Reversed-phase depletion coupled with hydrophilic affinity enrichment for the selective isolation of N-linked glycopeptides by using Click OEG-CD matrix

Selective enrichment of glycopeptides is of great importance for protein glycosylation analysis using mass spectrometry since the signals of glycopeptides could be severely suppressed by the coexisting non-glycosylated peptides in the protein digest. In the present work, a strategy for N-linked glycopeptide enrichment through reversed-phase depletion coupled with hydrophilic affinity enrichment by applying the customized matrix named Click OEG-CD is developed. Compared with single hydrophilic interaction liquid chromatography (HILIC) mode, the strategy exhibited remarkably higher selectivity for N-linked glycopeptides. As many as 22, 18, and eight glycopeptides were detected in the glycopeptide fraction enriched with the strategy from the digests of human immunoglobulin G, horseradish peroxidase and bovine ribonuclease B, respectively. In addition, the strategy also showed high glycosylation microheterogeneity coverage for the enrichment of human α₁-acid glycoprotein glycopeptides. More than 170 glycopeptides covering all the glycosylation sites were detected in the enriched fraction. The revered-phase liquid chromatography depletion coupled with HILIC enrichment strategy by using Click OEG-CD matrix is expected to show more potential in further applications in glycosylation analysis.     

新型四肽亲水材料用于糖肽的高效富集

本研究采用原子转移自由聚合(Atom-transfer radical-polymerization, ATRP)法合成了一种新型四肽亲水作用色谱材料 (Poly-DAPD),用于糖肽的选择性富集.通过氮气吸脱附、热重分析和X射线光电子能谱等技术进行表征,结果表明,四肽已成功接枝到硅球上.固相萃取富集实验表明,合成的亲水材料对牛胎球蛋白(Fetuin)糖肽富集选择性高;与商品化ZIC-HILIC材料相比,Poly-DAPD材料富集掺有5摩尔倍数牛血清蛋白(BSA)的Fetuin样品时,在获得的糖肽数目及抗干扰性能方面都更具优势.此Poly-DAPD材料可进一步用于不同糖蛋白的糖基化分析研究.     

两性离子双功能化超亲水金属有机框架纳米复合材料的制备及其用于糖肽的选择性富集

蛋白糖基化是生物体中普遍发生且重要的生物学过程,其参与多种分子生物学的功能和途径,是临床诊断重要的生物标志物.但是,糖肽因其丰度低、离子化效率低、糖链异质性等难点,使糖蛋白分析一直面临巨大的挑战.因此,研究合成了一种新型的两性离子双功能化纳米金(AuGC)修饰的超亲水性沸石咪唑骨架(ZIF-8)纳米复合材料(AuGC/...     

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。     

Rapid glycopeptide enrichment and N-glycosylation site mapping strategies based on amine-functionalized magnetic nanoparticles

Glycoproteins secreted or expressed on the cell surface at specific pathophysiological stages are well-recognized disease biomarkers and therapeutic targets. While mapping of specific glycan structures can be performed at the level of released glycans, site-specific glycosylation and identification of specific protein carriers can only be determined by analysis of glycopeptides. A key enabling step in mass spectrometry (MS)-based glycoproteomics is the ability to selectively or non-selectively enrich for the glycopeptides from a total pool of a digested proteome for MS analysis since the highly heterogeneous glycopeptides are usually present at low abundance and ionize poorly compared with non-glycosylated peptides. Among the most common approaches for non-destructive and non-glycan-selective glycopeptide enrichment are strategies based on various forms of hydrophilic interaction liquid chromatography (HILIC). We present here a variation of this method using amine-derivatized Fe₃O₄ nanoparticles, in concert with in situ peptide N-glycosidase F digestion for direct matrix-assisted laser desorption/ionization–mass spectrometry analysis of N-glycosylation sites and the released glycans. Conditions were also optimized for efficient elution of the enriched glycopeptides from the nanoparticles for on-line nanoflow liquid chromatography–MS/MS analysis. Successful applications to single glycoproteins as well as total proteomic mixtures derived from biological fluids established the unrivaled practical versatility of this method, with enrichment efficiency comparable to other HILIC-based methods.     

Recent advances in hydrophilic interaction liquid chromatography (HILIC) for structural glycomics

This review presents recent progress in employing hydrophilic interaction liquid chromatography (HILIC) for glycan and glycopeptides analysis. After an introduction of this technique, the following themes are addressed: (i) implementation of HILIC in large-scale studies for analyzing the human plasma N-glycome; (ii) the use of HILIC UPLC (ultrahigh pressure liquid chromatography) for fast high-resolution runs and its successful application with online MS for glycan and glycopeptide analysis; (iii) high-throughput profiling using HILIC solid-phase extraction in combination with MS detection; (iv) HILIC sample preparation for CE and CGE; (v) the latest glycoproteomic approaches implementing HILIC separation; (vi) future perspectives of HILIC including its use in large-scale glycoproteomics studies such as the analysis of entire glycoproteomes at the glycopeptide level.     

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。     

Synthesis and evaluation of carboxymethyl chitosan for glycopeptide enrichment

Glycans are known to be involved in a variety of biological processes throughout human physiology. Mass spectrometry has demonstrated itself as powerful analytical tool for quantitative and structural characterization of glycans. Studying these molecules at the glycopeptide level however, offers distinct advantages, namely the ability to characterize both the glycan and peptide fragments simultaneously, and moreover the ability to assign site specific heterogeneity. In light of this, peptides often dominate the spectrum and hinder the ionization efficiency of glycopeptides. For this reason, enrichment protocols prior to downstream MS analysis need to be developed. Here, we discuss the synthesis and use of carboxymethyl chitosan (CMCH) to enrich glycopeptides from a 12 protein mixture for MS analysis. This protocol was compared to a commercially available glycopeptide enrichment kit offered by EMD Millipore through the use of tandem mass tags (TMT) for relative quantification. Using this approach, we identified 98 unique N-linked glycopeptides and observed, that CMCH was able to enrich more sialylation than the commercial kit. In addition, we observed a trend based on TMT reporter ratios with respect to increasing sialylation. This corroborated that this stationary phase was exhibiting a mixed-mode enrichment through both hydrophilic interaction liquid chromatography (HILIC) and weak anion exchange (WAX) principles.     

Selective enrichment of glycopeptides for mass spectrometry analysis using C18 fractionation and titanium dioxide chromatography

Comprehensive glycoprotein characterization based on mass spectrometry (MS) is challenging because of low concentration of glycopeptides and suppression effect of abundant non-glycosylated peptides in MS. Therefore, it is vital to enrich glycopeptides before MS analysis. A new method was developed to selectively enrich glycopeptides from complex sample by coupling C18 fractionation with titanium dioxide (TiO(2)) enrichment. The new method allows to selectively enrich N-linked glycopeptides with various glycan forms and different sequence lengths. Compared with single TiO(2) method, the established method demonstrated higher glycopeptide selectivity and higher glycosylation heterogeneity coverage. Further application of this method to mixture of non-glycosylated protein and glycoprotein digests at different levels reveals the feasibility of enrichment of tryptic glycopeptides from simple proteomics samples.     

完整糖基化肽段的富集与质谱解析新技术研究进展

蛋白质糖基化是生物体内最重要的翻译后修饰之一,在蛋白质稳定性、细胞内和细胞间信号转导、激素活化或失活和免疫调节等生理过程和病理进程中发挥重要作用.而异常的蛋白质糖基化往往和多种疾病的发生发展密切相关,目前应用于临床检测的多种肿瘤生物标志物大多属于糖蛋白或者糖抗原.因此在组学层次系统分析蛋白质糖基化的变化对阐明生物体内糖...     

One-pot synthesis of magnetic colloidal nanocrystal clusters coated with chitosan for selective enrichment of glycopeptides

Selective enrichment of glycopeptides prior to the mass spectrometry (MS) analysis is essential due to ion suppression effect during ionization caused by the co-presence of non-glycosylated peptides. Among the enrichment approaches, hydrophilic interaction liquid chromatography (HILIC) based on magnetic separation has become a popular method in recent years. As the conventional synthesis procedures of these materials are tedious and time-consuming with at least four steps. Herein, magnetic colloidal nanocrystal clusters coated with chitosan (Fe₃O₄@CS MCNCs) have been successfully prepared by a simple one-pot method. The resulting Fe₃O₄@CS MCNCs demonstrated an excellent ability for glycopeptide enrichment with high selectivity, low detection limit and high binding capacity. Furthermore, in the analysis of real complicated biological sample, 283 unique N-glycosylation sites corresponding to 175 glycosylated proteins were identified in three replicate analyses of 45 μg protein sample extracted from HeLa cells, indicating the great potential in detection and identification of low abundant glycopeptides in glycoproteome analysis.     

基于固相萃取的微量糖蛋白糖基化修饰表征技术的建立

结合自制亲水固相萃取富集柱和生物质谱鉴定技术,实现了糖基化蛋白质核糖核酸酶B的糖含量测定、糖基化位点确认、聚糖富集及结构表征,以及不同糖型相对丰度分析。结果表明:其糖含量8.47%,糖基化位点为34位的Asn,糖链主要为5种高甘露糖型结构(Man5-9GlcNAc2)。所建立的HILIC富集技术,有利于针对微量生物样本,如生物工程药物糖蛋白及重要功能糖蛋白,开展位点特异性糖链结构解析,为糖蛋白质的药效或功能研究提供线索。     

Design and fabrication of highly hydrophilic magnetic material by anchoring l-cysteine onto chitosan for efficient enrichment of glycopeptides

Hydrophilic interaction liquid chromatography(HILIC) has been recognized as an effective strategy for glycopeptide enrichment. Hydrophilic materials pave the way to solve the limit of low enrichment capacity and poor selectivity. The present study is the first attempt to combine chitosan(CS) and L-cysteine(L-Cys) to design a novel hydrophilic material focusing on glycopeptide enrichment. CS containing a large number of hydrophilic amino and hydroxyl groups has unique chemical properties, which m...     

利用亲水富集策略分析人血浆中N-糖基化蛋白及N-糖类型

蛋白质的N-糖基化是最重要的翻译后修饰之一,许多已知的血浆肿瘤诊断标志物及治疗靶标都是N-糖基化蛋白。针对血浆的糖蛋白质组研究有利于发现新的蛋白标志物。然而,血浆蛋白质浓度分布的动态范围非常宽,且同一位点上的糖链存在微观不均一性,影响了血浆中糖蛋白的鉴定效率。本文利用亲水材料ZIC-HILIC制备亲水富集柱分别对人血浆中的N-糖链和N-糖肽进行富集,并结合碱性反相色谱进行肽段的预分离和高准确度质谱分析,最终在健康人的血浆中鉴定到了299个糖基化蛋白、637个糖基化位点,并识别出31种不同的糖型。在这些鉴定到的糖基化位点中,新发现有107个N-糖基化位点(占总位点数的16.8%)。本方法操作简单,可以有效富集N-糖肽和N-糖,为在血浆中寻找糖蛋白和糖链生物标志物提供了可靠的手段。     

Facile preparation of hydrophilic glutathione modified magnetic nanomaterials for specific enrichment of glycopeptides

Glutathione modified magnetic nanoparticles (Fe₃O₄@Au-GSH) were synthesized through a simple process and exploited to enrich glycopeptides from complex samples.     

Facile preparation of hydrophilic glutathione modified magnetic nanomaterials for specific enrichment of glycopeptides

Investigations of glycosylated proteins or peptides and their related biological pathways provide new possibilities for illuminating the physiological and pathological mechanisms of glycosylation modification. However, open-ended and in-depth analysis of glycoproteomics is usually subjected to the low-abundance of glycopeptides, heterogeneous glycans, and a variety of interference molecules. In order to alleviate the influence of these obstacles, effective preconcentration of glycopeptides are indispensable. Here, we employed a hydrophilic interaction liquid chromatography (HILIC)-based method to universally capture glycopeptides. Glutathione modified magnetic nanoparticles (Fe₃O₄@Au-GSH) were synthesized through a simple process and exploited to enrich glycopeptides from complex samples. The prepared materials showed excellent ability to trap glycopeptides from standard glycoproteins digests, low detection limit (10 fmol/μL), and good selectivity (HRP:BSA = 1:100). These results indicated that glutathione-based magnetic nanoparticles synthesized in this work had great potential for glycopeptides enrichment.     

A procedure for the analysis of site‐specific and structure‐specific fucosylation in alpha‐1‐antitrypsin

A MS‐based methodology has been developed for analysis of core‐fucosylated versus antennary‐fucosylated glycosites in glycoproteins. This procedure is applied to the glycoprotein alpha‐1‐antitrypsin (A1AT), which contains both core‐ and antennary‐fucosylated glycosites. The workflow involves digestion of intact glycoproteins into glycopeptides, followed by double digestion with sialidase and galactosidase. The resulting glycopeptides with truncated glycans were separated using an off‐line HILIC (hydrophilic interaction liquid chromatography) separation where multiple fractions were collected at various time intervals. The glycopeptides in each fraction were treated with PNGase F and then divided into halves. One half of the sample was applied for peptide identification while the other half was processed for glycan analysis by derivatizing with a meladrazine reagent followed by MS analysis. This procedure provided site‐specific identification of glycosylation sites and the ability to distinguish core fucosylation and antennary fucosylation via a double digestion and a mass profile scan. Both core and antennary fucosylation are shown to be present on various glycosites in A1AT.     

Click maltose as an alternative to reverse phase material for desalting glycopeptides

Desalting peptides before mass spectrometry analysis is important because salts lead to adduct formation, increased chemical noise and ion suppression effect. A high concentration of salt can clog nanoelectrospray ionization (ESI) emitters. The reverse phase C18 material is commonly used to desalt peptides because of its high binding capacity. However, peptides with high hydrophilicity, such as glycopeptides, are not retained well on this material, resulting in the loss of peptide information. To improve the efficiency of glycopeptide desalting, we introduced a hydrophilic interaction chromatography (HILIC)-based material named click maltose. Four glycoproteins, horseradish peroxidase (HRP), human serum immunoglobulin G (IgG), bovine ribonuclease B (RNase B), and α-1 acid glycoprotein (AGP) were chosen as models and their glycopeptides were desalted with click maltose, AQ C18, Empore C18 and ZipTip C18. Click maltose as a HILIC material exhibited better performance than the other three C18 materials for both number of targeted glycopeptides and their corresponding intensities. In addition, accurate glycopeptide profiling was achieved with click maltose desalting regardless of peptide lengths and glycan types.     

Determination of N-Glycopeptides by Hydrophilic Interaction Liquid Chromatography and Porous Graphitized Carbon Chromatography with Mass Spectrometry Detection

An offline two-dimensional chromatographic method based on the combination of hydrophilic interaction liquid chromatography (HILIC) and porous graphitized carbon (PGC) chromatography was developed for the separation and purification of glycopeptides. The high selectivity of HILIC and PGC isolated high-purity isomers of N-glycopeptides from ribonuclease B. N-Glycopeptides were first separated from nonglycosylated peptides, and N-glycopeptides were sorted into fractions through the first-dimensional HILIC according to their monosaccharides. Further separation of the glycopeptide isomers in each fraction was achieved using second-dimensional PGC. Structural differences of the glycopeptide isomers were further enzymatically hydrolyzed with peptide-N-glycosidase F. The glycan structure were elucidated by matrix assisted laser desorption ionization tandem quadrupole time-of-flight mass spectrometry. The established procedure allows the isolation of glycopeptide or glycan standards from natural sources.     

Gold nanoparticles immobilized hydrophilic monoliths with variable functional modification for highly selective enrichment and on-line deglycosylation of glycopeptides

The poly (glycidyl methacrylate-co-poly (ethylene glycol) diacrylate) monoliths modified with gold nanoparticles, with advantages of enhanced reactive sites, good hydrophilicity and facile modification, were prepared as the matrix, followed by variable functionalization with cysteine and PNGase F for glycopeptide enrichment and on-line deglycosylation respectively. By the cysteine functionalized monolithic column, glycopeptides could be efficiently and selectively enriched with good reproducibility based on hydrophilic interaction chromatography (HILIC). Furthermore, the enrichment was specially achieved in weak alkaline environment, with 10 mM NH₄HCO₃ as the elution buffer, compatible with deglycosylation conditions. Therefore, the glycopeptides could be on-line deglycosylated with high efficiency and throughput by directly coupling the PNGase F functionalized monolithic column with the enrichment column during elution without the requirement of buffer exchange and pH adjustment. By such a method, within only 70-min pretreatment, 196 N-linked glycopeptides, corresponding to 122 glycoproteins, could be identified from 5 μg of human plasma with 14 high-abundant proteins removed, and the N-linked glycopeptides occupied 81% of all identified peptides, achieving to the best of our knowledge, the highest selectivity of HILIC-based methods. All the results demonstrated the high efficiency, selectivity and throughput of our proposed strategy for the large scale glycoproteome analysis.