spectrometry is necessary. (Besinon et al, reference 2, "novel Materials for N-glycopeptide/glycoprotein separation Enrichment," chemical Advances. 2019,31, 996-.
To date, methods for glycopeptide enrichment include the following classes: hydrophilic interaction liquid chromatography, lectin affinity, boronic acid affinity chromatography and hydrazide chemistry. (literature 4 Zhengtong et al, "progress in the research of enrichment of smart polymer-based materials with phosphorylated peptides and glycopeptides". chromatograph. 2021,39, 15-25; literature 5Yao, et al, "Recent advances in media materials for sample preparation in proteomics research." TrAC Trends in Analytical chemistry.2018,99, 88-100). The hydrophilic interaction liquid chromatography (HILIC) is to separate and enrich glycopeptide by utilizing the fact that the hydrophilic interaction force of the adsorbent is stronger than that of glycopeptide and non-glycopeptide. It can be widely used because of indiscriminate enrichment of glycopeptides and no damage to the glycopeptide saccharide structure.
At present, most HILIC hydrophilic materials are prepared by derivatizing or grafting hydrophilic groups (including carboxyl, amino, hydroxyl, zwitterionic molecules and the like) on a substrate, and the preparation process is complicated, has multiple steps and is long-lasting, so that a new preparation method with simple exploration procedures is always a research hotspot (document 6Zhang, et al, "One-step synthesis of hydrophilic microspheres for high selectivity reaction of N-linked glyceropeptides.
Disclosure of Invention
The invention aims to provide a zwitterion-containing hydrophilic microsphere material and a preparation method thereof, which can be used as a hydrophilic interaction chromatographic adsorbent for separating and enriching glycopeptides in a biological sample.
To achieve the above object, the following process can be performed:
adding 200-800 mg of 3- [ N, N-dimethyl- [2- (2-methylpropane-2-enoyloxy) ethyl ] ammonium ] propane-1-sulfonic acid inner salt, 50-600 mg of N, N-methylene bisacrylamide and 1-10 mL of water into a container, and dissolving to form a uniform and transparent solution; continuously adding 10-60 mL of n-heptane, 200-600 mg of span 80 and 50-400 mg of Tween 80, and mixing; then, adding 10-100 mg of thermal initiator sodium persulfate, and stirring for 10-40 min under mechanical stirring at the rotating speed of 100-300 r/min to uniformly disperse the sodium persulfate in the reaction liquid; then raising the temperature to 60-80 ℃, and continuing stirring for 4-8 hours to complete the polymerization reaction; and finally, washing the obtained microsphere material with an ethanol/water (1/1-1/2, v/v) mixed solvent for 3-5 times, and drying in vacuum at the temperature of 60-80 ℃ for 8-24 hours. The zwitterion hydrophilic microsphere can be used for separating and enriching glycopeptides in a biological sample.
The invention has the following advantages:
(1) the hydrophilic microsphere with the surface containing zwitterions can be prepared by only one-step reaction, the production process is simple, and the reaction conditions are mild.
(2) The prepared material can be directly used as an adsorbent of hydrophilic interaction chromatography without late grafting or derivation, and glycopeptide in a biological sample is separated and enriched.
Drawings
FIG. 1 is a schematic diagram of the preparation of hydrophilic microspheres containing zwitterions.
FIG. 2 is a comparison graph of Fourier transform-infrared spectra of hydrophilic zwitterions-containing microspheres and monomers from which they were prepared.
FIG. 3 is a scanning electron microscope image of helium ions of hydrophilic microspheres. FIGS. 3 a-b: the hydrophilic microspheres I containing zwitterions prepared in example 1; FIGS. 3 c-d: the zwitterion-containing hydrophilic microspheres II prepared in example 2; FIGS. 3 e-f: the microspheres prepared in example 3 were free of zwitterions.
FIG. 4 shows the nitrogen adsorption/desorption curve and the pore size distribution of the microspheres. FIGS. 4 a-b: the zwitterion-containing hydrophilic microspheres I prepared in example 1; FIGS. 4 c-d: the zwitterion-containing hydrophilic microspheres II prepared in example 2; FIGS. 4 e-f: example 3 prepared zwitterion-free microspheres.
FIG. 5 is a comparison graph of mass spectra of the hydrophilic microspheres before and after enrichment of human immunoglobulin G (IgG) enzymatic hydrolysate. FIG. 5a is before enrichment; FIG. 5b shows that the hydrophilic microspheres I prepared in example 1 are adsorbents; FIG. 5c shows that the hydrophilic microspheres II prepared in example 2 are adsorbents; 5d represents that the microspheres without zwitterion prepared in the embodiment 3 are adsorbents; FIG. 5e is a mass spectrum of commercial Vensui HILIC material enriched with adsorbent. Asterisks (—) represent glycopeptides.
Description of the attached tables
Table 1 shows the mass-to-charge ratio and glycoform composition of glycopeptide enriched from IgG enzymatic hydrolysate of hydrophilic microsphere I prepared in example 1.
Table 2 shows the mass-to-charge ratio and the glycoform composition of glycopeptide enriched from the IgG enzymatic hydrolysate by the hydrophilic microsphere II prepared in example 2.
Table 3 shows the mass-to-charge ratio and glycoform composition of glycopeptides enriched in IgG enzymatic hydrolysate by the zwitterion-free microspheres prepared in example 3.
Table 4 shows the mass-to-charge ratio and the glycoform composition of glycopeptide enriched from IgG enzymatic hydrolysate by the commercial hydrophilic material in comparative example 1.
Detailed Description
Example 1 preparation and use of zwitterionic hydrophilic microspheres I
Preparation of zwitterion-containing hydrophilic microspheres:
in a container, 558.7mg of 3- [ N, N-dimethyl- [2- (2-methylprop-2-enoyloxy) ethyl ] ammonium ] propane-1-sulfonic acid inner salt, 92.5mg of N, N-methylenebisacrylamide and 3mL of water were added and dissolved to form a uniform and transparent solution; then, continuously adding 20mL of n-heptane, 300mg of span 80 and 200mg of Tween 80, and mixing; then, 50mg of thermal initiator sodium persulfate is added, and the mixture is stirred for 20min under the mechanical stirring at the rotating speed of 300r/min so that the sodium persulfate is uniformly dispersed in the reaction liquid; then raising the temperature to 70 ℃, and continuing stirring for 6 hours to complete the polymerization reaction; finally, the obtained microsphere material is washed 5 times by using an ethanol/water (1/1, v/v) mixed solvent and dried for 12 hours in vacuum at the temperature of 60 ℃ to obtain the hydrophilic microsphere I containing zwitterion.
Preparation of human immunoglobulin G (IgG) enzymatic hydrolysate sample: 2.0mg of IgG was dissolved in 1.0mL of urea containing 8M and 100mM NH 4 HCO 3 In an aqueous solution of a denaturing agent. Then 20 mul of aqueous solution of dithiothreitol with the concentration of 1M is added, after centrifugation at 8000r/min for 20min, the temperature is kept for 2h at 37 ℃. In a light-shielding stateNext, 7.4mg of iodoacetamide was added thereto and the mixture was reacted for 35min under dark conditions. After removal, 7.0mL of Tris-HCl buffer was added to dilute the urea to 1M. Adding trypsin according to the enzyme-protein mass ratio of 1:25, reacting in a water bath at 37 ℃ for 18h, desalting the obtained enzymolysis liquid, freeze-drying, and storing in a refrigerator at-20 ℃ for later use.
Enrichment of glycosylated peptides: first, 200. mu.L of a sample solution (ACN/H) was added 2 O/TFA, 75:24:1, v/v/v) the zwitterionic hydrophilic microsphere I material obtained above was equilibrated twice, each time with shaking at room temperature for 15min, centrifuged, and the supernatant removed. 200 μ L of the loading solution (ACN/H) containing 10 μ g of IgG enzymolysis solution 2 O/TFA, 75:24:1, v/v/v) was added to the material, and the sample was shaken at room temperature for 30min, centrifuged, and the supernatant was removed. Then sampling liquid (ACN/H) 2 O/TFA, 75:24:1, v/v/v) two rinses (200. mu.L. times.2) with shaking for 15min each, centrifugation, and removal of the supernatant to remove non-glycopeptide and other impurities. Then 100. mu.L of eluent (ACN/H) was added 2 O/TFA, 30:69:1, v/v/v) and shaking at room temperature for 15min, centrifuging, taking the supernatant, and performing matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/MS) analysis by using MALDI TOF 5800 mass spectrometry.
Product characterization
Fourier transform-Infrared Spectroscopy of the zwitterionic hydrophilic microspheres prepared in example 1 and the monomers prepared therefrom the monomer 3- [ N, N-dimethyl- [2- (2-methylprop-2-enyloxy) ethyl ] is shown in FIG. 2]Ammonium salt]The wave number of the propane-1-sulfonic acid inner salt is 1035cm -1 And 957cm -1 Are each-SO 3 Stretching vibration absorption peak and-N of middle S ═ O bond + (CH 3 ) 2 Absorption peaks of stretching vibration of C-N bond in the medium, whose wave numbers in the zwitterionic microspheres respectively correspond to 1034cm -1 And 963cm -1 (ii) a The wave number of the cross-linking agent N, N-methylene-bisacrylamide is 1655cm -1 And 1538cm -1 Respectively representing an amide IC ═ O stretching vibration peak and an amide II-N-H stretching vibration peak, and the wave numbers of the two peaks in the zwitterionic microspheres are respectively 1651cm -1 And 1538cm -1 (ii) a The median wave number of the two monomers is 1625cm -1 And 1634cm -1 Peak of stretching vibration of C ═ C bond, however1620cm in the material -1 In conclusion, the zwitterionic hydrophilic microsphere is prepared from the monomers and the crosslinking agent, and the microsphere has the zwitterionic hydrophilic group.
As shown in FIGS. 3a and 3b, the helium ion scanning electron microscope shows that the material is spherical and has a particle size of 10 to 20 μm. The nitrogen physical adsorption/desorption experiment result (figure 4a) shows that the specific surface area of the hydrophilic microsphere containing zwitterion is 34.7m 2 g -1 And contains mesopores of about 3.7nm (FIG. 4 b).
Product application
The prepared microsphere has zwitter-ion hydrophilic groups, so that the microsphere can be directly used as an adsorbent for hydrophilic interaction chromatography to separate and enrich glycopeptides in a biological sample. FIG. 5 is a comparison graph of mass spectra of human immunoglobulin G enzymatic hydrolysate samples before and after enrichment. As shown in FIG. 5a, most of the peak signals with higher intensity in the spectra before enrichment were non-glycopeptide, and the glycopeptide signals were almost suppressed, and only two significant glycopeptide signals were observed. After the hydrophilic microsphere I containing zwitterion is used for enrichment, as shown in fig. 5b, the non-glycopeptide peak is obviously reduced, the glycopeptide peak signal is obviously enhanced, and 18 typical glycopeptide signal peaks (shown in table 1, the signal-to-noise ratio is 10/1) can be detected, which shows that the microsphere has high enrichment effect and selectivity on glycopeptide.
Example 2 preparation and use of hydrophilic microspheres II containing zwitterions
The preparation process, conditions and glycopeptide enrichment step are the same as those in example 1, and the difference from example 1 is that in the preparation process, the mass of the cross-linking agent N, N-methylene bisacrylamide in the reaction solution is increased to 308.3mg, and the rest of the preparation process and conditions and the glycopeptide enrichment step are the same as those in example 1.
Product characterization
As shown in FIGS. 3c and 3d, the helium ion scanning electron microscope of the obtained microsphere shows that the material is spherical in appearance and has a particle size of 5-10 μm. The results of the nitrogen physical adsorption/desorption experiments (FIG. 4c) show that the specific surface area of the microspheres is 87.4m 2 g -1 Containing mesopores of about 6.6nm (FIG. 4d). These results show that the content of the crosslinking agent N, N-methylene-bisacrylamide in the reaction solution has a great influence on the diameter and the pore channel distribution of the microspheres.
Product application
As shown in fig. 5c, although 23 glycopeptides can be enriched from the human immunoglobulin G enzymatic hydrolysate by using the hydrophilic microsphere II as the adsorbent (the signal-to-noise ratio is 10/1 as shown in table 2), the interference of the non-glycopeptide peak is not negligible, which indicates that the enrichment efficiency of the microsphere on glycopeptides is high, but the selectivity is not strong, which is lower than that of the hydrophilic microsphere I prepared in example 1, and the reason may be that the content of zwitterion in the prepared microsphere is reduced and the hydrophilicity is reduced due to the increase of the cross-linking agent in the reaction solution.
Example 3 preparation and use of a zwitterion-free Material
Preparation of a material containing no zwitterion:
the preparation process, conditions and glycopeptide enrichment step are the same as those in example 1, and are different from example 1 in that in the preparation process, the functional monomer 3- [ N, N-dimethyl- [2- (2-methylpropane-2-enoyloxy) ethyl ] ammonium ] propane-1-sulfonic acid inner salt in the reaction liquid is removed (namely, is not added), and the rest of the preparation process and conditions are the same as those in example 1 in the glycopeptide enrichment step.
Product characterization
As shown in FIGS. 3e and 3f, the obtained material was bulk and amorphous. The results of the nitrogen physical adsorption/desorption experiments (FIG. 4e) show that the specific surface area of the material is 192.4m 2 g -1 And contains mesopores of about 5.1nm (FIG. 4 f). This result indicates that the functional monomer 3- [ N, N-dimethyl- [2- (2-methylprop-2-enoyloxy) ethyl]Ammonium salt]The appearance and the pore distribution of the propane-1-sulfonic acid inner salt can be influenced in the synthesis process of the material.
Product application
As shown in fig. 5d, the material is used as an adsorbent, only 20 glycopeptides are enriched from the human immunoglobulin enzymatic hydrolysate (table 3 shows that the signal-to-noise ratio is 10/1), the non-sugar interference is more obvious than that in example 1, and the signal intensity is lower than the glycopeptide enrichment effect of the zwitterionic hydrophilic microspheres prepared in examples 1 and 2, which indicates that the addition of zwitterions can improve the enrichment efficiency and selectivity of the material on glycopeptides.
Comparative example 1
Enrichment of glycopeptides using commercial hydrophilic materials
For comparison with example 1, commercial hydrophilic material (Venusil HILIC, 10 μm) was purchased from borna aigel technologies ltd, and glycopeptide in human immunoglobulin hydrolysate was enriched using the same enrichment process as in example 1. The result is shown in fig. 5e, the glycopeptide peak signal intensity is low and non-sugar interference is observed, 19 glycopeptides can be detected (table 4 shows that the signal to noise ratio is 10/1), which is lower than the result of example 1, indicating that the glycopeptide enrichment efficiency and selectivity are not as good as those of the hydrophilic microsphere I prepared in example 1.
The results show that the microsphere containing zwitterion prepared by the invention has high enrichment efficiency and selectivity on glycopeptide. The preparation method is simple to operate, can be completed once, and can be directly used as an adsorbent for hydrophilic interaction chromatography without secondary modification.
TABLE 1 molecular weight and glycoform composition of glycopeptide in IgG-enriched enzymatic hydrolysate in example 1
N # represents a glycosylation site; hex mannose; HexNac N-acetylglucosamine; fuc is fucose.
TABLE 2 EXAMPLE 2 molecular weight and glycoform composition of glycopeptides in IgG-enriched enzymatic hydrolysate
N # represents a glycosylation site; hex mannose; HexNac is N-acetylglucosamine; fuc is fucose.
TABLE 3 molecular weight and glycoform composition of glycopeptide in IgG-enriched enzymatic hydrolysate in example 3
N # represents a glycosylation site; hex mannose; HexNac is N-acetylglucosamine; fuc is fucose.
TABLE 4 molecular weight and glycoform composition of glycopeptide in IgG enzymolysis solution enriched in comparative example
N # represents a glycosylation site; hex mannose; HexNac is N-acetylglucosamine; fuc is fucose.
Claims (7)
1. A preparation method of hydrophilic microspheres containing zwitterions comprises the steps of utilizing 3- [ N, N-dimethyl- [2- (2-methylpropane-2-enoyloxy) ethyl ] ammonium ] propane-1-sulfonic acid inner salt (MSA) as a functional monomer, utilizing N, N-methylene Bisacrylamide (BIS) as a cross-linking agent, and carrying out reverse suspension polymerization by adopting a thermal initiation mode in the presence of a stabilizer to prepare the hydrophilic microspheres containing the zwitterions.
2. The method for preparing hydrophilic microspheres containing zwitterions according to claim 1, wherein:
3- [ N, N-dimethyl- [2- (2-methylpropane-2-enoyloxy) ethyl ] ammonium ] propane-1-sulfonic acid inner salt is used as a functional monomer;
n, N-methylene bisacrylamide is taken as a cross-linking agent;
sodium persulfate is used as a thermal initiator;
non-ionic surfactant span 80 and tween 80 are used as a mixed stabilizer;
taking a mixed solvent of n-heptane and water as a continuous medium, and carrying out reversed phase suspension polymerization at the temperature of 60-80 ℃ to obtain the hydrophilic microsphere material containing zwitterions in one step.
3. The production method according to claim 1 or 2, characterized in that: the hydrophilic microspheres containing zwitterions may be prepared as follows:
adding 200-800 mg of 3- [ N, N-dimethyl- [2- (2-methylpropane-2-enoyloxy) ethyl ] ammonium ] propane-1-sulfonic acid inner salt, 50-600 mg of N, N-methylene bisacrylamide and 1-10 mL of water into a container, and dissolving to form a uniform and transparent solution; continuously adding 10-60 mL of n-heptane, 200-600 mg of span 80 and 50-400 mg of Tween 80, and mixing; then, adding 10-100 mg of thermal initiator sodium persulfate, and stirring for 10-40 min under mechanical stirring at the rotating speed of 100-300 r/min to uniformly disperse the sodium persulfate in the reaction liquid; then raising the temperature to 60-80 ℃, and continuing stirring for 4-8 hours to complete the polymerization reaction; and finally, washing the obtained microsphere material with an ethanol/water (1/1-1/2, v/v) mixed solvent for 3-5 times, and drying in vacuum at the temperature of 60-80 ℃ for 8-24 hours.
4. Hydrophilic microspheres containing zwitterions, obtainable by a process according to any one of claims 1 to 3.
5. Use of the zwitter-ion-containing hydrophilic microspheres of claim 4 as an adsorbent or filler for hydrophilic interaction chromatography.
6. Use according to claim 5, characterized in that: the hydrophilic microsphere material containing zwitterions has good hydrophilic performance, can be directly used as an adsorbent or a filler of hydrophilic interaction chromatography, and is used for separating and enriching glycopeptides in a biological sample.
7. Use according to claim 6, characterized in that: the biological sample is one or more than two of human and/or animal body fluid, tissue, cell enzymolysis liquid and the like.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110234609.5A CN115007119A (en) | 2021-03-03 | 2021-03-03 | Zwitterion-containing hydrophilic microsphere and preparation and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110234609.5A CN115007119A (en) | 2021-03-03 | 2021-03-03 | Zwitterion-containing hydrophilic microsphere and preparation and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN115007119A true CN115007119A (en) | 2022-09-06 |
Family
ID=83064749
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110234609.5A Pending CN115007119A (en) | 2021-03-03 | 2021-03-03 | Zwitterion-containing hydrophilic microsphere and preparation and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115007119A (en) |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100092526A1 (en) * | 2008-09-26 | 2010-04-15 | Nanobio Corporation | Nanoemulsion therapeutic compositions and methods of using the same |
| CN102940980A (en) * | 2012-10-22 | 2013-02-27 | 暨南大学 | Preparation method and application for hydrophilic organic polymer liquid phase monolithic chromatographic column |
| CN103037913A (en) * | 2010-06-09 | 2013-04-10 | 森普鲁斯生物科学公司 | Non-fouling, anti-microbial, anti-thrombogenic graft-from compositions |
| CN105693906A (en) * | 2015-01-20 | 2016-06-22 | 于乐 | Zwitterionic polymer microspheres and preparing method thereof |
| CN109304101A (en) * | 2017-07-28 | 2019-02-05 | 中国科学院宁波材料技术与工程研究所 | A kind of bisexual ion purification high intensity anti-pollution forward osmosis membrane and preparation method thereof |
| CN110437472A (en) * | 2019-08-12 | 2019-11-12 | 浙江工业大学 | A kind of high-intensitive, salt response double-network hydrogel and its application |
-
2021
- 2021-03-03 CN CN202110234609.5A patent/CN115007119A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100092526A1 (en) * | 2008-09-26 | 2010-04-15 | Nanobio Corporation | Nanoemulsion therapeutic compositions and methods of using the same |
| CN103037913A (en) * | 2010-06-09 | 2013-04-10 | 森普鲁斯生物科学公司 | Non-fouling, anti-microbial, anti-thrombogenic graft-from compositions |
| CN102940980A (en) * | 2012-10-22 | 2013-02-27 | 暨南大学 | Preparation method and application for hydrophilic organic polymer liquid phase monolithic chromatographic column |
| CN105693906A (en) * | 2015-01-20 | 2016-06-22 | 于乐 | Zwitterionic polymer microspheres and preparing method thereof |
| CN109304101A (en) * | 2017-07-28 | 2019-02-05 | 中国科学院宁波材料技术与工程研究所 | A kind of bisexual ion purification high intensity anti-pollution forward osmosis membrane and preparation method thereof |
| CN110437472A (en) * | 2019-08-12 | 2019-11-12 | 浙江工业大学 | A kind of high-intensitive, salt response double-network hydrogel and its application |
Non-Patent Citations (1)
| Title |
|---|
| 夏宇正等: "《精细高分子化工及应用》", 化学工业出版社, pages: 31 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Bertolla et al. | Solvent-responsive molecularly imprinted nanogels for targeted protein analysis in MALDI-TOF mass spectrometry | |
| Terracciano et al. | Selective binding and enrichment for low‐molecular weight biomarker molecules in human plasma after exposure to nanoporous silica particles | |
| US20090101581A1 (en) | Immobilized titanium ion affinity chromatography material and its preparation and application | |
| Xu et al. | Boronic acid-functionalized detonation nanodiamond for specific enrichment of glycopeptides in glycoproteome analysis | |
| Fu et al. | Preparation of glutathione-functionalized zwitterionic silica material for efficient enrichment of sialylated N-glycopeptides | |
| CN110152624B (en) | Microporous polymer coated hydrophilic resin and application thereof in glycopeptide enrichment | |
| Pan et al. | Brush polymer modified and lectin immobilized core–shell microparticle for highly efficient glycoprotein/glycopeptide enrichment | |
| Tian et al. | Glutathione-modified ordered mesoporous silicas for enrichment of N-linked glycopeptides by hydrophilic interaction chromatography | |
| Zhang et al. | Preparation of poly (vinylphenylboronic acid) chain grafted poly (glycidylmethacrylate‐co‐ethylenedimethacrylate) beads for the selective enrichment of glycoprotein | |
| CN106432644A (en) | Hydrophilic polymer functional magnetic nanospheres as well as preparation method and application thereof | |
| Zhang et al. | Deep eutectic solvents-based polymer monolith incorporated with titanium dioxide nanotubes for specific recognition of proteins | |
| Lin et al. | Preparation of iminodiacetic acid functionalized silica capillary trap column for on-column selective enrichment of N-linked glycopeptides | |
| CN115007119A (en) | Zwitterion-containing hydrophilic microsphere and preparation and application thereof | |
| CN113721028A (en) | Synthetic method and application of GO @ CS @ ZIF-8 foam material | |
| CN116813920A (en) | Preparation method and application of beta-cyclodextrin functionalized covalent organic framework composite material | |
| CN110961083B (en) | Hydrophilic chitosan integral material and preparation and application thereof | |
| CN108948303B (en) | Material based on porous polyacylsemicarbazide, preparation method and application | |
| CN115007113B (en) | Carbon dot-doped chitosan film and preparation and application thereof | |
| JP2007017445A (en) | Filler for chromatography | |
| Zou et al. | Novel multifunctional chitosan-GMA-IDA-Cu (II) nanospheres for high dynamic range characterization of the human plasma proteome | |
| CN111111626B (en) | Zwitterion modified hydrophilic carbon sphere, preparation thereof and application thereof in glycopeptide enrichment | |
| CN111748121B (en) | Glutathione-modified hybrid material and preparation and application thereof | |
| CN110871055B (en) | Preparation method and application of molecular engram polymer silica gel microsphere with glutamic acid as end group and fresh polypeptide surface | |
| WO2016028075A1 (en) | Monoclonal antibody based online phosphoprotein proteomics analysis method using microbore hollow fiber enzymatic reactor-tandem mass spectrometry | |
| CN113145088B (en) | Hydrophilic integral material and preparation and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |