糖蛋白中糖链的结构研究

糖蛋白中糖链的结构研究王晨，田庚元（中国科学院上海有机化学研究所，２０００３２）糖蛋白是指以共价键与糖连结的蛋白质，但一般不包括蛋白聚糖这一类化合物。在过去几十年里，糖蛋白的研究侧重于蛋白部分，对糖链的作用缺乏足够的重视。而近十几年来，越来越多的研究...     

代谢组学中的分离分析科学*

代谢组学从概念的提出到现在已经有十多年的时间,它已被广泛应用于药物开发、疾病、微生物和植物等生命科学各个领域的研究,成为系统生物学不可或缺的一部分。代谢组学关注生物体系新陈代谢网络下游的小分子,分离分析科学是代谢组学技术平台的重要组成部分,与其它分析化学技术共同肩负着生物样本中代谢物全组分定性、定量的重任。本文结合我们研究组的工作,主要介绍气相色谱、液相色谱以及毛细管电泳等分离分析技术在代谢组中的应用和进展,以期对读者有所启迪。     

基于质谱的糖蛋白/糖链定量技术研究进展

糖基化修饰是一种重要的蛋白质翻译后修饰。糖基化修饰的蛋白质在生命体内具有重要的生物学功能。研究糖蛋白含量以及蛋白上糖链变化对于阐明糖基化修饰的功能具有重要的意义,也是当今的研究热点。本文就糖蛋白和糖链定量方法的研究进展和应用做了简要概述。     

N-糖组富集方法研究进展

糖基化是一种重要的蛋白质翻译后修饰,使蛋白质具有更多样的功能.N-糖基化作为蛋白质主要的糖基化类型之一,与许多生命活动密切相关,在疾病发生发展中起关键作用.因此,研究糖蛋白N-糖链具有重要意义.生物质谱技术是糖组学研究的重要工具之一,然而,由于糖链结构复杂、丰度低及质谱离子化效率低等原因,基于质谱的N-糖组学研究还面临...     

微量样本中N-糖组分析技术的发展与应用

针对糖组学分析面临的初始样本量需求较高的技术挑战,该研究发展了一种微量样本N-糖链制备技术(GPAT),通过在移液枪头(Tip)中分别装填C_(18)和HILIC填料,实现了一站式原位蛋白消化、N-糖链释放和富集。与目前普遍采用的基于过滤辅助样品制备(FASP)技术的N-糖组分析策略相比,使用GPAT技术可以实现微量免疫球蛋白G(IgG)和人肝癌HepG2细胞提取蛋白的原位N-糖链的释放和富集,样本起始量减少90%,可以从10μg IgG和10μg的HepG2细胞蛋白中分别检测到20条和39条N-糖链。从6例健康人(3例男性,3例女性)尿液中提取的10μg尿蛋白中检测到49条N-糖链。该方法实现了微量复杂蛋白样本N-糖链简便、快速的定量分析策略,为进一步的糖组学方法推广应用奠定了基础。     

花生致敏糖蛋白Ara h1糖链决定簇的质谱分析

以花生种子总蛋白及其主要致敏糖蛋白Ara h1为研究对象,采用"一釜法"对蛋白上的糖链进行释放并同时进行衍生化标记,通过C18固相萃取柱纯化,以电喷雾质谱(ESI-MS)、多级串联质谱(MSn)和亲水性液相色谱-质谱联用(HILIC-MS)进行结构解析和定量分析.结果表明,蛋白Ara h1共有10条N-糖链,其中7条为高甘露糖型,2条为木糖修饰,另外1条为与过敏原相关的核心α1,3-Fuc修饰N-糖链,其含量约占总糖链的12.45%.     

微量生物样品中糖蛋白N-糖链电喷雾电离质谱分析前处理方法的建立

基于电喷雾电离质谱检测技术,建立了一种可靠、高效、简单,适合于微量糖蛋白N-糖链解离、富集纯化的方法.以糖蛋白牛胰核糖核酸酶(Rib B)和卵清白蛋白(OVA)为模型蛋白直接酶解,比较了4种方法纯化酶解样品的效果.比较了直接酶解和经过聚偏氟乙烯(PVDF)膜富集后酶解微量复杂生物来源样品胎牛血清的酶解效果,最终建立了微量生物样品中糖蛋白N-糖链的质谱分析前处理方法.采用PVDF膜吸附复杂生物样品中的糖蛋白,N-糖苷酶F(PNGase F)酶直接在膜上完成糖链释放(37℃,24 h),采用微晶纤维素柱结合石墨碳柱对糖链进行富集纯化,用于微克级胎牛血清和健康人血清中N-糖链质谱分析的前处理.本方法通用性好,在微量生物样品糖链质谱分析检测的前处理方面具有一定应用价值.     

基于质谱技术的代谢组学研究及其在中国的发展

代谢组学是关于生物系统代谢物组成及变化规律的科学,是系统生物学的重要组成部分.质谱技术是目前代谢组学研究中最主要的分析手段之一,广泛应用于代谢组学各个领域.本文阐述了基于质谱技术的代谢组学方法及其应用,重点介绍和评论了近年来我国在该领域取得的进步和成果,并对基于质谱技术的代谢组学研究目前存在的问题及未来的发展进行了分析与展望.     

流感病毒糖蛋白糖链的作用和功能研究

流感病毒是近几年的研究热点之一.糖链在流感病毒生活周期中发挥重要作用,例如宿主细胞表面的唾液酸化糖链是病毒侵染细胞时的特异性受体,宿主决定的病毒糖蛋白糖链结构影响病毒的宿主范围和毒力.本文从糖组学角度综述糖链在甲型流感病毒生活周期中的重要作用,着重阐述病毒血凝素糖基化的影响因素及其对病毒宿主范围、毒力的影响和在病毒演化...     

基于质谱技术研究黑色素瘤血清特异性O-糖链

对接种和未接种B16黑色素瘤细胞的C57小鼠进行血清O-糖链比较糖组学研究,寻找黑色素瘤血清特异性O-糖链.小鼠血清10μL,β-消除反应释放O-糖链.反应混合物经石墨化炭黑固相萃取小柱(GCC SPE)分离纯化后,用于MALDI-Qit-TOF-MS分析.通过Launchpad软件采集并输出质谱数据,MATLAB进行数据解析,找到了10个稳定出现的差异糖链质谱峰.利用串联质谱分析了其中5个主要差异糖链的结构.     

Recent Advances in the Chemical Biology of N-Glycans

Asparagine-linked N-glycans on proteins have diverse structures, and their functions vary according to their structures. In recent years, it has become possible to obtain high quantities of N-glycans via isolation and chemical/enzymatic/chemoenzymatic synthesis. This has allowed for progress in the elucidation of N-glycan functions at the molecular level. Interaction analyses with lectins by glycan arrays or nuclear magnetic resonance (NMR) using various N-glycans have revealed the molecular basis for the recognition of complex structures of N-glycans. Preparation of proteins modified with homogeneous N-glycans revealed the influence of N-glycan modifications on protein functions. Furthermore, N-glycans have potential applications in drug development. This review discusses recent advances in the chemical biology of N-glycans.     

Universal proteolysis and MSn for N‐ and O‐ glycan branching analysis

The continually growing list of critical glycosylation‐related processes has made analytical methodology for detailed glycan characterization an area of increasing interest. Glycosylation is a post translational modification of unsurpassed complexity due to the variety of compositions and linkages formed by these biopolymers. Structural characterization of glycan isomers has been achieved using ion trap mass spectrometry and MSⁿ of released, permethylated glycans. However, N‐ and O‐glycans require different sample preparation strategies; and release of the glycans may be hindered, result in degradation of the glycan, and/or produce limited yields of permethylated product. In the current report, we demonstrate universal proteolysis of both N‐ and O‐linked glycoproteins to individual glycoamino acids. These samples were shown to be directly amenable to permethylation and MSⁿ analysis for isomeric structural determination. Universal proteolysis and permethylation provides an identical sample preparation strategy for both classes of glycans that avoids potential pitfalls of commonly used release methods. This methodology should be applicable to all glycoproteins and serve as an alternative to glycan release for MSⁿ branching analysis. Published 2013. This article is a U.S. Government work and is in the public domain in the USA.     

Computational Modeling of O-Linked Glycan Biosynthesis in CHO Cells

Glycan biosynthesis simulation research has progressed remarkably since 1997, when the first mathematical model for N-glycan biosynthesis was proposed. An O-glycan model has also been developed to predict O-glycan biosynthesis pathways in both forward and reverse directions. In this work, we started with a set of O-glycan profiles of CHO cells transiently transfected with various combinations of glycosyltransferases. The aim was to develop a model that encapsulated all the enzymes in the CHO transfected cell lines. Due to computational power restrictions, we were forced to focus on a smaller set of glycan profiles, where we were able to propose an optimized set of kinetics parameters for each enzyme in the model. Using this optimized model we showed that the abundance of more processed glycans could be simulated compared to observed abundance, while predicting the abundance of glycans earlier in the pathway was less accurate. The data generated show that for the accurate prediction of O-linked glycosylation, additional factors need to be incorporated into the model to better reflect the experimental conditions.     

铁皮石斛原球茎多糖DCPP3c-1的分离纯化及结构初步分析

铁皮石斛原球茎粗多糖(DCPP)经阴离子交换纤维素柱(DEAE-2)和凝胶柱(Sephadex G-200)依次层析,分离纯化得灰色多糖DCPP3c-1.其纯度经比旋光度法、柱层析、紫外扫描检测,组分和结构经薄层层析、高效液相色谱、红外光谱及高碘酸钠氧化等试验分析.结果显示DCPP3c-1为均一组分,相对分子质量为72.4 ku.由甘露糖、鼠李糖、半乳糖醛酸、葡萄糖、半乳糖和阿拉伯糖组成,其分子物质的量之比为1.120 4:1:1.046:23.354:3.828:1.046.分子中1→6残基占14%,1→2或1→4残基占40.7%,1→3键占45.3%.红外光谱显示其具有多糖特征吸收峰,并存在吡喃糖苷键.DCPP3c-1是首次从液体悬浮培养的原球茎中分离得到的新型酸性杂多糖组分.     

微流控芯片系统中固液双相分离富集技术

发展微流控芯片系统中的分离富集技术，是微全分析系统向集成化、自动化和便携化发展必须突破的瓶颈之一，正逐步成为微全分析系统研究和应用领域的前沿和热点。本文针对重要而且应用广泛的固液双相分离富集技术，详细介绍了过滤式、膜分离式、固相萃取式等不同分离富集操作模式在微流控芯片系统中的应用，对每一种操作模式的特点、研究现状、存在的问题和发展趋势进行了综述。     

Versatile metal–organic framework-functionalized magnetic graphene nanoporous composites: As deft matrix for high-effective extraction and purification of the N-linked glycans

The highly selective enrichment of N-linked glycans from complex biological sample is still very important but challenging task due to the ultra-low abundance, complicated structures and strong ion suppress effect caused by distractors such as proteins, peptides and salts. Here, we firstly present a novel metal–organic frameworks (MOFs)-functionalized magnetic nanoporous carbon-graphene composites (C-magG@ZIF-8) synthesized through a smart process. The obtained materials enjoy the unique properties including strong magnetic responsiveness, a large sum of graphitized carbon pore, remarkable biocompatibility and large specific surface area. By virtue of these unique properties, the C-magG@ZIF-8 composites displayed excellent selectivity and sensitivity, good recyclability and incredible size exclusion ability (roughly 2000 times) in the N-linked glycans analysis. Furthermore, 48 N-linked glycans were clearly identified from the normal human serum treated with the C-magG@ZIF-8. There is reason to believe that our smart strategy offers new possibilities for preparing the MOFs-functionalized composites for large-scale characterization of glycoproteomics by mass spectrometry analysis.     

基于质谱的蛋白质O-糖基化分析研究进展

蛋白质的O-糖基化是一种重要的蛋白质翻译后修饰,它和N-糖基化一样是蛋白质糖基化修饰的主要形式。蛋白质的O-糖基化对蛋白质的结构功能有重要的影响,因此分析蛋白质的O-糖基化具有重要的生物学意义。蛋白质O-糖基化分析包含4个方面的内容:(1)鉴定O-糖基化蛋白质的种类; (2)鉴定糖基化位点; (3)鉴定糖链结构; (4)糖链的定量分析。由于缺少保守的O-糖基化氨基酸特征序列,缺乏通用的糖苷酶以及O-糖链结构的复杂性等原因,基于质谱的蛋白质O-糖基化的分析目前仍处于方法开发阶段。本文主要介绍基于质谱的O-糖基化蛋白质的分析方法学在近期取得的一些进展,包括以下4个方面:O-糖蛋白/多肽的富集、O-糖链的解离、O-糖链的结构分析及O-糖基化定量分析。     

超疏水表面的研究进展

表面的浸润性是决定材料应用的一个重要性质,许多物理化学过程,如吸附、润滑、黏合、分散和摩擦等均与表面的浸润性密切相关.近年来,由于超疏水表面在自清洁表面、微流体系统和生物相容性等方面的潜在应用,有关超疏水表面的研究引起了极大的关注.本文综述了超疏水表面研究的新进展:简单介绍了表面浸润性的表征手段和影响因素,归纳了超疏水表面的制备方法和相关的理论分析,对超疏水表面研究的发展进行了展望.     

Isolation,purification, and identification of the main phenolic compounds from leaves of celery (Apium graveolens L. var. dulce Mill./Pers.)

We developed a simple and meaningful preparative method for the separation and purification of the main phenolic compounds from the leaves of celery (Apium graveolens L. var. dulce Mill./Pers.) and we established an accurate and specific analytical method for the identification of the main phenolic compounds from celery leaves. The crude extract from celery leaves was prefractioned by polyamide resin to enrich the phenolic compounds. They were then purified further by preparative high‐performance liquid chromatography, and seven main phenolic compounds were obtained: including chlorogenic acid, luteolin 7‐O‐β‐d‐ apiofuranosyl(1→2)‐β‐d‐ glucopyranoside, luteolin 7‐O‐β‐d‐ glucopyranoside, apiin, chrysoeriol 7‐O‐β‐d‐ apiofuranosyl(1→2)‐β‐d‐ glucopyranoside, luteolin 7‐O‐[β‐d‐ apiofuranosyl(1→2)‐(6′′‐O‐malonyl)]‐β‐d‐ glucopyranoside, and apigenin 7‐O‐[β‐d‐ apiofuranosyl(1→2)‐(6′′‐O‐malonyl)]‐β‐d‐ glucopyranoside. Their purities were measured by using high‐performance liquid chromatography, and their chemical structures were confirmed using UV spectrophotometry, ultra high performance liquid chromatography with quadrupole time‐of‐flight tandem mass spectrometry, and NMR spectroscopy. Our studies indicate that preparative high‐performance liquid chromatography combined with polyamide resin is a simple and meaningful preparative method for the separation and purification of phenolic compounds from the leaves of celery or other plants, and the use of UV spectrophotometry, ultra high performance liquid chromatography with quadrupole time‐of‐flight tandem mass spectrometry, and NMR spectroscopy is an accurate and specific analytical method for the identification of phenolic compounds.     

固体脂质纳米粒作为药物载体

固体脂质纳米粒是一种新型的纳米给药系统.本文综述了固体脂质纳米粒作为药物载体的研究进展,包括固体脂质纳米粒的组分选择、制备方法、表面修饰,并且对物化性质分析、给药途径以及载药和释放进行了讨论.