基于多级质谱的蛋白质组定量新方法新技术进展

定量蛋白质组学已经成为蛋白质组学的一个重要分支,以生物质谱为核心的定量蛋白质组方法日益发展。按照定量所依据的质谱信号来源于一级质谱谱图还是多级质谱谱图可以将定量蛋白质组方法分为一级质谱定量和多级质谱定量。本文主要综述基于多级质谱的定量方法和技术进展,分析比较了这些方法的优缺点,并对基于多级质谱的定量方法发展进行了展望。     

定量蛋白质组学分析方法

精确测量多个不同生理或病理条件下生物样本中蛋白质表达量的变化是定量蛋白质组学(quantitative proteomics)研究的重要内容。与传统的蛋白质定量方法(见表1)相比,组学规模的蛋白质定量可以实现在一次实验中对成百上千个蛋白质的定量测定和比较分析,为规模化发现和验证疾病诊断的生物标志物以及发展新的药物靶标提供了重要手段。     

基于稳定同位素标记的蛋白质组学定量方法研究进展

定量蛋白质组学已经成为后基因时代的重要研究方向之一。目前该领域的研究主要采用无标记定量方法和稳定同位素标记定量法。其中,基于稳定同位素标记的蛋白质组定量方法发展非常迅速,已为生命科学研究提供了重要的技术支撑。本文分析了基于稳定同位素标记的蛋白质组学定量方法,包括相对定量方法和绝对定量方法,并对其发展进行了展望。     

基于ICP-MS的蛋白质定量方法*

蛋白质组学已经成为生命科学研究中最为活跃的领域之一。研究蛋白质的生物功能,不但需要高通量的鉴定蛋白质,还需要定量分析动态变化的蛋白质,即定量蛋白质组学研究。蛋白质的定量研究有助于发现新的生物功能,并可以用于疾病的预警和药物靶点的发现。现有的定量蛋白质组学研究主要利用同位素标记结合生物质谱（电喷雾电离质谱ESI-MS,基质辅助激光解吸电离质谱MALDI-MS）技术而实现。近年来电感耦合等离子体质谱（ICP-MS）作为ESI-MS和MALDI-MS的补充,越来越多地应用于蛋白质的定量分析,特别是蛋白质的绝对定量分析。ICP-MS是检测生物分子中痕量元素的理想工具,具有灵敏度高、动态范围广,不易受基体的影响等优点。本文将讨论基于ICP-MS的分析方法,及其在蛋白质定量分析和免疫分析中的部分成功应用。     

基于稳定同位素标记与质谱分析的蛋白质定量算法研究进展

蛋白质定量研究已成为蛋白质组学的热点,它是疾病相关生物标志物发现的重要途径.基于稳定同位素标记的质谱分析技术是蛋白质定量最常用的方法之一.随着实验方法的发展和改进,定量数据处理方法也在不断更新与完善.一般来说,定量数据处理包括四步:搜库鉴定、图谱定量信息提取与计算、肽段丰度比计算和蛋白质丰度比计算及差异显著性分析,其中后三步是数据处理的核心.目前,后三步中每步都有多种可选算法,这些算法一般都是针对特定实验技术而提出的,缺乏深入的工作对它们进行系统比较和优化.为此,在总结目前主要实验技术方法的基础上,论述了定量算法的现状和存在问题,并针对一些问题提出了可行的解决办法.     

分离分析技术在蛋白质组学研究中应用的新进展

蛋白质组学研究的核心技术之一是分离分析方法。该综述重点评述了分离分析技术在蛋白质组学研究,即在蛋白质组表达谱构建、翻译后修饰蛋白质组研究、蛋白质复合体和相互作用研究、蛋白质组定量研究中应用的新进展,介绍了各种分离分析方法的优点、应用范围和有待解决的问题。引用文献89篇。     

蛋白质组学定量的技术与方法研究进展

蛋白质组学是一门在整体水平上研究细胞内蛋白质组成及其活动规律的新兴学科。定量蛋白质组学是指通过某种方法或技术，对生物样品(细胞、组织或体液等)在某些过程中蛋白质的含量进行比较分析。近几年来，定量蛋白质组的技术发展很快，稳定同位素标记技术的提出，为准确定量在细胞或组织体系中发挥重要功能的低丰度蛋白质提供了一个理想的方法。本文综述了蛋白质组定量分析技术及其最新的研究进展。     

化学生物学新前沿——化学蛋白质组学

随着包括人类在内的主要模式生物的基因组计划的完成,生命科学的研究重心转向蛋白质组的研究--在对应基因组的整体蛋白质水平上系统研究调控细胞生命活动的蛋白质.化学蛋白质组学是化学生物学在后基因组时代的最新发展:化学蛋白质组学利用化学小分子为工具和手段,以基于靶蛋白质功能的新战略探测体内蛋白质组,是新一代的功能蛋白质组学.本文综述了化学蛋白质组学的最新进展、有关技术及其在生物医学和药物研发等方面的应用,并对化学蛋白质组学的发展趋势和前景进行了讨论.     

HPLC/ICP-MS定量分析蛋白质

定量蛋白质组学已经成为蛋白质组学的重要组成部分,也给分析化学带来了巨大的机遇和挑战.已有的定量蛋白质组学方法主要利用同位素标记结合生物质谱技术而完成定量分析.与生物质谱不同,电感耦合等离子体质谱(ICP-MS)可以分析周期表中大部分元素,而且元素在ICP-MS中的响应与其所处的分子环境无关.如果蛋白质已被鉴定,或者其元素含量已知,就可以通过分析蛋白质中某一元素而实现对蛋白质的绝对定量分析~([1]).     

选择性反应监测技术在蛋白质组学研究中的进展及应用

选择性反应监测（SRM）技术作为一种重要的定向蛋白质分析技术,通过选择性检测特定母离子和子离子来排除非目标组分的干扰,增强了检测灵敏度和定量准确度,具有选择性高、重复性好、灵敏度高、动态范围宽等优点,已被广泛应用于定量蛋白质组学研究,在生命科学领域发挥着至关重要的作用。本文从分析通量、检测灵敏度、定量方法以及相关软件资源4个方面,对近期SRM技术的研究进展进行了综述。然后,对SRM技术在蛋白质组学研究包括生物标志物验证、蛋白质翻译后修饰研究、生物工程以及信号通路分析等领域中的应用进行了概述。最后,本文对SRM技术的应用以及发展前景进行了展望。     

Selected reaction monitoring applied to proteomics

Selected reaction monitoring (SRM) performed on triple quadrupole mass spectrometers has been the reference quantitative technique to analyze small molecules for several decades. It is now emerging in proteomics as the ideal tool to complement shotgun qualitative studies; targeted SRM quantitative analysis offers high selectivity, sensitivity and a wide dynamic range. However, SRM applied to proteomics presents singularities that distinguish it from small molecules analysis. This review is an overview of SRM technology and describes the specificities and the technical aspects of proteomics experiments. Ongoing developments aiming at increasing multiplexing capabilities of SRM are discussed; they dramatically improve its throughput and extend its field of application to directed or supervised discovery experiments.     

基于质谱技术蛋白质定量方法的研究进展

质谱技术已经成为目前蛋白质鉴定的重要工具。定量分析细胞内蛋白质组的动态变化,是当前研究蛋白质功能、揭示细胞生物机理、寻找疾病蛋白标记物和药物靶标的迫切需要。本文综述了基于质谱技术蛋白质定量的策略、方法和应用等方面近年来的进展,评述了几种蛋白质质谱定量方法的特点和应用潜力。     

Minimal deuterium isotope effects in quantitation of dimethyl-labeled complex proteomes analyzed with capillary zone electrophoresis/mass spectrometry

Stable heavy-isotope labeling is commonly used in quantitative proteomics. Several common techniques incorporate deuterium (²H) as the heavy isotopic label using reductive amination with formaldehyde. Compared with alternatives, dimethyl labeling reagents are inexpensive and the labeling chemistry is simple and rapid. However, the substitution of hydrogen by deuterium can introduce subtle changes in peptides’ polarities, leading to a shift in chromatographic retention times between deuterated and nondeuterated peptides that can lead to quantification deviations. Capillary zone electrophoresis has emerged as a complementary separation for ESI–MS-based proteomics, including targeted and quantitative approaches. The extent to which the deuterium isotope effect impacts CZE-based proteomics, which separates peptides based on their S/N ratios, has not been investigated. To address this issue, CZE was used to analyze dimethyl labeled E. coli tryptic digests in 100 min single-shot analyses. The median migration time shift was 0.1 s for light versus heavy labeled peptides, which is 2.5% of the peak width. For comparison, nUHPLC–ESI–MS/MS was used to analyze the same sample. In UPLC, deuterated peptides tended to elute earlier than nondeuterated peptides, with a retention shift of 3 s for light versus heavy labeled peptides, which is roughly half the peak width. This shift in separation time did not have a significant effect on quantitation for either method for equal mixing ratios of the light-intermediate-heavy isotope labeled samples.     

Recent liquid chromatographic-(tandem) mass spectrometric applications in proteomics

Conventional proteomics makes use of two-dimensional gel electrophoresis followed by mass spectrometric analysis of tryptic fragments derived from in-gel digestion of proteins. Although being a very strong technique capable of separating and visualizing hundreds of proteins, 2D-gel electrophoresis has some well-documented disadvantages as well. More recently, liquid chromatographic-(tandem) mass spectrometric techniques have been developed to overcome some of the shortcomings of 2D-gel electrophoresis. In this review we have described several recent applications of liquid chromatography-(tandem) mass spectrometry in the field of proteomics and especially in the field of membrane proteomics, quantitative proteomics and in the analysis of post-translational modifications.     

Werkzeug für die Chemische Biologie. Semisynthese

Semisynthetic techniques have greatly contributed to the rapid development of Chemical Biology in recent years. In this regard the semisynthesis of complex modified proteins as well as the selective derivatization of natural products has evolved into more than mere proof‐of‐principle concepts but powerful tools to probe protein functions. This technology provides a solid basis for further investigations on proteomics and qualitative and quantitative cell biology. The interdisciplinary charter bridging chemistry and biology is the hallmark of semisynthesis. It can be expected that its scientific impact will further increase in the future.     

Current trends in quantitative proteomics – an update

Proteins can provide insights into biological processes at the functional level, so they are very promising biomarker candidates. The quantification of proteins in biological samples has been routinely used for the diagnosis of diseases and monitoring the treatment. Although large‐scale protein quantification in complex samples is still a challenging task, a great amount of effort has been made to advance the technologies that enable quantitative proteomics. Seven years ago, in 2009, we wrote an article about the current trends in quantitative proteomics. In writing this current paper, we realized that, today, we have an even wider selection of potential tools for quantitative proteomics. These tools include new derivatization reagents, novel sampling formats, new types of analyzers and scanning techniques, and recently developed software to assist in assay development and data analysis. In this review article, we will discuss these innovative methods, and their current and potential applications in proteomics. Copyright © 2017 John Wiley & Sons, Ltd.