基于叠氮基-氰基点击化学的苯硼酸亲和硅胶的制备及其在糖蛋白/糖肽选择性富集中的应用

硼亲和色谱法在糖肽/糖蛋白选择性富集中的应用趋于成熟。硼酸亲和材料的选择性，生物相容性，制备过程是否简便均是开发新型苯硼酸功能化材料需要考虑的问题。该研究立足硼酸亲和材料开发的关键问题，设计并开发了一种新型苯硼酸亲和硅胶（TCNBA）。该材料采用基于叠氮基-氰基的无铜催化点击化学方法进行合成，生物相容性好，制备方法简便。红外光谱和X射线光电子能谱图表征结果证明材料合成成功。TCNBA的糖肽富集选择性利用基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）进行评价，结果表明，TCNBA能够分别从辣根过氧化物酶（HRP）和免疫球蛋白G（IgG）酶解液中鉴定出13个和11个糖肽；以HRP和牛血清白蛋白（BSA）酶解液混合物（物质的量比1：10）作为研究对象，富集后能够鉴定出5个糖肽。TCNBA的糖蛋白富集选择性利用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳法（SDS-PAGE）进行评价，以HRP、IgG、核糖核酸酶B（RNaseB）作为考察对象，结果表明，TCNBA对糖蛋白具有较好的富集选择性。以实际样品人血清为测试对象验证TCNBA在实际生物样品中的应用价值。结果显示，富集后非糖蛋白得到较大程度去除，糖蛋白得以富集。所制备的材料和方法具有大规模实际蛋白质样品分离处理的应用前景。     

多孔有机骨架材料对顺式二醇化合物富集分离的研究进展

基于在碱性环境下硼酸能与顺式二醇化合物可逆共价结合形成稳定的五元或六元环酯,而在酸性环境下环酯开环释放顺式二醇化合物这一特性,设计合成高效、高选择性、高富集性能的硼亲和材料的研究备受关注。近年来,许多研究工作者合成了各种类型的硼亲和材料,应用于高选择性富集顺式二醇化合物。金属有机骨架(MOFs)和共价有机骨架(COFs)由于具有孔径可调、高孔隙率、高比表面积、骨架结构可调和化学及热稳定性良好等特点,被广泛应用于色谱分离和样品前处理领域。为赋予MOFs和COFs材料对顺式二醇化合物的富集选择性,各种不同结构和不同种类的硼酸修饰的MOFs和COFs被合成出来。该综述主要是对近几年来80余篇源于科学引文索引关于硼酸功能化MOFs和COFs的种类、合成方法及其应用文章的总结,包括“金属配体-片段共组装”“合成后修饰”和“自下而上”的硼酸功能化多孔材料的修饰策略,以及硼酸功能化MOFs和COFs的种类,介绍了其在化学分析和生物分析领域的发展概况和应用前景,客观评价了硼酸功能化MOFs和COFs的区别和优缺点。该文旨在让研究人员能够充分了解近几年硼酸功能化多孔有机骨架材料的研究现状、掌握合成思路和方法,为其应用提供一定的理论指导和技术支撑,为加快硼酸功能化多孔有机骨架材料的商业化脚步贡献绵薄之力。     

用于富集低分子量糖蛋白的多功能磁性纳米材料的制备

低分子量糖蛋白被认为是发现疾病生物标志物的宝库。特异性的萃取吸附剂对这一类化合物的萃取和富集是必不可少的。硼亲和材料在近年来取得了很大的发展,但专门用于选择性富集低分子量糖蛋白的硼亲和材料目前鲜有报道。该文提出了具有多种功能的磁性纳米颗粒(MNPs),用于低分子量糖蛋白的选择性捕获。该多功能磁性纳米颗粒是用硼酸功能化聚合物网络包裹的磁性纳米复合物。该多功能磁性纳米材料是利用磁性的纳米颗粒内核通过在其表面修饰苯硼酸功能团的聚丙烯酸高分子网络链制备得到。该材料不仅具有常规磁性材料在磁分离方面的基本优势,还能提供三重预先设计的先进功能:1)尺寸排阻效应,去除高分子量蛋白质的干扰;2)对低分子量糖蛋白的选择性萃取;3)保护捕获到的低分子量糖蛋白不被降解和污染。该材料的选择性萃取功能来自于硼酸配基与糖蛋白的顺式二醇部分的亲和性,而尺寸限制效应和保护功能则依赖于磁性纳米颗粒表面修饰的聚合物网络,允许低分子量化合物选择性通过。通过实验验证了这些预设的功能,且通过改变聚合物链长可以调节限径效应的阈值。这种多功能磁性纳米复合物可以进一步发展成有前景的纳米探针,不仅可以选择性捕获低分子量糖蛋白,还可以选择性捕获核苷和聚糖等其他具有重要生物学意义的顺式二醇分子。因此,该文报道的材料制备策略为从复杂样品中选择性萃取靶标化合物的多功能吸附剂的设计和合成提供了新思路。     

小尺度4-巯基苯硼酸修饰纳米金粒子用于大鼠肝脏糖蛋白选择性富集和质谱鉴定研究

报道了利用硼氢化钠还原一步法合成小尺度巯基苯硼酸修饰的纳米金材料（尺寸约为3 nm）,合成方法简便快捷.利用纳米金表面的硼酸基团与糖蛋白/糖肽中顺式邻二羟基的酯化反应,成功实现了糖肽和糖蛋白的选择性富集.利用该材料对糖蛋白HRP酶解产物进行富集,经富集后糖肽质谱峰的信号强度提高近百倍;将该材料用于大鼠肝脏中糖蛋白的富集...     

基于纳米粒子的糖蛋白/糖肽分离富集方法

蛋白质糖基化是一种重要的蛋白质翻译后修饰方式,糖基化对蛋白质的结构和功能有着非常重要的影响。在血清或者组织提取液中,一些低浓度的糖蛋白/糖肽是具有高度临床灵敏性和特异性的生物标记物,这些生物分子可能对疾病发生机理探讨、疾病标记物发现及蛋白类新药开发提供重要信息。由于糖蛋白/糖肽的丰度低,从复杂的生物样品中高选择性富集糖蛋白/糖肽一直是糖蛋白组学的难点和重点。纳米结构的材料因其大比表面积、丰富的活性亲和位点和特殊结构,已经广泛应用于糖蛋白/糖肽的分离富集中。本文对基于金、SiO₂、TiO₂、Fe₃O₄、金刚石和聚合物纳米粒子为载体的糖蛋白/糖肽分离富集方法的研究进展作了简要概述,并且阐明了糖蛋白/糖肽分离富集方法所面临的挑战,最后,对其未来发展方向做了展望。     

重组含糖识别结构域的人源半乳糖凝集素-3在糖蛋白/糖肽富集中的应用

植物凝集素是广泛使用的糖蛋白富集和识别材料,动物凝集素则较少被尝试用于糖蛋白富集。基于人源半乳糖凝集素-3的糖识别结构域（CRD）,设计了两种重组凝集素：Gal3C （一个CRD）和Tetra-Gal3C （四重串联CRD）。通过将两种凝集素固定于链霉亲和素琼脂糖小球上,构造了富集糖蛋白的重组凝集素亲和柱。使用凝胶电泳、免疫印迹以及生物质谱技术对重组凝集素的生物特征及其糖蛋白富集能力进行了表征与评价,发现两种类型的重组凝集素对糖蛋白/糖肽都有良好的富集效果,并具有较高的特异性和灵敏度。相对于Gal3C而言,Tetra-Gal3C由于具有四重串联的CRD结构域,表现出更高的糖蛋白/糖肽富集能力。该凝集素亲和柱成功用于人肝癌细胞系HepG2的糖蛋白富集,表明重组凝集素具有从复杂生物样本中选择性识别和富集糖蛋白/糖肽的能力。     

高容量硼酸亲和磁性纳米粒子的制备及其在邻羟基生物分子富集中的应用

硼亲和吸附法是分离富集邻羟基物质的重要方法。为提高吸附剂的吸附容量,本研究采用聚乙烯亚胺修饰磁性纳米粒子,增加粒子表面引发剂的密度,再通过表面引发原子转移自由基聚合法(SI-ATRP)将3-丙烯酰基苯硼酸原位聚合在纳米粒子表面,制备了一种高密度聚合物分子刷型的硼酸亲和磁性纳米粒子吸附剂。采用磁性分散固相萃取法对非邻羟基和邻羟基物质的混合液进行富集,发现此吸附剂对邻羟基小分子和生物大分子具有良好的吸附特异性;采用吸附等温线法测定了吸附剂的吸附容量,发现此吸附剂对邻苯二酚、腺苷和卵清蛋白的吸附容量分别为(151±32)μmol/g、(123±18)μmol/g和1.5μmol/g,远高于传统吸附剂的吸附容量。采用所制备的硼酸亲和磁性纳米粒子对尿液中4种核苷和蛋清中的糖蛋白进行萃取,结果表明,此吸附剂能够有效去除生物样品中的干扰物,且对核苷具有较高的萃取回收率(83.8%~108.7%,RSD15%),对糖蛋白有特异性富集作用,说明此吸附剂在生物样品的选择性富集中具有良好应用潜能。     

功能化磁性纳米材料在糖蛋白及糖肽富集中的研究进展

蛋白质糖基化作为最重要的翻译后修饰之一,在生物体诸如细胞信号转导、蛋白质翻译调控、免疫应答等诸多生命过程中发挥重要作用。此外,蛋白质的异常糖基化还与肿瘤等疾病的发生发展密切相关,这为以糖蛋白为目标的疾病生物标志物的发现提供了可能。尽管质谱已经成为糖蛋白质组学的重要分析工具,但糖肽的低丰度和低电离效率使得其直接质谱分析仍面临挑战。在糖蛋白质组学研究中,从复杂的生物样品中富集糖蛋白和糖肽是重要的环节。磁性固相萃取(MSPE)是一种操作简单、成本低和萃取效率高的样品预处理方法。在磁性固相萃取中,磁性吸附剂是影响萃取效果的关键,将功能化磁性纳米材料作为吸附剂进行糖蛋白质组学研究已经得到广泛应用。该文综述了糖分子、离子液体、凝集素、硼酸亲和配体、金属有机框架、共价有机骨架等功能化磁性纳米材料的制备及其在糖蛋白及糖肽富集中的应用。上述功能化磁性纳米材料具有高比表面积、大量作用位点等特点,其富集机理包括亲水相互作用色谱、凝集素亲和作用色谱、硼酸化学法和肼化学法等,主要应用于血清、血浆、细胞、组织、唾液等样品的糖蛋白和糖肽的富集。该文引用了近十年来发表的约90篇源于科学引文索引(SCI)与中文核心期刊的相关论文,并于文末对磁性纳米材料在糖蛋白和糖肽富集领域的发展趋势进行了展望。     

共价有机骨架功能材料及其在糖肽选择性富集中的应用

蛋白质糖基化是生物体中最重要的翻译后修饰手段之一,糖蛋白/糖肽的有效分离和富集成为目前糖蛋白组学研究的首要问题。对于复杂的生物样本,糖蛋白的数量较少,酶解后大量高丰度非糖基化修饰肽的存在,使得低丰度糖肽的检测更加困难。因此,需要一些手段来有效地富集糖肽以提高其检测丰度,发展高选择性的糖肽富集材料及方法就成为在分子水平上有效地监测糖蛋白或糖肽的重要途径。相对于传统的糖肽富集材料,共价有机骨架材料具有比表面积大和可修饰位点丰富的优点,在糖肽富集领域具有很大的应用潜力。该文制备了一种新型的共价有机骨架材料(O-T-D-COFs),利用1,3,5-三(4-氨苯基)苯和2,5-二甲氧基苯-1,4-二甲醛作为反应单体通过共聚缩合反应生成的席夫碱构成了材料的框架,对合成后的中间体材料进行氧化处理,从而提高材料的富集性能。利用扫描电镜、透射电镜、红外光谱和固体核磁等表征技术对材料的结构进行了表征,并将其应用于糖肽的选择性富集。分别对富集过程的上样条件、淋洗条件、洗脱条件进行了优化,结合质谱检测技术,从人血清免疫球蛋白G酶解液中观察到32个明显的糖肽信号峰。通过模拟复杂样本体系验证材料富集选择性,在人血清免疫球蛋白G和牛血清白蛋白的酶解液混合物摩尔比达到1∶50时,该材料仍然保持了良好的选择性。此外,还考察了材料的检测限、富集容量、回收率等富集性能,及在实际样品中的应用潜力。以人血清免疫球蛋白G为评价对象,O-T-D-COFs具有较低的检测限(2.5 fmol/μL)、较高的富集容量(120 mg/g),及较好的富集回收率(103.5%±6.6%、101.5%±10.4%)。在血清样品中富集到来自53个N-糖蛋白中的86个N-糖肽序列,并鉴定到了94个N-糖基化位点。这些结果都表明,该材料在糖肽富集领域有较好的应用前景。     

免疫亲和富集-荧光光谱法检测牛血清白蛋白的研究

本文通过戊二醛法合成免疫磁球(IMMs),采用荧光电镜、透射电镜和振动样品磁强计对其进行表征。合成的IMMs为单分散的球形结构,粒径约为510 nm,具有超顺磁性(饱和磁化强度为40.44 emu/g)。基于此,以牛血清白蛋白(BSA)作为蛋白质模型,建立了免疫亲和富集-荧光光谱法用于目标蛋白质的分离与检测。考察并优化了影响免疫亲和富集的因素。在最佳实验条件下,IMMs对目标蛋白质的最大吸附容量为107.7μg/g,可重复使用10次以上。将该方法应用于人血清基质中BSA的测定,加标回收率不低于89.3%,由基质辅助激光解吸电离飞行时间质谱仪测得分离后的BSA纯度较高。结果表明,该方法适用于人血清样品中蛋白质的选择性分离与检测。     

Mass spectrometry signal amplification for ultrasensitive glycoprotein detection using gold nanoparticle as mass tag combined with boronic acid based isolation strategy

We describe a novel method for rapid and ultrasensitive detection of intact glycoproteins without enzymatic pretreatment which was commonly used in proteomic research. This method is based on using gold nanoparticle (AuNP) as signal tag in laser desorption/ionization mass spectrometry (LDI-MS) analysis combined with boronic acid assisted isolation strategy. Briefly speaking, target glycoproteins were firstly isolated from sample solution with boronic acid functionalized magnetic microparticles, and then the surface modified gold nanoparticles were added to covalently bind to the glycoproteins. After that, these AuNP tagged glycoproteins were eluted from magnetic microparticles and applied to LDI-MS analysis. The mass signal of AuNP rather than that of glycoprotein was detected and recorded in this strategy. Through data processing of different standard glycoproteins, we have demonstrated that the signal of AuNP could be used to quantitatively represent glycoprotein. This method allows femtomolar detection of intact glycoproteins. We believe that the successful validation of this method on three different kinds of glycoproteins suggests the potential use for tracking trace amount of target glycoproteins in real biological samples in the near future.     

Selective glycoprotein detection through covalent templating and allosteric click-imprinting

Many glycoproteins are intimately linked to the onset and progression of numerous heritable or acquired diseases of humans, including cancer. Indeed the recognition of specific glycoproteins remains a significant challenge in analytical method and diagnostic development. Herein, a hierarchical bottom-up route exploiting reversible covalent interactions with boronic acids and so-called click chemistry for the fabrication of glycoprotein selective surfaces that surmount current antibody constraints is described. The self-assembled and imprinted surfaces, containing specific glycoprotein molecular recognition nanocavities, confer high binding affinities, nanomolar sensitivity, exceptional glycoprotein specificity and selectivity with as high as 30 fold selectivity for prostate specific antigen (PSA) over other glycoproteins. This synthetic, robust and highly selective recognition platform can be used in complex biological media and be recycled multiple times with no performance decrement.     

Selective extraction and enrichment of glycoproteins based on boronate affinity SPME and determination by CIEF-WCID

In this study, a new thin-film boronic acid coating was developed for solid-phase microextraction (SPME) followed by capillary isoelectric focusing with whole-column imaging detection (CIEF-WCID). Boronate functionalized particles of phenylboronic acid (PBA) and 3-aminophenylboronic acid (3-aPBA) were utilized as boronate affinity solid phase coating on thin-film stainless steel blades for selective extraction and enrichment of glycoproteins. The process of extraction and elution could be easily controlled by adjusting pH. To test specificity, asialofetuin and lactoferrin were selected as glycoproteins test molecules, while BSA and myoglobin were used as control non-glycoproteins in this study. The boronate affinity coating was characterized. The effect of buffer, pH, extraction profiles and elution profiles were investigated. The developed method was successfully applied to extract glycoproteins from standard buffer, PBS, human plasma and 10-fold diluted human blood using two kinds of boronate affinity blades. Boronate affinity SPME could be a promising tool for selective extraction and enrichment of low-abundance glycoproteins in real biological samples.     

Facile fabrication of hydrophilic PAA-Ti/TiO2 nanocomposite for selective enrichment and detection of phosphopeptides from complex biological samples

Highly selective enrichment of trace phosphorylated proteins or peptides from complex biological samples is of profound significance for the discovery of disease biomarkers in biological systems. In this study, a novel affinity material has been synthesized to improve the enrichment specificity for phosphopeptides by using PAAS as coupling molecule. In the resulting materials, highly abundant titanium is available for selective enrichment of phosphopeptides, with plenty of carboxylate groups that can inhibit nonspecific adsorption. The enrichment results demonstrated that the hydrophilic PAA-Ti/TiO₂ composite possesses excellent selectivity for phosphopeptides even at a very low molar ratio of phosphopeptides/non-phosphopeptides (1:1000), extreme sensitivity (the detection limit was at the fmol level), and high recovery of phosphopeptides (as high as 78%). Moreover, the as-prepared nanocomposite provides effective enrichment of phosphopeptides from real samples (mouse liver), showing great potential in the detection of low-abundance phosphopeptides in biological samples.     

Novel functionalized poly(glycidyl methacrylate‐co‐ethylene dimethacrylate) microspheres for the solid‐phase extraction of glycopeptides/glycoproteins

Novel 3‐aminophenylboronic acid functionalized poly(glycidyl methacrylate‐co‐ethylene dimethacrylate) microspheres were prepared for the solid‐phase extraction of glycopeptides/glycoproteins. The adsorption efficiency, maximum adsorption capacity, and specific recognition of the microspheres to glycoprotein were investigated. The results indicated excellent adsorption of glycoproteins by the microspheres, which are attributed to the well‐defined boronic acid brushes on the microsphere surfaces. Furthermore, a solid‐phase extraction microcolumn filled with the microspheres was used to efficiently enrich glycopeptides from enzymatic hydrolysates from human serum samples. The mass spectrometry results demonstrated that the method is suitable for the separation and enrichment of glycopeptides/glycoproteins from complex biological samples.     

Highly specific purification of N-glycans using phosphate-based derivatization as an affinity tag in combination with Ti-SPE enrichment for mass spectrometric analysis

N-linked protein glycosylation is involved in regulation of a wide variety of cellular processes and associated with numerous diseases. Highly specific identification of N-glycome remains a challenge while its biological significance is acknowledged. The relatively low abundance of glycan in complex biological mixtures, lack of basic sites for protonation, and suppression by other highly abundant proteins/peptides lead to the particularly poor detection sensitivity of N-glycans in the MS analysis. Therefore, the highly specific purification procedure becomes a crucial step prior to MS analysis of the N-glycome. Herein, a novel N-glycans enrichment approach based on phosphate derivatization combined with Ti⁴⁺-SPE (solid phase extraction) was developed. Briefly, in this strategy, N-glycans were chemically labeled with a phospho-group at their reducing ends, such that the Ti⁴⁺-SPE microspheres were able to capture the phospho-containing glycans. The enrichment method was developed and optimized using model oligosaccharides (maltoheptaose DP7 and sialylated glycan A1) and also glycans from a standard glycoprotein (asialofetuin, ASF). This method experimentally showed high derivatization efficiency (almost 100%), excellent selectivity (analyzing DP7 in the digests of bovine serum albumin at a mass ratio of 1:100), high enriching recovery (90%), good reproducibility (CV<15%) as well as high sensitivity (LOD at fmol level). At last, the proposed method was successfully applied in the profiling of N-glycome in human serum, in which a total of 31 N-glycan masses were identified.     

氧化石墨烯固定化凝集素的制备及在糖蛋白/糖肽富集中的应用

生物体内蛋白质的糖基化修饰调控着细胞识别、细胞黏附和迁移以及免疫应答等多种生理过程,并与多种人类重大疾病的发生、发展密切相关。因此对蛋白质糖基化修饰的鉴定,不仅能够为生物学机理研究提供重要信息,对疾病诊断标志物和治疗靶标的发现也至关重要。然而在复杂生物体系中,大多数糖蛋白为低丰度蛋白质,其含量与现有质谱仪器的检测灵敏度之间存在较大差距,所以对含有不同糖型结构的糖蛋白进行全面/高效的富集,是实现高灵敏度糖蛋白鉴定的必由之路。凝集素富集作为一种有效的糖蛋白富集方法,已在糖蛋白质组学研究中得到了广泛的应用。针对现有凝集素功能化材料存在负载量偏低以及富集效率有限等问题,我们制备了两种以氧化石墨烯(GO)为载体的新型固定化凝集素,利用GO比表面积大,功能基团含量高,分散性、化学稳定性好等特点,实现了高负载量的凝集素固定(GO-ConA 2.073 mg/mg, RSD=1.0%; GO-WGA 1.908 mg/mg, RSD=0.14%)。同时考察了材料的可重复使用性与稳定性:每隔3天测一次同一GO-lectin材料对对应糖蛋白的富集效果,可以看出材料合成两周内富集效果都>200 μg/mg。将该GO-lectin成功应用于糖蛋白、糖肽的选择性富集,在糖蛋白质组学研究中体现出良好的应用潜力。     

半胱氨酸基麦芽糖修饰亲水硅胶的制备及其对糖肽的富集

糖蛋白与糖肽在复杂生物体系中丰度一般较低,为了在糖蛋白质组学研究中深入和全面分析鉴定糖基化位点与糖链,通常需要进行富集前处理操作。该文设计并合成了一种半胱氨酸基麦芽糖修饰亲水硅胶分离介质(Cys-Mal@SiO_2),将其装填入固相萃取柱中制备新型亲水固相萃取柱。在以人免疫球蛋白G酶解液为样品进行富集鉴定时,Cys-Mal@SiO_2鉴定糖肽的质谱信号强度和信噪比比半胱氨酸修饰硅胶(Cys@SiO_2)、麦芽糖修饰硅胶(Mal@SiO_2)和商品化ZIC-HILIC更高。在复杂的鼠肝蛋白质提取物的糖蛋白质组学分析中,Cys-Mal@SiO_2共鉴定出1 551条糖肽,属于466个糖蛋白的906个N-糖基化位点,比Cys@SiO_2和Mal@SiO_2鉴定糖肽数、蛋白质数和N-糖基化位点数分别多211、67、127个和289、76、193个。将Cys-Mal@SiO_2成功应用于低丰度糖肽的选择性富集与鉴定,在糖蛋白质组学研究中体现出良好的应用潜力。