利用亲水富集策略分析人血浆中N-糖基化蛋白及N-糖类型

蛋白质的N-糖基化是最重要的翻译后修饰之一,许多已知的血浆肿瘤诊断标志物及治疗靶标都是N-糖基化蛋白。针对血浆的糖蛋白质组研究有利于发现新的蛋白标志物。然而,血浆蛋白质浓度分布的动态范围非常宽,且同一位点上的糖链存在微观不均一性,影响了血浆中糖蛋白的鉴定效率。本文利用亲水材料ZIC-HILIC制备亲水富集柱分别对人血浆中的N-糖链和N-糖肽进行富集,并结合碱性反相色谱进行肽段的预分离和高准确度质谱分析,最终在健康人的血浆中鉴定到了299个糖基化蛋白、637个糖基化位点,并识别出31种不同的糖型。在这些鉴定到的糖基化位点中,新发现有107个N-糖基化位点(占总位点数的16.8%)。本方法操作简单,可以有效富集N-糖肽和N-糖,为在血浆中寻找糖蛋白和糖链生物标志物提供了可靠的手段。     

基于质谱的蛋白质O-糖基化分析

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.     

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。     

糖组学研究中糖蛋白糖链结构分析技术

遗传信息由DNA传递至蛋白质,再经蛋白质翻译后糖基化修饰形成糖蛋白.与DNA、蛋白质相比,糖蛋白糖链结构更加多样,功能更加复杂,在一些重大的生理、病理事件中发挥着重要调节作用;而糖链如此复杂的功能是由其多样的结构决定的,糖链结构是糖组学研究的重要内容.本文就近年来糖组学研究中糖蛋白样品的提取分离、糖链释放及结构分析的基本方法及相关技术进展作了简要介绍.     

基于质谱的糖蛋白/糖链定量技术研究进展

糖基化修饰是一种重要的蛋白质翻译后修饰。糖基化修饰的蛋白质在生命体内具有重要的生物学功能。研究糖蛋白含量以及蛋白上糖链变化对于阐明糖基化修饰的功能具有重要的意义,也是当今的研究热点。本文就糖蛋白和糖链定量方法的研究进展和应用做了简要概述。     

用于N-连接完整糖肽鉴定的糖基化蛋白质组学数据处理方法最新研究亮点北大核心CSCD

蛋白质糖基化与疾病的发生发展密切相关,临床上使用的大多数肿瘤标志物是糖基化蛋白质。在组学层次上进行位点特异性糖型的分析对发现新型疾病标志物、提高基于蛋白质糖基化的精准医学研究水平等具有重要作用。色谱-质谱联用技术在糖蛋白的分离分析研究中得到了广泛的应用。基于液相色谱-串联质谱(liquid chromatography-tandem mass spectrometry,LC-MS/MS)的完整糖肽鉴定已成为研究蛋白质上位点特异性糖链修饰的主要手段,其主要优势在于分析过程中可以同时揭示蛋白位点与糖链修饰的信息,从而在组学层次实现规模化的蛋白糖基化分析。     

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.     

糖蛋白/糖肽的分离富集方法*

蛋白质糖基化是一种重要的翻译后修饰,糖基化对蛋白质的结构和功能有着重要的影响。目前,作为蛋白质组学的一个组成部分,糖蛋白质组学是备受关注的研究热点。而从复杂的生物样品体系中富集糖蛋白/ 糖肽是蛋白质糖基化研究的重点和难点,本文就糖蛋白/糖肽分离富集方法的研究进展和应用作了简要概述。这些方法包括常用的凝集素亲和法、硼酸法、肼化学法和亲水作用法,还包括分子筛法、强阳离子交换法等新方法。     

Ionic catch and release oligosaccharide synthesis (ICROS)

A general and efficient chromatography-free ionic-liquid-supported "catch-and-release" strategy for oligosaccharide synthesis (ICROS) is reported. The methodology is compatible with current glycosylation strategies and amenable to protecting group manipulations. A series of β-(1→6) and β-(1→2)-linked glycan structures have been prepared to showcase the versatility of the strategy.     

Correlating Glycoforms of DC‐SIGN with Stability Using a Combination of Enzymatic Digestion and Ion Mobility Mass Spectrometry

The immune scavenger protein DC‐SIGN interacts with glycosylated proteins and has a putative role in facilitating viral infection. How these recognition events take place with different viruses is not clear and the effects of glycosylation on the folding and stability of DC‐SIGN have not been reported. Herein, we report the development and application of a mass‐spectrometry‐based approach to both uncover and characterise the effects of O‐glycans on the stability of DC‐SIGN. We first quantify the Core 1 and 2 O‐glycan structures on the carbohydrate recognition and extracellular domains of the protein using sequential exoglycosidase sequencing. Using ion mobility mass spectrometry, we show how specific O‐glycans, and/or single monosaccharide substitutions, alter both the overall collision cross section and the gas‐phase stability of the DC‐SIGN isoforms. We find that rather than the mass or length of glycoprotein modifications, the stability of DC‐SIGN is better correlated with the number of glycosylation sites.     

Analysis of glycans derived from glycoconjugates by capillary electrophoresis-mass spectrometry

The high structural variation of glycan derived from glycoconjugates, which substantially increases with the molecular size of a protein, contributes to the complexity of glycosylation patterns commonly associated with glycoconjugates. In the case of glycoproteins, such variation originates from the multiple glycosylation sites of proteins and the number of glycan structures associated with each site (microheterogeneity). The ability to comprehensively characterize highly complex mixture of glycans has been analytically stimulating and challenging. Although the most powerful MS and MS/MS techniques are capable of providing a wealth of structural information, they are still not able to readily identify isomeric glycan structures without high-order MS/MS (MS(n) ). The analysis of isomeric glycan structures has been attained using several separation methods, including high-pH anion-exchange chromatography, hydrophilic interaction chromatography and GC. However, CE and microfluidics CE (MCE) offer high separation efficiency and resolutions, allowing the separation of closely related glycan structures. Therefore, interfacing CE and MCE to MS is a powerful analytical approach, allowing potentially comprehensive and sensitive analysis of complex glycan samples. This review describes and discusses the utility of different CE and MCE approaches in the structural characterization of glycoproteins and the feasibility of interfacing these approaches to MS.     

Biomimetic synthesis of the tumor-associated (2,3)-sialyl-T antigen and its incorporation into glycopeptide antigens from the mucins MUC1 and MUC4

Glycoproteins on epithelial tumor cells often exhibit aberrant glycosylation profiles. The incomplete formation of the glycan side chains resulting from a down-regulated glucosamine transfer and a premature sialylation results in additional peptide epitopes, which become accessible to the immune system in mucin-type glycoproteins. These cancer-specific structure alterations are considered to be a promising basis for selective immunological attack on tumor cells. Among the tumor-associated saccharide antigens, the (2,3)-sialyl-T antigen has been identified as the most abundant glycan, found in several different carcinoma cell lines. According to a linear biomimetic strategy, the (2,3)-sialyl-T antigen was synthesized by a stepwise glycan chain extension of a protected galactosamine-threonine precursor. For the construction of immunostimulating antigens combining both peptide and saccharide motifs, this antigen was incorporated into glycopeptide partial structures from the mucins MUC1 and MUC4 by sequential solid-phase synthesis.     

Dephosphorylation of intact glycoprotein to greatly improve digestion efficiency coupled with matrix-assisted laser desorption/ionization–Fourier transform ion cyclotron resonance mass spectrometric analysis

Sialylation is essential for a variety of cellular functions. Herein, we used bovine fetuin with three potential N-linked glycosylation sites containing complex-type glycan structures, four potential O-linked glycosylation sites and six potential phosphorylation sites as a model compound to develop a highly-efficient digestion strategy for sialylated glycoproteins and efficient enrichment strategy for sialylated glycopeptides using titanium dioxide. The former according to the process of alkaline phosphatase digestion followed by tryptic digestion and then proteinase K digestion could greatly improve the enzymatic efficiency on fetuin, and the latter could obviously enhance the enrichment efficiency for multisialylated glycopeptides using phosphoric acid solution as elution buffer. The mass spectra of the enriched glycopeptides derived from fetuin reveal that several series of the ion clusters with mass difference of 291 Da correspond to the presence of multisialylated glycopeptides. In addition, the approach was applied to characterize the sialylated status of α2-macroglobulin and transferrin, respectively, from the sera of healthy subjects and sex- and age-matched patients with thyroid cancer, and their spectra indicate that the change in the amount of the glycoforms containing different number of sialic acid (SA) residues from one glycosylation site may be used to differentiate between healthy subjects and cancer cases.     

Glycosylation characterization of recombinant human erythropoietin produced in glycoengineered Pichia pastoris by mass spectrometry

Glycosylation plays a critical role in the in vivo efficacy of both endogenous and recombinant erythropoietin (EPO). Using mass spectrometry, we characterized the N‐/O‐linked glycosylation of recombinant human EPO (rhEPO) produced in glycoengineered Pichia pastoris and compared with the glycosylation of Chinese hamster ovary (CHO) cell‐derived rhEPO. While the three predicted N‐linked glycosylation sites (Asn24, Asn38 and Asn83) showed complete site occupancy, Pichia‐ and CHO‐derived rhEPO showed distinct differences in the glycan structures with the former containing sialylated bi‐antennary glycoforms and the latter containing a mixture of sialylated bi‐, tri‐ and tetra‐antennary structures. Additionally, the N‐linked glycans from Pichia‐produced rhEPO were similar across all three sites. A low level of O‐linked mannosylation was detected on Pichia‐produced rhEPO at position Ser126, which is also the O‐linked glycosylation site for endogenous human EPO and CHO‐derived rhEPO. In summary, the mass spectrometric analyses revealed that rhEPO derived from glycoengineered Pichia has a highly uniform bi‐antennary N‐linked glycan composition and preserves the orthogonal O‐linked glycosylation site present on endogenous human EPO and CHO‐derived rhEPO. Copyright © 2013 John Wiley & Sons, Ltd.     

Glycosylation analysis of interleukin-23 receptor: elucidation of glycosylation sites and characterization of attached glycan structures

Interleukin-23 (IL-23) is a heterodimeric cytokine, a central factor in chronic/autoimmune inflammation. It signals through a heterodimeric receptor consisting of IL-23r, which is heavily glycosylated. The structural characterization of IL-23r has not been reported. In this work, glycosylation profiles of soluble recombinant human IL-23r (rhIL-23r) were established using mass spectrometry (MS), which included defining glycosylation sites, degree of glycosylation occupancy of each site and structure of attached oligosaccharides. Specifically, precursor ion scan of oxonium ion protonated N-acetylglucosamine (GlcNAc(+)) (m/z 204) was performed using a triple quadrupole MS instrument to locate the retention time of glycopeptides. Both the glycopeptides and their corresponding deglycosylated forms in each collected HPLC fraction were studied by liquid chromatography-tandem mass spectrometry (LC-MS/MS) (LTQ-Orbitrap) for glycosylation site profiling. The attached glycan structures were elucidated by collision-induced dissociation (CID) fragmentation of target glycopeptides in combination with accurate mass measurement. Eight glycosylation sites were identified on IL-23r (Asn24, Asn209, Asn239, Asn157, Asn118, Asn250, Asn58 and Asn6). Most of the glycosylation sites were > 95% occupied except Asn250 and Asn6. Those two sites were 88% and 45% occupied by estimation from trypsin digestion and were 55% and 42% occupied from LysC digestion. Multiple glycoforms were observed in IL-23r. Most of them were bi-, tri- or tetra-antennary complex type structures with fucose and sialic acid. High mannose and hybrid type glycans were only observed on Asn157. The structural characterization on IL-23r glycosylation provides useful information for better understanding of the biological function of IL-23r.     

Mass spectrometry characterization for chemoenzymatic glycoprotein synthesis

The current project describes the chemoenzymatic modification of bovine ribonuclease B (RNase B) to contain a single glycosylation site with a known glycan. A reactive disaccharide oxazoline derivative was synthesized and stereospecifically added to deglycosylated RNase B through endo-β-N-acetylglucosaminidase M catalyzed chemoenzymatic transglycosylation. Oxazoline formation conditions were optimized using mass spectrometry, and the product verified based on its collision-induced dissociation (CID) mass spectrum. Enzymatic removal of native glycans as well as formation of the desired homogeneous product was also monitored using mass spectrometry. LC-MS(n) using four sequential rounds of CID was used to verify that the original glycosylation site had been reorganized to contain the new glycan. The techniques described herein are not limited to this analyte or glycan and should be amenable to the synthesis of numerous homogeneous glycoconjugates with judicious choice of enzyme/substrate combinations. The combined use of chemoenzymatic synthesis and mass spectrometry-based characterization shows promise for the development of homogeneous glycoprotein reference materials. A well-defined glycoprotein standard containing a single glycan of known composition, linkage and stereochemistry would be of great value for the comparison and evaluation of glycoprotein analysis techniques.     

Distinctive MS/MS Fragmentation Pathways of Glycopeptide‐Generated Oxonium Ions Provide Evidence of the Glycan Structure

Post‐translational glycosylation of proteins play key roles in cellular processes and the site‐specific characterisation of glycan structures is critical to understanding these events. Given the challenges regarding identification of glycan isomers, glycoproteomic studies generally rely on the assumption of conserved biosynthetic pathways. However, in a recent study, we found characteristically different HexNAc oxonium ion fragmentation patterns that depend on glycan structure. Such patterns could be used to distinguish between glycopeptide structural isomers. To acquire a mechanistic insight, deuterium‐labelled glycopeptides were prepared and analysed. We found that the HexNAc‐derived m/z 126 and 144 oxonium ions, differing in mass by H₂O, had completely different structures and that high‐mannose N‐glycopeptides generated abundant Hex‐derived oxonium ions. We describe the oxonium ion decomposition mechanisms and the relative abundance of oxonium ions as a function of collision energy for a number of well‐defined glycan structures, which provide important information for future glycoproteomic studies.     

Differential Glycosite Profiling—A Versatile Method to Compare Membrane Glycoproteomes

Glycosylation is the most prevalent and varied form of post-translational protein modifications. Protein glycosylation regulates multiple cellular functions, including protein folding, cell adhesion, molecular trafficking and clearance, receptor activation, signal transduction, and endocytosis. In particular, membrane proteins are frequently highly glycosylated, which is both linked to physiological processes and of high relevance in various disease mechanisms. The cellular glycome is increasingly considered to be a therapeutic target. Here we describe a new strategy to compare membrane glycoproteomes, thereby identifying proteins with altered glycan structures and the respective glycosites. The workflow started with an optimized procedure for the digestion of membrane proteins followed by the lectin-based isolation of glycopeptides. Since alterations in the glycan part of a glycopeptide cause mass alterations, analytical size exclusion chromatography was applied to detect these mass shifts. N-glycosidase treatment combined with nanoUPLC-coupled mass spectrometry identified the altered glycoproteins and respective glycosites. The methodology was established using the colon cancer cell line CX1, which was treated with 2-deoxy-glucose—a modulator of N-glycosylation. The described methodology is not restricted to cell culture, as it can also be adapted to tissue samples or body fluids. Altogether, it is a useful module in various experimental settings that target glycan functions.