Plant proteomics: concepts, applications, and novel strategies for data interpretation

Proteomics is an essential source of information about biological systems because it generates knowledge about the concentrations, interactions, functions, and catalytic activities of proteins, which are the major structural and functional determinants of cells. In the last few years significant technology development has taken place both at the level of data analysis software and mass spectrometry hardware. Conceptual progress in proteomics has made possible the analysis of entire proteomes at previously unprecedented density and accuracy. New concepts have emerged that comprise quantitative analyses of full proteomes, database-independent protein identification strategies, targeted quantitative proteomics approaches with proteotypic peptides and the systematic analysis of an increasing number of posttranslational modifications at high temporal and spatial resolution. Although plant proteomics is making progress, there are still several analytical challenges that await experimental and conceptual solutions. With this review I will highlight the current status of plant proteomics and put it into the context of the aforementioned conceptual progress in the field, illustrate some of the plant-specific challenges and present my view on the great opportunities for plant systems biology offered by proteomics.     

Plant organelle proteomics: Collaborating for optimal cell function

Organelle proteomics describes the study of proteins present in organelle at a particular instance during the whole period of their life cycle in a cell. Organelles are specialized membrane bound structures within a cell that function by interacting with cytosolic and luminal soluble proteins making the protein composition of each organelle dynamic. Depending on organism, the total number of organelles within a cell varies, indicating their evolution with respect to protein number and function. For example, one of the striking differences between plant and animal cells is the plastids in plants. Organelles have their own proteins, and few organelles like mitochondria and chloroplast have their own genome to synthesize proteins for specific function and also require nuclear‐encoded proteins. Enormous work has been performed on animal organelle proteomics. However, plant organelle proteomics has seen limited work mainly due to: (i) inter‐plant and inter‐tissue complexity, (ii) difficulties in isolation of subcellular compartments, and (iii) their enrichment and purity. Despite these concerns, the field of organelle proteomics is growing in plants, such as Arabidopsis, rice and maize. The available data are beginning to help better understand organelles and their distinct and/or overlapping functions in different plant tissues, organs or cell types, and more importantly, how protein components of organelles behave during development and with surrounding environments. Studies on organelles have provided a few good reviews, but none of them are comprehensive. Here, we present a comprehensive review on plant organelle proteomics starting from the significance of organelle in cells, to organelle isolation, to protein identification and to biology and beyond. To put together such a systematic, in‐depth review and to translate acquired knowledge in a proper and adequate form, we join minds to provide discussion and viewpoints on the collaborative nature of organelles in cell, their proper function and evolution. © 2010 Wiley Periodicals, Inc., Mass Spec Rev 30:772–853, 2011     

The role of mass spectrometry in plant systems biology

Large-scale analyses of proteins and metabolites are intimately bound to advancements in MS technologies. The aim of these non-targeted "omic" technologies is to extend our understanding beyond the analysis of only parts of the system. Here, metabolomics and proteomics emerged in parallel with the development of novel mass analyzers and hyphenated techniques such as gas chromatography coupled to time-of-flight mass spectrometry (GC-TOF-MS) and multidimensional liquid chromatography coupled to mass spectrometry (LC-MS). The analysis of (i) proteins (ii) phosphoproteins, and (iii) metabolites is discussed in the context of plant physiology and environment and with a focus on novel method developments. Recently published studies measuring dynamic (quantitative) behavior at these levels are summarized; for these works, the completely sequenced plants Arabidopsis thaliana and Oryza sativa (rice) have been the primary models of choice. Particular emphasis is given to key physiological processes such as metabolism, development, stress, and defense. Moreover, attempts to combine spatial, tissue-specific resolution with systematic profiling are described. Finally, we summarize the initial steps to characterize the molecular plant phenotype as a corollary of environment and genotype.     

Prospective highlights of functional skin proteomics

Although a wide variety of protein profiles have been extensively constructed via proteomic analysis, the comprehensive proteomic profiling of the skin, which is considered to be the largest organ of the human body, is still far from complete. Our efforts to establish the functional skin proteome, a protein database describing the protein networks that underlie biological processes, has set in motion the identification and characterization of proteins expressed in the epidermis and dermis of the BALB/c mice. In this review, we will highlight various cutaneous proteins we have characterized and discuss their biological functions associated with skin distress, immunity, and cancer. This type of research into functional skin proteomics will provide a critical step toward understanding disease and developing successful therapeutic strategies.     

基于质谱的蛋白质组学方法新进展

蛋白质组学是后基因组时代的科研热点,其研究方向主要涵盖蛋白质表达谱、蛋白质翻译后修饰谱、蛋白质相互作用谱和单细胞蛋白质组等定性分析以及相对和绝对定量分析,现已广泛应用于生物、医药及病理研究中。由于质谱具有灵敏、准确、高通量等特点,是蛋白质组学研究必不可少的工具。本文综述了近年来基于质谱技术的蛋白质组鉴定及定量方法,并展望未来蛋白质组学研究的发展方向。     

Mass spectrometry in the proteome analysis of mature cereal kernels

In the last decade, the improved performance and versatility of the mass spectrometers together with the increasing availability of gene and genomic sequence database, led the mass spectrometry to become an indispensable tool for either protein and proteome analyses in cereals. Mass spectrometric works on prolamins have rapidly evolved from the determination of the molecular masses of proteins to the proteomic approaches aimed to a large‐scale protein identification and study of functional and regulatory aspects of proteins. Mass spectrometry coupled with electrophoresis, chromatographic methods, and bioinformatics tools is currently making significant contributions to a better knowledge of the composition and structure of the cereal proteins and their structure–function relationships. Results obtained using mass spectrometry, including characterization of prolamins, investigation of the gluten toxicity for coeliac patients, identification of proteins responsible of cereal allergies, determination of the protein pattern and its modification under environmental or stress effects, investigation of genetically modified varieties by proteomic approaches, are summarized here, to illustrate current trends, analytical troubles and challenges, and suggest possible future perspectives. © 2011 Wiley Periodicals, Inc. Mass Spec Rev 31:448–465, 2012     

Interphase chromosomes and the Rabl configuration: does genome size matter?

It is now well established that the cereals share a common gene order or gene synteny. However, the cereal species encompass an enormous range of genome size, with wheat being one of the largest and rice one of the smallest. Here we describe the current state of knowledge of interphase chromosome structure within the cereal species. In wheat and its close relatives, the interphase chromosomes adopt a highly regular Rabl configuration, with the two chromosome arms lying next to each other and the centromeres and telomeres located at opposite poles of the nuclei. By contrast, the chromosomes in most rice nuclei clearly do not show a Rabl configuration. Surprisingly, the chromosomes in the endoreduplicated xylem vessel cells of rice do adopt a Rabl configuration. To explain this observation, we propose that endoreduplication may occur immediately after chromosome segregation in these cells, and that the new chromatin interactions, particularly at the centromeres, in the endoreduplicated chromosomes may stabilize the anaphase chromosome configuration.     

Expression analysis of <i>OsSERK</i>, <i>OsLEC1</i> and <i>OsWOX4</i> genes in rice (<i>Oryza</i> sativa L.) callus during somatic embryo development

Somatic embryogenesis is an asexual reproduction process that occurs in many plant species, including rice. This process contains several totipotency markers such as Somatic Embryogenesis Receptor-like Kinase (SERK), Leafy Cotyledon1 (LEC1) and WUSCHEL-Related Homeobox4 (WOX4) and also a helpful model for embryo development and clones and transformations. Here, we report the gene expression during somatic embryo development correlates with regeneration frequency in 14 Javanica rice (pigmented and non-pigmented) using modifified N6 media supplemented with Kinetin (2.0 mg/L) and NAA (1.0 mg/L). Although there have been advances in understanding the genetic basis of somatic embryogenesis in other varieties, rice is still unexplored, especially during somatic embryo development. Moreover, for the formation of callus induction from immature embryos, 2,4-D (2.0 mg/L, 3.0 mg/L) was used. This study analysed the gene expression of OsSERK, OsWOX4 and OsLEC1 genes through RT-PCR analysis. Higher expression of the OsLEC1 gene indicates that their function may correlate in the in vitro with the high response of rice after transfer to regeneration media. This study found that rice varieties of pigmented rice (MS Pendek and Gogoniti II) and non-pigmented rice (Pandan Ungu) showed high regeneration frequency, showing higher OsLEC1 expression than other varieties because OsLEC1 promotes the maturation of somatic embryos in plant regeneration on day 14. However, the contrast with Genjah nganjuk may be effective because of other regulatory genes. RT-PCR analysis showed OsSERK had less expression level than OsLEC1 and OsWOX4 in the varieties, which correlate with the percentage of plant regeneration, but not for Gogoniti II. In conclusion, the higher percentage of plant regeneration correlates with the higher expression level of OsLEC1 at day 14 of media regeneration of rice.     

Intact-protein based sample preparation strategies for proteome analysis in combination with mass spectrometry

The complexity of tissue and cell proteomes and the vast dynamic range of protein abundance present a formidable challenge for analysis that no one analytical technique can overcome. As a result, there is a need to integrate technologies to achieve the high-resolution and high-sensitivity analysis of complex biological samples. The combined technologies of separation science and biological mass spectrometry (Bio-MS) are the current workhorse in proteomics, and are continuing to evolve to meet the needs for high sensitivity and high throughput. They are relied upon for protein quantification, identification, and analysis of post-translational modifications (PTMs). The standard technique of two dimensional poly-acrylamide gel electrophoresis (2D PAGE) offers relatively limited resolution and sensitivity for the simultaneous analysis of all cellular proteins, with only the most highly abundant proteins detectable in whole cell or tissue-derived samples. Hence, many alternative strategies are being explored. Numerous sample preparation procedures are currently available to reduce sample complexity and to increase the detectability of low-abundance proteins. Maintaining proteins intact during sample preparation has important advantages compared with strategies that digest proteins at an early step. These strategies include the ability to quantitate and recover proteins, and the assessment of PTMs. A review of current intact protein-based strategies for protein sample preparation prior to mass spectrometry (MS) is presented in the context of biomedically driven applications.     

Capillary electrophoresis-mass spectrometry as a powerful tool in biomarker discovery and clinical diagnosis: an update of recent developments

Proteome analysis has emerged as a powerful technology to decipher biological processes. One of the main goals is to discover biomarkers for diseases from tissues and body fluids. However, the complexity and wide dynamic range of protein expression present an enormous challenge to separation technologies and mass spectrometry (MS). In this review, we examine the limitations of proteomics, and aim towards the definition of the current key prerequisites. We focus on capillary electrophoresis coupled to mass spectrometry (CE-MS), because this technique continues to show great promise. We discuss CE-MS from an application point of view, and evaluate its merits and vices for biomarker discovery and clinical applications. Finally, we present several examples on the use of CE-MS to determine urinary biomarkers and implications for disease diagnosis, prognosis, and therapy evaluation.     

药物免修饰的药靶鉴定蛋白质组方法研究进展

药物靶蛋白的筛选是现代药物研发过程必不可少的步骤。传统的药物靶点筛选通常使用假设驱动的生物学实验进行药物靶点的逐个验证，近年来高通量、高分辨质谱技术的进步以及液相色谱-质谱联用技术的发展，促进了蛋白质组学技术在药物靶蛋白筛选领域的广泛应用。化学蛋白质组学技术在药物-蛋白相互作用研究领域已取得巨大突破，因其需要在药物小分子结构上进行化学衍生等问题，最近新兴的药物免修饰的靶蛋白筛选技术引起广泛关注。本文综述了近15年来药物免修饰（非化学修饰）的药物靶蛋白筛选方法的研究进展，包括细胞热位移分析（CETSA）、热蛋白质组分析（TPP）、蛋白质氧化速率稳定性分析（SPROX）、基于有机溶剂沉淀蛋白稳定性方法（SIP）、药物亲和相应靶点稳定性分析（DARTS）、脉冲酶解法（PP）、限制性酶解法（LiP）等，并比较了这些方法的优势、局限性以及应用场景。     

Mass spectrometry-based clinical proteomics: head-and-neck cancer biomarkers and drug-targets discovery

Mass spectrometry (MS)-based proteomics is a rapidly developing technology for both qualitative and quantitative analyses of proteins, and investigations into protein posttranslational modifications, subcellular localization, and interactions. Recent advancements in MS have made tremendous impact on the throughput and comprehensiveness of cancer proteomics, paving the way to unraveling deregulated cellular pathway networks in human malignancies. In turn, this knowledge is rapidly being translated into the discovery of novel potential cancer markers (PCMs) and targets for molecular therapeutics. Head-and-neck cancer is one of the most morbid human malignancies with an overall poor prognosis and severely compromised quality of life. Early detection and novel therapeutic strategies are urgently needed for more effective disease management. The characterizations of protein profiles of head-and-neck cancers and non-malignant tissues, with unprecedented sensitivity and precision, are providing technology platforms for identification of novel PCMs and drug targets. Importantly, low-abundance proteins are being identified and characterized, not only from the tumor tissues, but also from bodily fluids (plasma, saliva, and urine) in a high-throughput and unbiased manner. This review is a critical appraisal of recent advances in MS-based proteomic technologies and platforms for facilitating the discovery of biomarkers and novel drug targets in head-and-neck cancer. A major challenge in the discovery and verification of these cancer biomarkers is the typically limited availability of well-characterized and adequately stored clinical samples in tumor and sera banks, collected using recommended procedures, and with detailed information on clinical, pathological parameters, and follow-up. Most biomarker discovery studies use limited number of clinical samples and verification of cancer markers in large number of samples is beyond the scope of a single laboratory. The validation of these potential markers in large sample cohorts in multicentric studies is needed for their translation from the bench to the bedside.     

Mass spectrometry‐based proteomics: The road to lung cancer biomarker discovery

Lung cancer is the leading cause of cancer death in men and women in Western nations, and is among the deadliest cancers with a 5‐year survival rate of 15%. The high mortality caused by lung cancer is attributable to a late‐stage diagnosis and the lack of effective treatments. So, it is crucial to identify new biomarkers that could function not only to detect lung cancer at an early stage but also to shed light on the molecular mechanisms that underlie cancer development and serve as the basis for the development of novel therapeutic strategies. Considering that DNA‐based biomarkers for lung cancer showed inadequate sensitivity, specificity, and reproducibility, proteomics could represent a better tool for the identification of useful biomarkers and therapeutic targets for this cancer type. Among the proteomics technologies, the most powerful tool is mass spectrometry. In this review, we describe studies that use mass spectrometry‐based proteomics technologies to analyze tumor proteins and peptides, which might represent new diagnostic, prognostic, and predictive markers for lung cancer. We focus in particular on those findings that hold promise to impact significantly on the clinical management of this disease. © 2012 Wiley Periodicals, Inc., Mass Spec Rev 32:129–142, 2013