ANALYTICAL BIOCHEMISTRY
·
2024
·
IF 2.6
Cheng Li,Qinghui Chen,Jinqiao Rong,Houde He,Yu Lu,Yuxia Liu,Zhongfu Wang
As the main active glycoprotein of egg white, the biological functions of chicken ovomucin α- and β-subunit are closely related to the structure of glycans. However, the exact composition and structure of the subunit glycans are still unknown. We obtained highly pure chicken ovomucin α-subunit and β-subunit protein bands by the strategy combined with two-step isoelectric precipitation and SDS-PAGE gel electrophoresis. The ammonia-catalyzed one-pot procedure was then used to release and capture α-and β-subunit protein glycans with 1-phenyl- 3-Methyl-5-pyrazolone (PMP). The N / O -glycans of bis-PMP derivatives were purified and analyzed by LC-MS. More importantly, an effective dual modification was performed to accurately quantify neutral and sialylated O -glycans through methylamidation of sialic acid residues and simultaneously through carbonyl condensation reactions of reducing ends with PMP. We first showed that the α-subunit protein has only N -glycosylation modification, and the β-subunit only O -glycosylation, a total of 22 N -glycans and 20 O -glycans were identified in the α- and β-subunit, respectively. In addition, the complex N -glycan (47 %) and the sialylated O -glycan (77 %) are each major types of the above subunits. Such findings in this study provide a basis for studying the functional and biological activities of chicken ovomucin glycans.
研究领域:
生物化学
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糖生物学
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蛋白质组学
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DOI
PubMed
高影响因子
GLYCOBIOLOGY
·
2021
·Vol. 31
·期 8
·931-946
·
IF 4.313
Li Chunxia,Palma Angelina S,Zhang Pengtao,Zhang Yibing,Gao Chao,Silva Lisete M,Li Zhen,Trovão Filipa,Weishaupt Markus,Seeberger Peter H,Likhosherstov Leonid M,Piskarev Vladimir,Yu Jin,Westerlind Ulrika,Chai Wengang
Glycan microarrays have played important roles in detection and specificity assignment of glycan recognition by proteins. However, the size and diversity of glycan libraries in current microarray systems are small compared to estimated glycomes, and these may lead to missed detection or incomplete assignment. For microarray construction, covalent and noncovalent immobilization are the two types of methods used, but a direct comparison of results from the two platforms is required. Here we develop a chemical strategy to prepare lipid-linked probes from both naturally derived aldehyde-terminating and synthetic amino-terminating glycans that addresses the two aspects: expansion of sequence-defined glycan libraries and comparison of the two platforms. We demonstrate the specific recognition by plant and mammalian lectins, carbohydrate-binding modules and antibodies and the overall similarities from the two platforms. Our results provide new knowledge on unique glycan-binding specificities for the immune receptor Dectin-1 toward β-glucans and the interaction of rotavirus P[19] adhesive protein with mucin O-glycan cores.
研究领域:
生物化学
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免疫学
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分子生物学
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DOI
PubMed
高影响因子
APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY
·
2025
·
IF 3.3
Zhao Shanshan,Du Shouwen,Guan Yudong,Schlüter Hartmut
The structural instability and electronegativity of sialic acids make N-sialoglycan profiling challenging. We compared commonly used preparation strategies, including native, acetohydrazide-based derivatization, and permethylation, in N-glycomics with nMatch, an R-based bioinformatics tool that ensures full coverage of the detected N-glycans using mass spectrometry. We demonstrated that permethylation leads to efficient detection of not only N-sialoglycans but also other N-glycans and, therefore, provides a valuable reference for the selection of the appropriate N-glycan preparation strategy in future studies.
研究领域:
分析化学
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糖组学
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质谱技术
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生物信息学
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DOI
PubMed
高影响因子
ANALYTICA CHIMICA ACTA
·
2020
·
IF 5.977
Qihao Zhu,Weiran Mao,Chengli Zhang,Yuan Zhou,Zhiyong Tang,Chao Yu
Here, a novel electrochemiluminescence biosensor based on potential-resolved strategy was firstly prepared for the detection of dual targets α2,3-sialylated glycans and α2,6-sialylated glycans. This is the first time that Au@BSA microsphere was used to connect with luminol to enhance its ECL intensity, and it can generate ECL signals at positive potential. Zeolitic Imidazolate Framework-8 (ZIF-8) and Meso-tetra (4-carboxyphenyl) porphyrin (TCPP) were linked using a one-pot method to synthesize a novel luminescent ZIF (L-ZIF) named TZZ, which can emit ECL signals at negative potential. Moreover, magnetite microspheres were used to construct a sandwich-type biosensor to obtain higher sensitivity and reduce background signals. In addition, the biosensor manufactured directly in solution have a wider linear range than constructed on electrode because it has more available space than the electrode surface. Due to the above advantages, the prepared ECL biosensor exhibited high sensitivity, stability and broader linear range, even for practical analysis. Therefore, the prepared ECL biosensor will become a promising method for determination of α2,3-sialylated glycans and α2,6-sialylated glycans in clinical applications in the future. What is more, it provides a potential method for detection of other multi-targets.
研究领域:
分析化学
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生物医学工程
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传感技术
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DOI
PubMed
高影响因子
ANALYTICA CHIMICA ACTA
·
2022
·
IF 6.911
Yan Cai,Weifu Ren,Haiyan Wang,Qi Bian
Diabetic kidney disease (DKD) is a common and serious kidney-related complication of diabetic mellitus. Although albuminuria and estimated glomerular filtration rate (eGFR) are commonly used diagnostic biomarkers, they have limitations in the diagnosis of DKD due to the lack of specificity and sensitivity. Urinary N-glycans are emerging as novel biomarkers for predicting renal prognosis in DKD. However, most of the current N-glycan profiling methods for DKD were based on lectin affinity enrichment or hydrophilic interaction chromatography (HILIC) with optical detection, which provided limited N-glycome coverage in low throughput. Herein, we developed a novel N-glycan purification method, termed Ultrafast Glycoprotein Immobilization for Glycan extraction (UltraGIG), for in-depth profiling of urinary N-glycome in DKD using mass spectrometry (MS). In UltraGIG, proteins were rapidly (within 40 min) immobilized to resin with high efficiency (over 98%) through NHS-reactive chemistry , and then N-glycans were enzymatically released from the resin by PNGase F. Owing to good efficiency of immobilization of proteins to resin, the subsequent washing steps to remove proteins and other impurities could be easily performed. The UltraGIG showed good selectivity towards glycans (extracting N-glycans from the mixture of IgG and BSA at a 1:100 M ratio) and samples loss was minimized (detection sensitivity at fmol level). A total of 237 N-glycan compositions were identified from urine samples with DKD and healthy controls, which representing the largest data set of N-glycome from DKD. Compared to healthy controls, 3 N-glycans were up-regulated and 14 N-glycans were down-regulated in DKD patients. Collectively, UltraGIG offers a competitive sample purification method for in-depth analysis of urinary N-glycome by MS and provide new insights into biomarker study for the diagnosis of DKD.
研究领域:
医学
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生物化学
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DOI
PubMed
高影响因子
ANALYTICA CHIMICA ACTA
·
2020
·
IF 5.977
Sha Liu,Xiaoteng Jiang,Zhi Shang,Yin Ji,Huiyu Wang,Zeyuan Wang,Peng Wang,Yan Zhang,Hua Xiao
Glycoproteins are important biomarkers for cancers, while most glycoproteomics biomarkers suffering from low sensitivity and specificity due to their uncharacterized glycan structures. AZGP1 is a potential biomarker for salivary diagnostics of lung cancer, which is used as a model glycoprotein in this study for method development. We initially analyzed salivary N-glycoproteome by using lectin affinity chromatography and more than 300 N-glycoproteins were identified, including AZGP1. 7 gel spots of AZGP1 were resolved by two-dimensional gel electrophoresis and further confirmed by two-dimensional western blot as well as mass spectrometry. The isomeric glycan structures of AZGP1 in these spots were systematically characterized both at composition level and at structure level. Our results revealed 10 glycan compositions for salivary AZGP1, including core fucosylated glycans on Asn128 and sialylated glycans on Asn109 and Asn112. We further compared the glycan structures of salivary AZGP1 from lung cancer group and control group. Accordingly, 14 and 7 potential glycan structures were successfully revealed, respectively. In total, 15 glycan compositions and 22 potential glycan structures were identified and characterized for AZGP1, including some different structures with the same compositions. In particular, 5 potential glycan structures were identified as lung cancer unique signatures. Our developed strategy holds promise for thorough identification of glycan structures on a target glycoprotein biomarker. In-depth characterization of its glycan structures will ultimately enhance its sensitivity and specificity for cancer detection.
研究领域:
生物化学
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癌症研究
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蛋白质组学
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DOI
PubMed
高影响因子
ANALYTICAL CHEMISTRY
·
2011
·
IF 5.874
Hongqiang Qin,Liang Zhao,Ruibin Li,Ren’an Wu,Hanfa Zou
Many diseases are characterized by the changes of either glycan structure or glycosylation site of glycoproteins. The glycan profiling can provide the overview of glycosylation in despite of the absence of the glycosylation sites, which in turn simplifies the complexity of disease diagnosis. Herein, we describe a simple method to profile the N-linked glycans by MALDI-TOF MS with the enrichment using oxidized ordered mesoporous carbon, taking advantages of the size-exclusive effect of mesopore against proteins as well as the interaction between glycans and carbon. Twenty four N-linked glycans derived from ovalbumin could be efficiently detected with high signal-to-noise (S/N) ratios and sufficient peak intensities. In the analysis of complex serum samples, 32 N-linked glycans could be profiled, and 5 (4 core-fucosylated glycans) of them were distinguished from liver cancer and healthy samples.
研究领域:
分析化学
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生物医学
·
蛋白质组学
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产品关联
DOI
PubMed
高影响因子
Scientific Reports
·
2024
·Vol. 14
·期 1
·1-9
·
IF 3.8
Sun Yunze,Mi Zhuang,Chen Xiaoyu,Li Jian-Jun,Lu Jun,Shan Xinyan,Lu Xinghua,Du Yuguang
Glycans, nucleic acids and proteins are three major classes of natural biopolymers. The extremely high diversity of isomerization makes structural elucidation of glycans the most challenging job among three classes. In the past few years, the single molecule sensing technique based on nanopores has achieved great success in sequencing of DNA. Inspired by this, it is potential to sequence glycans in the similar manner. Herein, SiN x nanopores were used to identify nine common monosaccharides in mammals. Each monosaccharide showed characteristic blockage current, which roughly increased with the increase of its molecular weight. In order to distinguish nine monosaccharides, several machine learning models were trained and tested, of which the highest F1 value was 1. These results illustrated that nine common monosaccharides in mammals could be clearly identified and discriminate by our method combining solid-state nanopores and machine learning. As far as we know, this is the first report that monosaccharides can be sensed and distinguished by solid-state nanopores. Our work showed the great potential of solid-state nanopores in glycan sequencing, and would lay the foundation for solid-state nanopore-based glycan sequencing.
研究领域:
生物化学
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分子生物学
·
纳米技术
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产品关联
DOI
PubMed
高影响因子
ANALYTICA CHIMICA ACTA
·
2017
·
IF 4.95
Chang Wang,Yike Wu,Liang Zhang,Bi-Feng Liu,Yawei Lin,Xin Liu
Quantitative analysis of glycans is an emerging field in glycomic research. Herein we present a rapid and effective dual-labeling strategy, in the combination of isotopic derivatization of N -glycosylamine-based glycans by d0/d5-benzoyl chloride and methylamidation of sialic acids , to relatively quantify both neutral and sialylated N -glycans simultaneously by MALDI-MS. The derivatization efficiencies were increased by microwave-accelerated deglycosylation which not only largely reduce the time of glycoprotein deglycosylation but also inhibit the hydrolysis of intermediate glycosylamines produced by PNGase F digestion. Three model glycoproteins, including RNase B, bovine fetuin and IgG from human serum, were applied to validate this technique. Results showed that the glycans from microgram level of glycoprotein can be successfully quantified with high reproducibility and the whole time of analytical procedure was shortened to 4 h. Furthermore, this proposed method was applied for the comparative analysis of N -glycans from serum of healthy donors and multiple myeloma patients. It was found that five N -glycans may be as the potential biomarkers for rapid detection of early multiple myeloma, indicating the feasibility of this strategy in monitoring subtle quantitative differences of serum glycomics.
研究领域:
生物化学
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医学诊断
·
糖组学
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产品关联
DOI
PubMed
高影响因子
RSC Advances
·
2017
·Vol. 7
·期 57
·35687-35693
·
IF 3.108
Wenjie Gao,Yanhua Jiang,Zhihui Zhang,Yifang Zhang,Yanyan Liu,Yanhong Zhou,Xin Liu
Conventional protocols for cellular N-glycan profiling often need several time-consuming and labor-intensive steps, and a large amount of material (5–20 × 106 cells) is required. In this study, an optimized co-derivatization method was applied for convenient, rapid and highly-sensitive analysis of cellular N-glycan. Taking human cervical carcinoma cells (HeLa) as a model, the required amount for comprehensive cellular N-glycans analysis was reduced down to an original cell number of 105 and the total analysis time was decreased from several days to several hours. Good reproducibility of the method was also obtained with the CV averaging to less than 13.1%. In addition, compared to permethylation derivatization, more than 5-fold sensitivity improvement was achieved and more concise and clear mass spectra were obtained with the new developed method. As a preliminary study, this method was also successfully applied to reveal the difference in cellular N-glycan profiling of two heterogeneous live cell lines, human normal liver cells (L-02) and human hepatocyte carcinoma cells (HepG2), showing great potential for high-throughput analysis of N-glycans from limited cell samples, such as primary cell lines, cells obtained from cell sorting and small size cancer specimens.
研究领域:
生物化学
·
分析化学
·
医学研究
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DOI
高影响因子
CARBOHYDRATE RESEARCH
·
2018
·
IF 2.074
Ting Wang,Shen-Li Zheng,Li Liu,Josef Voglmeir
PNGases are crucial targets and valuable tools in analyzing asparagine-linked carbohydrate moieties (N-glycans) of glycoproteins . Activity tests of PNGases have been little improved since their discovery four decades ago, and still rely on observing deglycosylation patterns of glycoproteins or glycopeptides using SDS-PAGE or HPLC analysis. These techniques cannot be easily adapted for automated sampling and high-throughput procedures. Herein, we describe a PNGase activity assay which relies on the conversion of WST-1, a yellowish, water-soluble tetrazolium dye (sodium 2-(4-Iodophenyl)-3-(4-nitro-phenyl)-5-(2,4-disulfophenyl)-2H-tetrazolate), into a blue formazan dye. In this work, we showed that WST-1 could be reduced by N-glycans, which were enzymatically released from glycoprotein substrates. After optimization of the assay conditions, the robustness of the method was challenged by quantifying the activity of various PNGase isoforms at different purification stages using a microwell plate reader. Furthermore, the assay could be used to obtain steady-state kinetics of PNGase H + wild-type and mutant variants, which showed significant differences in their enzymatic reaction rates. The simplicity and robustness of this method might be of benefit for the detection of PNGase activity in routine applications of large amounts of samples.
研究领域:
生物化学
·
分子生物学
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DOI
PubMed
高影响因子
ANALYTICAL AND BIOANALYTICAL CHEMISTRY
·
2023
·Vol. 415
·期 23
·5575-5588
·
IF 4.3
Liu Yuanyuan,Han Yutong,Zhu Wenjie,Luo Qingming,Yuan Jing,Liu Xin
Glycosylation is one of the most common types of post-translational modifications in mammals. It is well known that N-glycans play a key role in cell adhesion, differentiation, synapsis, and myelination during the development of the mammalian central nervous system (CNS). Neuropathological symptoms (such as epilepsy and Alzheimer’s disease) are usually accompanied by N-glycosylation changes. In this study, we extracted N-glycan chains from eight regions of the mouse brain, and combined high-throughput, high-resolution matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) with the Fmoc N-hydroxysuccinimide ester (Fmoc-OSU) derivatization method to improve the sensitivity of glycan detection to characterize the total N-glycans in the mouse brain. A total of 96 N-glycan moieties were detected. An exhaustive examination of the relative abundance of N-glycans, coupled with a comparative analysis of differences, has uncovered discernible variations of statistical significance, including high mannose, fucosylated, sialylated, and galactosylated N-glycans. According to our investigations, a thorough and regionally specific cartography of glycans within the brain can facilitate the investigation of glycan-mediated mechanisms related to both the developmental trajectory and functional output of the brain. Additionally, this approach may serve as a basis for identifying potential biomarkers that are relevant to various brain-associated pathologies. Graphical Abstract
研究领域:
生物化学
·
神经科学
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分子生物学
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DOI
PubMed
高影响因子
危险品化学品经营许可证（带存储）