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
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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。
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