毛细管电泳技术在单克隆抗体药物分析中的应用

单克隆抗体药物在生物制药行业占有重要地位,是生物医药领域发展的主要方向。因此,单克隆抗体药物的质量控制已成为全球生物制药企业及法规机构关注的热点,对单克隆抗体药物精确表征的需求日益增加。毛细管电泳技术具有分离效率高、分析速度快、分离模式多、样品用量少等特点,已成为单克隆抗体药物分析和质量控制的重要手段。该文对毛细管凝胶电泳、毛细管等电聚焦、毛细管区带电泳等模式在单克隆抗体药物的纯度分析、等电点测定、电荷异质性分析和N-寡糖分析的应用进行综述,以期为国内单克隆抗体研究开发和生产的企事业单位提供技术参考。     

Discovery and application of 6π-azaelectrocyclization to natural product synthesis and synthetic biology

While elucidating the inhibitory mechanism of a hydrolytic enzyme by aldehyde-containing natural product,we discovered a reaction involving a rapid 6π-azaelectrocyclization of azatrienes generated from aldehyde with lysine residues.The electrocyclic reaction of the 1-azatriene system,a cyclization precursor,exhibited a significant substituent effect.Asymmetric chiral piperidine synthesis and a one-pot library synthesis of pyridines on solid-supports were applied to synthesize pyridine/indole alkaloid-type natural product.Additionally,we developed lysine-based labeling and engineering of biomolecules and living cells based on the rapid 6π-azaelectrocyclization.Both labels and oligosaccharide structures were introduced efficiently and selectively into surface lysines under the mild conditions;notable effects of N-glycans on proteins and living cells were visualized for the first time by PET and noninvasive fluorescence imaging.     

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。     

Synthesis of a LEC14 nonasaccharide, a core-fucosylated, biantennary N-glycan with a novel GlcNAc residue in the core region

The recently found core substitution of N-glycans termed LEC14 is characterized by a GlcNAc residue linked β(1,2) to the central β-mannoside. Starting from a pentasaccharide building block functionalized for core-fucosylated N-glycans the total synthesis of a protected LEC14 nonasaccharide was accomplished. The key step of the synthesis was the introduction of the additional β(1,2)-linked GlcNAc residue that was highly dependent on the solvent and appears to proceed via an amide acetal intermediate.     

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.     

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.     

A facile synthesis of a complex type N-glycan thiazoline as an effective inhibitor against the antibody-deactivating endo-β-N-acetylglucosaminidases

Endo-β-N-acetylglucosaminidases are a class of endoglycosidases that deglycosylate N-glycans from glycoproteins. We describe here a facile synthesis of a complex type N-glycan thiazoline as a new mechanism-based inhibitor for this class of enzymes. The synthesis started with the readily available sialoglycopeptide (SGP) and its conversion into the glycan thiazoline through several enzymatic and chemical reactions. The synthetic glycan thiazoline showed potent inhibitory activity against several endoglycosidases including the two antibody-deactivating enzymes, Endo-S and Endo-S2, from human pathogen Streptococcus pyogenes, which would be useful as tools for structural and functional studies of these enzymes.     

Design and Synthesis of Glycosylated Cholera Toxin B Subunit as a Tracer of Glycoprotein Trafficking in Organelles of Living Cells

Glycosylation of proteins is known to be essential for changing biological activity and stability of glycoproteins on the cell surfaces and in body fluids. Delivering of homogeneous glycoproteins into the endoplasmic reticulum (ER) and the Golgi apparatus would enable us to investigate the function of asparagine-linked (N-) glycans in the organelles. In this work, we designed and synthesized an intentionally glycosylated cholera toxin B-subunit (CTB) to be transported to the organelles of mammalian cells. The heptasaccharide, the intermediate structure of various complex-type N-glycans, was introduced to the CTB. The synthesized monomeric glycosyl-CTB successfully entered mammalian cells and was transported to the Golgi and the ER, suggesting the potential use of synthetic CTB to deliver and investigate the functions of homogeneous N-glycans in specific organelles of living cells.     

N-糖链唾液酸连接异构体的质谱分析方法研究进展

蛋白质在翻译过程中、翻译过程后会发生糖基化. 糖基化会以直接或间接的方式影响蛋白质的功能及其相互作用, 并与多种人类疾病有关, 其中, 唾液酸化N-糖链在一些重要的生理和病理过程中发挥关键作用. 已知的唾液酸与相邻单糖之间的连接方式包括α-2,3-、α-2,6-、α-2,8-、α-2,9-连接, 连接方式不同的唾液酸化N-糖链在细胞活动、生命体的生理和病理过程中的功能往往不同. 质谱技术是分析N-糖链的重要工具, 它能够快速和灵敏地检测N-糖链, 通过将色谱技术以及衍生化方法等与质谱联用可以实现对唾液酸化N-糖链及其连接异构体的分离和检测. 本文主要围绕α-2,3-和α-2,6-连接的唾液酸化N-糖链进行综述, 介绍它们的结构和在细胞活动及疾病中不同的功能, 并综述近年来基于质谱的唾液酸化N-糖链的连接异构体分析方法以及这些方法在生物医学领域的应用, 并对未来的生物医学研究提供新的思路和途径.