Modification-specific proteomics in plant biology

Post-translational modifications (PTMs) are involved in the regulation of a wide range of biological processes, and affect e.g. protein structure, activity and stability. Several hundred PTMs have been described in the literature, but relatively few have been studied using mass spectrometry and proteomics. In general, methods for PTM characterization are developed to study yeast and mammalian biology and later adopted to investigate plants. Our point of view is that it is advantageous to enrich for PTMs on the peptide level as part of a quantitative proteomics strategy to not only identify the PTM, but also to determine the functional relevance in the context of regulation, response to abiotic stress etc. Protein phosphorylation is the only PTM that has been studied extensively at the proteome wide level in plants using mass spectrometry based methods.     

植物膜蛋白质组学研究技术现状

植物膜蛋白质组学是当前植物科学研究的热点领域。本文概论了蛋白质组学在植物膜蛋白研究中的应用,包括双向电泳前膜蛋白样品的制备以及植物质膜、液泡膜和其他膜蛋白组分的蛋白质组学研究进展,并介绍了植物膜蛋白质组学相关的数据库,最后对其发展作了展望。     

Mass spectrometry for high throughput quantitative proteomics in plant research: lessons from thylakoid membranes

Proteomics seeks to monitor the flux of protein through cells under variable developmental and environmental influences as programmed by the genome. Consequently, it is necessary to measure changes in protein abundance and turnover rate as faithfully as possible. In the absence of non-invasive technologies, the majority of proteomics approaches involve destructive sampling at various time points to obtain 'snapshots' that periodically report the genomes's product. The work has fallen to separations technologies coupled to mass spectrometry, for high throughput protein identification. Quantitation has become the major challenge facing proteomics as the field matures. Because of the variability of day-to-day measurements of protein quantities by mass spectrometry, a common feature of quantitative proteomics is the use of stable isotope coding to distinguish control and experimental samples in a mixture that can be profiled in a single experiment. To address limitations with separation technologies such as 2D-gel electrophoresis, alternative systems are being introduced including multi-dimensional chromatography. Strategies that accelerate throughput for mass spectrometry are also emerging and the benefits of these 'shotgun' protocols will be considered in the context of the thylakoid membrane and photosynthesis. High resolution Fourier-transform mass spectrometry is bringing increasingly accurate mass measurements to peptides and a variety of gas-phase dissociation mechanisms are permitting 'top-down' sequencing of intact proteins. Finally, a versatile workflow for sub-cellular compartments including membranes is presented that allows for intact protein mass measurements, localization of post-translational modifications and relative quantitation or turnover measurement.     

Advances in qualitative and quantitative plant membrane proteomics

The membrane proteome consists of integral and membrane-associated proteins that are involved in various physiological and biochemical functions critical for cellular function. It is also dynamic in nature, where many proteins are only expressed during certain developmental stages or in response to environmental stress. These proteins can undergo post-translational modifications in response to these different conditions, allowing them to transiently associate with the membrane or other membrane proteins. Along with their increased size, hydrophobicity, and the additional organelle and cellular features of plant cells relative to mammalian systems, the characterization of the plant membrane proteome presents unique challenges for effective qualitative and quantitative analysis using mass spectrometry (MS) analysis. Here, we present the latest advancements developed for the isolation and fractionation of plant organelles and their membrane components amenable to MS analysis. Separations of membrane proteins from these enriched preparations that have proven effective are discussed for both gel- and liquid chromatography-based MS analysis. In this context, quantitative membrane proteomic analyses using both isotope-coded and label-free approaches are presented and reveal the potential to establish a wider-biological interpretation of the function of plant membrane proteins that will ultimately lead to a more comprehensive understanding of plant physiology and their response mechanisms.     

Targeted label-free quantitative analysis of secretory proteins from adipocytes in response to oxidative stress

Adipocytes are well known to release regulation factors associated with metabolic disorders. In particular, increased oxidative stress in adipocytes contributes to dysregulation of adipokine production. In this study, we applied relative quantitative proteomic analysis based on label-free multiple reaction monitoring (MRM) to discover biological changes of adipokines under oxidative stress. Among a total of 194 identified proteins, 8 proteins were selected and quantified between control and hydrogen peroxide (H₂O₂)-treated groups by label-free MRM quantification. The secretion levels of matrix metalloproteinase-2 (MMP-2), stromal cell-derived factor-1 (SDF-1, CXCL12), resistin, and complement factor D (CFD, adipsin) decreased, whereas the secretion levels of tissue inhibitor of metalloproteinase-2 (TIMP-2) and aldolase A increased. Here we suggest that our study with label-free quantitative analysis will contribute to the efficient quantitative analysis of targeted proteins in complex mixtures and specifically to a better understanding of changes of adipokines under oxidative stress.     

Biomedical applications of ion mobility-enhanced data-independent acquisition-based label-free quantitative proteomics

Mass spectrometry-based proteomics greatly benefited from recent improvements in instrument performance and the development of bioinformatics solutions facilitating the high-throughput quantification of proteins in complex biological samples. In addition to quantification approaches using stable isotope labeling, label-free quantification has emerged as the method of choice for many laboratories. Over the last years, data-independent acquisition approaches have gained increasing popularity. The integration of ion mobility separation into commercial instruments enabled researchers to achieve deep proteome coverage from limiting sample amounts. Additionally, ion mobility provides a new dimension of separation for the quantitative assessment of complex proteomes, facilitating precise label-free quantification even of highly complex samples. The present work provides a thorough overview of the combination of ion mobility and data-independent acquisition-based label-free quantification LC-MS and its applications in biomedical research.     

Miniaturized proteomics and peptidomics using capillary liquid separation and high resolution mass spectrometry

Knowledge of the protein and peptide content in a tissue or a body fluid is vital in many areas of medical and biomedical sciences. Information from proteomic and peptidomic studies may reveal alterations in expression due to, e.g., a disease and facilitate the understanding of the pathophysiology and the identification of biological markers. In this minireview, we discuss miniaturized proteomic and peptidomic approaches that have been applied in our laboratory in order to investigate the protein and peptide contents of body fluids (such as plasma, cerebrospinal and amniotic fluid), as well as extracted tissues. The methods involve miniaturized liquid separation, i.e., capillary liquid chromatography and capillary electrophoresis, combined with high resolution mass spectrometry (MS), i.e., Fourier transform ion cyclotron resonance MS. These approaches provide the opportunity to analyze samples of small volumes with high throughput, high sensitivity, good dynamic range and minimal sample handling. Also, the experiments are relatively easy to automate.     

Including shared peptides for estimating protein abundances: A significant improvement for quantitative proteomics

Inferring protein abundances from peptide intensities is the key step in quantitative proteomics. The inference is necessarily more accurate when many peptides are taken into account for a given protein. Yet, the information brought by the peptides shared by different proteins is commonly discarded. We propose a statistical framework based on a hierarchical modeling to include that information. Our methodology, based on a simultaneous analysis of all the quantified peptides, handles the biological and technical errors as well as the peptide effect. In addition, we propose a practical implementation suitable for analyzing large data sets. Compared to a method based on the analysis of one protein at a time (that does not include shared peptides), our methodology proved to be far more reliable for estimating protein abundances and testing abundance changes. The source codes are available at http://pappso.inra.fr/bioinfo/all_p/.     

Liquid chromatography-mass spectrometry-based quantitative proteomics

LC-MS-based quantitative proteomics has become increasingly applied to a wide range of biological applications due to growing capabilities for broad proteome coverage and good accuracy and precision in quantification. Herein, we review the current LC-MS-based quantification methods with respect to their advantages and limitations and highlight their potential applications.     

Multi-dimensional liquid phase based separations in proteomics

This review covers recent developments towards the implementation of multi-dimensional (MuD) liquid phase based systems for proteome investigations. Although two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) has been used as a standard approach in proteomics, its drawbacks including the limited dynamic range and molecular mass range, together with lack of on-line integration with biological mass spectrometery (Bio-MS) have limited its widespread use and applications in proteomics. In the meantime, various liquid-phase based multi-dimensional separation techniques have been explored. Especially, with the emergence of the combination of nanoflow capillary high-performance liquid chromatography (cHPLC) and Bio-MS, attention is again refocused on utilizing multi-dimensional liquid-phase based separation of proteins. Some remarkable applications of on-line analysis of intact proteins and on-column digested proteins, and the emergence of approaches such as multiple HPLC-electrospray ionization tandem MS and capillary array electrophoresis-matrix assisted laser desorption ionization MS, have stimulated thinking towards developing a automated multi-dimensional system (MuDSy) that integrates liquid phase based separation, digestion and identification of proteins in complex biological mixtures.     

Comparison of label-free and label-based strategies for proteome analysis of hepatoma cell lines

Within the past decade numerous methods for quantitative proteome analysis have been developed of which all exhibit particular advantages and disadvantages. Here, we present the results of a study aiming for a comprehensive comparison of ion-intensity based label-free proteomics and two label-based approaches using isobaric tags incorporated at the peptide and protein levels, respectively. As model system for our quantitative analysis we used the three hepatoma cell lines HepG2, Hep3B and SK-Hep-1. Four biological replicates of each cell line were quantitatively analyzed using an RPLC–MS/MS setup. Each quantification experiment was performed twice to determine technical variances of the different quantification techniques. We were able to show that the label-free approach by far outperforms both TMT methods regarding proteome coverage, as up to threefold more proteins were reproducibly identified in replicate measurements. Furthermore, we could demonstrate that all three methods show comparable reproducibility concerning protein quantification, but slightly differ in terms of accuracy. Here, label-free was found to be less accurate than both TMT approaches. It was also observed that the introduction of TMT labels at the protein level reduces the effect of underestimation of protein ratios, which is commonly monitored in case of TMT peptide labeling. Previously reported differences in protein expression between the particular cell lines were furthermore reproduced, which confirms the applicability of each investigated quantification method to study proteomic differences in such biological systems. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge.     

Recent advances in peptide separation by multidimensional liquid chromatography for proteome analysis

Shotgun proteomics dominates the field of proteomics. The foundations of the strategy consist of multiple rounds of peptide separation where chromatography provides the bedrock. Initially, the scene was relatively simple with the majority of strategies based on some types of ion exchange and reversed phase chromatography. The thirst to achieve comprehensivity, when it comes to proteome coverage and the global characterization of post translational modifications, has led to the introduction of several new separations. In this review, we attempt to provide a historical perspective to separations in proteomics as well as indicate the principles of their operation and rationales for their implementation. Furthermore, we provide a guide on what are the possibilities for combining different separations in order to increase peak capacity and proteome coverage. We aim to show how separations enrich the world of proteomics and how further developments may impact the field.     

肾脏蛋白质组学研究进展

随着现代分子生物学及基因工程技术的发展,蛋白质组学的研究有了长足进步。肾脏疾病作为世界范围内常见的重要疾病。针对肾脏结构、功能、病理生理方面的蛋白质组学研究日益受到重视和深入研究。本文综述了近年来蛋白质组学发展的相关重要新兴技术,以及应用这些技术在肾脏结构、功能、生理病理、分子标记物等各领域的肾脏的蛋白质组学取得的主要研究进展。     

丝状真菌蛋白质组学研究进展

丝状真菌不仅是致病菌,而且在异源表达工业酶、化学制品以及药物活性物质中发挥着越来越重要的作用。随着人类基因组计划的实施和推进,生命科学研究已进入了功能基因组时代,特别是蛋白质组学,在蛋白质水平对丝状真菌细胞生命过程中蛋白质功能和蛋白质之间的相互作用以及特殊条件下的变化机制进行研究,对生命的复杂活动进行深入而又全面的认识也为丝状真菌工业酶制剂和重组药物的开发提供广阔的创新空间。本文综述了蛋白质组学的研究内容和方法,总结了其在丝状真菌致病菌、抗生素产生菌和纤维素酶产生菌中的应用现状。不同层次的功能基因组学分析可以从各个角度掌握生物体的代谢网络和调控机制,本文还对蛋白质组学以及功能基因组学各部分内容的整合运用进行了展望。     

Characterising live cell behaviour: Traditional label-free and quantitative phase imaging approaches

Label-free imaging uses inherent contrast mechanisms within cells to create image contrast without introducing dyes/labels, which may confound results. Quantitative phase imaging is label-free and offers higher content and contrast compared to traditional techniques. High-contrast images facilitate generation of individual cell metrics via more robust segmentation and tracking, enabling formation of a label-free dynamic phenotype describing cell-to-cell heterogeneity and temporal changes. Compared to population-level averages, individual cell-level dynamic phenotypes have greater power to differentiate between cellular responses to treatments, which has clinical relevance e.g. in the treatment of cancer. Furthermore, as the data is obtained label-free, the same cells can be used for further assays or expansion, of potential benefit for the fields of regenerative and personalised medicine.     

寡糖色谱分离研究进展

糖类化合物一直被认为是生物结构的重要组成部分和能量来源。近年来的研究发现,糖类化合物特别是寡糖具有细胞识别等多种生物功能,因此引起了人们日益广泛的关注。寡糖的色谱分离是糖生物学中重要的研究领域之一,小型化和高通量制备可能会成为寡糖色谱分离的发展方向。对寡糖色谱分离方面的最新进展进行综述。     

Recent developments in the application of proteomics to the analysis of plant responses to heavy metals

Pollution of soils by heavy metals is an ever‐growing problem throughout the world, and is the result of human activities as well as geochemical weathering of rocks and other environmental causes such as volcanic eruptions, acid rain and continental dusts. Plants everywhere are continuously exposed to metal‐contaminated soils. The uptake of heavy metals not only constrains crop yields, but can also be a major hazard to the health of humans and to the entire ecosystem. Although analysis of gene expression at the mRNA level has enhanced our understanding of the response of plants to heavy metals, many questions regarding the functional translated portions of plant genomes under metal stress remain unanswered. Proteomics offers a new platform for studying complex biological functions involving large numbers and networks of proteins, and can serve as a key tool for revealing the molecular mechanisms that are involved in interactions between toxic metals and plant species. This review focuses on recent developments in the applications of proteomics to the analysis of the responses of plants to heavy metals; such studies provide a deeper understanding of protein responses and the interactions among the possible pathways that are involved in detoxification of toxic metals in plant cells. In addition, the challenges faced by proteomics in understanding the responses of plants to toxic metal are discussed, and some possible future strategies for meeting these challenges are proposed.     

Label-free quantitative proteomics reveals differentially regulated proteins in the latex of sticky diseased Carica papaya L. plants

Papaya meleira virus (PMeV) is so far the only described laticifer-infecting virus, the causal agent of papaya (Carica papaya L.) sticky disease. The effects of PMeV on the laticifers' regulatory network were addressed here through the proteomic analysis of papaya latex. Using both 1-DE- and 1D-LC-ESI-MS/MS, 160 unique papaya latex proteins were identified, representing 122 new proteins in the latex of this plant. Quantitative analysis by normalized spectral counting revealed 10 down-regulated proteins in the latex of diseased plants, 9 cysteine proteases (chymopapain) and 1 latex serine proteinase inhibitor. A repression of papaya latex proteolytic activity during PMeV infection was hypothesized. This was further confirmed by enzymatic assays that showed a reduction of cysteine-protease-associated proteolytic activity in the diseased papaya latex. These findings are discussed in the context of plant responses against pathogens and may greatly contribute to understand the roles of laticifers in plant stress responses.