Quantitative phosphoproteomics to characterize signaling networks

Reversible protein phosphorylation is involved in the regulation of most, if not all, major cellular processes via dynamic signal transduction pathways. During the last decade quantitative phosphoproteomics have evolved from a highly specialized area to a powerful and versatile platform for analyzing protein phosphorylation at a system-wide scale and has become the intuitive strategy for comprehensive characterization of signaling networks. Contemporary phosphoproteomics use highly optimized procedures for sample preparation, mass spectrometry and data analysis algorithms to identify and quantify thousands of phosphorylations, thus providing extensive overviews of the cellular signaling networks. As a result of these developments quantitative phosphoproteomics have been applied to study processes as diverse as immunology, stem cell biology and DNA damage. Here we review the developments in phosphoproteomics technology that have facilitated the application of phosphoproteomics to signaling networks and introduce examples of recent system-wide applications of quantitative phosphoproteomics. Despite the great advances in phosphoproteomics technology there are still several outstanding issues and we provide here our outlook on the current limitations and challenges in the field.     

Quantitative phosphoproteomic analysis of signaling network dynamics

Protein phosphorylation mediated cellular signaling is a highly regulated, dynamic process that controls many aspects of cellular biology. Over the past few years many methods have been developed to quantify temporal dynamics of protein phosphorylation, including mass spectrometry, which can be applied in both an unbiased, discovery mode and in a targeted mode to monitor specific phosphorylation sites. Other methods, such as kinase activity assays and antibody microarrays, have been applied to quantify central nodes in the signaling network, yielding intriguing biological insights. This review provides a concise overview of the latest advances in the quantitative analysis of signaling dynamics including a brief commentary on the future of the field.     

Quantitative phosphoproteomics--an emerging key technology in signal-transduction research

Protein phosphorylation is the most important type of reversible post-translational modification involved in the regulation of cellular signal-transduction processes. In addition to controlling normal cellular physiology on the molecular level, perturbations of phosphorylation-based signaling networks and cascades have been implicated in the onset and progression of various human diseases. Recent advances in mass spectrometry-based proteomics helped to overcome many of the previous limitations in protein phosphorylation analysis. Improved isotope labeling and phosphopeptide enrichment strategies in conjunction with more powerful mass spectrometers and advances in data analysis have been integrated in highly efficient phosphoproteomics workflows, which are capable of monitoring up to several thousands of site-specific phosphorylation events within one large-scale analysis. Combined with ongoing efforts to define kinase-substrate relationships in intact cells, these major achievements have considerable potential to assess phosphorylation-based signaling networks on a system-wide scale. Here, we provide an overview of these exciting developments and their potential to transform signal-transduction research into a technology-driven, high-throughput science.     

Interactome Mapping: Using Protein Microarray Technology to Reconstruct Diverse Protein Networks

A major focus of systems biology is to characterize interactions between cellular components, in order to develop an accurate picture of the intricate networks within biological systems. Over the past decade, protein microarrays have greatly contributed to advances in proteomics and are becoming an important platform for systems biology. Protein microarrays are highly flexible, ranging from large-scale proteome microarrays to smaller customizable microarrays, making the technology amenable for detection of a broad spectrum of biochemical properties of proteins. In this article, we will focus on the numerous studies that have utilized protein microarrays to reconstruct biological networks including protein-DNA interactions, posttranslational protein modifications (PTMs), lectin-glycan recognition, pathogen-host interactions and hierarchical signaling cascades. The diversity in applications allows for integration of interaction data from numerous molecular classes and cellular states, providing insight into the structure of complex biological systems. We will also discuss emerging applications and future directions of protein microarray technology in the global frontier.     

Toward quantitative phosphotyrosine profiling in vivo

Tyrosine phosphorylation is a dynamic reversible post-translational modification that regulates many aspects of cell biology. To understand how this modification controls biological function, it is necessary to not only identify the specific sites of phosphorylation, but also to quantify how phosphorylation levels on these sites may be altered under specific physiological conditions. Due to its sensitivity and accuracy, mass spectrometry (MS) has widely been applied to the identification and characterization of phosphotyrosine signaling across biological systems. In this review we highlight the advances in both MS and phosphotyrosine enrichment methods that have been developed to enable the identification of low level tyrosine phosphorylation events. Computational and manual approaches to ensure confident identification of phosphopeptide sequence and determination of phosphorylation site localization are discussed along with methods that have been applied to the relative quantification of large numbers of phosphorylation sites. Finally, we provide an overview of the challenges ahead as we extend these technologies to the characterization of tyrosine phosphorylation signaling in vivo. With these latest developments in analytical and computational techniques, it is now possible to derive biological insight from quantitative MS-based analysis of signaling networks in vitro and in vivo. Application of these approaches to a wide variety of biological systems will define how signal transduction regulates cellular physiology in health and disease.     

Phosphoproteomic approaches to elucidate cellular signaling networks

Protein phosphorylation is crucial in the regulation of signaling pathways that control various biological responses. Recent progress in diverse methodologies to investigate protein phosphorylation in complex biological samples has resulted in more rapid, detailed and quantitative analyses of signaling networks. In particular, advances in mass spectrometry (MS) have enabled the identification and quantification of thousands of both known and novel phosphorylation sites. Initial MS-based information can be complemented with a variety of recently developed and improved phosphoproteomic techniques. These include multiplexed microbead or kinase activity assays, flow cytometry based single-cell analysis, protein microarrays and interaction studies. The combination of multiple approaches, coupled with phenotypic response measurements, computational modeling and biochemical manipulations, will ultimately reveal the mechanistic regulation of signaling networks.     

Unremitting progresses for phosphoprotein synthesis

Phosphorylation, one of the important protein post-translational modifications, is involved in many essential cellular processes. Site-specifical and homogeneous phosphoproteins can be used as probes for elucidating the protein phosphorylation network and as potential therapeutics for interfering their involved biological events. However, the generation of phosphoproteins has been challenging owing to the limitation of chemical synthesis and protein expression systems. Despite the pioneering discoveries in phosphoprotein synthesis, over the past decade, great progresses in this field have also been made to promote the biofunctional exploration of protein phosphorylation largely. Therefore, in this review, we mainly summarize recent advances in phosphoprotein synthesis, which includes five sections: 1) synthesis of the nonhydrolyzable phosphorylated amino acid mimetic building blocks, 2) chemical total and semisynthesis strategy, 3) in-cell and in vitro genetic code expansion strategy, 4) the late-stage modification strategy, 5) nonoxygen phosphoprotein synthesis.     

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.     

Genome-wide analysis of signaling domain function

Approximately 2.5% of human gene products contain one or more small domains that drive interactions between proteins and other cellular components in cell signaling processes. The many interactions driven by these relatively simple domains are thought to cooperate with one another to yield complex signaling networks that allow very fine control of cell function. In principle, if we can understand all domain-mediated interactions it should be possible to model these networks. Genome-wide analysis of signaling domain interactions represents a first step in this direction, and several advances of this sort in yeast have been reported over the past year. These reports suggest, for some domains at least, that the prospect of generating 'wiring diagrams' with this simple approach is feasible.     

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.     

拟南芥中myo-肌醇-多磷酸合成与代谢及其信号

在酵母、真菌、动物和植物等真核生物中, 以myo-肌醇为基石通过不同位点的磷酸化形成各种myo-肌醇-多磷酸及其衍生物。过去10年的研究发现这些肌醇多磷酸参与了膜脂定向转运、蛋白结构稳定、离子通道调控、RNA转运以及DNA修复和染色质重塑等细胞生物学的基本进程。近些年在模式植物拟南芥(Arabidopsis thaliana)的研究中, 许多调控植物生长发育和环境胁迫应答的重要基因被发现, 并证实这些基因参与myo-肌醇-多磷酸的合成与代谢。该文概述了拟南芥中myo-肌醇-多磷酸合成与代谢的基因调控机理, 综述了不同肌醇多磷酸作为信号分子的功能, 提出肌醇多磷酸如同一类信息代码传递着植物细胞有序进程的基本指令。     

Proteoglycans: from structural compounds to signaling molecules

Our knowledge of proteoglycan biology has significantly expanded over the past decade with the discovery of a host of new members of this multifunctional family leading to their present classification into three major categories: (1) small leucine-rich proteoglycans, 2) modular proteoglycans, and 3) cell-surface proteoglycans. In addition to being structural proteins, proteoglycans play a major role in signal transduction with regulatory functions in various cellular processes. Being mostly extracellular, they are upstream of many signaling cascades and are capable of affecting intracellular phosphorylation events and modulating distinct pathways, including those driven by bone morphogenetic protein/transforming growth factor superfamily members, receptor tyrosine kinases, the insulin-like growth factor-I receptor, and Toll-like receptors. Mechanistic insights into the molecular and cellular functions of proteoglycans have revealed both the sophistication of these regulatory proteins and the challenges that remain in uncovering the entirety of their biological functions. This review aims to summarize the multiple functions of proteoglycans with special emphasis on their intricate composition and the newly described signaling events in which these molecules play a key role.     

Network analysis of epidermal growth factor signaling using integrated genomic, proteomic and phosphorylation data

To understand how integration of multiple data types can help decipher cellular responses at the systems level, we analyzed the mitogenic response of human mammary epithelial cells to epidermal growth factor (EGF) using whole genome microarrays, mass spectrometry-based proteomics and large-scale western blots with over 1000 antibodies. A time course analysis revealed significant differences in the expression of 3172 genes and 596 proteins, including protein phosphorylation changes measured by western blot. Integration of these disparate data types showed that each contributed qualitatively different components to the observed cell response to EGF and that varying degrees of concordance in gene expression and protein abundance measurements could be linked to specific biological processes. Networks inferred from individual data types were relatively limited, whereas networks derived from the integrated data recapitulated the known major cellular responses to EGF and exhibited more highly connected signaling nodes than networks derived from any individual dataset. While cell cycle regulatory pathways were altered as anticipated, we found the most robust response to mitogenic concentrations of EGF was induction of matrix metalloprotease cascades, highlighting the importance of the EGFR system as a regulator of the extracellular environment. These results demonstrate the value of integrating multiple levels of biological information to more accurately reconstruct networks of cellular response.     

Analytical methodologies for the detection and structural characterization of phosphorylated proteins

Phosphorylation of proteins is a frequent post-translational modification affecting a great number of fundamental cellular functions in living organisms. Because of its key role in many biological processes, much effort has been spent over the time on the development of analytical methodologies for characterizing phosphoproteins. In the past decade, mass spectrometry-based techniques have emerged as a viable alternative to more traditional methods of phosphorylation analysis, providing accurate information for a purified protein on the number of the occurring phosphate groups and their exact localization on the polypeptide sequence. This review summarizes the analytical methodologies currently available for the analysis of protein phosphorylation, emphasizing novel mass spectrometry (MS) technologies and dedicated biochemical procedures that have been recently introduced in this field. A formidable armamentarium is now available for selective enrichment, exaustive structural characterization and quantitative determination of the modification degree for phosphopeptides/phosphoproteins. These methodologies are now successfully applied to the global analysis of cellular proteome repertoire according a holistic approach, allowing the quantitative study of phosphoproteomes on a dynamic time-course basis. The enormous complexity of the protein phosphorylation pattern inside the cell and its dynamic modification will grant important challenges to future scientists, contributing significantly to deeper insights into cellular processes and cell regulation.     

Experimental and computational tools useful for (re)construction of dynamic kinase–substrate networks

The explosion of site‐ and context‐specific in vivo phosphorylation events presents a potentially rich source of biological knowledge and calls for novel data analysis and modeling paradigms. Perhaps the most immediate challenge is delineating detected phosphorylation sites to their effector kinases. This is important for (re)constructing transient kinase–substrate interaction networks that are essential for mechanistic understanding of cellular behaviors and therapeutic intervention, but has largely eluded high‐throughput protein‐interaction studies due to their transient nature and strong dependencies on cellular context. Here, we surveyed some of the computational approaches developed to dissect phosphorylation data detected in systematic proteomic experiments and reviewed some experimental and computational approaches used to map phosphorylation sites to their effector kinases in efforts aimed at reconstructing biological signaling networks.     

Construction of human activity‐based phosphorylation networks

The landscape of human phosphorylation networks has not been systematically explored, representing vast, unchartered territories within cellular signaling networks. Although a large number of in vivo phosphorylated residues have been identified by mass spectrometry (MS)‐based approaches, assigning the upstream kinases to these residues requires biochemical analysis of kinase‐substrate relationships (KSRs). Here, we developed a new strategy, called CEASAR, based on functional protein microarrays and bioinformatics to experimentally identify substrates for 289 unique kinases, resulting in 3656 high‐quality KSRs. We then generated consensus phosphorylation motifs for each of the kinases and integrated this information, along with information about in vivo phosphorylation sites determined by MS, to construct a high‐resolution map of phosphorylation networks that connects 230 kinases to 2591 in vivo phosphorylation sites in 652 substrates. The value of this data set is demonstrated through the discovery of a new role for PKA downstream of Btk (Bruton's tyrosine kinase) during B‐cell receptor signaling. Overall, these studies provide global insights into kinase‐mediated signaling pathways and promise to advance our understanding of cellular signaling processes in humans.     

Biological functions of the small leucine-rich proteoglycans: from genetics to signal transduction

The small leucine-rich proteoglycan (SLRP) family has significantly expanded in the past decade to now encompass five discrete classes, grouped by common structural and functional properties. Some of these gene products are not classical proteoglycans, whereas others have new and unique features. In addition to being structural proteins, SLRPs constitute a network of signal regulation: being mostly extracellular, they are upstream of multiple signaling cascades. They affect intracellular phosphorylation, a major conduit of information for cellular responses, and modulate distinct pathways, including those driven by bone morphogenetic protein/transforming growth factor beta superfamily members, receptor tyrosine kinases such as ErbB family members and the insulin-like growth factor I receptor, and Toll-like receptors. The wealth of mechanistic insights into the molecular and cellular functions of SLRPs has revealed both the sophistication of this family of regulatory proteins and the challenges that remain in uncovering the totality of their functions. This review is focused on novel biological functions of SLRPs with special emphasis on their protein cores, newly described genetic diseases, and signaling events in which SLRPs play key functions.     

Peptide arrays for kinome analysis: new opportunities and remaining challenges

Phosphorylation is the predominant mechanism of post-translational modification for regulation of protein function. With central roles in virtually every cellular process, and strong linkages with many diseases, there is a considerable interest in defining, and ultimately controlling, kinase activities. Investigations of human cellular phosphorylation events, which includes over 500 different kinases and tens of thousands of phosphorylation targets, represent a daunting challenge for proteomic researchers and cell biologists alike. As such, there is a priority to develop tools that enable the evaluation of cellular phosphorylation events in a high-throughput, and biologically relevant, fashion. Towards this objective, two distinct, but functionally related, experimental approaches have emerged; phosphoproteome investigations, which focus on the sub-population of proteins which undergo phosphorylation and kinome analysis, which considers the activities of the kinase enzymes mediating these phosphorylation events. Within kinome analysis, peptide arrays have demonstrated considerable potential as a cost-effective, high-throughput approach for defining phosphorylation-mediated signal transduction activity. In particular, a number of recent advances in the application of peptide arrays for kinome analysis have enabled researchers to tackle increasingly complex biological problems in a wider range of species. In this review, recent advances in kinomic analysis utilizing peptides arrays including several of the biological questions studied by our group, as well as outstanding challenges still facing this technology, are discussed.     

In the Beginning,There Was Protein Phosphorylation

The importance of reversible protein phosphorylation to cellular regulation cannot be overstated. In eukaryotic cells, protein kinase/phosphatase signaling pathways regulate a staggering number of cellular processes, including cell proliferation, cell death (apoptosis, necroptosis, necrosis), metabolism (at both the cellular and organismal levels), behavior and neurological function, development, and pathogen resistance. Although protein phosphorylation as a mode of eukaryotic cell regulation is familiar to most biochemists, many are less familiar with protein kinase/phosphatase signaling networks that function in prokaryotes. In this thematic minireview series, we present four minireviews that cover the important field of prokaryotic protein phosphorylation.