定量蛋白质组学分析方法及应用

蛋白质组学经历了近10年的发展,现在已经初具规模。但是由于它是动态地观察生物体不断变化的所有蛋白质,所以技术难度非常之大。为使研究简化并更具针对性,人们着重进行比较蛋白质组学的研究。为了具体量化这些蛋白质的变化产生了定量蛋白质组学,近几年各种标记技术的进步使得该学科得以迅猛发展。     

定量蛋白质组同位素标记技术及应用

定量蛋白质组学是对蛋白质组进行精确的定量和鉴定的学科,突破了传统蛋白质组研究集中于对蛋白质的分离和鉴定,着重于定性定量解析细胞蛋白质的动态变化信息,更真实地反映了细胞功能、过程机制等综合信息。以同位素为内标的质谱分析新技术的提出,显示出可同时自动鉴定和精确定量的能力,代表了目前定量蛋白质组研究的主要发展方向。对近年来定量蛋白质组学同位素标记技术和应用研究所取得的重要进展以及最新的发展动态进行了综述。     

Quantitative Proteomics Using Reductive Dimethylation for Stable Isotope Labeling

Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a method to accurately quantify protein expression differences between samples using mass spectrometry. ReDi labeling is performed using either regular (light) or deuterated (heavy) forms of formaldehyde and sodium cyanoborohydride to add two methyl groups to each free amine. Here we demonstrate a robust protocol for ReDi labeling and quantitative comparison of complex protein mixtures. Protein samples for comparison are digested into peptides, labeled to carry either light or heavy methyl tags, mixed, and co-analyzed by LC-MS/MS. Relative protein abundances are quantified by comparing the ion chromatogram peak areas of heavy and light labeled versions of the constituent peptide extracted from the full MS spectra. The method described here includes sample preparation by reversed-phase solid phase extraction, on-column ReDi labeling of peptides, peptide fractionation by basic pH reversed-phase (BPRP) chromatography, and StageTip peptide purification. We discuss advantages and limitations of ReDi labeling with respect to other methods for stable isotope incorporation. We highlight novel applications using ReDi labeling as a fast, inexpensive, and accurate method to compare protein abundances in nearly any type of sample.     

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。     

18O标记法在定量蛋白质组学中的应用

基于质谱技术去识别和检测蛋白质表达差异是一个热点,有助于生物过程和体系的分子机制的研究。近年来各种基于质谱技术的定量蛋白质组学研究方法发展较快,相对其他方法而言,18O标记法是一种较为理想、相对容易实现并且在不断完善的体外标记方法,最近在定量蛋白质组学研究中应用较多。现对18O标记法原理、特点以及技术方法的优化和应用进展进行综述。     

Quantitative Proteomics Using Isobaric Labeling: A Practical Guide

In the past decade, relative proteomic quantification using isobaric labeling technology has developed into a key tool for comparing the expression of proteins in biological samples. Although its multiplexing capacity and flexibility make this a valuable technology for addressing various biological questions, its quantitative accuracy and precision still pose significant challenges to the reliability of its quantification results. Here, we give a detailed overview of the different kinds of isobaric mass tags and the advantages and disadvantages of the isobaric labeling method. We also discuss which precautions should be taken at each step of the isobaric labeling workflow, to obtain reliable quantification results in large-scale quantitative proteomics experiments. In the last section, we discuss the broad applications of the isobaric labeling technology in biological and clinical studies, with an emphasis on thermal proteome profiling and proteogenomics.     

微生物蛋白质组学的定量分析

越来越多的微生物基因组序列数据为系统地研究基因的调节和功能创造了有利条件.由于蛋白质是具有生物功能的分子,蛋白质组学在微生物基因组的功能研究中异军突起、蓬勃发展.微生物蛋白质组学的基本原则是,用比较研究来阐明和理解不同微生物之间或不同生长条件下基因的表达水平.显而易见,定量分析技术是比较蛋白质组学中急需发展的核心技术.对蛋白质组学定量分析技术在微生物蛋白质组研究中的进展进行了综述.     

Protease- and Acid-catalyzed Labeling Workflows Employing 18O-enriched Water

Stable isotopes are essential tools in biological mass spectrometry. Historically, ¹⁸O-stable isotopes have been extensively used to study the catalytic mechanisms of proteolytic enzymes^1-3. With the advent of mass spectrometry-based proteomics, the enzymatically-catalyzed incorporation of ¹⁸O-atoms from stable isotopically enriched water has become a popular method to quantitatively compare protein expression levels (reviewed by Fenselau and Yao⁴, Miyagi and Rao⁵ and Ye et al.⁶). ¹⁸O-labeling constitutes a simple and low-cost alternative to chemical (e.g. iTRAQ, ICAT) and metabolic (e.g. SILAC) labeling techniques⁷. Depending on the protease utilized, ¹⁸O-labeling can result in the incorporation of up to two ¹⁸O-atoms in the C-terminal carboxyl group of the cleavage product³. The labeling reaction can be subdivided into two independent processes, the peptide bond cleavage and the carboxyl oxygen exchange reaction⁸. In our PALeO (protease-assisted labeling employing ¹⁸O-enriched water) adaptation of enzymatic ¹⁸O-labeling, we utilized 50% ¹⁸O-enriched water to yield distinctive isotope signatures. In combination with high-resolution matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF MS/MS), the characteristic isotope envelopes can be used to identify cleavage products with a high level of specificity. We previously have used the PALeO-methodology to detect and characterize endogenous proteases⁹ and monitor proteolytic reactions^10-11. Since PALeO encodes the very essence of the proteolytic cleavage reaction, the experimental setup is simple and biochemical enrichment steps of cleavage products can be circumvented. The PALeO-method can easily be extended to (i) time course experiments that monitor the dynamics of proteolytic cleavage reactions and (ii) the analysis of proteolysis in complex biological samples that represent physiological conditions. PALeO-TimeCourse experiments help identifying rate-limiting processing steps and reaction intermediates in complex proteolytic pathway reactions. Furthermore, the PALeO-reaction allows us to identify proteolytic enzymes such as the serine protease trypsin that is capable to rebind its cleavage products and catalyze the incorporation of a second ¹⁸O-atom. Such "double-labeling" enzymes can be used for postdigestion ¹⁸O-labeling, in which peptides are exclusively labeled by the carboxyl oxygen exchange reaction. Our third strategy extends labeling employing ¹⁸O-enriched water beyond enzymes and uses acidic pH conditions to introduce ¹⁸O-stable isotope signatures into peptides.     

蛋白质组研究新前沿：定量蛋白质组学

在过去几年里,蛋白质组研究取得了令人鼓舞的进展,2DE-MS途径的自动化,多维色谱整合串联质谱的使用,弥补了一些用双向凝胶电泳分离蛋白质的技术缺陷;从稳定同位素标记到ICAT战略的提出,为准确定量在细胞或组织中发挥重要调节功能的低丰度蛋白质提供了一个较为理想的方法。同时,蛋白质芯片技术的不断发展,也极大的丰富了定量蛋白质组学的研究。就定量蛋白质组学及其相关技术研究进展作一简要综述。     

细胞培养稳定同位素标记技术在蛋白质组学中的应用与进展

细胞培养稳定同位素标记技术（SILAC）是在细胞培养过程中,利用稳定同位素标记的氨基酸结合质谱技术,对蛋白表达进行定量分析的一种新技术。它不仅可以对蛋白质进行定性分析,还可通过质谱图上一对轻-重稳定同位素峰的比例来反映对应蛋白在不同状态下的表达水平,实现对蛋白质的精确定量。SILAC结合质谱技术在定量蛋白质组学中发挥了巨大的作用,其应用范围从细胞系扩展到亚细胞器、组织与动物整体水平,具体的应用策略也在不断完善发展。我们总结评述了SILAC技术在差异表达蛋白质组、蛋白质翻译后修饰、药物蛋白质组和蛋白质相互作用等方面的应用与进展。     

Trypsin is the primary mechanism by which the (18)O isotopic label is lost in quantitative proteomic studies

Labeling with (18)O is currently one of the most commonly used methods for incorporating a stable isotopic label into samples for comparative proteomic studies. In this approach, isotopic labeling involves the enzymatic digestion, typically performed with trypsin, of a protein population in (18)O-water, which incorporates the stable isotope into the C termini of the newly formed peptides. Although trypsin is often used to facilitate isotopic incorporation after digestion, it is typically overlooked that this same mechanism can lead to isotopic loss even under conditions such as low pH where it is assumed that trypsin is inactive. To examine the role that trypsin plays in isotopic loss, several experiments were performed on the rate of delabeling under conditions relevant to multidimensional proteomic experiments. Results from these studies demonstrate that enzyme-facilitated exchange of (18)O in the peptide with (16)O in the aqueous solvent was the major process by which the label is removed from the peptides, even under conditions of low pH and temperature where trypsin is thought to be inactive. This study brings the rapid, tryptic-facilitated exchange to the attention of laboratories using this scheme to prevent inaccuracies in quantitative labeling due to loss of the isotopic label.     

串联亲和纯化（TAP）技术在蛋白质组学中的应用

蛋白质是各种生命活动的主要执行者,因此构建蛋白质相互作用的网络图对于准确理解蛋白质功能、揭开各种细胞活动的奥秘十分重要.串联亲和纯化（TAP）,是近年来发展出来的一种能够快速研究在生理条件下蛋白质相互作用,揭示蛋白质复合体相互作用网络的新技术,已成为研究蛋白质组学的一个重要工具.随着该技术的不断完善,TAP技术在认识蛋白质相互作用的过程中必将发挥越来越重要的作用.     

定量蛋白质组学无标记定量方法的研究进展

依靠质谱技术的蛋白质组学快速发展,寻求速度快、重复性好以及准确度高的定量方法是该领域的一项艰巨任务,定量蛋白质组学分支领域应运而生．其中,无标记定量方法以其样品制备简单、耗材费用低廉以及结果数据分析便捷等优点渐露锋芒．无标记定量方法通常分为信号强度法和谱图计数法两大类．本文在这两种无标记定量方法计算原理的基础上,针对各种常用的无标记定量方法及最新进展做一个较为全面的介绍,并将详细讨论两类方法的异同点,以及目前蛋白质组学中无标记定量方法所面临的主要挑战,希望能为这一领域的研究人员在选择无标记定量方法时提供一个合理的参考．     

Identification of Protein Interaction Partners in Mammalian Cells Using SILAC-immunoprecipitation Quantitative Proteomics

Quantitative proteomics combined with immuno-affinity purification, SILAC immunoprecipitation, represent a powerful means for the discovery of novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants. Low affinity interactions can be preserved through the use of less-stringent buffer conditions and remain readily identifiable. This protocol discusses the labeling of tissue culture cells with stable isotope labeled amino acids, transfection and immunoprecipitation of an affinity tagged protein of interest, followed by the preparation for submission to a mass spectrometry facility. This protocol then discusses how to analyze and interpret the data returned from the mass spectrometer in order to identify cellular partners interacting with a protein of interest. As an example this technique is applied to identify proteins binding to the eukaryotic translation initiation factors: eIF4AI and eIF4AII.     

定量蛋白质组学分析PknG在分枝杆菌中的功能

丝氨酸苏氨酸蛋白激酶G(PknG)是分枝杆菌中一个类似于真核生物蛋白激酶C的蛋白质,对结核分枝杆菌的生长和新陈代谢等生理过程,以及结核分枝杆菌的耐药和在宿主细胞中的存活都起着重要的调节作用.本文在耻垢分枝杆菌(Mycobacterium smegmatis)mc2155中构建了过表达结核分枝杆菌PknG的重组菌株PknG-mc2155,并发现PknG-mc2155的生长速度慢于mc2155.应用化学修饰结合LC-LC-MS/MS的定量蛋白质组学方法,在mc2155和PknG-mc2155中鉴定到了176种有差异表达的蛋白,其中152种蛋白在PknG-mc2155中表达下调,24种蛋白表达上调.这些差异表达的蛋白参与了多个细胞过程,包括代谢、蛋白翻译等.基于这些结果,我们推测PknG-mc2155生长速度慢的原因是因为代谢相关酶如GlpK,ALD和DesA1等蛋白表达的下调;而Ag85A,Ag85C,SecA2等蛋白的上调则增强细菌的感染性;另外KatG蛋白的下调提示PknG的过表达增强了菌株的抗药性.代谢组学分析发现谷氨酸和谷氨酰胺在PknG-mc2155中的水平低于在mc2155中水平,证实了PknG影响谷氨酰胺的稳态平衡.利用蛋白质磷酸化分析,我们发现PknG的苏氨酸残基T-320上有一个自磷酸化修饰,而且在PknG-mc2155菌株中,也鉴定到gltA和glmM上的磷酸化修饰,显示gltA和glmM是PknG的底物.本研究为理解PknG的功能和作用机制提供了新的依据和解释,为深入研究PknG在结核分枝杆菌中的功能奠定了基础,我们的结果也表明蛋白质组学技术是系统研究细菌蛋白质功能的重要工具.     

Super-SILAC: current trends and future perspectives

Stable isotope labeling with amino acids in cell culture (SILAC) has risen as a powerful quantification technique in mass spectrometry (MS)–based proteomics in classical and modified forms. Previously, SILAC was limited to cultured cells because of the requirement of active protein synthesis; however, in recent years, it was expanded to model organisms and tissue samples. Specifically, the super-SILAC technique uses a mixture of SILAC-labeled cells as a spike-in standard for accurate quantification of unlabeled samples, thereby enabling quantification of human tissue samples. Here, we highlight the recent developments in super-SILAC and its application to the study of clinical samples, secretomes, post-translational modifications and organelle proteomes. Finally, we propose super-SILAC as a robust and accurate method that can be commercialized and applied to basic and clinical research.     

Absolute quantification strategies in proteomics based on mass spectrometry

The strong need for quantitative information in proteomics has fueled the development of mass spectrometry-based analytical methods that are able to determine protein abundances. This article reviews mass spectrometry experiments aimed at providing an absolute quantification of proteins. The experiments make use of the isotope-dilution concept by spiking a known amount of synthetic, isotope-labeled reference peptide into the analyte sample. Quantification is achieved by comparing the mass spectrometry signal intensities of the reference with an endogenous peptide that is generated upon proteolytic cleavage of the target protein. In an analogous manner, the level of post-translational modification at a distinct residue within a target protein can be determined. Among the strengths of absolute quantification are low detection limits reaching subfemtomole levels, a high dynamic range spanning approximately five orders of magnitude, low requirements for sample clean-up, and a fast and straightforward method development. Recent studies have demonstrated the compatibility of absolute quantification with various mass spectrometry readout techniques and sample purification steps such as 1D gel electrophoresis, size-exclusion chromatography, isoelectric peptide focusing, strong cation exchange and reversed phase or affinity chromatography. Under ideal conditions, quantification errors and coefficients of variation below 5% have been reported. However, the fact that at the start of the experiment the analyte is a protein and the internal standard is a peptide, severe quantification errors may result due to the selection of unsuitable reference peptides and/or imperfect protein proteolysis. Within the ensemble of mass spectrometry-based quantification methods, absolute quantification is the method of choice in cases where absolute numbers, many repetitive experiments or precise levels of post-translational modifications are required for a few, preselected species of interest. Consequently, prominent application areas include biomarker quantification, the study of post-translational modifications such as phosphorylation or ubiquitination and the comparison of concentrations of interacting proteins.     

Microbial minorities modulate methane consumption through niche partitioning

Microbes catalyze all major geochemical cycles on earth. However, the role of microbial traits and community composition in biogeochemical cycles is still poorly understood mainly due to the inability to assess the community members that are actually performing biogeochemical conversions in complex environmental samples. Here we applied a polyphasic approach to assess the role of microbial community composition in modulating methane emission from a riparian floodplain. We show that the dynamics and intensity of methane consumption in riparian wetlands coincide with relative abundance and activity of specific subgroups of methane-oxidizing bacteria (MOB), which can be considered as a minor component of the microbial community in this ecosystem. Microarray-based community composition analyses demonstrated linear relationships of MOB diversity parameters and in vitro methane consumption. Incubations using intact cores in combination with stable isotope labeling of lipids and proteins corroborated the correlative evidence from in vitro incubations demonstrating γ-proteobacterial MOB subgroups to be responsible for methane oxidation. The results obtained within the riparian flooding gradient collectively demonstrate that niche partitioning of MOB within a community comprised of a very limited amount of active species modulates methane consumption and emission from this wetland. The implications of the results obtained for biodiversity–ecosystem functioning are discussed with special reference to the role of spatial and temporal heterogeneity and functional redundancy.