基于电喷雾电离质谱的人肝癌细胞HepG2与正常肝细胞L02的N-糖链的定性定量比较

以培养的原发性肝癌细胞HepG2和正常肝细胞L02为研究对象,采用细胞裂解液提取总蛋白,用PNGase F酶解释放N-糖链,以微晶纤维素柱结合石墨碳柱纯化分离N-糖链,通过电喷雾电离质谱(ESI-MS)和串联质谱(MS/MS)对N-糖链进行序列鉴定,以β-环糊精为内标对2种细胞系的N-糖链进行了定量比较分析.结果表明,在肝癌细胞系HepG2和正常细胞系L02中共检测到26种N-糖链,与L02相比,HepG2的大多数高甘露糖型糖链、唾液酸化糖链和岩藻糖基化糖链的数量都明显升高,其中有15种糖链在数量上具有极显著性差异(p0.01),1种糖链具有显著性差异(p0.05).本研究为进一步探索肝癌中各类N-糖链的表达特点及发现早期肝癌糖链标志物提供了参考.     

微量生物样品中糖蛋白N-糖链电喷雾电离质谱分析前处理方法的建立

基于电喷雾电离质谱检测技术,建立了一种可靠、高效、简单,适合于微量糖蛋白N-糖链解离、富集纯化的方法.以糖蛋白牛胰核糖核酸酶(Rib B)和卵清白蛋白(OVA)为模型蛋白直接酶解,比较了4种方法纯化酶解样品的效果.比较了直接酶解和经过聚偏氟乙烯(PVDF)膜富集后酶解微量复杂生物来源样品胎牛血清的酶解效果,最终建立了微量生物样品中糖蛋白N-糖链的质谱分析前处理方法.采用PVDF膜吸附复杂生物样品中的糖蛋白,N-糖苷酶F(PNGase F)酶直接在膜上完成糖链释放(37℃,24 h),采用微晶纤维素柱结合石墨碳柱对糖链进行富集纯化,用于微克级胎牛血清和健康人血清中N-糖链质谱分析的前处理.本方法通用性好,在微量生物样品糖链质谱分析检测的前处理方面具有一定应用价值.     

基于ConA富集和LTQ-FT质谱鉴定的小鼠肝脏内质网N-糖蛋白质组研究

糖基化修饰是生物体内复杂和重要的蛋白质翻译后修饰方式之一.N-糖基化蛋白质在内质网中进行合成的过程中,所有的N-糖链都以甘露糖和葡萄糖结尾,而凝集素ConA对以甘露糖结尾的糖链有较高的亲和性,可以用来富集在内质网中合成的N-糖蛋白质.本文据此提出了一种基于内质网分离和凝集素ConA富集的复杂样品N-糖基化位点研究策略.通过使用高准确度的质谱线性离子阱-傅立叶变换回旋离子共振质谱对N-糖蛋白质进行鉴定,并对N-糖基化位点进行确定.我们采用模式生物C57BL/6J肝脏作为生物样本,在生物水平和质谱水平分别进行了3次重复,共鉴定了212个N-糖蛋白质的323个N-糖基化位点.在这些蛋白中,131个是Swissprot库中已确认的N-糖蛋白质.此方法富集的糖蛋白,糖型统一,有利于样品的分离和PNGaseF酶切作用,提高了鉴定的效率.对鉴定的212个N-糖蛋白质的定位和功能进行了分析,本文鉴定的N-糖蛋白质对现有的鼠肝N-糖蛋白质数据库进行了有效的补充.     

Pronase E酶解释放糖蛋白N-糖链的方法及荧光标记衍生物的LC-MS分析

建立了一种用非特异性酶链酶蛋白酶 E(Pronase E)从糖蛋白上释放N-糖链的方法. 以牛胰核糖核酸酶 B(Ribo B)和鸡白蛋白(Chicken Albumin)为材料, 用Pronase E代替N-糖苷酶 F(PNGase F)释放N-糖链. 当蛋白酶质量与糖蛋白质量比为1∶1时, 得到只带一个天冬氨酸(Asn)的闭环N-糖链, 称其为糖氨酸(glycan-Asn), 这样既为糖链引入了天然的-NH2活性基团, 同时还保持了糖链原有的还原端闭环结构. 以9-氯甲酸芴甲酯(Fmoc-Cl)为衍生试剂对解离后的糖氨酸进行衍生, 采用高效液相色谱-电喷雾质谱联用技术(HPLC-ESI/MS)对Fmoc-Cl糖氨酸衍生物进行分析, 建立了糖蛋白的Pronase E酶解、微量糖氨酸的Fmoc-Cl衍生以及糖氨酸衍生物的HPLC-ESI/MS分析方法, 该方法保持了N-糖链的天然结构, 便于以-NH2为功能基团进一步进行荧光标记、分离制备以及糖链与蛋白质的相互作用研究.     

基于MALDI-TOF质谱技术分析人宫颈癌 HeLa细胞N-糖链结构轮廓

以微量HeLa细胞(10⁷个)为对象, 经细胞裂解、还原羧甲基化、胰酶降解和Oasis-HLB柱提取分离得到总糖肽后, 用PNGase F酶解释放N-糖链. 对所得N-糖链用Sep-Pak C₁₈柱纯化后进行完全甲基化衍生, 再采用基质辅助激光解吸电离-飞行时间质谱(MALDI-TOF MS)分析HeLa细胞表面N-糖链的结构轮廓. 结果表明, 在获得的34种N-糖链中, 除高甘露糖型、二天线、三天线、四天线和五天线等N-糖链外, 还出现了在某种程度上与肿瘤发生转移相关的特殊平分型和Lewis结构. 利用MALDI-TOF MS技术可快速分析微量癌细胞表面N-糖链的结构轮廓, 为进一步寻找肿瘤糖链标志物及肿瘤的早期预防诊断提供技术支持.     

人肝癌Bel-7402细胞及Bel-7402/5-FU细胞的N-连接聚糖的差异分析

以培养的人肝癌细胞Bel-7402及其5-氟尿嘧啶(5-FU)耐药细胞(Bel-7402/5-FU)为研究对象,通过使用快速PNGase F酶切,结合TMPP-Ac-OSu和甲胺化共衍生方法,对两者的总蛋白和分泌蛋白的N-连接聚糖进行了基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)分析。从两种细胞总蛋白中共鉴定到56种N-连接聚糖,分泌蛋白中鉴定到38种N-连接聚糖。5-FU与Bel-7402相比,耐药细胞岩藻糖化唾液酸糖型在总蛋白和分泌蛋白中都显著升高;高甘露糖型N-聚糖在总蛋白中下降,而在分泌蛋白中显著增加。本研究为进一步探索肿瘤耐药提前诊断提供糖链标志物起到一定的参考作用,并为后续解决癌症治疗的耐药提供理论依据。     

双重衍生化-质谱法同时分析检测人血清中酸性与中性N-糖链

本文发展了一种新型"双重衍生化"策略,即首先以甲胺化衍生修饰酸性聚糖中的唾液酸残基,然后采用吉拉尔特试剂P(GP)对N-糖链还原端进行标记,最后以基质辅助激光解吸电离-飞行时间质谱(MALDI-TOF MS)进行检测。该衍生化方法不但保护了酸性聚糖中的唾液酸残基,而且大大提高了N-糖链的离子化效率。采用该方法,在25ng人血清样品中成功检测出16种N-糖链,其中包括9种酸性糖链和7种中性糖链,为中性、酸性N-糖链的同时检测提供了新的途径,并有望将其用于疾病相关糖链标志物的研究。     

人肝癌细胞HepG2与正常肝细胞L02的Ο-糖链的比较分析

以培养的原发性肝细胞癌HepG2细胞和正常肝细胞L02为研究对象,用细胞裂解液提取总蛋白,然后采用Carlson还原性β-消除法释放O-糖链,以阳离子交换柱结合C18柱纯化分离O-糖链,用电喷雾电离质谱( ESI-MS)和串联质谱( MS/MS)对O-糖链进行序列鉴定,以β-环糊精为内标对2种细胞系的O-糖链进行定量比较分析.结果表明,在肝癌细胞系HepG2中检测到10种O-糖链,正常细胞系L02中检测到9种O-糖链,其中9种O-糖链是2种细胞系中共有的,但HepG2中存在癌细胞中特有的缩短的O-糖链N1A1( NeuAc-GalNAc, sialyl Tn 抗原). t检验结果表明, HepG2与L02相比,在检测到的10种O-糖链中有5种的含量具有极显著性差异(P<0.01),2种的含量具有显著性差异(P<0.05).     

基于质谱的蛋白质O-糖基化分析研究进展

蛋白质的O-糖基化是一种重要的蛋白质翻译后修饰,它和N-糖基化一样是蛋白质糖基化修饰的主要形式。蛋白质的O-糖基化对蛋白质的结构功能有重要的影响,因此分析蛋白质的O-糖基化具有重要的生物学意义。蛋白质O-糖基化分析包含4个方面的内容:(1)鉴定O-糖基化蛋白质的种类; (2)鉴定糖基化位点; (3)鉴定糖链结构; (4)糖链的定量分析。由于缺少保守的O-糖基化氨基酸特征序列,缺乏通用的糖苷酶以及O-糖链结构的复杂性等原因,基于质谱的蛋白质O-糖基化的分析目前仍处于方法开发阶段。本文主要介绍基于质谱的O-糖基化蛋白质的分析方法学在近期取得的一些进展,包括以下4个方面:O-糖蛋白/多肽的富集、O-糖链的解离、O-糖链的结构分析及O-糖基化定量分析。     

基于Endo-M N175Q糖链转移反应与丙酮富集糖肽的LC-MS分析糖蛋白N-糖链

基于化学酶标记和丙酮富集糖肽方法,建立了一种可靠、有效、简单的糖蛋白N-糖链分析方法。以唾液酸糖肽(SGP)为模型糖肽,比较了样品中丙酮加入量对SGP的富集效果,最终选择加入样品体积5倍量的丙酮。用丙酮富集经胰蛋白处理的核糖核酸酶B(RNase B)酶解液中的糖肽,以富集分离得到的糖肽(糖基供体)和PDPZ-Boc-Asn-GlcNAc(糖基受体)作为酶反应底物,进行Endo-M N175Q的转糖基反应,得到PDPZBoc-Asn-GlcNAc-N-糖链标记物。采用YMC C18色谱柱为分析柱,10 mmol/L甲酸铵-乙腈为流动相梯度洗脱,经液相色谱-串联质谱(LC-MS)检测得到5种高甘露糖型糖链。结果表明,丙酮可有效地富集大量肽和少量糖肽混合溶液中的糖肽,Endo-M N175Q可将天然糖肽的糖链转移到PDPZ-Boc-Asn-GlcNAc受体上。将该方法应用于胎球蛋白N-糖链分析,检测到5种复杂型N-糖链。该研究为各种糖蛋白N-糖链检测提供了新的分析方法。     

Use of specific peptide biomarkers for quantitative confirmation of hidden allergenic peanut proteins Ara h 2 and Ara h 3/4 for food control by liquid chromatography–tandem mass spectrometry

A liquid chromatography–electrospray-tandem mass spectrometry (LC–ESI-MS–MS) method based on the detection of biomarker peptides from allergenic proteins was devised for confirming and quantifying peanut allergens in foods. Peptides obtained from tryptic digestion of Ara h 2 and Ara h 3/4 proteins were identified and characterized by LC–MS and LC–MS–MS with a quadrupole-time of flight mass analyzer. Four peptides were chosen and investigated as biomarkers taking into account their selectivity, the absence of missed cleavages, the uniform distribution in the Ara h 2 and Ara h 3/4 protein isoforms together with their spectral features under ESI-MS–MS conditions, and good repeatability of LC retention time. Because of the different expression levels, the selection of two different allergenic proteins was proved to be useful in the identification and univocal confirmation of the presence of peanuts in foodstuffs. Using rice crispy and chocolate-based snacks as model food matrix, an LC–MS–MS method with triple quadrupole mass analyzer allowed good detection limits to be obtained for Ara h2 (5 μg protein g⁻¹ matrix) and Ara h3/4 (1 μg protein g⁻¹ matrix). Linearity of the method was established in the 10–200 μg g⁻¹ range of peanut proteins in the food matrix investigated. Method selectivity was demonstrated by analyzing tree nuts (almonds, pecan nuts, hazelnuts, walnuts) and food ingredients such as milk, soy beans, chocolate, cornflakes, and rice crispy. Figure ESI-QTOF-MS mass spectrum of Ara h3/4 triptig digest     

Mass spectrometric analysis of the glycosphingolipid‐derived glycans from miniature pig endothelial cells and islets: identification of NeuGc epitope in pig islets

Glycosphingolipid (GSL) is a major component of the plasma membrane in eukaryotic cells that is involved directly in a variety of immunological events via cell‐to‐cell or cell‐to‐protein interactions. In this study, qualitative and quantitative analyses of GSL‐derived glycans on endothelial cells and islets from a miniature pig were performed and their glycosylation patterns were compared. A total of 60 and 47 sialylated and neutral GSL‐derived glycans from the endothelial cells and islets, respectively, were characterized by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) and collision‐induced fragmentation using positive‐ion electrospray ionization (ESI) ion‐trap tandem mass spectrometry (MS/MS). In accordance with previous immunohistochemistry studies, the α‐Gal‐terminated GSL was not detected but NeuGc‐terminated GSLs were newly detected from miniature pig islets. In addition, the neutral GSL‐derived glycans were relatively quantified by derivatization with carboxymethyl trimethylammonium hydrazide (so called Girard's T reagent) and MALDI‐TOF MS. The structural information of the GSL‐derived glycans from pig endothelial cells and islets suggests that special attention should be paid to all types of glycoconjugates expressed on pig tissues or cells for successful clinical xenotransplantation. Copyright © 2009 John Wiley & Sons, Ltd.     

银杏种子糖蛋白的SDS-PAGE分离及N-糖链的质谱分析

利用十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)分离了银杏种子中的糖蛋白组分, 进一步用氨水催化释放N-糖链, 并采用电喷雾离子质谱(ESI-MS)及在线液相色谱-质谱联用(LC-UV-MS/MS²)等技术对胶条上释放的N-糖链进行了定性定量分析. 结果表明, 从银杏种子中分离得到11种糖蛋白, 共检测到11条N-糖链, 包括高甘露糖型(4.88%)和复杂型(95.12%) 2种类型, 其中分子量为21000, 36000和50000的糖蛋白所释放的核心α-1,3-岩藻糖和β-1,2-木糖修饰的N-糖链所占比率较高, 分别为68.23%, 64.37%和75.09%. 本研究对进一步研究银杏糖蛋白功能具有重要意义.     

Determination of peanut allergens in cereal-chocolate-based snacks: metal-tag inductively coupled plasma mass spectrometry immunoassay versus liquid chromatography/electrospray ionization tandem mass spectrometry

A comparison of two methods for the identification and determination of peanut allergens based on europium (Eu)-tagged inductively coupled plasma mass spectrometry (ICP-MS) immunoassay and on liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) with a triple quadrupole mass analyzer was carried out on a complex food matrix like a chocolate rice crispy-based snack. The LC/MS/MS method was based on the determination of four different peptide biomarkers selective for the Ara h2 and Ara h3/4 peanut proteins. The performance of this method was compared with that of a non-competitive sandwich enzyme-linked immunosorbent assay (ELISA) method with ICP-MS detection of the metal used to tag the antibody for the quantitative peanut protein analysis in food. The limit of detection (LOD) and quantitation of the ICP-MS immunoassay were 2.2 and 5 microg peanuts g(-1) matrix, respectively, the recovery ranged from 86 +/- 18% to 110 +/- 4% and linearity was proved in the 5-50 microg g(-1) range. The LC/MS/MS method allowed us to obtain LODs of 1 and 5 microg protein g(-1) matrix for Ara h3/4 and Ara h2, respectively, thus obtaining significantly higher values with respect to the ELISA ICP-MS method, taking into account the different expression for concentrations. Linearity was established in the 10-200 microg g(-1) range of peanut proteins in the food matrix investigated and good precision (RSD <10%) was demonstrated. Both the two approaches, used for screening or confirmative purposes, showed the power of mass spectrometry when used as a very selective detector in difficult matrices even if some limitations still exist, i.e. matrix suppression in the LC/ESI-MS/MS procedure and the change of the Ag/Ab binding with matrix in the ICP-MS method.     

Immunochemical characterisation of structure and allergenicity of peanut 2S albumins using different formats of immunoassays

Proteins of the 2S albumin family, such as Ara h2 and Ara h6, are most frequently involved in peanut allergy. We have developed a reverse enzyme allergo-sorbent test (EAST) in which total serum IgE antibodies are first captured by immobilised anti-human IgE monoclonal antibodies, and then the binding of the anti-Ara h2 and anti-Ara h6 specific IgE to the corresponding labelled allergens is measured. This reverse immunoassay was used either as a direct EAST or as an EAST inhibition assay to study the interactions of whole peanut protein extract and purified Ara h2 and Ara h6 with IgE antibodies from peanut-allergic patients. Finally, we identified some IgE-binding epitopes on Ara h6 using a format of EAST in which the protein is immobilised in a particular, well defined, manner through interactions with specific monoclonal antibodies (mAbs) coated on the micro-plates. The fine specificity of those mAbs has been characterised at the epitope level, and their binding to the allergen thus masks a known particular epitope and makes it unavailable for recognition by IgE antibodies. The reverse EAST increased the ratio specific signal/background. It avoids interferences with competitors such as anti-peanut protein IgG antibodies and allows the study of the specificity and/or affinity of the interactions between IgE antibodies and Ara h2 or Ara h6 with a higher sensitivity and accuracy than the conventional EAST. The EAST results obtained when the allergens are presented by specific mAbs suggest that the homologous molecular domain(s) in peanut 2S albumins encompass major IgE epitope(s) and are strongly involved in peanut allergenicity.     

Poly(amidoamine) dendrimer‐coated magnetic nanoparticles for the fast purification and selective enrichment of glycopeptides and glycans

Glycosylated proteins modulate various important functions of organisms. To reveal the functions of glycoproteins, in‐depth characterization studies are necessary. Although mass spectrometry is a very efficient tool for glycoproteomic and glycomic studies, efficient sample preparation methods are required prior to analyses. In the study, poly(amidoamine) dendrimer‐coated magnetic nanoparticles were presented for the specific enrichment and fast purification of glycopeptides and glycans. The enrichment and purification performance of the developed method was evaluated both at the glycopeptide, and the glycan level using several standard glycoprotein digests and released glycan samples. The poly(amidoamine) dendrimer‐coated magnetic nanoparticles not only showed selective affinity (Immunoglobulin G/Bovine Serum Albumin, 1/10 by weight) to glycopeptides and released glycans but also good sensitivity (0.4 ng/µL for Immunoglobulin G) for glycoproteomic and glycomic applications. Thirty‐five glycopeptides of Immunoglobulin G were detected after enrichment with poly(amidoamine) dendrimer‐coated magnetic nanoparticles. In addition, 55 ¹⁸O tagged deamidated glycopeptides belonging to human plasma glycoproteome were confirmed. Finally, fifty 2‐aminobenzoic acid, and 30 procainamide‐labelled human plasma N‐glycans released from human plasma glycoproteins were determined after purifications. The results indicate that the proposed enrichment and purification method using poly(amidoamine) dendrimer‐coated magnetic nanoparticles could be simply adjusted to sample preparation methods.     

Selective and rapid immunomagnetic bead-based sample treatment for the liquid chromatography-electrospray ion-trap mass spectrometry detection of Ara h3/4 peanut protein in foods

Complex matrices commonly affect the sensitivity and selectivity of liquid chromatography-mass spectrometry (LC-MS) analysis. Thus, selective sample enrichment strategies are useful particularly to analyze organic biomarkers present in low abundance in samples. A selective immunomagnetic extraction procedure to isolate trace peanut allergen protein Ara h3/4 from breakfast cereals combined with microwave-assisted tryptic digestion and liquid chromatography-electrospray ion-trap tandem mass spectrometry (LC-ESI-IT-MS/MS) measurement was developed. Using protein A-coated magnetic bead (MB) support, anti-Ara h3/4 monoclonal antibodies (Abs) were used as selective capture molecules. The results obtained by LC-ESI-IT-MS/MS in terms of limit of detection (3mg peanuts/kg matrix) and a significantly reduced matrix effect demonstrated that the Ab-coated magnetic bead was very effective to selectively trap Ara h3/4 protein in breakfast cereals. The magnetic bead-based sample treatment followed by LC-IT-MS/MS method here developed can be proposed as very rapid and powerful confirmatory analytical method to verify the reliability of enzyme-linked immunosorbent assay (ELISA) screening methods, since the magnetic bead-LC-IT-MS/MS method combines good sensitivity to the identification capabilities of mass spectrometry.     

利用β-N-乙酰氨基葡萄糖苷酶快速酶切分析中国仓鼠卵巢细胞表达的西妥昔单抗抗原结合区的聚糖比例

西妥昔单抗具有较复杂的糖基化修饰,在抗原结合片段(Fab)和可结晶片段(Fc)的重链上都含有2个N-糖基化位点,其中Fab段的糖基化最为复杂,要研究清楚该位点的糖基化修饰,开发专一性切糖技术和稳定的聚糖比例分析方法是当前迫切需要解决的难题。以中国仓鼠卵巢(CHO)细胞表达的西妥昔单抗为研究对象,使用β-N-乙酰氨基葡萄糖苷酶(Endo F2)开发了一种快速Fab段聚糖释放的方法,利用超高效液相色谱-高分辨质谱(UPLC-HRMS)进行了定性和聚糖比例分析。第一步对抗体原液进行非变性酶切,抗体原液经超纯水稀释后,加入糖苷酶Endo F2进行酶切,通过质谱对质量数的解析,结果表明Endo F2酶切时间5 min, Fab段的聚糖就能完全切除,而Fc段的聚糖不受影响,实现了快速酶切,而且切糖具有很好的专一性。第二步对Fab段聚糖进行比例分析,将释放的聚糖经对氨基苯甲酰胺(2-AB)荧光标记后使用超高效液相色谱联用荧光检测器(FLR)进行检测,在亲水作用色谱(HILIC)柱上得到良好的分离并可以进行稳定地聚糖比例分析。3次独立试验结果表明,酶切后的质谱图基本一致,且聚糖的比例结果也基本一致,表明Endo F2酶切方法和聚糖比例分析方法都具有较好的稳定性和可靠性。此外,通过测定来自两个不同工艺生产的样品,数据显示两者的糖谱上具有非常明显的差异,表明利用开发的方法可以实现对抗体生产工艺进行监测研究,对抗体生产工艺的评估具有非常重要的意义。     

甲胺化衍生结合基于硅氢化物固定相的正相色谱用于单克隆抗体药物的N-糖基化表征

采用甲胺化衍生结合基于硅氢化物固定相的正相色谱（SiH-NPC）分析单抗的N-糖基化。样品经酶切、甲胺化衍生、纯化后由液相色谱-质谱进行分析。结果表明,相较于亲水相互作用色谱（HILIC）,SiH-NPC分离机制不同,使用常规的无盐流动相即可实现高分离度,避免污染质谱,色谱柱结构稳定,使用寿命长,更适合快速分析。结合唾液酸衍生方法,SiH-NPC在液相色谱-质谱联用鉴定酸性糖和糖异构体方面呈现显著优势,在生物制药行业中具有重要的应用潜力。     

Identification of Isomeric N-Glycans by Conformer Distribution Fingerprinting using Ion Mobility Mass Spectrometry

Glycans possess unparalleled structural complexity arising from chemically similar monosaccharide building blocks, configurations of anomeric linkages and different branching patterns, potentially giving rise to many isomers. This level of complexity is one of the main reasons that identification of exact glycan structures in biological samples still lags behind that of other biomolecules. Here, we introduce a methodology to identify isomeric N-glycans by determining gas phase conformer distributions (CDs) by measuring arrival time distributions (ATDs) using drift-tube ion mobility spectrometry-mass spectrometry. Key to the approach is the use of a range of well-defined synthetic glycans that made it possible to investigate conformer distributions in the gas phase of isomeric glycans in a systematic manner. In addition, we have computed CD fingerprints by molecular dynamics (MD) simulation, which compared well with experimentally determined CDs. It supports that ATDs resemble conformational populations in the gas phase and offer the prospect that such an approach can contribute to generating a library of CCS distributions (CCSDs) for structure identification.