基于固定化Pronase E酶与质谱的糖链结构分析方法

位点特异性糖链结构的解析,是糖蛋白质结构分析面临的巨大挑战。Pronase E蛋白水解酶,能够降解糖蛋白或糖肽中大部分的氨基酸序列,而保留糖链与少量氨基酸的序列,与色谱、质谱分析等联用,可以实现糖链结构的鉴定,同时,保留的氨基酸序列可以辅助实现修饰位点的识别,两者结合,可以获取位点特异性糖链结构的信息。但Pronase E酶解的缺点是,酶解效率较低,常需要较高浓度的蛋白酶。本实验将Pronase E固定化在基质上,固定化后的Pronase E具备较高的局部浓度,从而实现目标糖蛋白的快速高效酶解。采用核糖核酸酶B作为标准糖蛋白,优化了Pronase E酶切的方案,包括酶切时蛋白与酶的用量比、酶切时间、固定化Pronase E酶的有效贮存时间等;同时优化选择了糖链的富集方法,并对于基质辅助激光解吸飞行时间质谱分析中,糖链适合的基质进行对比选择,从而获得更好的糖链谱图及更为丰富的糖链结构信息。     

基于电喷雾电离质谱(ESI-MS)的丙酮稳定同位素标记对N-糖链的相对定量分析方法的研究

建立了一种基于电喷雾电离质谱的丙酮稳定同位素标记对N-糖链进行相对定量的研究方法. 与传统的PNGase F酶水解N-糖链的方法不同,采用非特异性蛋白酶Pronase E对N-糖蛋白进行处理,使N-糖蛋白被酶解为带有一个氨基酸的糖氨酸（Glycan-Asn）,为N-糖链引入了一个氨基活性基团,然后用丙酮对氨基进行标记. 用d₀/d₆丙酮对Ribo B标准糖蛋白的Pronase E酶解产物进行标记,考察了4对d₀/d₆丙酮标记的Glycan-Asn（Man₅～Man₈-Asn）在电喷雾电离质谱中的线性、动态范围以及重现性. 结果表明,在10倍动态范围内,相对定量方法有良好的线性关系（R=0.9981）和重现性（CV<8.7%）. 并将建立的方法应用于不同含量的鸡卵清白蛋白中,进一步验证了该方法的可行性. 研究结果表明,该方法能准确分析样品中N-糖链的含量,对不同样品中N-糖链进行相对定量. 该方法成本低廉,后处理方法简单方便,适于微量样品通量化分析,对差异糖组的研究有一定的意义.     

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.     

丝胶蛋白源Tn和T抗原的释放制备及质谱分析

以蚕茧丝胶蛋白源Tn抗原和T抗原为研究对象,采用“一釜法”、氨水催化法和链霉蛋白酶E(Pronase E)水解法对丝胶蛋白O-糖链进行释放,并通过固相萃取柱进行分级纯化,以高效液相色谱仪(HPLC)进行分离制备;利用电喷雾质谱(ESI-MS)、串联质谱(MS/MS)及在线液相色谱-质谱联用仪(Online LC-MS)进行了结构鉴定和定量分析.结果表明,丝胶蛋白中O-GalNAc含量为1.58μg/g, O-GalNAcGal含量为0.54μg/g,二者丰度比约为5.3∶1;并且氨水可高效释放出其还原性O-连接单糖GalNAc,通过液相色谱法可实现其精细分离纯化,而以等量Pronase E对丝胶蛋白水解可有效释放出Tn抗原和T抗原.     

β-伴大豆球蛋白的N-糖基化位点及其N-糖链的质谱分析

以β-伴大豆球蛋白(β-conglycinin)为研究对象,利用Trypsin和Pepsin酶对其进行水解,以Con A亲和层析柱富集纯化糖肽,并用糖苷酶PNGase F和Endo H分别酶解糖肽,再采用电喷雾质谱(ESI-MS)和串联质谱(MS/MS)对所得肽段的氨基酸序列及糖链的结构进行了分析,最后通过数据库检索对分析结果进行验证.结果表明,β-conglycinin具有5个N-糖基化位点,分别为α亚基的199和455位天冬酰胺(Asn),α'亚基的215和489位Asn及β亚基的326位Asn,且每个糖基化位点均被H5N2,H6N2,H7N2和H8N2这4种高甘露糖型N-糖链所修饰.本研究为各种糖蛋白的糖基化位点及其对应的糖链结构的鉴定分析提供了方法参考,并为深入理解大豆糖蛋白抗原表位的特异性及致敏机理提供了依据.     

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.     

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。     

花生致敏糖蛋白Ara h1糖链决定簇的质谱分析

以花生种子总蛋白及其主要致敏糖蛋白Ara h1为研究对象,采用"一釜法"对蛋白上的糖链进行释放并同时进行衍生化标记,通过C18固相萃取柱纯化,以电喷雾质谱(ESI-MS)、多级串联质谱(MSn)和亲水性液相色谱-质谱联用(HILIC-MS)进行结构解析和定量分析.结果表明,蛋白Ara h1共有10条N-糖链,其中7条为高甘露糖型,2条为木糖修饰,另外1条为与过敏原相关的核心α1,3-Fuc修饰N-糖链,其含量约占总糖链的12.45%.     

基因重组糖蛋白-人尿激酶原糖基化修饰的质谱测定

酶法结合生物质谱技术测定了基因重组糖蛋白-人尿激酶原(rhProUK)的N-糖含量、唾液酸含量分别为9％和5％。糖蛋白／肽先经Con A凝集素亲和富集,肽：N-糖苷酶F(PNGaseF)对N-糖基化位点进行特异性质量标记,然后利用Lc-MS/MS技术测定出N-糖基化位点在第302位天冬酰胺残基上。     

Two-step protease digestion and glycopeptide capture approach for accurate glycosite identification and glycoprotein sequence coverage improvement

A novel two-step protease digestion and glycopeptide capture approach has been developed. It is different from traditional tryptic digestion, glycopeptide enriching and identification approach in glycoproteomics. Here, proteins were first digested by Lys-C into relatively large peptides. Glycopeptides among them were selectively captured by hydrazide resin through oxidized glycans. After thorough washing steps, trypsin was used as a second protease to in situ release non-glycosylated part (named as LT-peptides) from glycopeptides. Subsequently, the remaining part of glycopeptides on resin was de-glycosylated by peptide-N-glycosidase F, and collected as DG-peptides. Finally, both LT- and DG-peptides could be analyzed by mass spectrometer, achieving glycoprotein and glycosite identification. The approach was applied to cell lysate after positive validation by a model glycoprotein: 143 N-glycoproteins identified from DG- and LT-fraction both. In those glycoproteins, 189 DG-peptide-revealed N-glycosites got further confirmation by neighboring LT-peptides, which, in the meantime, made 109 glycoproteins get improved sequence coverage with increase even up to 350% (averagely 79.4%). Through controllable release, separate identification and combined interpretation of non-glycopeptides (newly introduced LT-peptides here) and traditional de-glycopeptides, the approach could not only achieve routine N-glycosite identification, but also provide further proofs of N-glycosites and increase glycoprotein sequence coverage.     

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.     

Analysis of chicken and turkey ovalbumins by microchip electrophoresis combined with exoglycosidase digestion

The polypeptide and carbohydrate patterns of two glycoproteins, chicken ovalbumin (CO) and turkey ovalbumin (TO), were analyzed by microchip electrophoresis (ME), following digestion with proteases and exoglycosidases. Glycopeptides derived from ovalbumin were obtained by digestion with Pronase, followed by dialysis, and then separated by ME. Using CO as model, the method was developed to deduce the structure of glycans from glycoproteins by comparing the electropherograms of glycopeptides with and without digestion of exolycosidases. Applying the same approach, the structure of oligosaccharides linked to TO was determined. TO was found to contain high-mannose type oligosaccharides and oligosaccharides with terminal N-acetylglucosamine residues. The complete primary analysis of CO and TO by ME described in this paper provides a basis for an analysis of glycoproteins with an integrated microfluidic chip.     

Capillary zone electrophoresis of alpha 1-acid glycoprotein fragments from trypsin and endoglycosidase digestions

Capillary zone electrophoresis with fused-silica tubes having hydrophilic coating on the inner walls was evaluated in the separation of peptide and glycopeptide fragments from trypsin digestion of alpha 1-acid glycoprotein. Submapping of glycosylated and nonglycosylated tryptic fragments of the glycoprotein by capillary electrophoresis was facilitated by selective isolation of the glycopeptides on concanavalin A silica-based stationary phases prior to the electrophoretic run. In addition, the electrophoretic map and submaps of the whole tryptic digest and its concanavalin A fractions, respectively, allowed the elucidation of the microheterogeneity of the glycoprotein. Also, capillary zone electrophoresis proved suitable for the mapping of the oligosaccharide chains cleaved from the glycoproteins by endoglycosidase digestion. The oligosaccharides cleaved from human and bovine alpha 1-acid glycoprotein were analyzed after derivatization with 2-aminopyridine, which allowed their sensitive detection by on column UV absorption. The separation was best achieved when 0.1 M phosphate solution, pH 5.0, containing 50 mM tetrabutylammonium bromide was used as the running electrolyte. The effect of the organic salt on separation was attributed to ion-pair formation and/or hydrophobic interaction.     

伴刀豆球蛋白亲和色谱柱的制备及其在糖蛋白核糖核酸酶B结构分析中的应用

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。     

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.     

Modelling the electrophoretic migration behaviour of peptides and glycopeptides from glycoprotein digests in capillary electrophoresis-mass spectrometry

In this study, the classical semiempirical relationships between the electrophoretic mobility and the charge-to-mass ratio (m_e vs. q/M^α) were used to model the migration behaviour of peptides and glycopeptides originated from the digestion of recombinant human erythropoietin (rhEPO), a biologically and therapeutically relevant glycoprotein. The Stoke’s law (α = 1/3), the classical polymer model (α = 1/2) and the Offord’s surface law (α = 2/3) were evaluated to predict migration of peptides and glycopeptides, with and without sialic acids (SiA), in rhEPO digested with trypsin and trypsin–neuraminidase. The Stoke’s law resulted in better correlations for the set of peptides used to evaluate the models, while glycopeptides fitted better with the classical polymer model. Once predicted migration times with both models, it was easy to simulate their separation electropherogram. Results were later validated predicting migration and simulating separation of a different set of rhEPO glycopeptides and also human transferrin (Tf) peptides and glycopeptides. The excellent agreement between the experimental and the simulated electropherograms with rhEPO and Tf digests confirmed the potential applicability of this simple strategy to predict, in general, the peptide–glycopeptide electrophoretic map of any digested glycoprotein.     

麦胚凝集素亲和色谱富集糖肽过程中的肽键裂解

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。     

Ion mobility-mass correlation trend line separation of glycoprotein digests without deglycosylation

A high-throughput ion mobility mass spectrometer (IMMS) was used to rapidly separate and analyze peptides and glycopeptides derived from glycoproteins. Two glycoproteins, human α-1-acid glycoprotein and antithrombin III were digested with trypsin and subjected to electro-spray traveling wave IMMS analysis. No deglycosylation steps were performed; samples were complex mixtures of peptides and glycopeptides. Peptides and glycosylated peptides with different charge states (up to 4 charges) were observed and fell on distinguishable trend lines in 2-D IMMS spectra in both positive and negative modes. The trend line separation patterns matched between both modes. Peptide sequence was identified based on the corresponding extracted mass spectra and collision induced dissociation (CID) experiments were performed for selected compounds to prove class identification. The signal-to-noise ratio of the glycopeptides was increased dramatically with ion mobility trend line separation compared to non-trend line separation, primarily due to selection of precursor ion subsets within specific mobility windows. In addition, isomeric mobility peaks were detected for specific glycopeptides. IMMS demonstrated unique capabilities and advantages for investigating and separating glycoprotein digests in this study and suggests a novel strategy for rapid glycoproteomics studies in the future.     

Enzyme degradation, high performance liquid chromatography and liquid secondary ion mass spectrometry in the analysis of glycoproteins.

Analysis of small amounts of glycoproteins by high performance liquid chromatography (HPLC) and liquid secondary ion mass spectrometry (LSIMS) together with enzyme digestion has been investigated using fetuin as a model. Preliminary data indicates that 71% of the expected peptides were detected by LSIMS analysis of 200 pmol total digest. HPLC profiles of peptides and glycopeptides were obtained from 2 nmol of digest using a reversed phase (C18) column eluted in a solvent system containing TFA, water and acetonitrile. This has provided glycopeptides for subsequent oligosaccharide analysis. Strategies are reviewed for the chromatographic characterization of oligosaccharides following their release from glycopeptides by chemical and enzymatic procedures.