State-of-the-art in phosphoproteomics

Presently, phosphorylation of proteins is the most studied and best understood PTM. However, the analysis of phosphoproteins and phosphopeptides is still one of the most challenging tasks in contemporary proteome research. Since not every phosphoprotein is accessible by a certain method and identification of the phosphorylated amino acid residue is required in the majority of cases, various strategies for the detection and localization of phosphorylations have been developed. Identification and localization of protein phosphorylations is mostly done by MS nowadays but phosphoproteins and -peptides are often suppressed in comparison to the unphosphorylated species if measured in complex mixtures. Thus, the isolation of pure phosphopeptide samples is a main task. This review gives an overview over the most frequently used methods in isolation and detection of phosphoproteins and -peptides such as specific enrichment or separation strategies as well as the localization of the phosphorylated residues by various mass spectrometric techniques.     

Analytical strategies for phosphoproteomics

Protein phosphorylation is a key regulator of cellular signaling pathways. It is involved in most cellular events in which the complex interplay between protein kinases and protein phosphatases strictly controls biological processes such as proliferation, differentiation, and apoptosis. Defective or altered signaling pathways often result in abnormalities leading to various diseases, emphasizing the importance of understanding protein phosphorylation. Phosphorylation is a transient modification, and phosphoproteins are often very low abundant. Consequently, phosphoproteome analysis requires highly sensitive and specific strategies. Today, most phosphoproteomic studies are conducted by mass spectrometric strategies in combination with phospho‐specific enrichment methods. This review presents an overview of different analytical strategies for the characterization of phosphoproteins. Emphasis will be on the affinity methods utilized specifically for phosphoprotein and phosphopeptide enrichment prior to MS analysis, and on recent applications of these methods in cell biological applications.     

磷酸化蛋白质组学研究中磷酸化肽段富集与分离方法研究进展

对磷酸化蛋白质组（phosphoproteome）进行系统深入的研究依赖于高重复性和特异性的磷酸化肽段富集与分离方法。目前发展了多种不同原理的磷酸化肽段富集方法,它们往往具有不同的选择性和特异性,因此,根据不同的研究目的选择最适合的富集方法显得尤为重要。本文综述了基于亲和色谱法（affinity chromatography）、免疫沉淀法（immunoprecipitation）、化学衍生法（chemical derivatization）、色谱法（chromatography）和其他新发展方法的磷酸化肽段富集方法,详细介绍了各自的优缺点及相关的优化与改进策略。此外,还简单介绍了磷酸化肽段富集与预分方法的不同组合的研究进展。     

Quantitative phosphoproteomics strategies for understanding protein kinase-mediated signal transduction pathways

Protein phosphorylation is a central regulatory mechanism of cell signaling pathways. This highly controlled biochemical process is involved in most cellular functions, and defects in protein kinases and phosphatases have been implicated in many diseases, highlighting the importance of understanding phosphorylation-mediated signaling networks. However, phosphorylation is a transient modification, and phosphorylated proteins are often less abundant. Therefore, the large-scale identification and quantification of phosphoproteins and their phosphorylation sites under different conditions are one of the most interesting and challenging tasks in the field of proteomics. Both 2D gel electrophoresis and liquid chromatography-tandem mass spectrometry serve as key phosphoproteomic technologies in combination with prefractionation, such as enrichment of phosphorylated proteins/peptides. Recently, new possibilities for quantitative phosphoproteomic analysis have been offered by technical advances in sample preparation, enrichment, separation, instrumentation, quantification and informatics. In this article, we present an overview of several strategies for quantitative phosphoproteomics and discuss how phosphoproteomic analysis can help to elucidate signaling pathways that regulate various cellular processes.     

Quantitative phosphoproteomics strategies for understanding protein kinase-mediated signal transduction pathways

Protein phosphorylation is a central regulatory mechanism of cell signaling pathways. This highly controlled biochemical process is involved in most cellular functions, and defects in protein kinases and phosphatases have been implicated in many diseases, highlighting the importance of understanding phosphorylation-mediated signaling networks. However, phosphorylation is a transient modification, and phosphorylated proteins are often less abundant. Therefore, the large-scale identification and quantification of phosphoproteins and their phosphorylation sites under different conditions are one of the most interesting and challenging tasks in the field of proteomics. Both 2D gel electrophoresis and liquid chromatography-tandem mass spectrometry serve as key phosphoproteomic technologies in combination with prefractionation, such as enrichment of phosphorylated proteins/peptides. Recently, new possibilities for quantitative phosphoproteomic analysis have been offered by technical advances in sample preparation, enrichment, separation, instrumentation, quantification and informatics. In this article, we present an overview of several strategies for quantitative phosphoproteomics and discuss how phosphoproteomic analysis can help to elucidate signaling pathways that regulate various cellular processes.     

磷酸化蛋白质组研究中的富集策略

蛋白质的磷酸化与去磷酸化过程,调控着包括信号转换、基因表达、细胞周期等诸多细胞过程。因此,对蛋白质磷酸化修饰的分析是蛋白质组研究中的重要内容。但由于磷酸化蛋白的丰度较低,难以用质谱直接检测。为了解决这个问题,改善质谱对磷酸肽的信号响应,需要对磷酸化蛋白质或磷酸肽进行富集。目前主要的富集方法包括免疫沉淀、固相金属离子亲和色谱、金属氧化物/氢氧化物亲和色谱等。     

基于BP神经网络的京津冀城市群可持续发展综合评价

在综合分析了京津冀城市群各城市功能定位的基础上,构建了包含经济发展、社会发展、科技创新和生态环境4个子系统的城市可持续发展评价指标体系,运用2006—2015年的数据,采用熵值法和BP神经网络对京津冀城市群可持续发展能力进行非线性测度与分类,结果较为理想。结果表明:(1)北京和天津处于高可持续发展水平,可持续发展能力在空间上呈现出以京、津为中心随距离递减的趋势,最南端的邯郸和邢台处于低可持续发展水平;(2)北京可持续发展能力呈现下滑趋势,其他城市可持续发展能力逐年稳步上升,大城市可持续发展压力较大;(3)城市在不同子系统中存在各自的优劣势。各个子系统在可持续发展中均起到重要作用,城市宜结合各自子系统的优、劣势制定具有针对性的发展对策。     

Phosphoprotein analysis: from proteins to proteomes

Characterization of protein modification by phosphorylation is one of the major tasks that have to be accomplished in the post-genomic era. Phosphorylation is a key reversible modification occurring mainly on serine, threonine and tyrosine residues that can regulate enzymatic activity, subcellular localization, complex formation and degradation of proteins. The understanding of the regulatory role played by phosphorylation begins with the discovery and identification of phosphoproteins and then by determining how, where and when these phosphorylation events take place. Because phosphorylation is a dynamic process difficult to quantify, we must at first acquire an inventory of phosphoproteins and characterize their phosphorylation sites. Several experimental strategies can be used to explore the phosphorylation status of proteins from individual moieties to phosphoproteomes. In this review, we will examine and catalogue how proteomics techniques can be used to answer specific questions related to protein phosphorylation. Hence, we will discuss the different methods for enrichment of phospho-proteins and -peptides, and then the various technologies for their identification, quantitation and validation.     

Isolation of the Arabidopsis phosphoproteome using a biotin-tagging approach

Protein phosphorylation plays a key role in signal transduction in cells. Since phosphoproteins are present in low abundance, enrichment methods are required for their purification and analysis. Chemical derivatization strategies have been devised for enriching phosphoproteins and phosphopeptides. In this report, we employed a strategy that replaces the phosphate moieties on serine and threonine residues with a biotin-containing tag via a series of chemical reactions. Ribulose 1,5-bis-phosphate carboxylase/oxygenase (RUBISCO)-depleted protein extracts prepared from Arabidopsis seedlings were chemically modified for 'biotin-tagging'. The biotinylated (previously phosphorylated) proteins were then selectively isolated by avidin-biotin affinity chromatography, followed by two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). This led to the identification of 31 protein spots, representing 18 different proteins, which are implicated in a variety of cellular processes. Despite its current technical limitations, with further improvements in tools and techniques this strategy may be developed into a useful approach.     

A survey of the Arabidopsis thaliana mitochondrial phosphoproteome

Plant mitochondria play central roles in cellular energy production, metabolism and stress responses. Recent phosphoproteomic studies in mammalian and yeast mitochondria have presented evidence indicating that protein phosphorylation is a likely regulatory mechanism across a broad range of important mitochondrial processes. This study investigated protein phosphorylation in purified mitochondria from cell suspensions of the model plant Arabidopsis thaliana using affinity enrichment and proteomic tools. Eighteen putative phosphoproteins consisting of mitochondrial metabolic enzymes, HSPs, a protease and several proteins of unknown function were detected on 2‐DE separations of Arabidopsis mitochondrial proteins and affinity‐enriched phosphoproteins using the Pro‐Q Diamond phospho‐specific in‐gel dye. Comparisons with mitochondrial phosphoproteomes of yeast and mouse indicate that these three species share few validated phosphoproteins. Phosphorylation sites for seven of the eighteen mitochondrial proteins were characterized by titanium dioxide enrichment and MS/MS. In the process, 71 phosphopeptides from Arabidopsis proteins which are not present in mitochondria but found as contaminants in various types of mitochondrial preparations were also identified, indicating the low level of phosphorylation of mitochondrial components compared with other cellular components in Arabidopsis. Information gained from this study provides a better understanding of protein phosphorylation at both the subcellular and the cellular level in Arabidopsis.     

Phosphoproteins analysis in plants: a proteomic approach

The study of phosphoproteome on a global scale represents one of the challenges in the post-genomic era. Here, we propose an integrated procedure starting from the crude protein extract, that consists of sequential purification steps, and ending up in the identification of phosphorylation sites. This involves (i) an enrichment in phosphoproteins with a commercially available chromatography matrix, (ii) a 2-D gel analysis of the enriched fraction followed by the selective staining with the phosphospecific fluorescent dye Pro-Q Diamond, (iii) a phosphopeptide capture, from the tryptic lysate of 2-D spots, using IMAC micro-columns. In the end, the identification of the phosphoproteins and their corresponding phosphorylation sites were achieved by MALDI-TOF-TOF spectrometry. The method was applied to contrasting samples prepared from cell suspension cultures of Arabidopsis thaliana and roots of Medicago truncatula. The results obtained, demonstrated the robustness of the combination of two enrichment stages, sequentially at the protein and at the peptide levels, to analyse phosphoproteins in plants.     

A Rapid Screening Assay to Search for Phosphorylated Proteins in Tissue Extracts

Reversible protein phosphorylation is an essential mechanism in the regulation of diverse biological processes, nonetheless is frequently altered in disease. As most phosphoproteome studies are based on optimized in-vitro cell culture studies new methods are in need to improve de novo identification and characterization of phosphoproteins in extracts from tissues. Here, we describe a rapid and reliable method for the detection of phosphoproteins in tissue extract based on an experimental strategy that employs 1D and 2D SDS PAGE, Western immunoblotting of phosphoproteins, in-gel protease digestion and enrichment of phosphorpeptides using metal oxide affinity chromatography (MOAC). Subsequently, phosphoproteins are identified by MALDI-TOF-MS/MS with the CHCA-TL or DHB ML sample matrix preparation method and further characterized by various bioinformatic software tools to search for candidate kinases and phosphorylation-dependent binding motifs. The method was applied to mouse lung tissue extracts and resulted in an identification of 160 unique phosphoproteins. Notably, TiO₂ enrichment of pulmonary protein extracts resulted in an identification of additional 17 phosphoproteins and 20 phosphorylation sites. By use of MOAC, new phosphorylation sites were identified as evidenced for the advanced glycosylation end product-specific receptor. So far this protein was unknown to be phosphorylated in lung tissue of mice. Overall the developed methodology allowed efficient and rapid screening of phosphorylated proteins and can be employed as a general experimental strategy for an identification of phosphoproteins in tissue extracts.     

Plant phosphoproteomics: a long road ahead

Phosphoproteomics can be defined as the comprehensive study of protein phosphorylation by identification of the phosphoproteins, exact mapping of the phosphorylation sites, quantification of phosphorylation, and eventually, revealing their biological function. Its place in today's research is vitally important to address the most fundamental question - how the phosphorylation events control most, if not all, of the cellular processes in a given organism? Despite the immense importance of phosphorylation, the analysis of phosphoproteins on a proteome-wide scale remains a formidable challenge. Nevertheless, several technologies have been developed, mostly in yeast and mammals, to conduct a large-scale phosphoproteomic study. Some of these technologies have been successfully applied to plants with a few modifications, resulting in documentation of phosphoproteins, phosphorylation site mapping, identification of protein kinase substrates, etc. at the global level. In this review, we summarize in vitro and in vivo approaches for detection and analysis of phosphoproteins including protein kinases and we discuss the importance of phosphoproteomics in understanding plant biology. These approaches along with bioinformatics will help plant researchers to design and apply suitable phosphoproteomic strategies in helping to find answers to their biological questions.     

Phosphoproteomics strategies for the functional analysis of signal transduction

Protein phosphorylation is a key regulatory mechanism of cellular signalling processes. The analysis of phosphorylated proteins and the characterisation of phosphorylation sites under different biological conditions are some of the most challenging tasks in current proteomics research. Reduction of the sample complexity is one major step for the analysis of low-abundance kinase substrates, which can be achieved by various subcellular fractionation techniques. One strategy is the enrichment of phosphorylated proteins or peptides by immunoprecipitation or chromatography, e.g. immobilised metal affinity chromatography, prior to analysis. 2-DE gels are powerful tools for the analysis of phosphoproteins when combined with new multiplexing techniques like DIGE, phosphospecific stains, autoradiography or immunoblotting. In addition, several gel-free methods combining chromatography with highly sensitive MS have been successfully applied for the analysis of complex phosphoproteomes. Recently developed approaches like KESTREL or 'chemical genetics' and also protein microarrays offer new possibilities for the identification of specific kinase targets. This review summarises various strategies for the analyses of phosphoproteins with a special focus on the identification of novel kinase substrates.     

Exome localization of complex disease association signals

Background

Recent progress in high-throughput technologies has greatly contributed to the development of DNA methylation profiling. Although there are several reports that describe methylome detection of whole genome bisulfite sequencing, the high cost and heavy demand on bioinformatics analysis prevents its extensive application. Thus, current strategies for the study of mammalian DNA methylomes is still based primarily on genome-wide methylated DNA enrichment combined with DNA microarray detection or sequencing. Methylated DNA enrichment is a key step in a microarray based genome-wide methylation profiling study, and even for future high-throughput sequencing based methylome analysis.

Results

In order to evaluate the sensitivity and accuracy of methylated DNA enrichment, we investigated and optimized a number of important parameters to improve the performance of several enrichment assays, including differential methylation hybridization (DMH), microarray-based methylation assessment of single samples (MMASS), and methylated DNA immunoprecipitation (MeDIP). With advantages and disadvantages unique to each approach, we found that assays based on methylation-sensitive enzyme digestion and those based on immunoprecipitation detected different methylated DNA fragments, indicating that they are complementary in their relative ability to detect methylation differences.

Conclusions

Our study provides the first comprehensive evaluation for widely used methodologies for methylated DNA enrichment, and could be helpful for developing a cost effective approach for DNA methylation profiling.     

Enrichment of phosphorylated proteins from cell lysate using a novel phosphate-affinity chromatography at physiological pH

While phosphoproteins have attracted great interest toward the post-genome research (e.g. clinical diagnosis and drug design), there have been few procedures for the specific enrichment of native phosphoproteins from cells or tissues. Here, we describe a simple and efficient protocol to enrich phosphoproteins comprehensively from a complex mixture containing solubilized cellular proteins. This method is based on immobilized metal affinity chromatography using a phosphate-binding tag molecule (i.e. a dinuclear zinc(II) complex) attached on a highly cross-linked agarose. The binding, washing, and elution processes were all conducted without a detergent or a reducing agent at pH 7.5 and room temperature. An additive, 1.0 M CH3COONa, was necessary in the binding and washing buffers (0.10 M Tris-CH3COOH, pH 7.5) to prevent the nonphosphorylated protein from binding. The absorbed phosphoproteins were eluted using a mixed buffer solution (pH 7.5) consisting of 0.10 M Tris-CH3COOH, 10 mM NaH2PO4-NaOH, and 1.0 M NaCl. In this study, we demonstrate a typical example of phosphate-affinity chromatography using an epidermal growth factor-stimulated A431 cell lysate. The total time for the column chromatography (1 mL gel scale) was less than 1 h. The strong enrichment of the phosphoproteins into the elution fraction was evaluated using SDS-PAGE followed by Western blotting analysis.     

Immobilized metal affinity chromatography revisited: pH/acid control toward high selectivity in phosphoproteomics

Despite recent advances in instrumentation and analytical strategies for identification and quantitation of protein phosphorylation, a highly specific enrichment protocol is still a challenge in large-scale studies. Here, we report a simple pH/acid control method that addresses the poor specificity seriously criticized in IMAC. Detailed evaluation of the capture and release mechanism in IMAC revealed that pH, buffer and salt yield a complex interplay in enrichment of phosphopeptides, yet they play individual roles in recovery and specificity. A revised one-step IMAC method with low sample loss and high specificity can be rationally designed by controlling salt, pH and the structure and concentration of organic acid. Without methyl esterification, the one-step IMAC enrichment with single LC-MS/MS identified 386 phosphoproteins in 550 mug of non-small-cell lung cancer cell lysate with 96% specificity. Additional fractionation by SDS-PAGE from 4 mg of cell lysate revealed the comprehensive proteome map, identifying 2747 phosphorylation sites from 2360 nondegenerate phosphopeptides and 1219 phosphoproteins with a false discovery rate of 0.63%. To our knowledge, this pH/acid-controlled IMAC procedure provides higher specificity than any other one-step IMAC purification procedure. Furthermore, the simple and reproducible IMAC protocol can be adapted to other solid supports, fully automated or manual, for large-scale identification of the vastly under-explored phosphoproteome.     

Affinity separation and enrichment methods in proteomic analysis

Protein separation or enrichment is one of the rate-limiting steps in proteomic studies. Specific capture and removal of highly-abundant proteins (HAP) with large sample-handling capacities are in great demand for enabling detection and analysis of low-abundant proteins (LAP). How to grasp and enrich these specific proteins or LAP in complex protein mixtures is also an outstanding challenge for biomarker discovery and validation. In response to these needs, various approaches for removal of HAP or capture of LAP in biological fluids, particularly in plasma or serum, have been developed. Among them, immunoaffinity subtraction methods based upon polyclonal IgY or IgG antibodies have shown to possess unique advantages for proteomic analysis of plasma, serum and other biological samples. In addition, other affinity methods that use recombinant proteins, lectins, peptides, or chemical ligands have also been developed and applied to LAP capture or enrichment. This review discusses in detail the need to put technologies and methods in affinity subtraction or enrichment into a context of proteomic and systems biology as "Separomics" and provides a prospective of affinity-mediated proteomics. Specific products, along with their features, advantages, and disadvantages will also be discussed.     

Phosphoproteomic analysis of the mouse brain cytosol reveals a predominance of protein phosphorylation in regions of intrinsic sequence disorder

We analyzed the mouse forebrain cytosolic phosphoproteome using sequential (protein and peptide) IMAC purifications, enzymatic dephosphorylation, and targeted tandem mass spectrometry analysis strategies. In total, using complementary phosphoenrichment and LC-MS/MS strategies, 512 phosphorylation sites on 540 non-redundant phosphopeptides from 162 cytosolic phosphoproteins were characterized. Analysis of protein domains and amino acid sequence composition of this data set of cytosolic phosphoproteins revealed that it is significantly enriched in intrinsic sequence disorder, and this enrichment is associated with both cellular location and phosphorylation status. The majority of phosphorylation sites found by MS were located outside of structural protein domains (97%) but were mostly located in regions of intrinsic sequence disorder (86%). 368 phosphorylation sites were located in long regions of disorder (over 40 amino acids long), and 94% of proteins contained at least one such long region of disorder. In addition, we found that 58 phosphorylation sites in this data set occur in 14-3-3 binding consensus motifs, linear motifs that are associated with unstructured regions in proteins. These results demonstrate that in this data set protein phosphorylation is significantly depleted in protein domains and significantly enriched in disordered protein sequences and that enrichment of intrinsic sequence disorder may be a common feature of phosphoproteomes. This supports the hypothesis that disordered regions in proteins allow kinases, phosphatases, and phosphorylation-dependent binding proteins to gain access to target sequences to regulate local protein conformation and activity.