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蛋白质糖基化是一种广泛存在的重要的蛋白质翻译后修饰,糖基化肽在总酶解肽中占的比例不超过5%,这使得糖肽的分离富集成为糖蛋白质组学研究发展的关键技术之一。在诸多糖蛋白质组富集技术中,化学方法是富集技术的主导,本文从化学反应的角度介绍糖基化蛋白质富集的技术进展。富集过程按照连接和释放分别讨论,在连接过程中,重点介绍硼酸化学法、肼化学法、胺化学法和肟点击法;在释放过程中,以N-糖蛋白的酶释放法和O-糖蛋白的β-消除法为主导,一并介绍了最新的氧化断裂释放的化学法。最后,讨论总体富集策略的发展现状。该文以糖蛋白富集的共价反应为核心,分析不同方法的优缺点以及各技术在糖蛋白质组学研究中的应用和贡献。 相似文献
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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。 相似文献
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蛋白质的糖基化是最重要的翻译后修饰之一,与蛋白质结构和功能的关系密切。凝集素亲和色谱是蛋白质糖基化研究中很常用的工具,不同的凝集素可以对不同的单糖或寡糖有特异的富集作用。麦胚凝集素(WGA)由于其特异作用的糖型广泛存在而成为使用最多的凝集素之一。在本研究中,发现将WGA用于糖肽亲和富集会导致部分肽段的降解,从而导致后续的肽段序列分析的失败。本文用4种标准蛋白质对这种现象进行了验证,结果表明肽段的降解可以发生在多个位点,其中较多地发生在酪氨酸、苯丙氨酸及亮氨酸的羧基端。这一结果提示:在糖蛋白质组研究中,如果应用WGA富集糖肽并采用质谱进行鉴定,则采用半酶切或非特异性酶切的检索策略更为合适。 相似文献
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通过对硅胶基质进行化学改性键合伴刀豆球蛋白(Con A),制备了对糖蛋白具有特异亲和作用的亲和色谱固定相;该固定相非特异性吸附弱,对于糖蛋白和糖肽的分离效果良好。对亲和色谱的分离条件进行了优化,以标准糖蛋白核糖核酸酶B(RNase B)为模型,对其进行了纯化;用糖苷酶切除糖链,并对切除糖链前后的RNase B用胰蛋白酶酶解;用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)对亲和色谱分离得到的糖蛋白、糖链及糖肽进行了分析,确定了RNase B的一级结构、糖含量、糖基化位点及糖连接方式。该方法快速准确,适于糖蛋白和糖肽的分离表征。将其应用于血清中糖蛋白及酶解后血清中糖肽的分离富集,取得了很好的效果。 相似文献
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生物体内蛋白质的糖基化修饰调控着细胞识别、细胞黏附和迁移以及免疫应答等多种生理过程,并与多种人类重大疾病的发生、发展密切相关。因此对蛋白质糖基化修饰的鉴定,不仅能够为生物学机理研究提供重要信息,对疾病诊断标志物和治疗靶标的发现也至关重要。然而在复杂生物体系中,大多数糖蛋白为低丰度蛋白质,其含量与现有质谱仪器的检测灵敏度之间存在较大差距,所以对含有不同糖型结构的糖蛋白进行全面/高效的富集,是实现高灵敏度糖蛋白鉴定的必由之路。凝集素富集作为一种有效的糖蛋白富集方法,已在糖蛋白质组学研究中得到了广泛的应用。针对现有凝集素功能化材料存在负载量偏低以及富集效率有限等问题,我们制备了两种以氧化石墨烯(GO)为载体的新型固定化凝集素,利用GO比表面积大,功能基团含量高,分散性、化学稳定性好等特点,实现了高负载量的凝集素固定(GO-ConA 2.073 mg/mg, RSD=1.0%; GO-WGA 1.908 mg/mg, RSD=0.14%)。同时考察了材料的可重复使用性与稳定性:每隔3天测一次同一GO-lectin材料对对应糖蛋白的富集效果,可以看出材料合成两周内富集效果都>200 μg/mg。将该GO-lectin成功应用于糖蛋白、糖肽的选择性富集,在糖蛋白质组学研究中体现出良好的应用潜力。 相似文献
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蛋白质的N-糖基化修饰在多种生物过程中发挥着至关重要的作用,近年来的许多研究证实异常的蛋白质糖基化与多种疾病的发生发展密切相关,表明糖基化蛋白质具有较大的潜力成为新的生物标志物或者药物靶标。在样本的处理过程中,对N-糖基化肽段进行富集分离后再进行质谱分析已经成为糖蛋白质组学分析前的必要步骤。但是,由于复杂生物样本中N-糖基化肽段的丰度低和离子化效率差等问题,通过质谱鉴定N-糖肽仍然是一项艰巨的任务。研究通过将纳米金线(Au)、4-巯基苯硼酸(4-MPB)与超薄二维二硫化钼(2D-MoS2)进行反应,成功制备了一种用于富集蛋白质N-糖基化肽段的新型功能纳米复合材料(MoS2/Au/4-MPB)。二硫化钼纳米材料的层状结构可以为反应提供大量的可修饰位点,便于修饰纳米金线;功能基团4-巯基苯基硼酸对N-糖肽具有高度的选择性,可以对生物样品中N-糖基化肽段进行特异性富集。使用标准蛋白人免疫球蛋白G(IgG)和牛血清白蛋白(BSA)胰蛋白酶酶切产物对新型功能纳米材料的N-糖基化肽段的富集性能进行评估,其灵敏度达到5 fmol,选择性达到1:1000。将其用于生物样品中N-糖基化肽段的富集,从50 μg尿液外泌体蛋白胰蛋白酶酶切产物中共富集鉴定出768个N-糖肽,归属于377个蛋白质。这些结果表明该新型功能纳米复合材料对复杂生物样品中N-糖肽的选择富集有着巨大的应用潜力,为糖蛋白质组的研究提供了一种新方法。 相似文献
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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
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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
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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
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《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
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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