CN115212852B - 一种亲水膜层包覆的大孔树脂材料及其制备和应用 - Google Patents
一种亲水膜层包覆的大孔树脂材料及其制备和应用 Download PDFInfo
- Publication number
- CN115212852B CN115212852B CN202110399508.3A CN202110399508A CN115212852B CN 115212852 B CN115212852 B CN 115212852B CN 202110399508 A CN202110399508 A CN 202110399508A CN 115212852 B CN115212852 B CN 115212852B
- Authority
- CN
- China
- Prior art keywords
- macroporous resin
- film layer
- resin material
- hydrophilic film
- hydrophilic
- 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.)
- Active
Links
- 239000011347 resin Substances 0.000 title claims abstract description 75
- 229920005989 resin Polymers 0.000 title claims abstract description 75
- 239000000463 material Substances 0.000 title claims abstract description 52
- 238000002360 preparation method Methods 0.000 title abstract description 12
- 102000002068 Glycopeptides Human genes 0.000 claims abstract description 37
- 108010015899 Glycopeptides Proteins 0.000 claims abstract description 37
- 239000004005 microsphere Substances 0.000 claims abstract description 31
- 238000002013 hydrophilic interaction chromatography Methods 0.000 claims abstract description 20
- OUPZKGBUJRBPGC-UHFFFAOYSA-N 1,3,5-tris(oxiran-2-ylmethyl)-1,3,5-triazinane-2,4,6-trione Chemical compound O=C1N(CC2OC2)C(=O)N(CC2OC2)C(=O)N1CC1CO1 OUPZKGBUJRBPGC-UHFFFAOYSA-N 0.000 claims abstract description 15
- 238000006243 chemical reaction Methods 0.000 claims abstract description 11
- 239000003463 adsorbent Substances 0.000 claims abstract description 9
- 125000003700 epoxy group Chemical group 0.000 claims abstract description 8
- 239000000178 monomer Substances 0.000 claims abstract description 7
- 238000007151 ring opening polymerisation reaction Methods 0.000 claims abstract description 7
- 239000012472 biological sample Substances 0.000 claims abstract description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 20
- 239000011148 porous material Substances 0.000 claims description 15
- 239000002245 particle Substances 0.000 claims description 10
- 239000012528 membrane Substances 0.000 claims description 7
- 229940113115 polyethylene glycol 200 Drugs 0.000 claims description 5
- 229940080817 rotenone Drugs 0.000 claims description 5
- JUVIOZPCNVVQFO-UHFFFAOYSA-N rotenone Natural products O1C2=C3CC(C(C)=C)OC3=CC=C2C(=O)C2C1COC1=C2C=C(OC)C(OC)=C1 JUVIOZPCNVVQFO-UHFFFAOYSA-N 0.000 claims description 5
- VOZRXNHHFUQHIL-UHFFFAOYSA-N glycidyl methacrylate Chemical compound CC(=C)C(=O)OCC1CO1 VOZRXNHHFUQHIL-UHFFFAOYSA-N 0.000 claims description 4
- 239000007788 liquid Substances 0.000 claims description 2
- 210000001124 body fluid Anatomy 0.000 claims 1
- 239000010839 body fluid Substances 0.000 claims 1
- 239000000203 mixture Substances 0.000 claims 1
- DQJCDTNMLBYVAY-ZXXIYAEKSA-N (2S,5R,10R,13R)-16-{[(2R,3S,4R,5R)-3-{[(2S,3R,4R,5S,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy}-5-(ethylamino)-6-hydroxy-2-(hydroxymethyl)oxan-4-yl]oxy}-5-(4-aminobutyl)-10-carbamoyl-2,13-dimethyl-4,7,12,15-tetraoxo-3,6,11,14-tetraazaheptadecan-1-oic acid Chemical compound NCCCC[C@H](C(=O)N[C@@H](C)C(O)=O)NC(=O)CC[C@H](C(N)=O)NC(=O)[C@@H](C)NC(=O)C(C)O[C@@H]1[C@@H](NCC)C(O)O[C@H](CO)[C@H]1O[C@H]1[C@H](NC(C)=O)[C@@H](O)[C@H](O)[C@@H](CO)O1 DQJCDTNMLBYVAY-ZXXIYAEKSA-N 0.000 abstract description 12
- 238000000034 method Methods 0.000 abstract description 10
- QBPPRVHXOZRESW-UHFFFAOYSA-N 1,4,7,10-tetraazacyclododecane Chemical compound C1CNCCNCCNCCN1 QBPPRVHXOZRESW-UHFFFAOYSA-N 0.000 abstract description 4
- 239000011248 coating agent Substances 0.000 abstract description 2
- 238000000576 coating method Methods 0.000 abstract description 2
- 230000008569 process Effects 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 14
- 239000000523 sample Substances 0.000 description 8
- 239000001307 helium Substances 0.000 description 7
- 229910052734 helium Inorganic materials 0.000 description 7
- 229940098197 human immunoglobulin g Drugs 0.000 description 7
- 238000001000 micrograph Methods 0.000 description 7
- 238000001228 spectrum Methods 0.000 description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 6
- 102000007079 Peptide Fragments Human genes 0.000 description 6
- 108010033276 Peptide Fragments Proteins 0.000 description 6
- 230000002255 enzymatic effect Effects 0.000 description 6
- 239000000413 hydrolysate Substances 0.000 description 6
- 230000013595 glycosylation Effects 0.000 description 5
- 238000004949 mass spectrometry Methods 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 238000001840 matrix-assisted laser desorption--ionisation time-of-flight mass spectrometry Methods 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 239000004202 carbamide Substances 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 238000012512 characterization method Methods 0.000 description 3
- 238000003795 desorption Methods 0.000 description 3
- 238000006206 glycosylation reaction Methods 0.000 description 3
- 238000011068 loading method Methods 0.000 description 3
- 229910052757 nitrogen Inorganic materials 0.000 description 3
- 230000004481 post-translational protein modification Effects 0.000 description 3
- 102000004196 processed proteins & peptides Human genes 0.000 description 3
- 108090000765 processed proteins & peptides Proteins 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 238000001179 sorption measurement Methods 0.000 description 3
- 239000012798 spherical particle Substances 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 238000001291 vacuum drying Methods 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- WXTMDXOMEHJXQO-UHFFFAOYSA-N 2,5-dihydroxybenzoic acid Chemical compound OC(=O)C1=CC(O)=CC=C1O WXTMDXOMEHJXQO-UHFFFAOYSA-N 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 238000002441 X-ray diffraction Methods 0.000 description 2
- 229940098773 bovine serum albumin Drugs 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 150000004676 glycans Chemical class 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 238000002186 photoelectron spectrum Methods 0.000 description 2
- 229920001282 polysaccharide Polymers 0.000 description 2
- 239000005017 polysaccharide Substances 0.000 description 2
- 230000017854 proteolysis Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- FHOMNNOYQDOCJQ-VKKIDBQXSA-N (2S,3S)-1,4-bis(sulfanyl)butane-2,3-diol hydrate Chemical compound O.O[C@H](CS)[C@H](O)CS FHOMNNOYQDOCJQ-VKKIDBQXSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 208000024827 Alzheimer disease Diseases 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 206010061598 Immunodeficiency Diseases 0.000 description 1
- 208000029462 Immunodeficiency disease Diseases 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 208000012902 Nervous system disease Diseases 0.000 description 1
- 208000025966 Neurological disease Diseases 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000003044 adaptive effect Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000033077 cellular process Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 239000003398 denaturant Substances 0.000 description 1
- 238000011033 desalting Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000007888 film coating Substances 0.000 description 1
- 238000009501 film coating Methods 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 102000035122 glycosylated proteins Human genes 0.000 description 1
- 108091005608 glycosylated proteins Proteins 0.000 description 1
- 230000005934 immune activation Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000007813 immunodeficiency Effects 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000010262 intracellular communication Effects 0.000 description 1
- PGLTVOMIXTUURA-UHFFFAOYSA-N iodoacetamide Chemical compound NC(=O)CI PGLTVOMIXTUURA-UHFFFAOYSA-N 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 238000000707 layer-by-layer assembly Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 238000000696 nitrogen adsorption--desorption isotherm Methods 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 238000000164 protein isolation Methods 0.000 description 1
- 230000009145 protein modification Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000007142 ring opening reaction Methods 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
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
- B01J20/264—Synthetic macromolecular compounds derived from different types of monomers, e.g. linear or branched copolymers, block copolymers, graft copolymers
-
- 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
- B01J20/28019—Spherical, ellipsoidal or cylindrical
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J9/00—Working-up of macromolecular substances to porous or cellular articles or materials; After-treatment thereof
- C08J9/36—After-treatment
- C08J9/40—Impregnation
- C08J9/405—Impregnation with polymerisable compounds
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J2333/00—Characterised by the use of homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical, or of salts, anhydrides, esters, amides, imides, or nitriles thereof; Derivatives of such polymers
- C08J2333/04—Characterised by the use of homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical, or of salts, anhydrides, esters, amides, imides, or nitriles thereof; Derivatives of such polymers esters
- C08J2333/14—Characterised by the use of homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical, or of salts, anhydrides, esters, amides, imides, or nitriles thereof; Derivatives of such polymers esters of esters containing halogen, nitrogen, sulfur, or oxygen atoms in addition to the carboxy oxygen
Landscapes
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Organic Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Engineering & Computer Science (AREA)
- Materials Engineering (AREA)
- Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Polymers & Plastics (AREA)
- Solid-Sorbent Or Filter-Aiding Compositions (AREA)
Abstract
本发明涉及一种亲水膜层包覆的大孔树脂材料及其制备和应用。具体是以表面带有环氧基的大孔树脂微球为基质，以轮环藤宁(Cyclen)和异氰尿酸三缩水甘油酯(1,3,5‑triglycidyl isocyanurate，TGIC)为功能单体，利用环氧‑胺开环聚合反应，在大孔树脂微球表面形成亲水膜层，得到亲水膜层包覆的大孔树脂材料。该树脂微球可以直接用作亲水相互作用色谱(hydrophilic interaction chromatography，HILIC)的吸附剂，用来富集复杂生物样品中的糖肽。采用该包覆亲水膜的方法，操作过程简单，反应条件温和，所得材料作为HILIC吸附剂对糖肽具有很高的富集效率和选择性。
Description
技术领域
本发明涉及一种亲水膜层包覆的大孔树脂材料及其制备和应用，具体是以表面带有环氧基的大孔树脂微球为基质，轮环藤宁和异氰尿酸三缩水甘油酯为功能单体，利用环氧-胺开环聚合反应在大孔树脂微球表面形成亲水膜层，得到亲水膜层包覆的大孔树脂材料。该树脂微球可以用作HILIC的吸附剂，用来富集复杂生物样品中的糖肽。
背景技术
作为翻译后修饰的主要形式，蛋白质糖基化在调节许多关键的细胞生理过程中起着重要作用，如细胞内通讯、免疫反应、蛋白质翻译和细胞生长(文献1.郑鑫彤等."智能聚合物基材料富集磷酸化肽和糖肽的研究进展"色谱.2021,39,15-25；文献2.G.W. Hart,et.al."Glycomics hits the big time"Cell.2010,143,672–676)。糖基化被认为是众多已知蛋白质修饰中最重要的翻译后修饰之一，据报道许多蛋白质发生了糖基化。体内蛋白质的异常糖基化通常与某些疾病有关，如免疫缺陷、阿尔茨海默病、神经系统疾病和癌症等(文献3,M.A.Wolfert,et.al."Adaptive immune activation:glycosylationdoesmatter"Nat.Chem.Biol.9,2013,776–784)。目前，质谱已经成为表征蛋白质糖基化的最灵敏的分析方法之一。翻译后修饰产物(例如糖肽)丰度很低，且存在着强烈的背景干扰，很难直接用质谱进行分析，因此需要开发高效的富集材料和技术来选择性富集糖肽(文献4.温雪等."基于整体柱的糖基化蛋白质分离富集方法的研究进展"分析化学,2020,48,13-21)。
HILIC因其操作简单、重现性好而备受重视。现已经开发了多种亲水材料用于糖肽的富集。研究表明，与多糖结合的有机大孔聚合物微球对多肽的富集具有良好的效率和选择性(文献5.Z.C.Xiong,et.al."Layer-by-layer assembly of multilayerpolysaccharide coatedmagnetic nanoparticles for the selective enrichment ofglycopeptides"Chem.Commun.2013,49,9284–9286)。
发明内容
本发明涉及一种亲水膜层包覆的大孔树脂材料及其制备和应用，具体是以表面带有环氧基的大孔树脂微球为基质，轮环藤宁和异氰尿酸三缩水甘油酯为功能单体，利用环氧-胺开环聚合反应在大孔树脂微球表面形成亲水膜层，得到亲水膜层包覆的大孔树脂材料材料可以用作HILIC的的吸附剂，用来富集复杂生物样品中的糖肽，其结构示意如下。
亲水膜层包覆的大孔树脂材料的具体制备过程包括如下步骤：
在圆底烧瓶中将20～60mg轮环藤宁、40～110mg异氰尿酸三缩水甘油酯溶解在 20～30mL含30％～40％(体积比)聚乙二醇200的乙醇溶液中，然后加入200～600mg大孔树脂微球，在60～70℃温度下，以100～150rpm的转速机械搅拌3～5h，产物用乙醇洗涤2～4次，60～70℃真空干燥6～12h，得到亲水膜层包覆的大孔树脂材料。
本发明所涉及亲水膜层包覆的大孔树脂材料的制备，操作简单，反应条件温和。
本发明是以表面带有环氧基的大孔树脂微球为基质，以轮环藤宁(Cyclen)和异氰尿酸三缩水甘油酯(1,3,5-triglycidyl isocyanurate，TGIC)为功能单体，利用环氧-胺开环聚合反应，在大孔树脂微球表面形成亲水膜层，得到亲水膜层包覆的大孔树脂材料。该树脂微球可以直接用作亲水相互作用色谱(hydrophilic interaction chromatography，HILIC)的吸附剂，用来富集复杂生物样品中的糖肽。采用该包覆亲水膜的方法，操作过程简单，反应条件温和，所得材料作为HILIC吸附剂对糖肽具有很高的富集效率和选择性。
附图说明
图1.实施例中亲水膜层包覆的大孔树脂材料的制备示意图。
图2.实施例中亲水膜层包覆的大孔树脂材料的氦离子电镜图。(a)(b)为大孔树脂微球，(c)(d)为实施例1制备的亲水膜层包覆的大孔树脂材料-1，(e)(f)为实施例2 制备的亲水膜层包覆的大孔树脂材料-2，(g)(h)为实施例3制备的亲水膜层包覆的大孔树脂材料-3。
图3.实施例中亲水膜层包覆的大孔树脂材料的的氮气吸附脱附等温线图及孔径分布图， (a)(b)为实施例1中亲水膜层包覆的大孔树脂材料-1，(c)(d)为实施例2中亲水膜层包覆的大孔树脂材料-2，(e)(f)为实施例3中亲水膜层包覆的大孔树脂材料-3。
图4.实施例1中亲水膜层包覆的大孔树脂材料-1的X-衍射光电子能谱图。(a)全谱图 (b)N 1s高分辨能谱图。
图5.人免疫球蛋白G富集后MALDI-TOF-MS图。(a)实施例1制备的亲水膜层包覆的大孔树脂材料-1，(b)实施例2制备的亲水膜层包覆的大孔树脂材料-2，(c)实施例3 制备的亲水膜层包覆的大孔树脂材料-3，(d)商品化HILIC材料，。
图6.实施例1中人免疫球蛋白G/牛血清白蛋白混合酶解液1/10(质量比)富集前后的 MALDI-TOF-MS对比图。(a)富集前，(b)富集后。
具体实施方式
基于亲水膜层包覆的大孔树脂材料用于糖肽的分离富集。
实施例1
亲水膜层包覆的大孔树脂材料-1的制备：在圆底烧瓶中将24mg轮环藤宁、50mg 异氰尿酸三缩水甘油酯溶解在20mL含30％(体积比)聚乙二醇200的乙醇溶液中，然后加入300mg大孔树脂微球(商品名：甲基丙烯酸环氧丙酯微球，粒径10～100μm，购自上海超研生物科技有限公司，平均孔径为19.9nm，孔径分布在2.4～107.1nm之间，比表面积为95.62mg2/cm)，在60℃下，以150rpm的速率机械搅拌5h，产物用乙醇洗涤三次，60℃真空干燥12h，得到亲水膜层包覆的大孔树脂材料-1(粒径10.32～100.38 μm，膜层厚度320～380nm)。
酶解样品的制备：用1.0mL含有8mol/L的尿素和100mmol/L NH4HCO3的变性剂水溶液溶解2.0mg的人免疫球蛋白G；然后加入20μL浓度为1mol/L二硫苏糖醇水溶液，8000rpm离心20min后，在37℃恒温反应2h；在避光条件下加入7.4mg碘代乙酰胺，反应35min；取出后，加入7.0mL的Tris-HCl缓冲液，将尿素浓度稀释为1mol/L；按照酶与蛋白质量比为1：25加入胰蛋白酶，37℃水浴反应18h，获得的酶解液进行除盐，冻干后保存在-20℃的冰箱中备用。
糖肽的富集：首先，称取5mg亲水膜层包覆的大孔树脂材料-1于离心管中，用上样溶液(87％ACN，1％TFA，其余为水，v/v)平衡。然后将冻干后的蛋白质酶解液用200μL上样溶液复溶，并转移至装有亲水膜层包覆的大孔树脂材料-1的离心管中。在室温下振荡30min后，离心除去上清液。用上样溶液(87％ACN，1％TFA，其余为水， v/v)洗材料三次以除去非特异性吸附肽段。最后用100μL洗脱溶液(30％ACN，1％TFA，其余为水，v/v)将吸附到材料上的糖肽洗脱下来。洗脱液用MALDI-TOF MS分析。
MALDI-TOF MS分析过程：将0.5μL洗脱液滴加在MADLI靶上，晾干后，再将 0.5μL浓度为25mg/mL的2,5-二羟基苯甲酸溶液覆盖到样品点上，待完全晾干后，送入质谱仪进行分析。
材料合成与表征：
图1为实施例中亲水膜层包覆的大孔树脂材料-1的制备示意图。大孔树脂微球表面带有大量的环氧基团，可以与单体轮环藤宁发生环氧-胺开环聚合反应，将亲水基团引入大孔树脂微球表面，利用其亲水作用，该材料可作为HILIC的吸附剂富集样品溶液中的糖肽。
图2c、d为实施例1中亲水膜层包覆的大孔树脂材料-1的氦离子电镜图。该材料为球状颗粒，尺寸在10.32～100.38μm之间，膜层厚度在320～380nm之间(由于微球是多分散的，并且膜层比较薄，故在氦离子电镜图上看不出明显粒径变化)。氮气吸附脱附实验结果(图3a)显示，该材料的平均孔径为14.2nm，孔径分布在2.6～115.7nm之间，比表面积为135.48mg2/cm。X-衍射光电子能谱图可以用来表征材料中的元素，如图4 所示，亲水膜层包覆的大孔树脂材料-1的谱图中出现了N元素，N元素来源于环氧-胺开环修饰，这一结果表明环氧-胺开环聚合反应成功进行。
材料应用
为了考察亲水膜层包覆的大孔树脂材料-1的富集性能，我们将10μg人免疫球蛋白G酶解液作为样品，进行了糖肽富集。图5a显示，富集后没有明显的非糖肽的信号出现，谱图中明显的信号峰都是糖肽肽段。说明该亲水膜层包覆的大孔树脂材料的富集性能良好。
为了考察亲水膜层包覆的大孔树脂材料-1的特异性，我们将质量比为1/10的人免疫球蛋白G/牛血清白蛋白混合酶解液作为样品，进行了糖肽富集。富集前(图6a)检测到肽段几乎都是非糖肽，富集后(图6b)没有明显的非糖肽的信号出现，谱图中明显的信号峰都是糖肽肽段。这说明该亲水膜层包覆的大孔树脂材料对糖肽具有很高的选择性。
实施例2
亲水膜层包覆的大孔树脂材料-2的制备：在圆底烧瓶中将41mg轮环藤宁、85mg 异氰尿酸三缩水甘油酯溶解在20mL含30％(体积比)聚乙二醇200的乙醇溶液中，然后加入300mg大孔树脂微球(甲基丙烯酸环氧丙酯微球，粒径10～100μm，购自上海超研生物科技有限公司，平均孔径为19.9nm，孔径分布在2.4～107.1nm之间，比表面积为95.62mg2/cm)，在60℃下，以150rpm的转速机械搅拌5h，产物用乙醇洗涤三次，60℃真空干燥12h，得到亲水膜层包覆的大孔树脂材料-2(粒径10.52～100.57μm，膜层厚度520～570nm)。
糖肽富集与质谱分析过程同实施例1
材料合成与表征：
图2e、f为实施例2中亲水膜层包覆的大孔树脂材料-2的氦离子电镜图。该材料为球状颗粒，尺寸在10.52～100.57μm之间，膜层厚度520～570nm之间(由于微球是多分散的，并且膜层比较薄，故在氦离子电镜图上看不出明显粒径变化)。氮气吸附脱附实验结果(图3c)显示，该材料的平均孔径为14.5nm，孔径分布在2.6～115.1nm之间，比表面积为123.89mg2/cm。
材料应用
为了考察亲水膜层包覆的大孔树脂材料-2的富集性能，我们将10μg人免疫球蛋白G酶解液作为样品，进行了糖肽富集。图5b显示，富集后没有明显的非糖肽的信号出现，谱图中明显的信号峰都是糖肽肽段。说明该亲水膜层包覆的大孔树脂材料的富集性能良好。
实施例3
亲水膜层包覆的大孔树脂材料-3的制备：在圆底烧瓶中将53mg轮环藤宁、110mg异氰尿酸三缩水甘油酯溶解在20mL含30％(体积比)聚乙二醇200的乙醇溶液中，然后加入300mg大孔树脂微球(甲基丙烯酸环氧丙酯微球，粒径10～100μm，购自上海超研生物科技有限公司，平均孔径为19.9nm，孔径分布在2.4～107.1nm之间，比表面积为95.62mg2/cm)，在60℃下，以150rpm的转速机械搅拌5h，产物用乙醇洗涤三次，60℃真空干燥12h，得到亲水膜层包覆的大孔树脂材料-3(粒径10.74～100.79μm，膜层厚度740～790nm)。
糖肽富集与质谱分析过程同实施例1
材料合成与表征：
图2g、h为实施例3中亲水膜层包覆的大孔树脂材料-3的氦离子电镜图。该材料为球状颗粒，尺寸在10.74～100.79μm之间，膜层厚度740～790nm之间(由于微球是多分散的，并且膜层比较薄，故在氦离子电镜图上看不出明显粒径变化)。氮气吸附脱附实验结果(图3e)显示，该材料的平均孔径为15.0nm，孔径分布在2.1～101.1nm之间，比表面积为119.32mg2/cm。
材料应用
为了考察亲水膜层包覆的大孔树脂材料-3的富集性能，我们将10μg人免疫球蛋白G酶解液作为样品，进行了糖肽富集。图5c显示，富集后没有明显的非糖肽的信号出现，谱图中明显的信号峰都是糖肽肽段。说明该亲水膜层包覆的大孔树脂材料的富集性能良好。
对比例商业化HILIC材料用于糖基化肽的分离富集
商业化HILIC材料富集糖基化肽：糖肽富集与质谱分析过程同实施例1(商品名：Venusil HILIC，购自天津博艾杰尔科技有限公司)。
为了与亲水膜层包覆的大孔树脂材料富集性能进行对比。商品化的HILIC材料也被用来作为吸附剂，富集糖肽。图5d显示，虽然商品化的HILIC材料也可以从免疫球蛋白酶解液富集到糖肽，但是，非糖肽的信号峰不能被完全忽略。
以上结果表明以表面带有环氧基的大孔树脂微球为基质，轮环藤宁和异氰尿酸三缩水甘油酯为功能单体所制备的亲水膜层包覆的大孔树脂材料比商品化的HILIC材料对糖肽具有更高的富集效率及特异性。
Claims (3)
1.一种亲水膜层包覆的大孔树脂材料的应用，其特征在于：以表面带有环氧基的大孔树脂微球为基质，以轮环藤宁和异氰尿酸三缩水甘油酯为功能单体，利用环氧-胺开环聚合反应，在大孔树脂微球表面形成亲水膜层，得到的亲水膜层包覆的大孔树脂材料；能够直接用作HILIC的吸附剂，用来富集生物样品中的糖肽；
按如下步骤操作，在圆底烧瓶中将20~60 mg轮环藤宁、40~110 mg异氰尿酸三缩水甘油酯溶解在20~30 mL含体积比为30%~40%聚乙二醇200的乙醇溶液中，然后加入200~600 mg大孔树脂微球，在60~70 ℃温度下，以100~150 rpm的转速机械搅拌3~5 h，产物用乙醇洗涤2~4次，60~70 ℃真空干燥6~12 h，得到亲水膜层包覆的大孔树脂材料。
2.按照权利要求1所述的应用，其特征在于：
所述表面带有环氧基的大孔树脂微球为甲基丙烯酸环氧丙酯微球，粒径为10~100 μm，平均孔径为19.9 nm，孔径分布在2.4~107.1 nm之间。
3.根据权利要求1所述的应用，其特征在于：所述的生物样品为人体和/或动物的体液、组织或者细胞酶解液等中的一种或二种以上。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110399508.3A CN115212852B (zh) | 2021-04-14 | 2021-04-14 | 一种亲水膜层包覆的大孔树脂材料及其制备和应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110399508.3A CN115212852B (zh) | 2021-04-14 | 2021-04-14 | 一种亲水膜层包覆的大孔树脂材料及其制备和应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN115212852A CN115212852A (zh) | 2022-10-21 |
| CN115212852B true CN115212852B (zh) | 2024-04-09 |
Family
ID=83604505
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110399508.3A Active CN115212852B (zh) | 2021-04-14 | 2021-04-14 | 一种亲水膜层包覆的大孔树脂材料及其制备和应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115212852B (zh) |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5410043A (en) * | 1991-12-06 | 1995-04-25 | Schering Aktiengesellschaft | Process for the production of mono-N-substituted tetraaza macrocycles |
| JP2014218548A (ja) * | 2013-05-01 | 2014-11-20 | 富士フイルム株式会社 | 分子膜及び分子膜を積層した累積膜 |
| JP2014218444A (ja) * | 2013-05-01 | 2014-11-20 | 富士フイルム株式会社 | 分子膜の製造方法 |
| CN104307495A (zh) * | 2014-10-14 | 2015-01-28 | 常州大学 | 一种含有四氮杂环的新型螯合树脂及其制备方法 |
| CN109317116A (zh) * | 2018-09-26 | 2019-02-12 | 中国工程物理研究院核物理与化学研究所 | 一种复合树脂及其制备方法和对硝酸介质中钯的回收方法 |
| CN110152624A (zh) * | 2018-02-12 | 2019-08-23 | 中国科学院大连化学物理研究所 | 一种微孔聚合物包覆的亲水性树脂及其在糖肽富集中的应用 |
-
2021
- 2021-04-14 CN CN202110399508.3A patent/CN115212852B/zh active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5410043A (en) * | 1991-12-06 | 1995-04-25 | Schering Aktiengesellschaft | Process for the production of mono-N-substituted tetraaza macrocycles |
| JP2014218548A (ja) * | 2013-05-01 | 2014-11-20 | 富士フイルム株式会社 | 分子膜及び分子膜を積層した累積膜 |
| JP2014218444A (ja) * | 2013-05-01 | 2014-11-20 | 富士フイルム株式会社 | 分子膜の製造方法 |
| CN104307495A (zh) * | 2014-10-14 | 2015-01-28 | 常州大学 | 一种含有四氮杂环的新型螯合树脂及其制备方法 |
| CN110152624A (zh) * | 2018-02-12 | 2019-08-23 | 中国科学院大连化学物理研究所 | 一种微孔聚合物包覆的亲水性树脂及其在糖肽富集中的应用 |
| CN109317116A (zh) * | 2018-09-26 | 2019-02-12 | 中国工程物理研究院核物理与化学研究所 | 一种复合树脂及其制备方法和对硝酸介质中钯的回收方法 |
Non-Patent Citations (1)
| Title |
|---|
| "One-step preparation of cyclen-containing hydrophilic polymeric monolithic materials via epoxy-amine ring-opening reaction and their application in enrichment of N-glycopeptides";Chen Ma等;Talanta;第225卷;第1-8页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN115212852A (zh) | 2022-10-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106732409B (zh) | 磺酸基修饰的金属有机骨架纳米复合材料的合成方法及其应用 | |
| Ji et al. | Efficient enrichment of glycopeptides using metal–organic frameworks by hydrophilic interaction chromatography | |
| CN106512965A (zh) | 一种金属有机骨架纳米复合材料的合成方法及其应用 | |
| CN101054406A (zh) | 采用金属氧化物磁性微球分离富集磷酸化肽段的方法 | |
| CN110694589A (zh) | 一种金属有机骨架-硅基复合材料及其制备方法和应用 | |
| CN107607640A (zh) | 一种硼酸修饰的纳米复合材料的糖肽富集与质谱检测方法 | |
| Sheng et al. | Hydrophilic graphene oxide-dopamine-cationic cellulose composites and their applications in N-Glycopeptides enrichment | |
| CN108212131B (zh) | 一种磷酸肽固相萃取小柱及制备和应用 | |
| CN113721028B (zh) | 一种go@cs@zif-8泡沫材料的合成方法及应用 | |
| CN110152624A (zh) | 一种微孔聚合物包覆的亲水性树脂及其在糖肽富集中的应用 | |
| CN115212852B (zh) | 一种亲水膜层包覆的大孔树脂材料及其制备和应用 | |
| Chen et al. | Novel boronate material affords efficient enrichment of glycopeptides by synergized hydrophilic and affinity interactions | |
| Li et al. | Coupling of metal-organic frameworks-containing monolithic capillary-based selective enrichment with matrix-assisted laser desorption ionization-time-of-flight mass spectrometry for efficient analysis of protein phosphorylation | |
| CN111089969B (zh) | 一种n-糖链快速酶解释放和固相富集并质谱分析的方法 | |
| Dong et al. | Specific capture of phosphopeptides by Zr4+‐modified monolithic capillary column | |
| Ba et al. | Mesoporous covalent organic framework microspheres with dual-phase separation strategy for high-purity glycopeptide enrichment | |
| CN113318715B (zh) | 一种磷酸化肽吸附剂及其制备和应用 | |
| CN110575825A (zh) | 一种磷酸功能化和Ti-IMAC碳材料及其制备和应用 | |
| CN112844258B (zh) | 一种半胱氨酸修饰的纳米核壳硅胶材料及制备与应用 | |
| CN112552395A (zh) | 一种快速分离富集乳制品中乳铁蛋白的方法 | |
| Zhang et al. | Novel functionalized poly (glycidyl methacrylate‐co‐ethylene dimethacrylate) microspheres for the solid‐phase extraction of glycopeptides/glycoproteins | |
| Song et al. | Facile synthesis of carboxymethyl‐β‐cyclodextrin conjugated magnetic nanoparticles for selective enrichment of glycopeptides | |
| CN108329459B (zh) | 嗜硫亲和磁珠及其制备方法及其应用 | |
| CN101747449A (zh) | 一种纳米壳聚糖衍生物亲和介质,其制备方法和用途 | |
| Liu et al. | A rapid approach for fabricating boronic acid-functionalized plates for on-probe detection of glycoprotein and glycopeptide |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |