Application of Lectin Microarrays for Biomarker Discovery

Many proteins in living organisms are glycosylated. As their glycan patterns exhibit protein-, cell-, and tissue-specific heterogeneity, changes in the glycosylation levels could serve as useful indicators of various pathological and physiological states. Thus, the identification of glycoprotein biomarkers from specific changes in the glycan profiles of glycoproteins is a trending field. Lectin microarrays provide a new glycan analysis platform, which enables rapid and sensitive analysis of complex glycans without requiring the release of glycans from the protein. Recent developments in lectin microarray technology enable high-throughput analysis of glycans in complex biological samples. In this review, we will discuss the basic concepts and recent progress in lectin microarray technology, the application of lectin microarrays in biomarker discovery, and the challenges and future development of this technology. Given the tremendous technical advancements that have been made, lectin microarrays will become an indispensable tool for the discovery of glycoprotein biomarkers.     

One-pot solid-phase glycoblotting and probing by transoximization for high-throughput glycomics and glycoproteomics

The development of rapid and efficient methods for high-throughput protein glycomics is of growing importance because the glycoform-focused reverse proteomics/genomics strategy will greatly contribute to the discovery of novel biomarkers closely related to cellular development, differentiation, growth, and aging as well as a variety of diseases such as cancers and viral infection. Recently, we communicated that rapid and efficient purification of carbohydrates can be achieved by employing sugar-specific chemical ligation with aminooxy-functionalized polymers, which we termed "glycoblotting" (see S.-I. Nishimura et al., Angew. Chem. 2005, 117, 93-98; Angew. Chem. Int. Ed. 2005, 44, 91-96). The chemoselective blotting of oligosaccharides present in crude biological materials onto synthetic polymers relies on the unique oxime-bond formation between aminooxy group displayed on the supporting materials and aldehyde/ketone group at the reducing terminal of all oligosaccharides, thus enabling highly selective and rapid oligosaccharide purification. Aiming to improve the detection sensitivity of the released oligosaccharides, we introduce here a novel strategy for one-pot solid-phase glycoblotting and probing by transoximization. We found that oligosaccharides captured by the polymer supports via the oxime bond can be released in the presence of excess O-substituted aminooxy derivatives in a weakly acidic condition. The released oligosaccharides could be recovered as newly formed oxime derivatives of the O-substituted aminooxy compound added, thus demonstrating the simultaneous releasing and probing. In addition, we synthesized a novel aminooxy-functionalized monomer, N-[2-[2-(2-tert-butoxycarbonylaminooxyacetylamino-ethoxy)ethoxy]ethyl]-2-methacrylamide, which allows for the large-scale preparation of a versatile polymer characterized by its high stability, high blotting capacity, and easy use. The one-pot protocol allowed to profile 23 kinds of N-glycan chains of human serum glycoproteins. This concept was further applied for the glycopeptides analysis in a crude mixture followed by galactose oxidase treatment to generate free aldehyde group at the non-reducing terminal of oligosaccharide moiety of glycopeptides. Our technique may be implemented in existing biochemistry and molecular diagnostics laboratories because enriched oligosaccharides and glycopeptides by solid-phase transoximization with high-sensitive labeling reagents are widely applicable in a variety of common analytical methods using two-dimensional HPLC, LC/MS, and capillary electrophoresis as well as modern mass spectrometry.     

Glycoproteomics: Charting new territory in mass spectrometry and glycobiology

Glycosylation is an incredibly common and diverse post-translational modification that contributes widely to cellular health and disease. Mass spectrometry is the premier technique to study glycoproteins; however, glycoproteomics has lagged behind traditional proteomics due to the challenges associated with studying glycosylation. For instance, glycans dissociate by collision-based fragmentation, thus necessitating electron-based fragmentation for site-localization. The vast glycan heterogeneity leads to lower overall abundance of each glycopeptide, and often, ion suppression is observed. One of the biggest issues facing glycoproteomics is the lack of reliable software for analysis, which necessitates manual validation and serves as a massive bottleneck in data processing. Here, I will discuss each of these challenges and some ways in which the field is attempting to address them, along with perspectives on how I believe we should move forward.     

纳米二氧化硅影响人肺癌细胞的定量糖蛋白质组学研究

纳米二氧化硅(纳米SiO₂)是一种正在规模化生产的纳米材料, 无定型纳米SiO₂因其吸入和口服对生命体不会造成直接的危害被认为是生物安全的纳米材料, 已被广泛用于疾病诊断、生物分析和成像、药物载体等的研究中, 导致其进入人体的方式日益增多, 因此它对人体健康影响的研究对于其作为生物材料真正实现广泛应用尤为重要. 本文采用肼化学方法为基础的定量蛋白质组学对无定型纳米SiO₂进入人肺癌细胞后产生的影响进行了分析, 结果表明其进入细胞后, 导致细胞内的平衡状态发生变化, 从而影响了细胞内许多重要的蛋白质的表达水平. 部分跨膜蛋白质表达的变化对纳米SiO₂进入细胞的途径的阐明有一定的指导意义.     

糖蛋白/糖肽分离富集中的化学

糖蛋白在许多生命过程中有重要的作用.糖蛋白/糖肽在复杂生物样品中的低丰度以及糖链结构的微观不均一性使其分离富集成为糖蛋白质组学研究中的难点,本文将糖蛋白/糖肽分离富集过程中使用的化学方法根据所应用的化学反应或外源功能基团的种类分为:肼化学方法,氨化学方法,硼酸化学法, β-消除米氏加成化学法;并对各化学方法的原理和优缺点进行了描述和讨论.     

The Need for Community Standards to Enable Accurate Comparison of Glycoproteomics Algorithm Performance

Protein glycosylation that mediates interactions among viral proteins, host receptors, and immune molecules is an important consideration for predicting viral antigenicity. Viral spike proteins, the proteins responsible for host cell invasion, are especially important to be examined. However, there is a lack of consensus within the field of glycoproteomics regarding identification strategy and false discovery rate (FDR) calculation that impedes our examinations. As a case study in the overlap between software, here as a case study, we examine recently published SARS-CoV-2 glycoprotein datasets with four glycoproteomics identification software with their recommended protocols: GlycReSoft, Byonic, pGlyco2, and MSFragger-Glyco. These software use different Target-Decoy Analysis (TDA) forms to estimate FDR and have different database-oriented search methods with varying degrees of quantification capabilities. Instead of an ideal overlap between software, we observed different sets of identifications with the intersection. When clustering by glycopeptide identifications, we see higher degrees of relatedness within software than within glycosites. Taking the consensus between results yields a conservative and non-informative conclusion as we lose identifications in the desire for caution; these non-consensus identifications are often lower abundance and, therefore, more susceptible to nuanced changes. We conclude that present glycoproteomics softwares are not directly comparable, and that methods are needed to assess their overall results and FDR estimation performance. Once such tools are developed, it will be possible to improve FDR methods and quantify complex glycoproteomes with acceptable confidence, rather than potentially misleading broad strokes.     

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。     

Differential Glycosite Profiling—A Versatile Method to Compare Membrane Glycoproteomes

Glycosylation is the most prevalent and varied form of post-translational protein modifications. Protein glycosylation regulates multiple cellular functions, including protein folding, cell adhesion, molecular trafficking and clearance, receptor activation, signal transduction, and endocytosis. In particular, membrane proteins are frequently highly glycosylated, which is both linked to physiological processes and of high relevance in various disease mechanisms. The cellular glycome is increasingly considered to be a therapeutic target. Here we describe a new strategy to compare membrane glycoproteomes, thereby identifying proteins with altered glycan structures and the respective glycosites. The workflow started with an optimized procedure for the digestion of membrane proteins followed by the lectin-based isolation of glycopeptides. Since alterations in the glycan part of a glycopeptide cause mass alterations, analytical size exclusion chromatography was applied to detect these mass shifts. N-glycosidase treatment combined with nanoUPLC-coupled mass spectrometry identified the altered glycoproteins and respective glycosites. The methodology was established using the colon cancer cell line CX1, which was treated with 2-deoxy-glucose—a modulator of N-glycosylation. The described methodology is not restricted to cell culture, as it can also be adapted to tissue samples or body fluids. Altogether, it is a useful module in various experimental settings that target glycan functions.     

Hydrophilic interaction chromatography for the analysis of biopharmaceutical drugs and therapeutic peptides: A review based on the separation characteristics of the hydrophilic interaction chromatography phases

Applications of hydrophilic interaction chromatography for the analysis of biopharmaceutical drugs, i.e., glycosylated proteins represented by monoclonal antibodies are discussed in the manner of glycoproteomics. They can be analyzed using hydrophilic interaction chromatography in five different stages as (1) their intact forms, (2) their subunits, (3) N‐ and O‐glycopeptides digested by proteases, (4) N‐ and O‐glycans released from the glycoproteins or glycopeptides, and (5) monosaccharides. Hydrophilic interaction chromatography is a more useful tool in the order of (1) to (5). At the stages (4) and (5), quantitation of glycans and saccharides are also reported. Hydrophilic interaction chromatography is employed not only for analytical uses, but also pretreatment items as solid phase extraction, followed by reversed‐phase liquid chromatography separations. Comprehensive search results of these application of hydrophilic interaction chromatography are summarized in tables to show what kind of hydrophilic interaction chromatography columns are suitable for each step of analysis.Relationship of favored and less favored hydrophilic interaction chromatography columns and their separation characteristics such as hydrophilicity, and selectivity for structural difference, is also discussed. Analysis of the therapeutic peptides (not glycosylated) using hydrophilic interaction chromatography is summarized, too.     

糖蛋白/糖肽的分离富集方法*

蛋白质糖基化是一种重要的翻译后修饰,糖基化对蛋白质的结构和功能有着重要的影响。目前,作为蛋白质组学的一个组成部分,糖蛋白质组学是备受关注的研究热点。而从复杂的生物样品体系中富集糖蛋白/ 糖肽是蛋白质糖基化研究的重点和难点,本文就糖蛋白/糖肽分离富集方法的研究进展和应用作了简要概述。这些方法包括常用的凝集素亲和法、硼酸法、肼化学法和亲水作用法,还包括分子筛法、强阳离子交换法等新方法。