综述: N-连接完整糖肽的质谱分析策略和研究方法

蛋白质糖基化作为最普遍、最重要的蛋白质修饰,一直是组学研究的焦点之一．近十几年来,N-连接糖蛋白质组学研究普遍采用的方法是将糖链与所修饰的多肽分开进行分析．该策略虽降低了分析难度,却也丢失了糖链与蛋白质糖基化位点间重要的对应关系信息．近年来,完整糖肽的质谱分析策略和方法逐步建立起来．总体而言,要实现对完整糖肽的直接质谱分析,首先需要从复杂样品中富集完整糖肽以消除非糖基化多肽对完整糖肽分析的影响,然后在质谱分析中还需要根据糖肽特性调整相应质谱分析参数,最后在后续数据分析中还需要开发相应的分析软件以完成完整糖肽中多肽序列和糖链组成或结构的鉴定．本文即从以上三个主要方面系统阐述目前N-完整糖肽分析中常用的质谱和数据分析策略和方法,并进一步在糖肽谱图识别、母离子单同位素分子质量校正、数据库选择以及假阳性率评估和控制等方面都进行了逐一探讨．完整糖肽的直接质谱分析有助于获取糖链和糖基化位点间的对应关系信息,可为生物标志物发现和疾病致病机理等研究提供更有力的糖蛋白质组学研究工具．     

糖蛋白质组定量技术进展

蛋白质的糖基化是最重要和最普遍的蛋白质翻译后修饰之一,在生物体内起着极为重要的作用。糖蛋白质的量和（或）糖基化程度的改变以及糖链结构的改变等与许多疾病密切相关,因此定量糖蛋白质组研究已经成为一个新的热点。然而由于糖基化蛋白质所具有的独特特征,其定量面临严峻的挑战。糖蛋白质组学定量方法和技术的发展将为更好地研究糖基化蛋白质生物学功能起到重要作用。综述了基于生物质谱的糖蛋白质组定量研究的技术和方法,及其优缺点和未来的发展趋势。     

基于质谱技术的蛋白质组定量方法

对不同状态下的蛋白质在表达和修饰水平上进行精确定量,对于探索蛋白质的生物功能、发现疾病的生物标志物都具有重要意义,也是当前蛋白质组学的一个重要研究前沿。近年来,各种蛋白质组定量的新技术和新方法不断涌现,但仍面临着巨大挑战。本文就基于质谱技术的多种蛋白质组定量方法的基本原理、近几年的研究进展和应用进行评述。     

植物N-糖链检测技术研究进展

N-糖基化作为一种重要的蛋白质翻译后修饰,在胚胎发育、癌症发生发展及免疫防御等诸多复杂的生命活动中发挥着关键作用。近年来,基于质谱的N-糖链的检测及其定量研究在动物方面取得了显著进展,相比之下,植物N-糖基化及N-糖链检测的相关研究要远远滞后,这也是制约植物糖生物学研究发展的关键瓶颈问题之一。对蛋白质N-糖链的释放、定量策略、可视化检测及其在植物中的应用进展进行了归纳总结,以期为指导后续植物N-糖链及N-糖组的定性定量检测提供参考。     

基于质谱的植物蛋白质组学研究方法

基于质谱的植物蛋白质组学研究方法,从定性和定量蛋白质组学两个方向进行了归纳总结,并对近年来出现的靶向蛋白质组学、DIA/SWATH技术、化学蛋白质组学,以及多组学联合分析等蛋白质组学研究的新技术、新方法和新应用进行了综述。     

蛋白质糖基化修饰的研究方法及其应用

蛋白质糖基化是一种重要的翻译后修饰,它参与和调控生物体的许多生命活动。随着蛋白质组技术的不断发展,蛋白质糖基化研究越来越受到广泛的重视。本文介绍了蛋白质糖基化修饰的研究内容与方法,并综述了最近的研究进展。     

基于数据非依赖采集的蛋白质组质谱数据解析方法研究进展

数据非依赖采集（DIA）是蛋白质组学领域近年来快速发展的质谱采集技术，其通过无偏碎裂隔离窗口内的所有母离子采集二级谱图，理论上可实现蛋白质样品的深度覆盖，同时具有高通量、高重现性和高灵敏度的优点。现有的DIA数据采集方法可以分为全窗口碎裂方法、隔离窗口序列碎裂方法和四维DIA数据采集方法（4D-DIA）3大类。针对DIA数据的不同特点，主要数据解析方法包括谱库搜索方法、蛋白质序列库直接搜索方法、伪二级谱图鉴定方法和从头测序方法4大类。解析得到的肽段鉴定结果需要进行可信度评估，包括使用机器学习方法的重排序和对报告结果集合的假发现率估计两个步骤，实现对数据解析结果的质控。本文对DIA数据的采集方法、数据解析方法及软件和鉴定结果可信度评估方法进行了整理和综述，并展望了未来的发展方向。     

糖蛋白质组学的信息学资源和方法

糖基化修饰是生物体内最常见、最重要的蛋白质翻译后修饰之一.哺乳动物体内超过50%的蛋白质都会发生糖基化修饰.糖蛋白广泛分布于各种组织的细胞膜表面,执行着重要的生物学功能.随着高通量、高灵敏度和高分辨率的蛋白质组学时代的来临,许多基于串级质谱技术解析糖链结构的生物数据库和分析软件也亦应运而生.本文综述了目前文献中最常用的糖类生物信息学资源,包括各种糖蛋白的数据库以及质谱解析糖类的相关工具和新技术、新方法.     

超支化聚合物新材料富集糖肽方法的研究

目的：将自合成的超支化聚合物固定到氨基硅球上形成一种新型的糖肽富集材料,用于糖肽的高效分离与富集。方法：阴离子开环聚合法合成的超支化聚缩水甘油,经高碘酸氧化成为超支化聚甘油醛（hyperbranched polyglycerol-aldehydes,HPG-ALD）后固定于氨基硅球上,再用己二酸二酰肼修饰得到的酰肼材料应用于糖肽富集。结果：经扫描电镜（Scanning Electron Microscope,SEM）、热重分析（thermogravimetric analysis,TGA）等手段证明材料被成功合成。同时,使用制备成功的新材料来富集标准糖蛋白和糖肽都获得了良好的富集效果。结论：实验证明超支化缩水甘油酰肼材料（hyperpolymer-assisted hydrazide functionalized microspheres,HHMs）可用于低丰度糖蛋白/糖肽的富集。     

N-连接完整糖肽的质谱分析策略和研究方法

蛋白质糖基化作为最普遍、最重要的蛋白质修饰,一直是组学研究的焦点之一.近十几年来,N-连接糖蛋白质组学研究普遍采用的方法是将糖链与所修饰的多肽分开进行分析.该策略虽降低了分析难度,却也丢失了糖链与蛋白质糖基化位点间重要的对应关系信息.近年来,完整糖肽的质谱分析策略和方法逐步建立起来.总体而言,要实现对完整糖肽的直接质谱分析,首先需要从复杂样品中富集完整糖肽以消除非糖基化多肽对完整糖肽分析的影响,然后在质谱分析中还需要根据糖肽特性调整相应质谱分析参数,最后在后续数据分析中还需要开发相应的分析软件以完成完整糖肽中多肽序列和糖链组成或结构的鉴定.本文即从以上三个主要方面系统阐述目前N-完整糖肽分析中常用的质谱和数据分析策略和方法,并进一步在糖肽谱图识别、母离子单同位素分子质量校正、数据库选择以及假阳性率评估和控制等方面都进行了逐一探讨.完整糖肽的直接质谱分析有助于获取糖链和糖基化位点间的对应关系信息,可为生物标志物发现和疾病致病机理等研究提供更有力的糖蛋白质组学研究工具.     

Intact Glycopeptide Analysis of Influenza A/H1N1/09 Neuraminidase Revealing the Effects of Host and Glycosite Location on Site‐Specific Glycan Structures

Influenza H1N1 virus has posed a serious threat to human health. The glycosylation of neuraminidase (NA) could affect the infectivity and virulence of the influenza virus, but detailed site‐specific glycosylation information of NA is still missing. In this study, intact glycopeptide analysis is performed on an influenza NA (A/H1N1/California/2009) that is expressed in human 293T and insect Hi‐5 cells. The data indicate that three of four potential N‐linked glycosylation sites are glycosylated, including one partial glycosylation site from both cell lines. The NA expressed in human cells has more complex glycans than that of insect cells, suggesting the importance of selecting an appropriate expression system for the production of functional glycoproteins. Different types of glycans are identified from different glycosites of NA expressed in human cells, which implies the site‐dependence of glycosylation on NA. This study provides valuable information for the research of influenza virus as well as the functions of viral protein glycosylation.     

Data-independent oxonium ion profiling of multi-glycosylated biotherapeutics

The characterization of glycosylation is required for many protein therapeutics. The emergence of antibody and antibody-like molecules with multiple glycan attachment sites has rendered glycan analysis increasingly more complicated. Reliance on site-specific glycopeptide analysis is therefore necessary to fully analyze multi-glycosylated biotherapeutics. Established glycopeptide methodologies have generally utilized a priori knowledge of the glycosylation states of the investigated protein(s), database searching of results generated from data-dependent liquid chromatography–tandem mass spectrometry workflows, and extracted ion quantitation of the individual identified species. However, the inherent complexity of glycosylation makes predicting all glycoforms on all glycosylation sites extremely challenging, if not impossible. That is, only the “knowns” are assessed. Here, we describe an agnostic methodology to qualitatively and quantitatively assess both “known” and “unknown” site-specific glycosylation for biotherapeutics that contain multiple glycosylation sites. The workflow uses data-independent, all ion fragmentation to generate glycan oxonium ions, which are then extracted across the entirety of the chromatographic timeline to produce a glycan-specific “fingerprint” of the glycoprotein sample. We utilized both HexNAc and sialic acid oxonium ion profiles to quickly assess the presence of Fab glycosylation in a therapeutic monoclonal antibody, as well as for high-throughput comparisons of multi-glycosylated protein drugs derived from different clones to a reference product. An automated method was created to rapidly assess oxonium profiles between samples, and to provide a quantitative assessment of similarity.     

N-glycosylation Site Analysis Reveals Sex-related Differences in Protein N-glycosylation in the Rice Brown Planthopper (Nilaparvata lugens)

1. Download : Download high-res image (275KB)
2. Download : Download full-size image

Highlights

• •N-glycosylation site analysis of the hemipteran pest insect Nilaparvata lugens.
• •Differential N-glycosylation of proteins is observed between male and female adults.
• •Sex-specific glycoproteins are involved in insect reproduction.

     

Affinity Selection and Mass Spectrometry-Based Strategies to Identify Lead Compounds in Combinatorial Libraries

The screening of diverse libraries of small molecules created by combinatorial synthetic methods is a recent development which has the potential to accelerate the identification of lead compounds in drug discovery. We have developed a direct and rapid method to identify lead compounds in libraries involving affinity selection and mass spectrometry. In our strategy, the receptor or target molecule of interest is used to isolate the active components from the library physically, followed by direct structural identification of the active compounds bound to the target molecule by mass spectrometry. In a drug design strategy, structurally diverse libraries can be used for the initial identification of lead compounds. Once lead compounds have been identified, libraries containing compounds chemically similar to the lead compound can be generated and used to optimize the binding characteristics. These strategies have also been adopted for more detailed studies of protein–ligand interactions.     

Large-scale Identification of N-linked Intact Glycopeptides in Human Serum using HILIC Enrichment and Spectral Library Search

1. Download : Download high-res image (200KB)
2. Download : Download full-size image

Highlights

• •This study proposed a spectral library search method to accurately identify N-linked glycopeptides in human serum through LC-MS/MS with pMatchGlyco software.
• •The identification depth of serum N-linked intact glycopeptides and glycoproteins was increased by combination of acetonitrile precipitation, HILIC enrichment and high-pH RPLC fractionation.
• •22,677 unique serum N-linked intact glycopeptides corresponding to 526 N-linked glycoproteins were identified with N-glycosylation motif-specific FDR control.
• •This study revealed the great microheterogeneity of N-linked glycoproteins in serum.

     

基于电感耦合等离子体质谱的单细胞分析

单细胞分析可以获得细胞在微环境中准确的个体信息,对于研究细胞的信号传导、生理病理和疾病的早期诊断等具有十分重要的意义.近年来,基于电感耦合等离子体质谱(ICP-MS)的单细胞分析方法开始得到越来越多的应用.本文综述了基于ICP-MS的单细胞分析方法及其在免疫分析、疾病诊断、药物筛选、纳米分析等方面的部分应用,并对基于ICP-MS的单细胞分析方法做出总结和展望.     

Glycosylation profiling to evaluate glycoprotein immunogens against HIV-1

Introduction: Much of the efforts to develop a vaccine against the human immunodeficiency virus (HIV) have focused on the design of recombinant mimics of the viral attachment glycoprotein (Env). The leading immunogens exhibit native-like antigenic properties and are being investigated for their ability to induce broadly neutralizing antibodies (bNAbs). Understanding the relative abundance of glycans at particular glycosylation sites on these immunogens is important as most bNAbs have evolved to recognize or evade the dense coat of glycans that masks much of the protein surface. Understanding the glycan structures on candidate immunogens enables triaging between native-like conformations and immunogens lacking key structural features as steric constraints limit glycan processing. The sensitivity of the processing state of a particular glycan to its structural environment has led to the need for quantitative glycan profiling and site-specific analysis to probe the structural integrity of immunogens.

Areas covered: We review analytical methodologies for HIV immunogen evaluation and discuss how these studies have led to a greater understanding of the structural constraints that control the glycosylation state of the HIV attachment and fusion spike.

Expert commentary: Total composition and site-specific glycosylation profiling are emerging as standard methods in the evaluation of Env-based immunogen candidates.