基于超滤膜辅助的糖蛋白全O-连接糖链的富集和MALDI-TOF/TOF质谱结构解析

哺乳动物中约有50%以上的蛋白质都发生了糖基化修饰.连接在丝氨酸或苏氨酸上的O-连接糖链是常见的蛋白质糖基化修饰方式之一,其主要功能是维持与其连接的蛋白质部分的空间构象,保护其免受蛋白酶水解及覆盖某些抗原决定簇.糖链结构的解析有助于更清楚地认识糖蛋白及其功能.本研究建立了一种基于超滤膜辅助(FASP)富集细胞、血清和尿液中糖蛋白全O-连接糖链的方法,根据糖蛋白与其糖链结构之间的分子质量差异,利用10 KD超滤膜富集蛋白质样品中由β消除反应释放的全O-连接糖链,将糖链甲基化修饰后再使用MALDI-TOF/TOF-MS进行解析,同时利用二级质谱进行结构确认.通过上述方法可从标准糖蛋白mucin、细胞、血清和尿液样本中分别鉴定到83、29、33和85种O-连接糖链结构,利用该方法可以从复杂样品中富集和解析糖蛋白全O-连接糖链,实现快速、高效、高通量地解析糖蛋白O-连接糖链的目的.     

基于FFPE组织切片的膀胱癌N-连接糖链原位酶解及分析

目的 研究膀胱癌FFPE组织切片的N-连接糖链,发现膀胱癌FFPE肿瘤组织的异常N-连接糖链修饰情况。方法 发展基于FFPE组织切片原位提取N-连接糖链的实验流程。通过PNGase F酶切FFPE组织解释放N-连接糖链。对N-连接糖链自由端进行全甲基化修饰。通过MALDI-TOF/TOF-MS检测N-连接糖链的相对含量。进行数据库匹配,确定N-连接糖链的可能糖型。ROC分析用于预测显著差异N-连接糖链作为预测膀胱癌生物标志物的准确度。结果 MALDI-TOF/TOF-MS检测泛甲基化修饰N-连接糖链的数据显示,在16例膀胱癌患者的肿瘤和癌旁组织的3次重复实验中,肿瘤组织中蛋白质高甘露糖型N2H6、N2H7、N2H8、N2H9和复杂型N5H6F1糖链修饰水平显著上升,同时高甘露糖型N2H5、杂合型N3H5以及复杂型N3H4、N4H4、N5H6F1S2糖链修饰水平显著下降。ROC分析显示,双天线型N-连接糖链N3H4（AUC=0.90）和N4H4（AUC=0.91）在单独或者共同区分膀胱癌患者肿瘤组织和癌旁组织中都具有很好的可靠性,可能成为膀胱癌的潜在生物标志物。结论 膀胱癌FFPE肿瘤组织中存在蛋白质异常N-糖基化修饰,N-连接糖链N3H4和N4H4或可成为膀胱癌的潜在生物标志物。     

视黄酸对皮肤上皮基底培养细胞表面膜糖蛋白N-连接型糖链结构的影响

维生素A类化合物对糖蛋白N-连接型糖链(简称N-糖链)结构的影响,近年来在研究其作用机制中颇受重视。本文研究视黄酸(RA)对大鼠皮肤上皮基底培养细胞表面膜糖蛋白糖链结构作用,发现RA促进N-糖链合成,使~3H-甘露糖掺入糖链量增加43.5％,RA可改变N-糖链的类型,促进复杂型N-糖链合成,表现为增加三、四天线复杂型N-糖链合成而不是二天线;RA还使含分叉性GIeNAc和核心Fuc的百分比上升。本文还用细胞电泳方法研究膜表面唾液酸相对量,发现RA可引起唾液酸含量下降。结果提示RA对N-糖链结构的影响,是其多种生物学作用的可能途径之一。     

糖蛋白的研究进展

糖蛋白是由糖链与多肽链以多种形式共价修饰而形成的一类重要生理活性物质.糖蛋白在生物体内种类繁多,分布广泛,具有重要的功能.糖蛋白的性质及功能和糖链的结构有关,因此糖蛋白中糖链的结构及作用机制研究成为生物学基础理论的课题之一.就近年来糖蛋白研究中糖蛋白样品的提取分离、糖链释放及结构分析的技术方法及研究领域作了简要介绍.     

植物N-糖链检测技术研究进展

N-糖基化作为一种重要的蛋白质翻译后修饰,在胚胎发育、癌症发生发展及免疫防御等诸多复杂的生命活动中发挥着关键作用。近年来,基于质谱的N-糖链的检测及其定量研究在动物方面取得了显著进展,相比之下,植物N-糖基化及N-糖链检测的相关研究要远远滞后,这也是制约植物糖生物学研究发展的关键瓶颈问题之一。对蛋白质N-糖链的释放、定量策略、可视化检测及其在植物中的应用进展进行了归纳总结,以期为指导后续植物N-糖链及N-糖组的定性定量检测提供参考。     

不同转移潜能膀胱癌细胞糖组相对定量分析

膀胱癌是发生在膀胱黏膜组织上的一种恶性肿瘤,是泌尿系统中最常见的恶性肿瘤,早期(非肌层浸润型膀胱癌)阶段的诊断和治疗是降低膀胱癌死亡率的最有效方式.肿瘤的发生过程与糖链表达的改变有着密切的关系,而定量分析膀胱癌发生过程中糖链的表达变化尚未有研究.本研究以2株人膀胱正常上皮细胞系(HCV29、HUCV1),1株非肌层浸润性膀胱癌细胞系(KK47),和3株浸润性膀胱癌细胞系(YTS1、J82、T24)为研究材料,应用本室建立的利用乙酰肼修饰糖链唾液酸,以及[12C6]-和[13C6]-苯胺同位素修饰糖链还原性末端技术,然后利用基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS),进行膀胱上皮细胞不同病理状态的糖组相对定量分析.从6株细胞中共鉴定出52种N-连接糖链结构,并定量分析了不同类型的糖链在不同细胞中的分布差异,发现唾液酸化、岩藻糖化的N-连接糖链在膀胱癌肿瘤细胞恶化过程中呈现显著升高的趋势,同时平分型糖链和高甘露糖型N-连接糖链也呈表达升高趋势,说明这些糖链结构的表达变化与膀胱癌发生关系密切,从而有助于进一步阐明膀胱癌发生过程中糖链相关的分子机理.     

糖蛋白质组学的信息学资源和方法

糖基化修饰是生物体内最常见、最重要的蛋白质翻译后修饰之一.哺乳动物体内超过50%的蛋白质都会发生糖基化修饰.糖蛋白广泛分布于各种组织的细胞膜表面,执行着重要的生物学功能.随着高通量、高灵敏度和高分辨率的蛋白质组学时代的来临,许多基于串级质谱技术解析糖链结构的生物数据库和分析软件也亦应运而生.本文综述了目前文献中最常用的糖类生物信息学资源,包括各种糖蛋白的数据库以及质谱解析糖类的相关工具和新技术、新方法.     

综述: N-连接完整糖肽的质谱分析策略和研究方法

蛋白质糖基化作为最普遍、最重要的蛋白质修饰,一直是组学研究的焦点之一．近十几年来,N-连接糖蛋白质组学研究普遍采用的方法是将糖链与所修饰的多肽分开进行分析．该策略虽降低了分析难度,却也丢失了糖链与蛋白质糖基化位点间重要的对应关系信息．近年来,完整糖肽的质谱分析策略和方法逐步建立起来．总体而言,要实现对完整糖肽的直接质谱分析,首先需要从复杂样品中富集完整糖肽以消除非糖基化多肽对完整糖肽分析的影响,然后在质谱分析中还需要根据糖肽特性调整相应质谱分析参数,最后在后续数据分析中还需要开发相应的分析软件以完成完整糖肽中多肽序列和糖链组成或结构的鉴定．本文即从以上三个主要方面系统阐述目前N-完整糖肽分析中常用的质谱和数据分析策略和方法,并进一步在糖肽谱图识别、母离子单同位素分子质量校正、数据库选择以及假阳性率评估和控制等方面都进行了逐一探讨．完整糖肽的直接质谱分析有助于获取糖链和糖基化位点间的对应关系信息,可为生物标志物发现和疾病致病机理等研究提供更有力的糖蛋白质组学研究工具．     

N-糖基化位点鉴定方法和非经典N-糖基化序列

蛋白质的N-糖基化修饰是生物体调控蛋白质在组织和细胞中的定位、功能、活性、寿命和多样性的一种普遍的翻译后方式。N-糖基化位点是理解糖链功能的重要前提之一。应用新的糖蛋白、糖肽富集技术和质谱技术,科学家们在不同组织中完成了对N-糖基化位点的鉴定。此外,不同于经典三联子的N-糖基化序列的发现使人们对N-糖基化过程的认识向纵深发展。     

二维电泳和质谱技术鉴定肝癌血清差异表达的N-连接糖蛋白

缺乏有效的早期诊断方法是导致肝细胞癌(hepatocellular carcinoma, HCC)预后极差的主要原因之一.蛋白质异常糖基化与恶性肿瘤细胞侵袭、转移等生物学过程关系密切,人体内至少有50%的蛋白质发生了糖基化修饰.本实验采用IgY12去除血清高丰度蛋白、多植物凝集素亲和层析技术分别从20例肝癌和年龄、性别匹配的20例非癌慢性肝病患者血清中纯化N 连接糖蛋白、二维电泳分析差异表达的蛋白质斑点,质谱检测、生物信息学等技术鉴定了18个差异表达的糖蛋白和/或其异质体（12种高表达和6种低表达）.ExPASy数据库比对结果表明,本实验鉴定的糖蛋白质分子含有至少1个已报道的N 糖基化位点.这些差异表达的糖蛋白属于急性期反应蛋白,分别具有蛋白酶抑制、生物转运、凝血和纤溶等功能,表明肝癌的发生发展过程中机体产生的急性期反应物可能是潜在的肝癌血清标志物.     

An alpha-numeric code for representing N-linked glycan structures in secreted glycoproteins

Advances in high-throughput techniques have led to the creation of increasing amounts of glycome data. The storage and analysis of this data would benefit greatly from a compact notation for describing glycan structures that can be easily stored and interpreted by computers. Towards this end, we propose a fixed-length alpha-numeric code for representing N-linked glycan structures commonly found in secreted glycoproteins from mammalian cell cultures. This code, GlycoDigit, employs a pre-assigned alpha-numeric index to represent the monosaccharides attached in different branches to the core glycan structure. The present branch-centric representation allows us to visualize the structure while the numerical nature of the code makes it machine readable. In addition, a difference operator can be defined to quantitatively differentiate between glycan structures for further analysis. The usefulness and applicability of GlycoDigit were demonstrated by constructing and visualizing an N-linked glycosylation network.     

The carbohydrate moiety of α-galactosidase from Trichoderma reesei

a-Galactosidase from Trichoderma reesei is a glycoprotein that contains O- and N-linked carbohydrate chains. There are 6 O-linked glycans per protein molecule that are linked to serine and threonine and can be released by b-elimination. Among these are monomers: D-glucose, D-mannose, and D-galactose; dimers: a1-6 D-mannopyranosyl- a-D-glycopyranoside and a1-6 D-glucopyranosyl- a-D-galactopyranoside and one trimer: a-D-glucopyranosyl- a1-2 D-mannopyranosyl- a1-6 D-galac-topyranoside. N-linked glycans are of the mannose-rich type and may be released by treating the protein with Endo- b-N-acetyl glycosaminidase F or by hydrozinolysis. The enzyme was deglycosylated with Endo- b- N-acetyl glycosaminidase F as well as with a number of exoglycosidases that partially remove the terminal residues of O-linked glycans. The effect of enzymatic deglycosylation on the properties of a-galactosidase has been considered. The effects of tunicamycin and 2-deoxyglucose on the secretion and glycosylation of the enzyme during culture growth have been analysed. The presence of two glycoforms of a-glactosidase differing in the number of N-linked carbohydrate chains and the microheterogeneity of the carbohydrate moiety of the enzyme are described. This revised version was published online in August 2006 with corrections to the Cover Date.     

Structural study of N-linked sugar chains of sheep erythrocyte membrane glycoproteins

Our previous study showed that non-reducing terminal galactose residues of N-linked sugar chains present in sheep erythrocyte membrane glycoproteins are important for rosette formation with T lymphoblastic cells [Ogasawara et al. (1995) Immunol Lett 48: 35–38]. As a first step to elucidate the significant structures of sugar chains involved in rosette formation, we analysed N-linked sugar chains released from the membrane glycoproteins by hydrazinolysis. The oligosaccharides were labeled with NaB3H4 and fractionated using columns of Aleuria aurantia lectin-Sepharose, MonoQ and Bio-Gel P-4. Structural analyses of oligosaccharides by sequential exoglycosidase digestion in combination with methylation analysis revealed that the membrane glycoproteins contain bi- (19%), tri- (33%), and tetraantennary (44%) complex-type oligosaccharides and that the oligosaccharides having exposed galactose residues amount to 40% of the total.     

Ovarian cancer is associated with changes in glycosylation in both acute-phase proteins and IgG

Ovarian cancer is the fourth most common cancer in women in the Western world. In a pilot scale study, we highlight changes in the total serum glycome of patients with advanced ovarian cancer that might shed insight into disease pathogenesis. These changes include increases in levels of core fucosylated, agalactosyl biantennary glycans (FA2) and sialyl Lewis x (SLe(x)). To investigate further which proteins contribute to these alterations, we developed technology to analyze simultaneously the glycosylation of protein glycoforms contained in single spots excised from a 2D gel (<1 ng protein). The acute-phase proteins, haptoglobin, alpha1-acid glycoprotein, and alpha1-antichymotrypsin from patients contained elevated levels of subsets of glycoforms containing SLe(x). We also established that IgG heavy chains from patients contained twice the level of FA2 compared with healthy controls. Serum CA125 is the only biomarker that is used routinely, and there is a need for complementary markers that will improve both sensitivity and specificity. There was some preliminary indication that combinations of changes in the serum glycome might improve the separation of ovarian cancer and benign tumors; however, a larger study using data receiver operating characteristic curves will be required to draw any firm conclusions.     

Advanced assessment of the physicochemical characteristics of Remicade® and Inflectra® by sensitive LC/MS techniques

In this study, we demonstrate the utility of ultra-performance liquid chromatography coupled to mass spectrometry (MS) and ion-mobility spectrometry (IMS) to characterize and compare reference and biosimilar monoclonal antibodies (mAbs) at an advanced level. Specifically, we focus on infliximab and compared the glycan profiles, higher order structures, and their host cell proteins (HCPs) of the reference and biosimilar products, which have the brand names Remicade® and Inflectra®, respectively. Overall, the biosimilar attributes mirrored those of the reference product to a very high degree. The glycan profiling analysis demonstrated a high degree of similarity, especially among the higher abundance glycans. Some differences were observed for the lower abundance glycans. Glycans terminated with N-glycolylneuraminic acid were generally observed to be at higher normalized abundance levels on the biosimilar mAb, while those possessing α-linked galactose pairs were more often expressed at higher levels on the reference molecule. Hydrogen deuterium exchange (HDX) analyses further confirmed the higher-order similarity of the 2 molecules. These results demonstrated only very slight differences between the 2 products, which, interestingly, seemed to be in the area where the N-linked glycans reside. The HCP analysis by a 2D-UPLC IMS-MS approach revealed that the same 2 HCPs were present in both mAb samples. Our ability to perform these types of analyses and acquire insightful data for biosimilarity assessment is based upon our highly sensitive UPLC MS and IMS methods.     

Interaction of N-linked glycans, having multivalent GlcNAc termini, with GM3 ganglioside

GM3 ganglioside interacts specifically with complex-type N-linked glycans having multivalent GlcNAc termini, as shown for (1) and (2) below. (1) Oligosaccharides (OS) isolated from ConA-non-binding N-linked glycans of ovalbumin, whose structures were identified as penta-antennary complex-type with bisecting GlcNAc, having five or six GlcNAc termini (OS B1, B2), or bi-antennary complex-type having two GlcNAc termini (OS I). OS I is a structure not previously described. (2) Multi-antennary complex-type N-linked OS isolated from fetuin, treated by sialidase followed by β-galactosidase, having three or four GlcNAc termini exposed. These OS, conjugated to phosphatidylethanolamine (PE), showed clear interaction with ³H-labeled liposomes containing GM3, when various doses of OS-PE conjugate were adhered by drying to multi-well polystyrene plates. Interaction was clearly observed only with liposomes containing GM3, but not LacCer, Gb4, or GalNAcα1-3Gb4 (Forssman antigen). GM3 interaction with PE conjugate of OS B1 or B2 was stronger than that with PE conjugate of OS I. GM3 interacted clearly with PE conjugate of N-linked OS from desialylated and degalactosylated fetuin, but not native fetuin. No binding was observed to cellobiose-PE conjugate, or to OS-PE conjugate lacking GlcNAc terminus. Thus, GM3, but not other GSL liposomes, interacts with various N-linked OS having multiple GlcNAc termini, in general. These findings suggest that the concept of carbohydrate-to-carbohydrate interaction can be extended to interaction of specific types of N-linked glycans with specific GSLs. Natural occurrence of such interaction to define cell biological phenomena is under investigation. All solvent ratios are by volume. An erratum to this article can be found at     

Transient CHO expression platform for robust antibody production and its enhanced N-glycan sialylation on therapeutic glycoproteins

Large-scale transient expression in mammalian cells is a rapid protein production technology often used to shorten overall timelines for biotherapeutics drug discovery. In this study we demonstrate transient expression in a Chinese hamster ovary (CHO) host (ExpiCHO-S™) cell line capable of achieving high recombinant antibody expression titers, comparable to levels obtained using human embryonic kidney (HEK) 293 cells. For some antibodies, ExpiCHO-S™ cells generated protein materials with better titers and improved protein quality characteristics (i.e., less aggregation) than those from HEK293. Green fluorescent protein imaging data indicated that ExpiCHO-S™ displayed a delayed but prolonged transient protein expression process compared to HEK293. When therapeutic glycoproteins containing non-Fc N-linked glycans were expressed in transient ExpiCHO-S™, the glycan pattern was unexpectedly found to have few sialylated N-glycans, in contrast to glycans produced within a stable CHO expression system. To improve N-glycan sialylation in transient ExpiCHO-S™, we co-transfected galactosyltransferase and sialyltransferase genes along with the target genes, as well as supplemented the culture medium with glycan precursors. The authors have demonstrated that co-transfection of glycosyltransferases combined with medium addition of galactose and uridine led to increased sialylation content of N-glycans during transient ExpiCHO-S™ expression. These results have provided a scientific basis for developing a future transient CHO system with N-glycan compositions that are similar to those profiles obtained from stable CHO protein production systems. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2724, 2019     

Review of Glycosylation Engineering of Biopharmaceuticals: Methods and Protocols

The glycoform profile of a glycoprotein is non-templated, i.e., is not encoded within the genome or otherwise predetermined; however, it is estimated that ~50% of human genes having an open reading frame encode a –N-X-S/T- amino acid sequence, where X represents any amino acid other than proline, that comprises a potential site (sequon) for N-linked glycosylation of the translated protein. N-linked glycosylation is both a co- and post-translational modification. The complex oligosaccharide GlcNAc2Man9Glu3 may be added at a –N-X-S/T- sequon as the polypeptide chain emerges from the ribosome tunnel. Local secondary structure determines whether oligosaccharide is added and the extent of addition. Higher occupancy is observed for –N-X-T- sequons than at –N-X-S- sequons, and the efficiency of addition can be further influenced by adjacent amino acid residues.