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1.
Chunli Fang Zhichao Xiong Hongqiang Qin Guang Huang Jing Liu Mingliang Ye Shun Feng Hanfa Zou 《Analytica chimica acta》2014
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. 相似文献
2.
《中国化学快报》2019,30(12):2181-2185
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. 相似文献
3.
《中国化学快报》2023,34(3):107619
β-Cyclodextrin (β-CD) based materials have attracted great attention in the separation of hydrophilic glycopeptides due to the abundant hydroxyl groups in its exterior. However, the current materials based on β-CD generally has complex synthesis process and harsh experimental conditions, on the other hand, the interior cavity of β-CD is hydrophobic and is harmful to capture glycopeptides. Herein, a novel hydrophilic material based on β-CD was engineered via a self-assembly process utilizing l-cysteine (l-Cys) or glutathione (GSH) derived adamantane for highly efficient glycopeptide enrichment. It is the first attempt to make use of the hydrophobic interior cavity of β-CD for hydrophilic glycopeptide capture. Taking advantages of strong hydrophilicity and superparamagnetism, the as-prepared materials possess low detection limit, high selectively, and excellent reusability when employed to glycopeptide enrichment. In addition, the feasibility of the hydrophilic material based on β-CD was verified by enriching glycopeptides from human serum and saliva samples. This study provides a heuristic strategy for the application of β-CD-based self-assembly materials in the enrichment of glycopeptides. Importantly, this strategy certified a possible that the change of glycopeptide enrichment sites through host-guest interaction between β-CD and adamantane derivatives with different functional groups. 相似文献
4.
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. 相似文献
5.
Tohru Ikegami 《Journal of separation science》2019,42(1):130-213
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. 相似文献
6.
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. 相似文献
7.
《中国化学快报》2022,33(10):4695-4699
Both glycosylation and phosphorylation exert crucial rule in multitudinous biological processes. For in-depth profiling of glycosylation and phosphorylation, a magnetic metal oxide is effectively coupled with inherently hydrophilic mesoporous channels (denoted as Fe3O4@TiO2@mSiO2-TSG). Based on the mechanism of hydrophilic interaction liquid chromatography (HILIC) and metal oxide affinity chromatography (MOAC), the Fe3O4@TiO2@mSiO2-TSG nanomaterial shows high capacity for simultaneously enriching glycopeptides and phosphopeptides. With human saliva collected in successive four days as practical biological sample, endogenous glycopeptides and phosphopeptides are efficiently enriched. Further gene ontology analysis reveals that the identified endogenous glycopeptides and phosphopeptides participate in diverse molecular functions and biological processes. This strategy is anticipated to promote variation analysis of salivary post-translational modifications. 相似文献
8.
Yu Liang Ci Wu Qun Zhao Qi Wu Bo Jiang Yejing Weng Zhen Liang Lihua Zhang Yukui Zhang 《Analytica chimica acta》2015
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. 相似文献
9.
10.
As a low molecular weight protein with the ability of binding metal ions and high inducibility, metallothionein (MT) is often regarded as an important biomarker for assessment of heavy metal pollution in water environment. In the light of that the traditional process of enrichment and identification is time-consuming and complicated, we prepared a core-shell nanoparticle, gold-coated iron oxide nanoparticles (Fe3O4@Au NPs) herein. It possessed the advantages of fast response to magnetic fields and optical properties attributing to Fe3O4 and Au nanoparticles, respectively. The Fe3O4@Au nanoparticles could be used to enrich MT simply through Au–S interaction, and the purified proteins were determined by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/MS). The results showed that the Fe3O4@Au nanoparticles could directly enrich MT from complex solutions and the detection limit could be as low as 10 fg mL?1. 相似文献
11.
Guang Huang Zhichao Xiong Hongqiang Qin Jun Zhu Zhen Sun Yi Zhang Xiaojun Peng Junjie ou Hanfa Zou 《Analytica chimica acta》2014
Zwitterionic hydrophilic interaction chromatography (ZIC-HILIC) materials have been increasingly attractive in glycopeptide enrichment. However, the traditional ZIC-HILIC materials are modified with monolayer zwitterionic molecules on the surface, therefore, the hydrophilicity, detection sensitivity and loading capacity are limited. In this work, we synthesized novel silica nanoparticles with uniform poly(2-(methacryloyloxy)ethyl)dimethyl-(3-sul-fopropyl)ammonium hydroxide (PMSA) brushes grafted onto the surface via reversible addition-fragmentation chain transfer (RAFT) polymerization (denoted as SiO2-RAFT@PMSA). The resulting SiO2-RAFT@PMSA nanoparticles demonstrated low detection limit (10 fmol) and high recovery yield (over 88%) for glycopeptide enrichment from tryptic digest of human IgG. The SiO2-RAFT@PMSA nanoparticles were further applied for the analysis of mouse liver glycoproteome, a total number of 303 unique N-glycosylation sites corresponding to 185 glycoproteins was reliably profiled in three replicate nano-LC–MS/MS runs. Significantly, more glycopeptides were identified than those of nanoparticles, monolayer MSA molecules modified SiO2@single-MSA and nonuniform multi-layer PMSA brushes coated SiO2@PMSA, as well as commercial ZIC@HILIC beads and Click Maltose beads. The excellent performance of SiO2-RAFT@PMSA nanoparticles results from the non-fouling property, a large quantity of functional molecules and suitable link arms provided by uniform PMSA brushes, as well as efficient interaction between glycopeptides and uniform PMSA brushes. It is concluded that the synthesized SiO2-RAFT@PMSA nanoparticles exhibit great potential in glycoproteome analysis. Moreover, this strategy to modify nanopaticles with uniform polymer brushes via RAFT polymerization can also be explored to design other types of materials for bioseparation application. 相似文献
12.
硼亲和色谱法在糖肽/糖蛋白选择性富集中的应用趋于成熟。硼酸亲和材料的选择性,生物相容性,制备过程是否简便均是开发新型苯硼酸功能化材料需要考虑的问题。该研究立足硼酸亲和材料开发的关键问题,设计并开发了一种新型苯硼酸亲和硅胶(TCNBA)。该材料采用基于叠氮基-氰基的无铜催化点击化学方法进行合成,生物相容性好,制备方法简便。红外光谱和X射线光电子能谱图表征结果证明材料合成成功。TCNBA的糖肽富集选择性利用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)进行评价,结果表明,TCNBA能够分别从辣根过氧化物酶(HRP)和免疫球蛋白G(IgG)酶解液中鉴定出13个和11个糖肽;以HRP和牛血清白蛋白(BSA)酶解液混合物(物质的量比1:10)作为研究对象,富集后能够鉴定出5个糖肽。TCNBA的糖蛋白富集选择性利用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳法(SDS-PAGE)进行评价,以HRP、IgG、核糖核酸酶B(RNaseB)作为考察对象,结果表明,TCNBA对糖蛋白具有较好的富集选择性。以实际样品人血清为测试对象验证TCNBA在实际生物样品中的应用价值。结果显示,富集后非糖蛋白得到较大程度去除,糖蛋白得以富集。所制备的材料和方法具有大规模实际蛋白质样品分离处理的应用前景。 相似文献
13.
利用种子生长法制备了磁性Fe2O3/Au/Ag复合纳米粒子,采用UV-vis和SEM对其光学性质以及表面结构的变化进行了表征.通过调节硝酸银的用量,制备了一系列具有不同Ag壳层厚度和表面结构的双金属外壳纳米粒子.以苯硫酚(TP)为探针分子,研究了不同Ag壳厚度的磁性纳米粒子的表面增强拉曼散射(SERS)活性.结果表明其SERS活性与表面结构的改变有关,在同时出现Ag和Au光学性质的Fe2O3/Au/Ag复合纳米粒子表面可观察到最强的SERS效应,这与表面的针孔效应以及Ag和Au之间的耦合增强作用有关.考察了Fe2O3/Au/Ag复合纳米粒子的磁富集作用,并利用SERS原位监测磁富集溶液中低浓度TP的能力,研究结果表明通过磁富集可提高SERS检测限,并且Fe2O3/Au/Ag的磁富集能力较Fe2O3/Au弱,但前者SERS信号较强. 相似文献
14.
An electrochemical approach for the sensitive detection of sequence-specific DNA has been developed. Horseradish peroxidase (HRP) assembled on the Fe3O4 nanoparticles (NPs) were utilized as signal amplification sources. High-content HRP was adsorbed on the Fe3O4 NPs via layer-by-layer (LbL) technique to prepare HRP-functionalized Fe3O4 NPs. Signal probe and diluting probe were then immobilized on the HRP-functionalized Fe3O4 NPs through the bridge of Au NPs. Thereafter, the resulting DNA-Au-HRP-Fe3O4 (DAHF) bioconjugates were successfully anchored to the gold nanofilm (GNF) modified electrode surface for the construction of sandwich-type electrochemical DNA biosensor. The electrochemical behaviors of the prepared biosensor had been investigated by the cyclic voltammetry (CV), chronoamperometry (i-t), and electrochemical impedance spectroscopy (EIS). Under optimal conditions, the proposed strategy could detect the target DNA down to the level of 0.7 fmol with a dynamic range spanning 4 orders of magnitude and exhibited excellent discrimination to two-base mismatched DNA and non-complementary DNA sequences. 相似文献
15.
Characterization of protein glycosylation requires highly specific methods for the enrichment of glycopeptides because of their sub-stoichiometric glycosylation-site occupancy. The hydrophilic affinity based strategy has attracted more attention, owing to its broad glycan specificity, good reproducibility, and compatibility with mass spectrometric (MS) analysis. Several polar matrices have emerged for hydrophilic interaction chromatography (HILIC) approaches, including sepharose, cellulose, ZIC-HILIC and titania. Here, we present the solid-phase extraction (SPE) utility of zirconia coated mesoporous silica (ZrO(2)/MPS) microspheres for glycopeptide isolation prior to MS analysis. The high specificity of this SPE approach was demonstrated by the enrichment of glycopeptides from the digests of model glycoproteins in HILIC mode. ZrO(2)/MPS microspheres show superior selectivity and glycosylation heterogeneity coverage for glycopeptide enrichment to conventional sepharose. Furthermore, digested mixtures of the phosphoprotein α-casein and IgG were also treated with ZrO(2)/MPS HILIC SPE materials, which exhibited that glycopeptides could be effectively enriched with interference from phosphorylated peptides. 相似文献
16.
Glutathione modified magnetic nanoparticles (Fe3O4@Au-GSH) were synthesized through a simple process and exploited to enrich glycopeptides from complex samples. 相似文献
17.
First, attapulgite‐Fe3O4 magnetic filler (ATP‐Fe3O4) was prepared by using a chemical precipitation method. Subsequently, graphite oxide (GO) was prepared through Hummer method, and then reduced GO (RGO) was prepared through GO reduced by chitosan (CS). Finally, a series of WPU‐RGO/ATP‐Fe3O4/CS composites were prepared by introduced RGO/ATP‐Fe3O4/CS to waterborne polyurethane. The structure and properties were characterized by scanning electron microscopy (SEM), Fourier transform infrared (FTIR), X‐ray diffraction (XRD), vibrating sample magnetometry (VSM), thermogravimetric analysis TGA, conductivity test, and tensile test. The experimental results indicated that thermal stability and tensile strength of nanocomposites were improved with the increase of the content of RGO/ATP‐Fe3O4/CS. Meanwhile, with the increase of the RGO/ATP‐Fe3O4/CS content, the electrical and magnetic properties of WPU‐RGO/ATP‐Fe3O4/CS composites were improved. When the content of RGO/ATP‐Fe3O4/CS was 8 wt%, the electrical conductivity and the saturation magnetic strength of WPU‐RGO/ATP‐Fe3O4/CS composites were 3.1 × 10?7 S·cm?1 and 1.38 emu/g, respectively. WPU‐RGO/ATP‐Fe3O4/CS composites have excellent electrical and magnetic properties. 相似文献
18.
Superparamagnetic pH‐sensitive multilayer hybrid hollow microspheres for targeted controlled release
Bin Mu Peng Liu Yun Dong Chunyin Lu Xueli Wu 《Journal of polymer science. Part A, Polymer chemistry》2010,48(14):3135-3144
The superparamagnetic multilayer hybrid hollow microspheres have been fabricated using the layer‐by‐layer assembly technique by the electrostatic interaction between the polyelectrolyte cation chitosan (CS) and the hybrid anion citrate modified ferroferric oxide nanoparticles (Fe3O4‐CA) onto the sacrificial polystyrene sulfonate microspheres templates after etching the templates by dialysis. The saturation magnetization and magnetite contents of the superparamagnetic multilayer hybrid hollow microspheres were 32.46 emu/g and 51.3%, respectively. The hybrid hollow microspheres showed pH‐sensitive characteristics. The adsorption and release of the basic dye (methylene blue) were applied to investigate the interaction between the amino groups of CS and the carboxyl groups of the Fe3O4‐CA nanoparticles in different pH media. The superparamagnetic pH‐sensitive multilayer hybrid hollow microspheres are expected to be used for the targeted controlled release of drugs or in diagnostics. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 48: 3135–3144, 2010 相似文献
19.
A sandwich-type electrochemical immunosensor for the detection of carbohydrate antigen 19-9 (CA 19-9) antigen based on the immobilization of primary antibody (Ab1) on three dimensional ordered macroporous magnetic (3DOMM) electrode, and the direct electrochemistry of horseradish peroxidase (HRP) that was used as both the label of secondary antibody (Ab2) and the blocking reagent. The 3DOMM electrode was fabricated by introducing core–shell Au–SiO2@Fe3O4 nanospheres onto the surface of three dimensional ordered macroporous (3DOM) Au electrode via the application of an external magnet. Au nanoparticles functionalized SBA-15 (Au@SBA-15) was conjugated to the HRP labeled secondary antibody (HRP-Ab2) through the Au–SH or Au–NH3+ interaction, and HRP was also used as the block reagent. The formation of antigen–antibody complex made the combination of Au@SBA-15 and 3DOMM exhibit remarkable synergistic effects for accelerating direct electron transfer (DET) between HRP and the electrode. Under the optimal conditions, the DET current signal increased proportionally to CA 19-9 concentration in the range of 0.05 to 15.65 U mL−1 with a detection limit of 0.01 U mL−1. Moreover, the immunosensor showed high selectivity, good stability, satisfactory reproducibility and regeneration. Importantly, the developed method was used to assay clinical serum specimens, achieving a good relation with those obtained from the commercialized electrochemiluminescent method. 相似文献
20.
Au nanoparticles (Au NPs) play a vital role in heterogeneous catalytic reactions. However, pristine Au NPs usually suffer from poor selectivity and difficult recyclability. In this work, Fe3O4‐Au@CeO2 hybrid nanofibers were prepared via a simple one‐pot redox reaction between HAuCl4 and Ce (NO3)3 in the presence of Fe3O4 nanofibers. CeO2 shell was uniformly coated on the surface of Fe3O4 nanofibers to form a unique core‐shell structure, while Au NPs were encapsulated inside the CeO2 shell. The as‐prepared Fe3O4‐Au@CeO2 hybrid nanofibers have been proved to be positively surface charged due to the formation of CeO2 shell, enabling them to be good candidates for predominant selective catalytic activity towards the degradation of negatively charged organic dyes. In addition, the Fe3O4‐Au@CeO2 hybrid nanofibers showed magnetic properties, offering them excellent recyclable usability. This work presents a facile and effective solution to prepare magnetic noble metal/metal oxide hybrid nanomaterials with unique chemical structure and surface characteristic for promising applications in heterogeneous catalysis. 相似文献