基于质谱的定量蛋白质组学技术发展现状

蛋白质组定量分析技术是支撑蛋白质组学研究的关键技术之一,随着蛋白质组定量分析技术的发展,基于质谱的定量蛋白质组学已成为蛋白质组学研究的重要分支。蛋白质组学定量技术可分为非靶向定量和靶向定量两类,靶向定量技术有MRM和PRM模式,非靶向定量技术有非标记定量和体内外标记定量模式,目前使用最多的同位素标记试剂是i TRAQ和TMT。蛋白质组定量技术按数据采集模式还可分DDA和DIA两类。通过对国内外相关文献收集和分析,系统介绍了蛋白质组质谱定量技术的主要特点和发展现状,旨在为生命科学研究者更好地应用定量蛋白质组学技术提供帮助。     

基于液相色谱质谱联用的蛋白质组非标记定量研究策略的建立及应用

定量蛋白质组研究是蛋白质组研究的热点和难点,而液相色谱质谱技术已经被广泛地应用于蛋白质的定性和定量研究.该研究建立和优化了一种基于液相色谱质谱联用技术的蛋白质组非标记定量方法,并对两种肽段质谱检测计数的归一化算法进行了比较,结果发现ASC法要优于Rsc法.最后,将建立的方法应用于肝癌细胞模型HepG2和HepG2-HBx细胞系的差异蛋白质组表达研究.质谱鉴定结果用聚类分析软件cluster3.0进行分析,最后鉴定出107个重叠蛋白,其中9个蛋白质表达上调(Ratio>1.75),6个蛋白质表达下调(Ratio<0.5),这些蛋白质均与肝癌发生和恶化密切相关.结果表明,该技术操作简单、方便,具有较高的灵敏度和动态范围,利用该方法进行差异蛋白质组研究和发现生物标志物在理论和临床上具有十分重要的意义.     

质谱MRM技术在蛋白质组学研究中的应用

质谱多反应监测(multiple reaction monitoring,MRM)技术是一种基于已知信息或假定信息有针对性地获取数据,进行质谱信号采集的技术,具有灵敏、准确和特异等优点.在基于蛋白组学的生物标志物研究、蛋白质翻译后修饰.定量蛋白质组和蛋白质相互作用等研究领域的应用逐渐受到重视.该文概述了该技术在蛋白质组学研究中的应用特点及其最新应用进展.     

iTRAQ技术在真菌研究中的应用进展

iTRAQ技术是一种新的、功能强大的、可以最多同时比较8种不同样品中蛋白质相对或绝对含量的蛋白质组学方法,结合多维液相色谱和串联质谱分析,iTRAQ技术已成为差异蛋白质组学定量研究的主要工具之一。而真菌的致病作用是多种蛋白质共同参与的真菌?宿主相互作用的复杂过程,因此整体、定量地分析真菌致病过程中的差异表达蛋白质谱,对于研究真菌的致病机制具有重要作用。该文重点就iTRAQ技术在真菌研究中的应用进展进行综述。     

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。     

稳定同位素化学标记结合质谱技术在定量蛋白质组学中的应用

传统的蛋白质组定量策略主要是通过双向凝胶电泳来进行相对定量。由于该方法不能对相对分子质量极高或极低、等电点极酸或极碱和含量低的蛋白质以及膜蛋白质等进行有效分离和检测,所以已不能适应目前蛋白质组研究深入发展的需要。近年来,定量蛋白质组学的发展主要是以同位素亲和标签试剂为代表的、以质谱检测为核心的稳定同位素化学标记方法。稳定同位素化学标记结合质谱技术,使定量蛋白质组的分析更趋简单、准确和快速,具有良好的发展前景。本文对稳定同位素化学标记结合质谱技术在定量蛋白质组学中的研究进展进行了评述。     

质谱技术及其在后基因组时代中的应用

蛋白质组学的建立开辟了功能基因组学研究的新领域,为研究蛋白质水平的生命活动展现了更为崭新的思路和广阔的前景.质谱技术能准确测量肽和蛋白质的相对分子质量、氨基酸序列及翻译后修饰,成为连接蛋白质与基因的重要技术.质谱技术联合蛋白质组学多角度、深层次探索生命系统分子本质成为现阶段生命科学研究领域.简要综述了肽和蛋白质等生物大分子质谱分析的原理、方式和应用,并对其发展前景做出展望.     

蛋白质水解酶和样品预处理优化技术在蛋白质组学研究中的应用

蛋白质组学是全景式鉴定、定量蛋白质,并研究蛋白质功能的学科。基于高分辨质谱的鸟枪法蛋白组学研究技术首先利用不同的位点特异性蛋白酶对复杂蛋白质样品进行酶解,进而利用质谱获得蛋白质相关的定性和定量信息。为了获得高质量的质谱信息,前期的样品处理和质谱数据采集同样重要。本文对蛋白组学中常用的Trypsin、Lys-C、Glu-C等位点特异性蛋白酶的酶切特点进行了总结,并综述了目前常用的几种酶切组合策略和样本预处理技术在提高蛋白质组学研究效率中的作用。     

蛋白质结构与相互作用研究新方法——交联质谱技术

蛋白质的空间结构信息以及蛋白质间的相互作用信息对于研究蛋白质的功能有重要意义.研究蛋白质结构与相互作用的传统技术,如核磁共振技术、X射线晶体衍射技术等,对于蛋白质的纯度、结晶性和绝对量均有比较高的要求,限制了其广泛应用.交联质谱技术是近十多年来发展起来的新技术,它将质谱技术与交联技术相结合,在研究蛋白质结构与相互作用方面具有速度快、成本小、蛋白质各方面性状要求低等优势.本文就交联质谱技术各个环节的技术方法加以综述,包括交联质谱实验分离富集技术、常见交联剂特性、交联质谱数据库搜索算法、结果验证研究和交联质谱技术的应用等方面,并展望了该研究方向未来的发展.     

iTRAQ多重化学标记串联质谱技术在比较蛋白质组学中的应用

研究不同生理和病理条件下细胞内蛋白质的含量和状态变化是比较蛋白质组学的核心内容。要揭示上述动态过程往往需要进行多个样品的同步比较分析。近年来,在体内和体外同位素标记基础上,用多维液相色谱分离多肽,进而用串联质谱进行相对定量的分析方法已成为高通量比较蛋白质组研究的主要手段之一。该文就目前唯一一种可以进行四重蛋白质样品同步比较的iTRAQ标记-串联质谱分析技术进行综述。     

Surface view of the lateral organization of lipids and proteins in lung surfactant model systems—A ToF-SIMS approach

The lateral organization of domain structures is an extremely significant aspect of biomembrane research. Chemical imaging by mass spectrometry with its recent advancement in sensitivity and lateral resolution has become a highly promising tool in biological research. In this review, we focus briefly on the instrumentation, working principle and important concepts related to time-of-flight secondary ion mass spectrometry followed by an overview of lipid/protein fragmentation patterns and chemical mapping. The key issues addressed are the applications of time-of-flight secondary ion mass spectrometry in biological membrane research. Additionally, we briefly review our recent investigations based on time-of-flight secondary ion mass spectrometry to unravel the lateral distribution of lipids and surfactant proteins in lung surfactant model systems as an example that highlights the importance of fluidity and ionic conditions on lipid phase behavior and lipid-protein interactions.     

Mass spectrometry-based immunoassays for the next phase of clinical applications

Recent applications of affinity mass spectrometry into clinical laboratories brought a renewed interest in immunoaffinity mass spectrometry as a more specific affinity method capable of selectively targeting and studying protein biomarkers. In mass spectrometry-based immunoassays, proteins are affinity retrieved from biological samples via surface-immobilized antibodies, and are then detected via mass spectrometric analysis. The assays benefit from dual specificity, which is brought about by the affinity of the antibody and the protein mass readout. The mass spectrometry aspect of the assays enables single-step detection of protein isoforms and their individual quantification. This review offers a comprehensive review of mass spectrometry-based immunoassays, from historical perspectives in the development of the immunoaffinity mass spectrometry, to current applications of the assays in clinical and population proteomic endeavors. Described in more detail are two types of mass spectrometry-based immunoassays, one of which incorporates surface plasmon resonance detection for protein quantification. All mass spectrometry-based immunoassays offer high-throughput targeted protein investigation, with clear implications in clinical research, encompassing biomarker discovery and validation, and in diagnostic settings as the next-generation immunoassays.     

Mass spectrometry-based immunoassays for the next phase of clinical applications

Recent applications of affinity mass spectrometry into clinical laboratories brought a renewed interest in immunoaffinity mass spectrometry as a more specific affinity method capable of selectively targeting and studying protein biomarkers. In mass spectrometry-based immunoassays, proteins are affinity retrieved from biological samples via surface-immobilized antibodies, and are then detected via mass spectrometric analysis. The assays benefit from dual specificity, which is brought about by the affinity of the antibody and the protein mass readout. The mass spectrometry aspect of the assays enables single-step detection of protein isoforms and their individual quantification. This review offers a comprehensive review of mass spectrometry-based immunoassays, from historical perspectives in the development of the immunoaffinity mass spectrometry, to current applications of the assays in clinical and population proteomic endeavors. Described in more detail are two types of mass spectrometry-based immunoassays, one of which incorporates surface plasmon resonance detection for protein quantification. All mass spectrometry-based immunoassays offer high-throughput targeted protein investigation, with clear implications in clinical research, encompassing biomarker discovery and validation, and in diagnostic settings as the next-generation immunoassays.     

Overcoming the dynamic range problem in mass spectrometry-based shotgun proteomics

Protein profiling using mass spectrometry technology has emerged as a powerful method for analyzing large-scale protein-expression patterns in cells and tissues. However, a number of challenges are present in proteomics research, one of the greatest being the high degree of protein complexity and huge dynamic range of proteins expressed in the complex biological mixtures, which exceeds six orders of magnitude in cells and ten orders of magnitude in body fluids. Since many important signaling proteins have low expression levels, methods to detect the low-abundance proteins in a complex sample are required. This review will focus on the fundamental fractionation and mass spectrometry techniques currently used for large-scale shotgun proteomics research.     

Overcoming the dynamic range problem in mass spectrometry-based shotgun proteomics

Protein profiling using mass spectrometry technology has emerged as a powerful method for analyzing large-scale protein-expression patterns in cells and tissues. However, a number of challenges are present in proteomics research, one of the greatest being the high degree of protein complexity and huge dynamic range of proteins expressed in the complex biological mixtures, which exceeds six orders of magnitude in cells and ten orders of magnitude in body fluids. Since many important signaling proteins have low expression levels, methods to detect the low-abundance proteins in a complex sample are required. This review will focus on the fundamental fractionation and mass spectrometry techniques currently used for large-scale shotgun proteomics research.     

Detecting outlier peptides in quantitative high-throughput mass spectrometry data

Quantitative high-throughput mass spectrometry has become an established tool to measure relative gene expression proteome-wide. The output of such an experiment usually consists of a list of expression ratios (fold changes) for several thousand proteins between two conditions. However, we observed that individual peptide fold changes may show a significantly different behavior than other peptides from the same protein and that these differences cannot be explained by imprecise measurements. Such outlier peptides can be the consequence of several technical (misidentifications, misquantifications) or biological (post-translational modifications, differential regulation of isoforms) reasons. We developed a method to detect outlier peptides in mass spectrometry data which is able to delineate imprecise measurements from real outlier peptides with high accuracy when the true difference is as small as 1.4 fold. We applied our method to experimental data and investigated the different technical and biological effects that result in outlier peptides. Our method will assist future research to reduce technical bias and can help to identify genes with differentially regulated protein isoforms in high throughput mass spectrometry data.     

生物质谱及其在蛋白质组学研究中的应用

生物质谱是蛋白质组学研究必不可少的关键技术。近年来,生物质谱在鉴定通量、分辨率和灵敏度等方面均有质的飞跃,从而促进了蛋白质组研究各个领域的飞速发展。本文就生物质谱技术的原理、技术和仪器发展现状,及其在蛋白质组学研究中的应用进展作一简要的综述。     

Modification-specific proteomics: characterization of post-translational modifications by mass spectrometry

Post-translational modifications generate tremendous diversity, complexity and heterogeneity of gene products, and their determination is one of the main challenges in proteomics research. Recent developments in mass spectrometry based approaches for systematic, qualitative and quantitative determination of modified proteins promise to bring new insights on the dynamics and spatio-temporal control of protein activities by post-translational modifications, and reveal their roles in biological processes and pathogenic conditions. Combinations of affinity-based enrichment and extraction methods, multidimensional separation technologies and mass spectrometry are particularly attractive for systematic investigation of post-translationally modified proteins in proteomics.     

Post-translational Modifications and Mass Spectrometry Detection

In this review, we provide a comprehensive bibliographic overview of the role of mass spectrometry and the recent technical developments in the detection of post-translational modifications (PTMs). We briefly describe the principles of mass spectrometry for detecting PTMs and the protein and peptide enrichment strategies for PTM analysis, including phosphorylation, acetylation and oxidation. This review presents a bibliographic overview of the scientific achievements and the recent technical development in the detection of PTMs is provided. In order to ascertain the state of the art in mass spectrometry and proteomics methodologies for the study of PTMs, we analyzed all the PTM data introduced in the Universal Protein Resource (UniProt) and the literature published in the last three years. The evolution of curated data in UniProt for proteins annotated as being post-translationally modified is also analyzed. Additionally, we have undertaken a careful analysis of the research articles published in the years 2010 to 2012 reporting the detection of PTMs in biological samples by mass spectrometry.