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.     

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.     

Quantitative phosphoproteomics

Cells are highly responsive to their environment. One of the main strategies used by cells in signal transduction is protein phosphorylation, a reversible modification that regulates numerous biological processes. Misregulation of phosphorylation-mediated processes is often implicated in many human diseases and cancers. A global and quantitative analysis of protein phosphorylation provides a powerful new approach and has the potential to reveal new insights in signaling pathways. Recent technological advances in high resolution mass spectrometers and multidimensional liquid chromatography, combined with the use of stable isotope labeling of proteins, have led to the application of quantitative phosphoproteomics to study in vivo signal transduction events on a proteome-wide scale. Here we review recent advancements in quantitative phosphoproteomic technologies, discuss their potentials, and identify areas for future development. A key objective of proteomic technology is its application to addressing biological questions. We will therefore describe how current quantitative phosphoproteomic technology can be used to study the molecular basis of phosphorylation events in the DNA damage response.     

Quantitative phosphoproteomics: New technologies and applications in the DNA damage response

Cells are highly responsive to their environment. One of the main strategies used by cells in signal transduction is protein phosphorylation, a reversible modification that regulates numerous biological processes. Misregulation of phosphorylation-mediated processes is often implicated in many human diseases and cancers. A global and quantitative analysis of protein phosphorylation provides a powerful new approach and has the potential to reveal new insights in signaling pathways. Recent technological advances in high resolution mass spectrometers and multidimensional liquid chromatography, combined with the use of stable isotope labeling of proteins, have led to the application of quantitative phosphoproteomics to study in vivo signal transduction events on a proteome-wide scale. Here we review recent advancements in quantitative phosphoproteomic technologies, discuss their potentials and identify areas for future development. A key objective of proteomic technology is its application to addressing biological questions. We will therefore describe how current quantitative phosphoproteomic technology can be used to study the molecular basis of phosphorylation events in the DNA damage response.Key words: proteomics, mass spectrometry, DNA damage response, phosphorylation, HILIC, SILAC     

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.     

Toward a hypothesis-free understanding of how phosphorylation dynamically impacts protein turnover

The turnover measurement of proteins and proteoforms has been largely facilitated by workflows coupling metabolic labeling with mass spectrometry (MS), including dynamic stable isotope labeling by amino acids in cell culture (dynamic SILAC) or pulsed SILAC (pSILAC). Very recent studies including ours have integrated themeasurement of post-translational modifications (PTMs) at the proteome level (i.e., phosphoproteomics) with pSILAC experiments in steady state systems, exploring the link between PTMs and turnover at the proteome-scale. An open question in the field is how to exactly interpret these complex datasets in a biological perspective. Here, we present a novel pSILAC phosphoproteomic dataset which was obtained during a dynamic process of cell starvation using data-independent acquisition MS (DIA-MS). To provide an unbiased “hypothesis-free” analysis framework, we developed a strategy to interrogate how phosphorylation dynamically impacts protein turnover across the time series data. With this strategy, we discovered a complex relationship between phosphorylation and protein turnover that was previously underexplored. Our results further revealed a link between phosphorylation stoichiometry with the turnover of phosphorylated peptidoforms. Moreover, our results suggested that phosphoproteomic turnover diversity cannot directly explain the abundance regulation of phosphorylation during cell starvation, underscoring the importance of future studies addressing PTM site-resolved protein turnover.     

Proteomic analysis of phosphorylation in cancer

Constitutive activity of kinases is known to be crucial for a tumor to maintain its malignant phenotype, a phenomenon which is often referred to as oncogene addiction. The in-depth analysis of aberrant signaling pathways by the analysis of protein phosphorylation has become feasible through recent advances in proteomics technology. In this article we will review developments in the field of phosphoproteomics and its application in cancer research. The most widely used technologies for the generic enrichment of phosphopeptides are discussed as well as targeted approaches for the analysis of a specific subset of phosphopeptides. Validation experiments of phosphorylation sites using targeted mass spectrometry are also explained. Finally, we will highlight applications of phosphoproteomic technology in cancer research using cell lines and tissue.     

Regulation of Protein Kinase C function by phosphorylation on conserved and non-conserved sites

Protein Kinase C (PKC) is a family of serine/threonine kinases whose function is influenced by phosphorylation. In particular, three conserved phosphorylation sites known as the activation-loop, the turn-motif and the hydrophobic-motif play important roles in controlling the catalytic activity, stability and intracellular localisation of the enzyme. Prevailing models of PKC phosphorylation suggest that phosphorylation of these sites occurs shortly following synthesis and that these modifications are required for the processing of newly-transcribed PKC to the mature (but still inactive) form; phosphorylation is therefore a priming event that enables catalytic activation in response to lipid second messengers. However, many studies have also demonstrated inducible phosphorylation of PKC isoforms at these sites following stimulation, highlighting that our understanding of PKC phosphorylation and its impact on enzymatic function is incomplete. Furthermore, inducible phosphorylation at these sites is often interpreted as catalytic activation, which could be misleading for some isoforms. Recent studies that include systems-wide phosphoproteomic profiling of cells has revealed a host of additional (and in many cases non-conserved) phosphorylation sites on PKC family members that influence their function. Many of these may in fact be more suitable than previously described sites as surrogate markers of catalytic activation. Here we discuss the role of phosphorylation in controlling PKC function and outline our current understanding of the mechanisms that regulate these phosphorylation sites.     

Strategies for quantitation of phosphoproteomic data

Recent developments in phosphoproteomic sample-preparation techniques and sensitive mass spectrometry instrumentation have led to large-scale identifications of phosphoproteins and phosphorylation sites from highly complex samples. This has facilitated the implementation of different quantitation strategies in order to study the biological role of protein phosphorylation during disease progression, differentiation or during external stimulation of a cellular system. In this article, a brief summary of the most popular strategies for phosphoproteomic studies is given; however, the main focus will be on different quantitation strategies. Methods for metabolic labeling, chemical modification and label-free quantitation and their applicability or inapplicability in phosphoproteomic studies are discussed.     

Fyn promotes phosphorylation of collapsin response mediator protein 1 at tyrosine 504, a novel,isoform‐specific regulatory site

In vertebrates the collapsin response mediator proteins (CRMPs) are encoded by five highly related genes. CRMPs are cytosolic phosphoproteins abundantly expressed in developing and mature mammalian brains. CRMPs are best understood as effectors of Semaphorin 3A signaling regulating growth cone collapse in migratory neurons. Phosphorylation in the carboxyl‐terminal regulatory domain of CRMPs by several serine/threonine kinases has been described. These phoshorylation events appear to function, at least in part, to disrupt the interaction of CRMPs with tubulin heterodimers. In a large‐scale phosphoproteomic analysis of murine brain, we recently identified a number of in vivo tyrosine phosphorylation sites on CRMP isoforms. Using biochemical approaches and quantitative mass spectrometry we demonstrate that one of these sites, CRMP1 tyrosine 504 (Y504), is a primary target of the Src family of tyrosine kinases (SFKs), specifically Fyn. Y504 is adjacent to CDK5 and GSK‐3β sites that regulate the interaction of CRMPs with tubulin. Although Y504 is highly conserved among vertebrate CRMP1 orthologs, a residue corresponding to Y504 is absent in CRMP isoforms 2–5. This suggests an isoform‐specific regulatory role for CRMP1 Y504 phosphorylation and may help explain the observation that CRMP1‐deficient mice exhibit neuronal migration defects not compensated for by CRMPs 2–5. J. Cell. Biochem. 111: 20–28, 2010. © 2010 Wiley‐Liss, Inc.     

磷酸化组氨酸研究进展

蛋白质磷酸化是生物体内一种广泛存在的蛋白质翻译后修饰形式,这种氨基酸与磷酸基团共价连接的修饰模式对蛋白质结构和功能起到了重要调节作用.目前天然蛋白质中发现的可磷酸化位点主要有9种氨基酸残基,其中包括以磷酰胺连接的磷酸化组氨酸.虽然该磷酸化形式在原核生物与真核生物中都起到了重要的调节作用,但对于其生物学功能的研究长期存在技术困难.由于磷酸化组氨酸本身不同于其他磷酸化氨基酸的化学性质,如存在异构体、化学不稳定等,其在传统的研究方法中容易发生水解去磷酸化.随着现代生物化学与分子生物学技术的不断进步,人们针对含有磷酸化组氨酸的蛋白质构建了新的制备、分离与表征策略,本领域也因此开始迅速发展.本文从磷酸化组氨酸的化学结构入手,分析其两种异构体的主要理化性质与化学反应特性,并概述了基于此发展的新型化学生物学研究手段以及对于磷酸化组氨酸生物功能的研究进展.     

Phosphoproteomics of human platelets: A quest for novel activation pathways

Besides their role in hemostasis, platelets are also highly involved in the pathogenesis and progression of cardiovascular diseases. Since important and initial steps of platelet activation and aggregation are regulated by phosphorylation events, a comprehensive study aimed at the characterization of phosphorylation-driven signaling cascades might lead to the identification of new target proteins for clinical research. However, it becomes increasingly evident that only a comprehensive phosphoproteomic approach may help to characterize functional protein networks and their dynamic alteration during physiological and pathophysiological processes in platelets. In this review, we discuss current methodologies in phosphoproteome research including their potentials as well as limitations, from sample preparation to classical approaches like radiolabeling and state-of-the-art mass spectrometry techniques.     

Phosphoproteome-based kinase activity profiling reveals the critical role of MAP2K2 and PLK1 in neuronal autophagy

Recent studies have demonstrated that dysregulation of macroautophagy/autophagy may play a central role in the pathogenesis of neurodegenerative disorders, and the induction of autophagy protects against the toxic insults of aggregate-prone proteins by enhancing their clearance. Thus, autophagy has become a promising therapeutic target against neurodegenerative diseases. In this study, quantitative phosphoproteomic profiling together with a computational analysis was performed to delineate the phosphorylation signaling networks regulated by 2 natural neuroprotective autophagy enhancers, corynoxine (Cory) and corynoxine B (Cory B). To identify key regulators, namely, protein kinases, we developed a novel network-based algorithm of in silico Kinome Activity Profiling (iKAP) to computationally infer potentially important protein kinases from phosphorylation networks. Using this algorithm, we observed that Cory or Cory B potentially regulated several kinases. We predicted and validated that Cory, but not Cory B, downregulated a well-documented autophagy kinase, RPS6KB1/p70S6K (ribosomal protein S6 kinase, polypeptide 1). We also discovered 2 kinases, MAP2K2/MEK2 (mitogen-activated protein kinase kinase 2) and PLK1 (polo-like kinase 1), to be potentially upregulated by Cory, whereas the siRNA-mediated knockdown of Map2k2 and Plk1 significantly inhibited Cory-induced autophagy. Furthermore, Cory promoted the clearance of Alzheimer disease-associated APP (amyloid β [A4] precursor protein) and Parkinson disease-associated SNCA/α-synuclein (synuclein, α) by enhancing autophagy, and these effects were dramatically diminished by the inhibition of the kinase activities of MAP2K2 and PLK1. As a whole, our study not only developed a powerful method for the identification of important regulators from the phosphoproteomic data but also identified the important role of MAP2K2 and PLK1 in neuronal autophagy.     

Phosphoproteome analysis of the pathogenic bacterium Helicobacter pylori reveals over-representation of tyrosine phosphorylation and multiply phosphorylated proteins

Increasing evidence shows that protein phosphorylation on serine (Ser), threonine (Thr) and tyrosine (Tyr) residues is a major regulatory post-translational modification in the bacteria. To reveal the phosphorylation state in the Gram-negative pathogenic bacterium Helicobacter pylori, we carried out a global and site-specific phosphoproteomic analysis based on TiO(2) -phosphopeptide enrichment and high-accuracy LC-MS/MS determination. Eighty-two phosphopeptides from 67 proteins were identified with 126 phosphorylation sites, among which 79 class I sites were determined to have a distribution of 42.8:38.7:18.5% for the Ser/Thr/Tyr phosphorylation, respectively. The H. pylori phosphoproteome is characterized by comparably big size, high ratio of Tyr phosphorylation, high abundance of multiple phosphorylation sites in individual phosphopeptides and over-representation of membrane proteins. An interaction network covering 28 phosphoproteins was constructed with a total of 163 proteins centering on the major H. pylori virulence factor VacA, indicating that protein phosphorylation in H. pylori may be delicately controlled to regulate many aspects of the metabolic pathways and bacterial virulence.     

Phosphoproteomics in Arabidopsis: moving from empirical to predictive science

Although protein phosphorylation is integral to the regulation of protein function in diverse biological responses, relatively little is currently known about the rules that govern phosphorylation in plants. This review will discuss how the data acquired by evolving phosphoproteomic methods are beginning to fill the gaps in our knowledge. In addition, ways are suggested in which new quantitative methods in conjunction with extrapolating from genomic data may provide a strategy to predict components of signalling networks that may be co-ordinately regulated.     

Human Protein Reference Database and Human Proteinpedia as resources for phosphoproteome analysis

Human Protein Reference Database (HPRD) is a rich resource of experimentally proven features of human proteins. Protein information in HPRD includes protein-protein interactions, post-translational modifications, enzyme/substrate relationships, disease associations, tissue expression, and subcellular localization of human proteins. Although, protein-protein interaction data from HPRD has been widely used by the scientific community, its phosphoproteome data has not been exploited to its full potential. HPRD is one of the largest documentations of human phosphoproteins in the public domain. Currently, phosphorylation data in HPRD comprises of 95,016 phosphosites mapped on to 13,041 proteins. Additionally, enzyme-substrate reactions responsible for 5930 phosphorylation events were also documented. Significant improvements in technologies and high-throughput platforms in biomedical investigations led to an exponential increase of biological data and phosphoproteomic data in recent years. Human Proteinpedia, a community annotation portal developed by us, has also contributed to the significant increase in phosphoproteomic data in HPRD. A large number of phosphorylation events have been mapped on to reference sequences available in HPRD and Human Proteinpedia along with associated protein features. This will provide a platform for systems biology approaches to determine the role of protein phosphorylation in protein function, cell signaling, biological processes and their implication in human diseases. This review aims to provide a composite view of phosphoproteomic data pertaining to human proteins in HPRD and Human Proteinpedia.     

玉米早期花药蛋白质组和磷酸化蛋白质组分析

蛋白质磷酸化修饰是调控其功能的一种重要方式。植物有性生殖过程在农作物产量形成和物种繁衍过程中起着重要作用。作为植物雄性生殖器官的花药,其正常生长发育对于保证形成功能性配子(花粉)以及完成双受精过程至关重要。本研究以重要农作物玉米(B73)为材料,利用Nano UHPLC-MS/MS质谱技术对玉米早期发育的花药在蛋白质组和磷酸化蛋白质组水平进行全面分析,以探究玉米花药发育过程中的蛋白调控网络和磷酸化修饰调控网络。在蛋白质组学分析中,共鉴定到了3 016个多肽,匹配到1 032个蛋白质上。通过Map Man分析,预测到了一些和花药发育相关的蛋白质,例如受体激酶(GRMZM2G082823_P01、GRMZM5G805485_P01等)。另外,在磷酸化蛋白质组学研究中,通过对Ti O2亲和层析富集到的磷酸化多肽进行质谱分析,检测到了257个磷酸化多肽,匹配到210个蛋白质上。我们的数据揭示了玉米花药发育过程中的223个磷酸化位点。与已发现的玉米中的86个磷酸化蛋白质(植物蛋白磷酸化数据库(P3DB):http://www.p3db.org/organism.php)相比,其中203个磷酸化蛋白和218个磷酸化位点为首次揭示。进一步生物信息学分析表明:磷酸化在14-3-3蛋白质、激酶、磷酸酶、转录因子、细胞周期和染色质结构相关的蛋白质介导的玉米早期花药发育过程中起着重要的调控作用。总之,本研究首次在蛋白质组学和磷酸化蛋白质组学水平研究了玉米早期花药发育的蛋白质调控网络,不仅丰富了玉米蛋白质和磷酸化修饰蛋白质数据库,并为利用遗传学和生物化学手段深入研究玉米花药发育的分子调控机理提供了基础。     

Evidence for a role of protein phosphorylation in the maintenance of the cnidarian–algal symbiosis

The endosymbiotic relationship between cnidarians and photosynthetic dinoflagellate algae provides the foundation of coral reef ecosystems. This essential interaction is globally threatened by anthropogenic disturbance. As such, it is important to understand the molecular mechanisms underpinning the cnidarian–algal association. Here we investigated phosphorylation‐mediated protein signalling as a mechanism of regulation of the cnidarian–algal interaction, and we report on the generation of the first phosphoproteome for the coral model system Aiptasia. Mass spectrometry‐based phosphoproteomics using data‐independent acquisition allowed consistent quantification of over 3,000 phosphopeptides totalling more than 1,600 phosphoproteins across aposymbiotic (symbiont‐free) and symbiotic anemones. Comparison of the symbiotic states showed distinct phosphoproteomic profiles attributable to the differential phosphorylation of 539 proteins that cover a broad range of functions, from receptors to structural and signal transduction proteins. A subsequent pathway enrichment analysis identified the processes of “protein digestion and absorption,” “carbohydrate metabolism,” and “protein folding, sorting and degradation,” and highlighted differential phosphorylation of the “phospholipase D signalling pathway” and “protein processing in the endoplasmic reticulum.” Targeted phosphorylation of the phospholipase D signalling pathway suggests control of glutamate vesicle trafficking across symbiotic compartments, and phosphorylation of the endoplasmic reticulum machinery suggests recycling of symbiosome‐associated proteins. Our study shows for the first time that changes in the phosphorylation status of proteins between aposymbiotic and symbiotic Aiptasia anemones may play a role in the regulation of the cnidarian–algal symbiosis. This is the first phosphoproteomic study of a cnidarian–algal symbiotic association as well as the first application of quantification by data‐independent acquisition in the coral field.     

Accurate prediction of kinase-substrate networks using knowledge graphs

Phosphorylation of specific substrates by protein kinases is a key control mechanism for vital cell-fate decisions and other cellular processes. However, discovering specific kinase-substrate relationships is time-consuming and often rather serendipitous. Computational predictions alleviate these challenges, but the current approaches suffer from limitations like restricted kinome coverage and inaccuracy. They also typically utilise only local features without reflecting broader interaction context. To address these limitations, we have developed an alternative predictive model. It uses statistical relational learning on top of phosphorylation networks interpreted as knowledge graphs, a simple yet robust model for representing networked knowledge. Compared to a representative selection of six existing systems, our model has the highest kinome coverage and produces biologically valid high-confidence predictions not possible with the other tools. Specifically, we have experimentally validated predictions of previously unknown phosphorylations by the LATS1, AKT1, PKA and MST2 kinases in human. Thus, our tool is useful for focusing phosphoproteomic experiments, and facilitates the discovery of new phosphorylation reactions. Our model can be accessed publicly via an easy-to-use web interface (LinkPhinder).