Site-specific Phosphorylation Dynamics Research Articles

Phosphoproteomic Analysis of Protein Phosphorylation Networks in Tetrahymena thermophila, a Model Single-celled Organism

Tetrahymena thermophila is a widely used unicellular eukaryotic model organism in biological research and contains more than 1000 protein kinases and phosphatases with specificity for Ser/Thr/Tyr residues. However, only a few dozen phosphorylation sites in T. thermophila are known, presenting a major obstacle to further understanding of the regulatory roles of reversible phosphorylation in this organism. In this study, we used high-accuracy mass-spectrometry-based proteomics to conduct global and site-specific phosphoproteome profiling of T. thermophila. In total, 1384 phosphopeptides and 2238 phosphorylation sites from 1008 T. thermophila proteins were identified through the combined use of peptide prefractionation, TiO2 enrichment, and two-dimensional LC-MS/MS analysis. The identified phosphoproteins are implicated in the regulation of various biological processes such as transport, gene expression, and mRNA metabolic process. Moreover, integrated analysis of the T. thermophila phosphoproteome and gene network revealed the potential biological functions of many previously unannotated proteins and predicted some putative kinase-substrate pairs. Our data provide the first global survey of phosphorylation in T. thermophila using a phosphoproteomic approach and suggest a wide-ranging regulatory scope of this modification. The provided dataset is a valuable resource for the future understanding of signaling pathways in this important model organism.

Global Impact of Salmonella Pathogenicity Island 2-secreted Effectors on the Host Phosphoproteome

During the late stages of infection, Salmonella secretes numerous effectors through a type III secretion system that is encoded within Salmonella pathogenicity island 2 (SPI2). Despite the importance of SPI2 as a major virulence factor leading to the systemic spread of the bacteria and diseases, a global view of its effects on host responses is still lacking. Here, we measured global impacts of SPI2 effectors on the host phosphorylation and protein expression levels in RAW264.7 and in HeLa cells, as macrophage and nonphagocytic models of infection. We observe that SPI2 effectors differentially modulate the host phosphoproteome and cellular processes (e.g. protein trafficking, cytoskeletal regulation, and immune signaling) in a host cell-dependent manner. Our unbiased approach reveals the involvement of many previously unrecognized proteins, including E3 ligases (HERC4, RanBP2, and RAD18), kinases (CDK, SIK3, and WNK1), and histones (H2B1F, H4, and H15), in late stages of Salmonella infection. Furthermore, from this phosphoproteome analysis and other quantitative screens, we identified HSP27 as a direct in vitro and in vivo molecular target of the only type III secreted kinase, SteC. Using biochemical and cell biological assays, we demonstrate that SteC phosphorylates multiple sites in HSP27 and induces actin rearrangement through this protein. Together, these results provide a broader landscape of host players contributing to specific processes/pathways mediated by SPI2 effectors than was previously appreciated.

Elucidation of B Cell Antigen Receptor Signaling by Global Phosphoproteomics Reveals Novel Signaling Pathways and Regulatory Mechanisms

AbstractAbstract 1056B cell antigen receptors (BCR) transmit information via posttranslational protein modifications, most importantly protein phosphorylation. BCR signaling is a complex process that regulates differentiation and activation responses of the B-lineage. Dysregulation of BCR signaling can induce malignant transformation that can result in the onset of leukemia or lymphoma. To date our understanding of normal- versus malignant B cell biology points to differences in properties of BCR signaling that may be amenable to selective therapeutic targeting. However, comprehensive knowledge about BCR signaling pathways is still lacking which makes it difficult to understand the molecular events that drive B cell function and malignant transformation. To globally characterize BCR signaling, we have applied quantitative phosphoproteomic techniques that allowed us to identify and quantify about 10000 phosphorylation sites as a function of BCR stimulus, time, and subcellular location. Our phosphoproteome approach uncovered many novel BCR signaling effectors and their site-specific phosphorylation dynamics. The majority of phosphorylation events occured on serine/threonine residues, while only about one percent was detected at tyrosine residues. Interestingly, most of the identified proteins contain multiple phosphorylation sites showing different kinetics upon BCR stimulation, suggesting that they serve as signal integrators. Moreover, our functional analysis revealed that certain phosphorylation sites in adapter proteins are not only relevant for regulating the onset but also the termination of BCR signaling. Such signal termination is at least partly achieved by coupling the phosphotyrosine-dependent formation of BCR signalosomes to the kinetically flexible cytosolic Ca2+ concentration. In summary, our analysis revealed the complex composition of BCR signaling pathways and their regulation, and hence provides a basis for the identification of potential BCR-associated drug targets in the future. Disclosures:No relevant conflicts of interest to declare.

Quantitative Phosphoproteomic Analysis Reveals a Role for Serine and Threonine Kinases in the Cytoskeletal Reorganization in Early T Cell Receptor Activation in Human Primary T Cells

Protein phosphorylation-dephosphorylation events play a primary role in regulation of almost all aspects of cell function including signal transduction, cell cycle, or apoptosis. Thus far, T cell phosphoproteomics have focused on analysis of phosphotyrosine residues, and little is known about the role of serine/threonine phosphorylation in early activation of the T cell receptor (TCR). Therefore, we performed a quantitative mass spectrometry-based analysis of the global phosphoproteome of human primary T cells in response to 5 min of TCR activation with anti-CD3 antibody. Combining immunoprecipitation with an antiphosphotyrosine antibody, titanium dioxide phosphopeptide enrichment, isobaric tag for the relative and absolute quantitation methodology, and strong cation exchange separation, we were able to identify 2814 phosphopeptides. These unique sites were employed to investigate the site-specific phosphorylation dynamics. Five hundred and seventeen phosphorylation sites showed TCR-responsive changes. We found that upon 5 min of stimulation of the TCR, specific serine and threonine kinase motifs are overrepresented in the set of responsive phosphorylation sites. These phosphorylation events targeted proteins with many different activities and are present in different subcellular locations. Many of these proteins are involved in intracellular signaling cascades related mainly to cytoskeletal reorganization and regulation of small GTPase-mediated signal transduction, probably involved in the formation of the immune synapse.

PTMScan Direct: Identification and Quantification of Peptides from Critical Signaling Proteins by Immunoaffinity Enrichment Coupled with LC-MS/MS

Proteomic studies of post-translational modifications by metal affinity or antibody-based methods often employ data-dependent analysis, providing rich data sets that consist of randomly sampled identified peptides because of the dynamic response of the mass spectrometer. This can complicate the primary goal of programs for drug development, mutational analysis, and kinase profiling studies, which is to monitor how multiple nodes of known, critical signaling pathways are affected by a variety of treatment conditions. Cell Signaling Technology has developed an immunoaffinity-based LC-MS/MS method called PTMScan Direct for multiplexed analysis of these important signaling proteins. PTMScan Direct enables the identification and quantification of hundreds of peptides derived from specific proteins in signaling pathways or specific protein types. Cell lines, tissues, or xenografts can be used as starting material. PTMScan Direct is compatible with both SILAC and label-free quantification. Current PTMScan Direct reagents target key nodes of many signaling pathways (PTMScan Direct: Multipathway), serine/threonine kinases, tyrosine kinases, and the Akt/PI3K pathway. Validation of each reagent includes score filtering of MS/MS assignments, filtering by identification of peptides derived from expected targets, identification of peptides homologous to expected targets, minimum signal intensity of peptide ions, and dependence upon the presence of the reagent itself compared with a negative control. The Multipathway reagent was used to study sensitivity of human cancer cell lines to receptor tyrosine kinase inhibitors and showed consistent results with previously published studies. The Ser/Thr kinase reagent was used to compare relative levels of kinase-derived phosphopeptides in mouse liver, brain, and embryo, showing tissue-specific activity of many kinases including Akt and PKC family members. PTMScan Direct will be a powerful quantitative method for elucidation of changes in signaling in a wide array of experimental systems, combining the specificity of traditional biochemical methods with the high number of data points and dynamic range of proteomic methods.

Phosphosite Mapping of P-type Plasma Membrane H+-ATPase in Homologous and Heterologous Environments

Phosphorylation is an important posttranslational modification of proteins in living cells and primarily serves regulatory purposes. Several methods were employed for isolating phosphopeptides from proteolytically digested plasma membranes of Arabidopsis thaliana. After a mass spectrometric analysis of the resulting peptides we could identify 10 different phosphorylation sites in plasma membrane H(+)-ATPases AHA1, AHA2, AHA3, and AHA4/11, five of which have not been reported before, bringing the total number of phosphosites up to 11, which is substantially higher than reported so far for any other P-type ATPase. Phosphosites were almost exclusively (9 of 10) in the terminal regulatory domains of the pumps. The AHA2 isoform was subsequently expressed in the yeast Saccharomyces cerevisiae. The plant protein was phosphorylated at multiple sites in yeast, and surprisingly, seven of nine of the phosphosites identified in AHA2 were identical in the plant and fungal systems even though none of the target sequences in AHA2 show homology to proteins of the fungal host. These findings suggest an unexpected accessibility of the terminal regulatory domain of plasma membrane H(+)-ATPase to protein kinase action.

CDK-Dependent Potentiation of MPS1 Kinase Activity Is Essential to the Mitotic Checkpoint

Accurate chromosome segregation relies upon a mitotic checkpoint that monitors kinetochore attachment toward opposite spindle poles before enabling chromosome disjunction [1]. The MPS1/TTK protein kinase is a core component of the mitotic checkpoint that lies upstream of MAD2 and BubR1 both at the kinetochore and in the cytoplasm [2, 3]. To gain insight into the mechanisms underlying the regulation of MPS1 kinase, we undertook the identification of Xenopus MPS1 phosphorylation sites by mass spectrometry. We mapped several phosphorylation sites onto MPS1 and we show that phosphorylation of S283 in the noncatalytic region of MPS1 is required for full kinase activity. This phosphorylation potentiates MPS1 catalytic efficiency without impairing its affinity for the substrates. By using Xenopus egg extracts depleted of endogenous MPS1 and reconstituted with single point mutants, we show that phosphorylation of S283 is essential to activate the mitotic checkpoint. This phosphorylation does not regulate the localization of MPS1 to the kinetochore but is required for the recruitment of MAD1/MAD2, demonstrating its role at the kinetochore. Constitutive phosphorylation of S283 lowers the number of kinetochores required to hold the checkpoint, which suggests that CDK-dependent phosphorylation of MPS1 is essential to sustain the mitotic checkpoint when few kinetochores remain unattached.

Large Scale Phosphoproteome Profiles Comprehensive Features of Mouse Embryonic Stem Cells

Embryonic stem cells are pluripotent and capable of unlimited self-renewal. Elucidation of the underlying molecular mechanism may contribute to the advancement of cell-based regenerative medicine. In the present work, we performed a large scale analysis of the phosphoproteome in mouse embryonic stem (mES) cells. Using multiplex strategies, we detected 4581 proteins and 3970 high confidence distinct phosphosites in 1642 phosphoproteins. Notably, 22 prominent phosphorylated stem cell marker proteins with 39 novel phosphosites were identified for the first time by mass spectrometry, including phosphorylation sites in NANOG (Ser-65) and RE1 silencing transcription factor (Ser-950 and Thr-953). Quantitative profiles of NANOG peptides obtained during the differentiation of mES cells revealed that the abundance of phosphopeptides and non-phosphopeptides decreased with different trends. To our knowledge, this study presents the largest global characterization of phosphorylation in mES cells. Compared with a study of ultimately differentiated tissue cells, a bioinformatics analysis of the phosphorylation data set revealed a consistent phosphorylation motif in human and mouse ES cells. Moreover, investigations into phosphorylation conservation suggested that phosphoproteins were more conserved in the undifferentiated ES cell state than in the ultimately differentiated tissue cell state. However, the opposite conclusion was drawn from this conservation comparison with phosphosites. Overall, this work provides an overview of phosphorylation in mES cells and is a valuable resource for the future understanding of basic biology in mES cells.

Evolutionary Constraints of Phosphorylation in Eukaryotes, Prokaryotes, and Mitochondria

High accuracy mass spectrometry has proven to be a powerful technology for the large scale identification of serine/threonine/tyrosine phosphorylation in the living cell. However, despite many described phosphoproteomes, there has been no comparative study of the extent of phosphorylation and its evolutionary conservation in all domains of life. Here we analyze the results of phosphoproteomics studies performed with the same technology in a diverse set of organisms. For the most ancient organisms, the prokaryotes, only a few hundred proteins have been found to be phosphorylated. Applying the same technology to eukaryotic species resulted in the detection of thousands of phosphorylation events. Evolutionary analysis shows that prokaryotic phosphoproteins are preferentially conserved in all living organisms, whereas-site specific phosphorylation is not. Eukaryotic phosphosites are generally more conserved than their non-phosphorylated counterparts (with similar structural constraints) throughout the eukaryotic domain. Yeast and Caenorhabditis elegans are two exceptions, indicating that the majority of phosphorylation events evolved after the divergence of higher eukaryotes from yeast and reflecting the unusually large number of nematode-specific kinases. Mitochondria present an interesting intermediate link between the prokaryotic and eukaryotic domains. Applying the same technology to this organelle yielded 174 phosphorylation sites mapped to 74 proteins. Thus, the mitochondrial phosphoproteome is similarly sparse as the prokaryotic phosphoproteomes. As expected from the endosymbiotic theory, phosphorylated as well as non-phosphorylated mitochondrial proteins are significantly conserved in prokaryotes. However, mitochondrial phosphorylation sites are not conserved throughout prokaryotes, consistent with the notion that serine/threonine phosphorylation in prokaryotes occurred relatively recently in evolution. Thus, the phosphoproteome reflects major events in the evolution of life.

Sequential Multiplex Analyte Capturing for Phosphoprotein Profiling

Microarray-based sandwich immunoassays can simultaneously detect dozens of proteins. However, their use in quantifying large numbers of proteins is hampered by cross-reactivity and incompatibilities caused by the immunoassays themselves. Sequential multiplex analyte capturing addresses these problems by repeatedly probing the same sample with different sets of antibody-coated, magnetic suspension bead arrays. As a miniaturized immunoassay format, suspension bead array-based assays fulfill the criteria of the ambient analyte theory, and our experiments reveal that the analyte concentrations are not significantly changed. The value of sequential multiplex analyte capturing was demonstrated by probing tumor cell line lysates for the abundance of seven different receptor tyrosine kinases and their degree of phosphorylation and by measuring the complex phosphorylation pattern of the epidermal growth factor receptor in the same sample from the same cavity.

Integrated Phosphoproteomics Analysis of a Signaling Network Governing Nutrient Response and Peroxisome Induction

Phosphorylation of proteins is a key posttranslational modification in cellular signaling, regulating many aspects of cellular responses. We used a quantitative, integrated, phosphoproteomics approach to characterize the cellular responses of the yeast Saccharomyces cerevisiae to the fatty acid oleic acid, a molecule with broad human health implications and a potent inducer of peroxisomes. A combination of cryolysis and urea solubilization was used to minimize the opportunity for reorientation of the phosphoproteome, and hydrophilic interaction liquid chromatography and IMAC chemistries were used to fractionate and enrich for phosphopeptides. Using these approaches, numerous phosphorylated peptides specific to oleate-induced and glucose-repressed conditions were identified and mapped to known signaling pathways. These include several transcription factors, two of which, Pip2p and Cst6p, must be phosphorylated for the normal transcriptional response of fatty acid-responsive loci encoding peroxisomal proteins. The phosphoproteome data were integrated with results from genome-wide assays studying the effects of signaling molecule deletions and known protein-protein interactions to generate a putative fatty acid-responsive signaling network. In this network, the most highly connected nodes are those with the largest effects on cellular responses to oleic acid. These properties are consistent with a scale-free topology, demonstrating that scale-free properties are conserved in condition-specific networks.

Quantitative Site-specific Phosphorylation Dynamics of Human Protein Kinases during Mitotic Progression

Reversible protein phosphorylation is a key regulatory mechanism of mitotic progression. Importantly, protein kinases themselves are also regulated by phosphorylation-dephosphorylation processes; hence, phosphorylation dynamics of kinases hold a wealth of information about phosphorylation networks. Here, we investigated the site-specific phosphorylation dynamics of human kinases during mitosis using synchronization of HeLa suspension cells, kinase enrichment, and high resolution mass spectrometry. In biological triplicate analyses, we identified 206 protein kinases and more than 900 protein kinase phosphorylation sites, including 61 phosphorylation sites on activation segments, and quantified their relative abundances across three specific mitotic stages. Around 25% of the kinase phosphorylation site ratios were found to be changed by at least 50% during mitotic progression. Further network analysis of jointly regulated kinase groups suggested that Cyclin-dependent kinase- and mitogen-activated kinase-centered interaction networks are coordinately down- and up-regulated in late mitosis, respectively. Importantly, our data cover most of the already known mitotic kinases and, moreover, identify attractive candidates for future studies of phosphorylation-based mitotic signaling. Thus, the results of this study provide a valuable resource for cell biologists and provide insight into the system properties of the mitotic phosphokinome.

Site-specific Phosphorylation Dynamics of the Nuclear Proteome during the DNA Damage Response

To investigate the temporal regulation of the DNA damage response, we applied quantitative mass spectrometry-based proteomics to measure site-specific phosphorylation changes of nuclear proteins after ionizing radiation. We profiled 5204 phosphorylation sites at five time points following DNA damage of which 594 sites on 209 proteins were observed to be regulated more than 2-fold. Of the 594 sites, 372 are novel phosphorylation sites primarily of nuclear origin. The 594 sites could be classified to distinct temporal profiles. Sites regulated shortly after radiation were enriched in the ataxia telangiectasia mutated (ATM) kinase SQ consensus sequence motif and a novel SXXQ motif. Importantly, in addition to induced phosphorylation, we identified a considerable group of sites that undergo DNA damage-induced dephosphorylation. Together, our data extend the number of known phosphorylation sites regulated by DNA damage, provides so far unprecedented temporal dissection of DNA damage-modified phosphorylation events, and elucidate the cross-talk between different types of post-translational modifications in the dynamic regulation of a multifaceted DNA damage response.

Shotgun Proteomics Analysis of Hibernating Arctic Ground Squirrels

Mammalian hibernation involves complex mechanisms of metabolic reprogramming and tissue protection. Previous gene expression studies of hibernation have mainly focused on changes at the mRNA level. Large scale proteomics studies on hibernation have lagged behind largely because of the lack of an adequate protein database specific for hibernating species. We constructed a ground squirrel protein database for protein identification and used a label-free shotgun proteomics approach to analyze protein expression throughout the torpor-arousal cycle during hibernation in arctic ground squirrels (Urocitellus parryii). We identified more than 3,000 unique proteins from livers of arctic ground squirrels. Among them, 517 proteins showed significant differential expression comparing animals sampled after at least 8 days of continuous torpor (late torpid), within 5 h of a spontaneous arousal episode (early aroused), and 1-2 months after hibernation had ended (non-hibernating). Consistent with changes at the mRNA level shown in a previous study on the same tissue samples, proteins involved in glycolysis and fatty acid synthesis were significantly underexpressed at the protein level in both late torpid and early aroused animals compared with non-hibernating animals, whereas proteins involved in fatty acid catabolism were significantly overexpressed. On the other hand, when we compared late torpid and early aroused animals, there were discrepancies between mRNA and protein levels for a large number of genes. Proteins involved in protein translation and degradation, mRNA processing, and oxidative phosphorylation were significantly overexpressed in early aroused animals compared with late torpid animals, whereas no significant changes at the mRNA levels between these stages had been observed. Our results suggest that there is substantial post-transcriptional regulation of proteins during torpor-arousal cycles of hibernation.

Enrichment and Site Mapping of O-Linked N-Acetylglucosamine by a Combination of Chemical/Enzymatic Tagging, Photochemical Cleavage, and Electron Transfer Dissociation Mass Spectrometry

Numerous cellular processes are regulated by the reversible addition of either phosphate or O-linked beta-N-acetylglucosamine (O-GlcNAc) to nuclear and cytoplasmic proteins. Although sensitive methods exist for the enrichment and identification of protein phosphorylation sites, those for the enrichment of O-GlcNAc-containing peptides are lacking. Reported here is highly efficient methodology for the enrichment and characterization of O-GlcNAc sites from complex samples. In this method, O-GlcNAc-modified peptides are tagged with a novel biotinylation reagent, enriched by affinity chromatography, released from the solid support by photochemical cleavage, and analyzed by electron transfer dissociation mass spectrometry. Using this strategy, eight O-GlcNAc sites were mapped from a tau-enriched sample from rat brain. Sites of GlcNAcylation were characterized on important neuronal proteins such as tau, synucleins, and methyl CpG-binding protein 2.

A New Approach for Quantitative Phosphoproteomic Dissection of Signaling Pathways Applied to T Cell Receptor Activation

Reversible protein phosphorylation plays a pivotal role in the regulation of cellular signaling pathways. Current approaches in phosphoproteomics focus on analysis of the global phosphoproteome in a single cellular state or of receptor stimulation time course experiments, often with a restricted number of time points. Although these studies have provided some insights into newly discovered phosphorylation sites that may be involved in pathways, they alone do not provide enough information to make precise predictions of the placement of individual phosphorylation events within a signaling pathway. Protein disruption and site-directed mutagenesis are essential to clearly define the precise biological roles of the hundreds of newly discovered phosphorylation sites uncovered in modern proteomics experiments. We have combined genetic analysis with quantitative proteomic methods and recently developed visual analysis tools to dissect the tyrosine phosphoproteome of isogenic Zap-70 tyrosine kinase null and reconstituted Jurkat T cells. In our approach, label-free quantitation using normalization to copurified phosphopeptide standards is applied to assemble high density temporal data within a single cell type, either Zap-70 null or reconstituted cells, providing a list of candidate phosphorylation sites that change in abundance after T cell stimulation. Stable isotopic labeling of amino acids in cell culture (SILAC) ratios are then used to compare Zap-70 null and reconstituted cells across a time course of receptor stimulation, providing direct information about the placement of newly observed phosphorylation sites relative to Zap-70. These methods are adaptable to any cell culture signaling system in which isogenic wild type and mutant cells have been or can be derived using any available phosphopeptide enrichment strategy.

SysPTM: A Systematic Resource for Proteomic Research on Post-translational Modifications

With the rapid expansion of protein post-translational modification (PTM) research based on large-scale proteomic work, there is an increasing demand for a suitable repository to analyze PTM data. Here we present a curated, web-accessible PTM data base, SysPTM. SysPTM provides a systematic and sophisticated platform for proteomic PTM research equipped not only with a knowledge base of manually curated multi-type modification data but also with four fully developed, in-depth data mining tools. Currently, SysPTM contains data detailing 117,349 experimentally determined PTM sites on 33,421 proteins involving nearly 50 PTM types, curated from public resources including five data bases and four web servers and more than one hundred peer-reviewed mass spectrometry papers. Protein annotations including Pfam domains, KEGG pathways, GO functional classification, and ortholog groups are integrated into the data base. Four online tools have been developed and incorporated, including PTMBlast, to compare a user's PTM dataset with PTM data in SysPTM; PTMPathway, to map PTM proteins to KEGG pathways; PTMPhylog, to discover potentially conserved PTM sites; and PTMCluster, to find clusters of multi-site modifications. The workflow of SysPTM was demonstrated by analyzing an in-house phosphorylation dataset identified by MS/MS. It is shown that in SysPTM, the role of single-type and multi-type modifications can be systematically investigated in a full biological context. SysPTM could be an important contribution to modificomics research. SysPTM is freely available online at www.sysbio.ac.cn/SysPTM.

In Silico Analysis of Phosphoproteome Data Suggests a Rich-get-richer Process of Phosphosite Accumulation over Evolution

Recent phosphoproteome analyses using mass spectrometry-based technologies have provided new insights into the extensive presence of protein phosphorylation in various species and have raised the interesting question of how this protein modification was gained evolutionarily on such a large scale. We investigated this issue by using human and mouse phosphoproteome data. We initially found that phosphoproteins followed a power-law distribution with regard to their number of phosphosites: most of the proteins included only a few phosphosites, but some included dozens of phosphosites. The power-law distribution, unlike more commonly observed distributions such as normal and log-normal distributions, is considered by the field of complex systems science to be produced by a specific rich-get-richer process called preferential attachment growth. Therefore, we explored the factors that may have promoted the rich-get-richer process during phosphosite evolution. We conducted a bioinformatics analysis to evaluate the relationship of amino acid sequences of phosphoproteins with the positions of phosphosites and found an overconcentration of phosphosites in specific regions of protein surfaces and implications that in many phosphoproteins these clusters of phosphosites are activated simultaneously. Multiple phosphosites concentrated in limited spaces on phosphoprotein surfaces may therefore function biologically as cooperative modules that are resistant to selective pressures during phosphoprotein evolution. We therefore proposed a hypothetical model by which the modularization of multiple phosphosites has been resistant to natural selection and has driven the rich-get-richer process of the evolutionary growth of phosphosite numbers.

Comparative Phosphoproteomics of Zebrafish Fyn/Yes Morpholino Knockdown Embryos

The coordinated movement of cells is indispensable for normal vertebrate gastrulation. Several important players and signaling pathways have been identified in convergence and extension (CE) cell movements during gastrulation, including non-canonical Wnt signaling. Fyn and Yes, members of the Src family of kinases, are key regulators of CE movements as well. Here we investigated signaling pathways in early development by comparison of the phosphoproteome of wild type zebrafish embryos with Fyn/Yes knockdown embryos that display specific CE cell movement defects. For quantitation we used differential stable isotope labeling by reductive amination of peptides. Equal amounts of labeled peptides from wild type and Fyn/Yes knockdown embryos were mixed and analyzed by on-line reversed phase TiO(2)-reversed phase LC-MS/MS. Phosphorylated and non-phosphorylated peptides were quantified, and significant changes in protein expression and/or phosphorylation were detected. We identified 348 phosphoproteins of which 69 showed a decrease in phosphorylation in Fyn/Yes knockdown embryos and 72 showed an increase in phosphorylation. Among these phosphoproteins were known regulators of cell movements, including Adducin and PDLIM5. Our results indicate that quantitative phosphoproteomics combined with morpholino-mediated knockdowns can be used to identify novel signaling pathways that act in zebrafish development in vivo.

Artifactual Sulfation of Silver-stained Proteins

Sulfation and phosphorylation are post-translational modifications imparting an isobaric 80-Da addition on the side chain of serine, threonine, or tyrosine residues. These two post-translational modifications are often difficult to distinguish because of their similar MS fragmentation patterns. Targeted MS identification of these modifications in specific proteins commonly relies on their prior separation using gel electrophoresis and silver staining. In the present investigation, we report a potential pitfall in the interpretation of these modifications from silver-stained gels due to artifactual sulfation of serine, threonine, and tyrosine residues by sodium thiosulfate, a commonly used reagent that catalyzes the formation of metallic silver deposits onto proteins. Detailed MS analyses of gel-separated protein standards and Escherichia coli cell extracts indicated that several serine, threonine, and tyrosine residues were sulfated using silver staining protocols but not following Coomassie Blue staining. Sodium thiosulfate was identified as the reagent leading to this unexpected side reaction, and the degree of sulfation was correlated with increasing concentrations of thiosulfate up to 0.02%, which is typically used for silver staining. The significance of this artifact is discussed in the broader context of sulfation and phosphorylation site identification from in vivo and in vitro experiments.