Quantitative phosphoproteomics by mass spectrometry: past, present, and future

Protein phosphorylation-mediated signaling networks regulate much of the cellular response to external stimuli, and dysregulation in these networks has been linked to multiple disease states. Significant advancements have been made over the past decade to enable the analysis and quantification of cellular protein phosphorylation events, but comprehensive analysis of the phosphoproteome is still lacking, as is the ability to monitor signaling at the network level while comprehending the biological implications of each phosphorylation site. In this review we highlight many of the technological advances over the past decade and describe some of the latest applications of these tools to uncover signaling networks in a variety of biological settings. We finish with a concise discussion of the future of the field, including additional advances that are required to link protein phosphorylation analysis with biological insight.     

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.     

Phosphorylation is required for myosin regulatory light chain (PmMRLC) to control yellow head virus infection in shrimp hemocytes

The cellular signal-transduction process is largely controlled by protein phosphorylation. Shrimp infected with yellow head virus show dramatic changes in their hemocyte phosphoproteomic patterns, and aberrant activation of phosphorylation-based signaling networks has been implicated in a number of diseases. In this study, we focused on phosphorylation of Penaeus monodon myosin regulatory light chain (PmMRLC) that is induced at an early hour post YHV infection and is concomitant with cellular actin remodeling. In shrimp cell cultures, this phosphorylation was inhibited by the myosin light chain kinase (MLCK) inhibitors ML-7 and ML-9, suggesting that PmMLC phosphorylation is MLCK pathway-dependent. Blocking PmMRLC phosphorylation resulted in increased replication of YHV and reduction of phagocytic activities of shrimp hemocytes called semigranular cells (SGC) and granular cells (GC). Injection of MLCK inhibitors prior to YHV challenge resulted in dose-dependent elevation in quantity of YHV-positive GC and cytoplasmic YHV protein, coincident with high shrimp mortality. Altogether, we demonstrated that PmMRLC phosphorylation increases after YHV infection in shrimp and that inhibition of the phosphorylation leads to increased YHV replication, reduced hemocyte phagocytic activity (probably through actin remodeling) and subsequent shrimp death. Thus, further studies on the MLCK activation pathway may lead to new strategies in development and implementation of therapy for YHV infections in shrimp.     

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.     

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.     

Combination of chemical genetics and phosphoproteomics for kinase signaling analysis enables confident identification of cellular downstream targets

Delineation of phosphorylation-based signaling networks requires reliable data about the underlying cellular kinase-substrate interactions. We report a chemical genetics and quantitative phosphoproteomics approach that encompasses cellular kinase activation in combination with comparative replicate mass spectrometry analyses of cells expressing either inhibitor-sensitive or resistant kinase variant. We applied this workflow to Plk1 (Polo-like kinase 1) in mitotic cells and induced cellular Plk1 activity by wash-out of the bulky kinase inhibitor 3-MB-PP1, which targets a mutant kinase version with an enlarged catalytic pocket while not interfering with wild-type Plk1. We quantified more than 20,000 distinct phosphorylation sites by SILAC, approximately half of which were measured in at least two independent experiments in cells expressing mutant and wild-type Plk1. Based on replicate phosphorylation site quantifications in both mutant and wild-type Plk1 cells, our chemical genetic proteomics concept enabled stringent comparative statistics by significance analysis of microarrays, which unveiled more than 350 cellular downstream targets of Plk1 validated by full concordance of both statistical and experimental data. Our data point to hitherto poorly characterized aspects in Plk1-controlled mitotic progression and provide a largely extended resource for functional studies. We anticipate the described strategies to be of general utility for systematic and confident identification of cellular protein kinase substrates.     

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.     

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.     

Insights from site-specific phosphoproteomics in bacteria

Recent advances in mass spectrometry allowed the charting of bacterial serine/threonine/tyrosine phosphoproteomes with unprecedented accuracy, including the acquisition of a large number of phosphorylation sites. Phosphorylated bacterial proteins are involved in some key housekeeping processes, and their phosphorylation is expected to play an important regulatory role. When coupled to stable isotope labeling by amino acids in cell culture (SILAC), high-resolution mass spectrometry allows the detection of changes in the occupancy of phosphorylation sites in response to various stimuli. This and similar approaches promise to lead bacterial phosphoproteomics into the era of systems biology, where the entire phosphorylation-based regulatory networks will be charted, modelled, and ultimately engineered to obtain desired properties.     

Proteomic analysis of cellular signaling

Protein phosphorylation events are key regulators of cellular signaling processes. In the era of functional genomics, rational drug design programs demand large-scale high-throughput analysis of signal transduction cascades. Significant improvements in the area of mass spectrometry-based proteomics have provided exciting opportunities for rapid progress toward global protein phosphorylation analysis. This review summarizes several recent advances made in the field of phosphoproteomics with an emphasis placed on mass spectrometry instrumentation, enrichment methods and quantification strategies. In the near future, these technologies will provide a tool that can be used for quantitative investigation of signal transduction pathways to generate new insights into biologic systems.     

Proteomic analysis of cellular signaling

Protein phosphorylation events are key regulators of cellular signaling processes. In the era of functional genomics, rational drug design programs demand large-scale high-throughput analysis of signal transduction cascades. Significant improvements in the area of mass spectrometry-based proteomics have provided exciting opportunities for rapid progress toward global protein phosphorylation analysis. This review summarizes several recent advances made in the field of phosphoproteomics with an emphasis placed on mass spectrometry instrumentation, enrichment methods and quantification strategies. In the near future, these technologies will provide a tool that can be used for quantitative investigation of signal transduction pathways to generate new insights into biologic systems.     

Recent findings and technological advances in phosphoproteomics for cells and tissues

Site-specific phosphorylation is a fast and reversible covalent post-translational modification that is tightly regulated in cells. The cellular machinery of enzymes that write, erase and read these modifications (kinases, phosphatases and phospho-binding proteins) is frequently deregulated in different diseases, including cancer. Large-scale studies of phosphoproteins – termed phosphoproteomics – strongly rely on the use of high-performance mass spectrometric instrumentation. This powerful technology has been applied to study a great number of phosphorylation-based phenotypes. Nevertheless, many technical and biological challenges have to be overcome to identify biologically relevant phosphorylation sites in cells and tissues. This review describes different technological strategies to identify and quantify phosphorylation sites with high accuracy, without significant loss of analysis speed and reproducibility in tissues and cells. Moreover, computational tools for analysis, integration and biological interpretation of phosphorylation events are discussed.     

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.     

Immunoaffinity profiling of tyrosine phosphorylation in cancer cells

Tyrosine kinases play a prominent role in human cancer, yet the oncogenic signaling pathways driving cell proliferation and survival have been difficult to identify, in part because of the complexity of the pathways and in part because of low cellular levels of tyrosine phosphorylation. In general, global phosphoproteomic approaches reveal small numbers of peptides containing phosphotyrosine. We have developed a strategy that emphasizes the phosphotyrosine component of the phosphoproteome and identifies large numbers of tyrosine phosphorylation sites. Peptides containing phosphotyrosine are isolated directly from protease-digested cellular protein extracts with a phosphotyrosine-specific antibody and are identified by tandem mass spectrometry. Applying this approach to several cell systems, including cancer cell lines, shows it can be used to identify activated protein kinases and their phosphorylated substrates without prior knowledge of the signaling networks that are activated, a first step in profiling normal and oncogenic signaling networks.     

Phosphoproteomics in bacteria: towards a systemic understanding of bacterial phosphorylation networks

Bacteria use protein phosphorylation to regulate all kinds of physiological processes. Protein phosphorylation plays a role in several key steps of the infection process of bacterial pathogens, such as adhesion to the host, triggering and regulation of pathogenic functions as well as biochemical warfare; scrambling the host signaling cascades and impairing its defense mechanisms. Recent phosphoproteomic studies indicate that the bacterial protein phosphorylation networks could be more complex than initially expected, comprising promiscuous kinases that regulate several distinct cellular functions by phosphorylating different protein substrates. Recent advances in protein labeling with stable isotopes in the field of quantitative mass spectrometry phosphoproteomics will enable us to chart the global phosphorylation networks and to understand the implication of protein phosphorylation in cellular regulation on the systems scale. For the study of bacterial pathogens, in particular, this research avenue will enable us to dissect phosphorylation-related events during different stages of infection and stimulate our efforts to find inhibitors for key kinases and phosphatases implicated therein.     

Phosphoproteomics in bacteria: towards a systemic understanding of bacterial phosphorylation networks

Bacteria use protein phosphorylation to regulate all kinds of physiological processes. Protein phosphorylation plays a role in several key steps of the infection process of bacterial pathogens, such as adhesion to the host, triggering and regulation of pathogenic functions as well as biochemical warfare; scrambling the host signaling cascades and impairing its defense mechanisms. Recent phosphoproteomic studies indicate that the bacterial protein phosphorylation networks could be more complex than initially expected, comprising promiscuous kinases that regulate several distinct cellular functions by phosphorylating different protein substrates. Recent advances in protein labeling with stable isotopes in the field of quantitative mass spectrometry phosphoproteomics will enable us to chart the global phosphorylation networks and to understand the implication of protein phosphorylation in cellular regulation on the systems scale. For the study of bacterial pathogens, in particular, this research avenue will enable us to dissect phosphorylation-related events during different stages of infection and stimulate our efforts to find inhibitors for key kinases and phosphatases implicated therein.     

Phosphoproteomic analysis identifies insulin enhancement of discoidin domain receptor 2 phosphorylation

The discoidin domain receptors (DDRs) are collagen binding receptor tyrosine kinases that play important roles in cell migration, invasion and adhesion. Crosstalk between growth factor signaling and components of the extracellular matrix are drivers of cellular function but the integrated signaling networks downstream of such crosstalk events have not been extensively characterized. In this report, we have employed mass spectrometry-based quantitative phosphotyrosine analysis to identify crosstalk between DDR2 and the insulin receptor. Our phosphoproteomic analysis reveals a cluster of phosphorylation sites in which collagen and insulin cooperate to enhance phosphotyrosine levels. Importantly, Y740 on the DDR2 catalytic loop was found in this cluster indicating that insulin acts to promote collagen I signaling by increasing the activity of DDR2. Furthermore, we identify two additional migration associated proteins that are candidate substrates downstream of DDR2 activation. Our data suggests that insulin promotes collagen I signaling through the upregulation of DDR2 phosphorylation which may have important consequences in DDR2 function in health and disease.     

Phosphoproteomic analysis of cells treated with longevity-related autophagy inducers

Macroautophagy is a self-cannibalistic process that enables cells to adapt to various stresses and maintain energy homeostasis. Additionally, autophagy is an important route for turnover of misfolded proteins and damaged organelles, with important implications in cancer, neurodegenerative diseases and aging. Resveratrol and spermidine are able to induce autophagy by affecting deacetylases and acetylases, respectively, and have been found to extend the life-span of model organisms. With the aim to reveal the signaling networks involved in this drug-induced autophagic response, we quantified resveratrol and spermidine-induced changes in the phosphoproteome using SILAC and mass spectrometry. The data were subsequently analyzed using the NetworKIN algorithm to extract key features of the autophagy-responsive kinase-substrate network. We found that two distinct sequence motifs were highly responsive to resveratrol and spermidine and that key proteins modulating the acetylation, phosphorylation, methylation and ubiquitination status were affected by changes in phosphorylation during the autophagic response. Essential parts of the apoptotic signaling network were subjected to post-translational modifications during the drug-induced autophagy response, suggesting potential crosstalk and balancing between autophagy and apoptosis. Additionally, we predicted cellular signaling networks affected by resveratrol and spermidine using a computational framework. Altogether, these results point to a profound crosstalk between distinct networks of post-translational modifications and provide a resource for future analysis of autophagy and cell death.