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Recent advances in combining flow cytometry and mass spectrometry have led to the development of mass cytometry, allowing for the interrogation of complex cell populations on an unprecedented scale. The volumes and high dimensionality of mass cytometry data pose significant challenges in terms of analysis and visualization. We implement a method called Radviz, where multidimensional single cell data can be visualized as a projection that maintains the original dimensions and data complexity whilst facilitating analysis and visualization. This enables identification of changes in populations, focusing the analysis on the most relevant aspect of large multidimensional datasets. To highlight the potential of Radviz, we profiled peripheral mononuclear blood cells (PBMCs) from three healthy donors and showed donor-specific differences in the number and composition of cell populations. In a second study, we explored the anti-inflammatory effects of two glucocorticoid receptor (GR) ligands (cpd6 and cpd11) compared to dexamethasone (Dex) on human primary macrophages. Standard analysis at the population level showed that cpd6 and cpd11 have an overall anti-inflammatory profile similar to that of Dex. CyTOF profiling and Radviz-driven analysis at the single cell level confirmed this observation, and identified a concentration-dependent effect of cpd6 that was not detected at the population level. Altogether, Radviz combines the strengths of a projection method, reducing the dimensionality of datasets, with that of a scatter plot, where the identity of each point can be inferred from the distance to the axis. This enables the visual exploration, analysis, and interpretation of complex, high dimensional data. © 2016 International Clinical Cytometry Society.

Death-associated protein kinase 2 (DAPK2) is a Ca(2+)/calmodulin-dependent Ser/Thr kinase that possesses tumor-suppressive functions and regulates programmed cell death, autophagy, oxidative stress, hematopoiesis, and motility. As only few binding partners of DAPK2 have been determined, the molecular mechanisms governing these biological functions are largely unknown. We report the identification of 180 potential DAPK2 interaction partners by affinity purification-coupled mass spectrometry, 12 of which are known DAPK binding proteins. A small subset of established and potential binding proteins detected in this screen was further investigated by bimolecular fluorescence complementation (BiFC) assays, a method to visualize protein interactions in living cells. These experiments revealed that α-actinin-1 and 14-3-3-β are novel DAPK2 binding partners. The interaction of DAPK2 with α-actinin-1 was localized at the plasma membrane, resulting in massive membrane blebbing and reduced cellular motility, whereas the interaction of DAPK2 with 14-3-3-β was localized to the cytoplasm, with no impact on blebbing, motility, or viability. Our results therefore suggest that DAPK2 effector functions are influenced by the protein's subcellular localization and highlight the utility of combining mass spectrometry screening with bimolecular fluorescence complementation to identify and characterize novel protein-protein interactions.

PURPOSE: Classical Hodgkin lymphoma (cHL) is characterized by a low percentage of tumor cells in a background of diverse, reactive immune cells. cHL cells commonly derive from preapoptotic germinal-center B cells and are characterized by the loss of B cell markers and the varying expression of other hematopoietic lineage markers. This phenotypic variability and the scarcity of currently available cHL-specific cell surface markers can prevent clear distinction of cHL from related lymphomas. EXPERIMENTAL DESIGN: We applied the Cell Surface Capture (CSC) technology to directly measure the pool of cell surface exposed proteins in four cHL and four non-Hodgkin lymphoma (NHL) cell lines. RESULTS: More than 1 000 membrane proteins, including 178 CD annotated proteins, were identified and allowed the generation of lymphoma surfaceome maps. The functional properties of identified cell surface proteins enable, but also limit the information exchange of lymphoma cells with their microenvironment. CONCLUSIONS AND CLINICAL RELEVANCE: Selected candidate proteins with potential diagnostic value were evaluated on a tissue microarray (TMA). Primary lymphoma tissue of 126 different B cell derived lymphoma cases were included in the TMA analysis. The TMA analysis indicated gamma-glutamyltranspeptidase 1 as a potential additional marker that can be included in a panel of markers for differential diagnosis of cHL versus NHL. This article is protected by copyright. All rights reserved.

Nucleic Acids Research, 37 (8), ISSN:1362-4962, ISSN:0301-5610

Targeted degradation of proteins through the ubiquitin-proteasome system (UPS) via the activities of E3 ubiquitin ligases regulates diverse cellular processes, and misregulation of these enzymes contributes to the pathogenesis of human diseases. One of the challenges facing the UPS field is to delineate the complete cohort of substrates for a particular E3 ligase. Advances in mass spectrometry and the development of antibodies recognizing the Lys-ϵ-Gly-Gly (diGly) remnant from ubiquitinated proteins following trypsinolysis have provided a tool to address this question. We implemented an inducible loss of function approach in combination with quantitative diGly proteomics to find novel substrates of HUWE1 (HECT, UBA, and WWE domain containing 1, E3 ubiquitin protein ligase), an E3 ligase implicated in cancer and intellectual disabilities. diGly proteomics results led to the identification of DNA damage-inducible transcript 4 (DDIT4) as a putative HUWE1 substrate. Cell-based assays demonstrated that HUWE1 interacts with and regulates ubiquitination and stability of DDIT4. Together these data suggest a model in which HUWE1 mediates DDIT4 proteasomal degradation. Our results demonstrate proof of concept that inducible knockdown of an E3 ligase in combination with diGly proteomics provides a potentially advantageous method for identifying novel E3 substrates that may help to identify candidates for therapeutic modulation in the UPS.

Scientific Reports, 7, ISSN:2045-2322

Recent advances in combining flow cytometry and mass spectrometry have led to the development of mass cytometry, allowing for the interrogation of complex cell populations on an unprecedented scale. The volumes and high dimensionality of mass cytometry data pose significant challenges in terms of analysis and visualization. We implement a method called Radviz, where multidimensional single cell data can be visualized as a projection that maintains the original dimensions and data complexity whilst facilitating analysis and visualization. This enables identification of changes in populations, focusing the analysis on the most relevant aspect of large multidimensional datasets. To highlight the potential of Radviz, we profiled peripheral mononuclear blood cells (PBMCs) from three healthy donors and showed donor-specific differences in the number and composition of cell populations. In a second study, we explored the anti-inflammatory effects of two glucocorticoid receptor (GR) ligands (cpd6 and cpd11) compared to dexamethasone (Dex) on human primary macrophages. Standard analysis at the population level showed that cpd6 and cpd11 have an overall anti-inflammatory profile similar to that of Dex. CyTOF profiling and Radviz-driven analysis at the single cell level confirmed this observation, and identified a concentration-dependent effect of cpd6 that was not detected at the population level. Altogether, Radviz combines the strengths of a projection method, reducing the dimensionality of datasets, with that of a scatter plot, where the identity of each point can be inferred from the distance to the axis. This enables the visual exploration, analysis, and interpretation of complex, high dimensional data. © 2016 International Clinical Cytometry Society.

The final step of B-cell maturation is to differentiate into plasma cells, a process that is accompanied by gross changes in subcellular organization to enable antibody secretion. To better understand this critical step in mounting a humoral immune response, we analyzed proteome dynamics during plasma cell differentiation with combined 2-DE/MS. Thirty-two identified protein spots changed in relative abundance when lipopolysaccharide (LPS)-stimulated primary B cells differentiated into antibody-secreting plasma cells. A correlative analysis of protein and transcript abundance suggested that one third of these proteins are post-transcriptionally regulated. Apart from ER-resident chaperones, lipid metabolic enzymes, and translation initiation factors, we identified several proteins that had not been previously studied in plasma cells. Among them is the transiently upregulated proteasome activator (PA) 28γ, a component of the putative nuclear proteasome. Additionally, we discovered that the non-canonical inflammatory cytokine high-mobility group box 1 (HMG1) was released from plasma cells into the extracellular milieu. This suggests a novel role for plasma cells as pro-inflammatory mediators, which has important implications for various autoimmune diseases and chronic inflammation.

Postreplicative mismatch repair (MMR) involves the concerted action of at least 20 polypeptides. Although the minimal human MMR system has recently been reconstituted in vitro, genetic evidence from different eukaryotic organisms suggests that some steps of the MMR process may be carried out by more than one protein. Moreover, MMR proteins are involved also in other pathways of DNA metabolism, but their exact role in these processes is unknown. In an attempt to gain novel insights into the function of MMR proteins in human cells, we searched for interacting partners of the MutL homologues MLH1 and PMS2 by tandem affinity purification and of PMS1 by large scale immunoprecipitation. In addition to proteins known to interact with the MutL homologues during MMR, mass spectrometric analyses identified a number of other polypeptides, some of which bound to the above proteins with very high affinity. Whereas some of these interactors may represent novel members of the mismatch repairosome, others appear to implicate the MutL homologues in biological processes ranging from intracellular transport through cell signaling to cell morphology, recombination, and ubiquitylation.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of autosomal-dominant forms of Parkinson's disease. LRRK2 is a modular, multidomain protein containing 2 enzymatic domains, including a kinase domain, as well as several protein-protein interaction domains, pointing to a role in cellular signaling. Although enormous efforts have been made, the exact pathophysiologic mechanisms of LRRK2 are still not completely known. In this study, we used a chemical genetics approach to identify LRRK2 substrates from mouse brain. This approach allows the identification of substrates of 1 particular kinase in a complex cellular environment. Several of the identified peptides are involved in the regulation of microtubule (MT) dynamics, including microtubule-associating protein (MAP)/microtubule affinity-regulating kinase 1 (MARK1). MARK1 is a serine/threonine kinase known to phosphorylate MT-binding proteins such as Tau, MAP2, and MAP4 at KXGS motifs leading to MT destabilization. In vitro kinase assays and metabolic-labeling experiments in living cells confirmed MARK1 as an LRRK2 substrate. Moreover, we also showed that LRRK2 and MARK1 are interacting in eukaryotic cells. Our findings contribute to the identification of physiologic LRRK2 substrates and point to a potential mechanism explaining the reported effects of LRRK2 on neurite morphology.

Molecular mechanisms of plasticity at GABAergic synapses are currently poorly understood. To identify signaling cascades that converge onto GABAergic postsynaptic density proteins, we performed MS analysis using gephyrin isolated from rat brain and identified multiple novel phosphorylation and acetylation residues on gephyrin. Here, we report the characterization of one of these phosphoresidues, Ser-268, which when dephosphorylated leads to the formation of larger postsynaptic scaffolds. Using a combination of mutagenesis, pharmacological treatment, and biochemical assays, we identify ERK as the kinase phosphorylating Ser-268 and describe a functional interaction between residues Ser-268 and Ser-270. We further demonstrate that alterations in gephyrin clustering via ERK modulation are reflected by amplitude and frequency changes in miniature GABAergic postsynaptic currents. We unravel novel mechanisms for activity- and ERK-dependent calpain action on gephyrin, which are likely relevant in the context of cellular signaling affecting GABAergic transmission and homeostatic synaptic plasticity in pathology.

Important aspects of photosynthetic electron transport efficiency in chloroplasts are controlled by protein phosphorylation. Two thylakoid-associated kinases, STN7 and STN8, have distinct roles in short- and long-term photosynthetic acclimation to changes in light quality and quantity. Although some substrates of STN7 and STN8 are known, the complexity of this regulatory kinase system implies that currently unknown substrates connect photosynthetic performance with the regulation of metabolic and regulatory functions. We performed an unbiased phosphoproteome-wide screen with Arabidopsis WT and stn8 mutant plants to identify unique STN8 targets. The phosphorylation status of STN7 was not affected in stn8 , indicating that kinases other than STN8 phosphorylate STN7 under standard growth conditions. Among several putative STN8 substrates, PGRL1-A is of particular importance because of its possible role in the modulation of cyclic electron transfer. The STN8 phosphorylation site on PGRL1-A is absent in both monocotyledonous plants and algae. In dicots, spectroscopic measurements with Arabidopsis WT, stn7 , stn8, and stn7 / stn8 double-mutant plants indicate a STN8-mediated slowing down of the transition from cyclic to linear electron flow at the onset of illumination. This finding suggests a possible link between protein phosphorylation by STN8 and fine-tuning of cyclic electron flow during this critical step of photosynthesis, when the carbon assimilation is not commensurate to the electron flow capacity of the chloroplast.

Postsynaptic scaffolding proteins ensure efficient neurotransmission by anchoring receptors and signaling molecules in synapse-specific subcellular domains. In turn, posttranslational modifications of scaffolding proteins contribute to synaptic plasticity by remodeling the postsynaptic apparatus. Though these mechanisms are operant in glutamatergic synapses, little is known about regulation of GABAergic synapses, which mediate inhibitory transmission in the CNS. Here, we focused on gephyrin, the main scaffolding protein of GABAergic synapses. We identify a unique phosphorylation site in gephyrin, Ser270, targeted by glycogen synthase kinase 3β (GSK3β) to modulate GABAergic transmission. Abolishing Ser270 phosphorylation increased the density of gephyrin clusters and the frequency of miniature GABAergic postsynaptic currents in cultured hippocampal neurons. Enhanced, phosphorylation-dependent gephyrin clustering was also induced in vitro and in vivo with lithium chloride. Lithium is a GSK3β inhibitor used therapeutically as mood-stabilizing drug, which underscores the relevance of this posttranslational modification for synaptic plasticity. Conversely, we show that gephyrin availability for postsynaptic clustering is limited by Ca 2+ -dependent gephyrin cleavage by the cysteine protease calpain-1. Together, these findings identify gephyrin as synaptogenic molecule regulating GABAergic synaptic plasticity, likely contributing to the therapeutic action of lithium.

Reversible protein phosphorylation ranks among the most important post-translational modifications that occurs in the cell. It is therefore highly relevant to elucidate the phosphorylation states of a given biological system, albeit challenging. Most notably the often low stoichiometry of phosphorylation is inherently incompatible with standard LC-MS analysis of a complex protein digest mixture, primarily due to the relative low dynamic range of current mass analyzers. Therefore a need for specific enrichment of phosphorylated peptides or proteins exists. Significant progress surrounding the biochemical analysis of reversible protein phosphorylation in the past years has led to the development of several new techniques to isolate or enrich phosphopeptides, particularly in large-scale analyses. This chapter deals with three such examples.

Background Members of the ErbB family of growth factor receptors are intricately linked with epithelial cell biology, development and tumourigenesis; however, the mechanisms involved in their downstream signalling are poorly understood. Indeed, it is unclear how signal specificity is achieved and the relative contribution each receptor has to specific gene expression. Methods Gene expression profiling of a human mammary luminal epithelial cell model of ErbB2-overexpression was carried out using cDNA microarrays with a common RNA reference approach to examine long-term overlapping and differential responses to EGF and heregulin beta1 treatment in the context of ErbB2 overexpression. Altered gene expression was validated using quantitative real time PCR and/or immunoblotting. One gene of interest was targeted for further characterisation, where the effects of siRNA-mediated silencing on IGF1-dependent signalling and cellular phenotype were examined and compared to the effects of loss of ErbB2 expression. Results 775 genes were differentially expressed and clustered in terms of their growth factor responsiveness. As well as identifying uncharacterized genes as novel targets of ErbB2-dependent signalling, ErbB2 overexpression augmented the induction of multiple genes involved in proliferation (e.g. MYC, MAP2K1, MAP2K3), autocrine growth factor signalling (VEGF, PDGF) and adhesion/cytoskeletal regulation (ZYX, THBS1, VCL, CNN3, ITGA2, ITGA3, NEDD9, TAGLN), linking them to the hyper-poliferative and altered adhesive phenotype of the ErbB2-overexpressing cells. We also report ErbB2-dependent down-regulation of multiple interferon-stimulated genes that may permit ErbB2-overexpressing cells to resist the anti-proliferative action of interferons. Finally, IGFBP3 was unique in its pattern of regulation and we further investigated a possible role for IGFBP3 down-regulation in ErbB2-dependent transformation through suppressed IGF1 signalling. We show that IGF1-dependent signalling and proliferation were enhanced in ErbB2-overexpressing cells, whilst loss of ErbB2 expression by siRNA silencing reduced IGF1 signalling. Furthermore, IGFBP3 knockdown resulted in basal ERK and Akt activation in luminal epithelial cells and increased invasiveness and anchorage-independent colony formation in SKBR3 cells. Conclusions These data show IGFBP3 as a negative regulator of transformation and that its down-regulation enhances IGF1-dependent signalling. They also show that ErbB2 can up-regulate IGF1-dependent signalling, possibly via the regulated expression of IGFBP3.

Immunophenotyping by flow cytometry or immunohistochemistry is a clinical standard procedure for diagnosis, classification, and monitoring of hematologic malignancies. Antibody-based cell surface phenotyping is commonly limited to cell surface proteins for which specific antibodies are available and the number of parallel measurements is limited. The resulting limited knowledge about cell surface protein markers hampers early clinical diagnosis and subclassification of hematologic malignancies. Here, we describe the mass spectrometry based phenotyping of 2 all-trans retinoic acid treated acute myeloid leukemia model systems at an unprecedented level to a depth of more than 500 membrane proteins, including 137 bona fide cell surface exposed CD proteins. This extensive view of the leukemia surface proteome was achieved by developing and applying new implementations of the Cell Surface Capturing (CSC) technology. Bioinformatic and hierarchical cluster analysis showed that the applied strategy reliably revealed known differentiation-induced abundance changes of cell surface proteins in HL60 and NB4 cells and it also identified cell surface proteins with very little prior information. The extensive and quantitative analysis of the cell surface protein landscape from a systems biology perspective will be most useful in the clinic for the improved subclassification of hematologic malignancies and the identification of new drug targets.

Fungi and bacteria are key players in the decomposition of leaf litter, but their individual contributions to the process and their interactions are still poorly known. We combined semi-quantitative proteome analyses (1-D PAGE-LC-MS/MS) with qualitative and quantitative analyses of extracellular degradative enzyme activities to unravel the respective roles of a fungus and a bacterium during litter decomposition. Two model organisms, a mesophilic Gram-negative bacterium (Pectobacterium carotovorum) and an ascomycete (Aspergillus nidulans), were grown in both, pure culture and co-culture on minimal medium containing either glucose or beech leaf litter as sole carbon source. P. carotovorum grew best in co-culture with the fungus, whereas growth of A. nidulans was significantly reduced when the bacterium was present. This observation suggests that P. carotovorum has only limited capabilities to degrade leaf litter and profits from the degradation products of A. nidulans at the expense of fungal growth. In accordance with this interpretation, our proteome analysis revealed that most of the extracellular biodegradative enzymes (i.e. proteases, pectinases, and cellulases) in the cultures with beech litter were expressed by the fungus, the bacterium producing only low levels of pectinases.

Reversible protein phosphorylation ranks among the most important post-translational modifications that occurs in the cell. It is therefore highly relevant to elucidate the phosphorylation states of a given biological system, albeit challenging. Most notably the often low stoichiometry of phosphorylation is inherently incompatible with standard LC-MS analysis of a complex protein digest mixture, primarily due to the relative low dynamic range of current mass analyzers. Therefore a need for specific enrichment of phosphorylated peptides or proteins exists. Significant progress surrounding the biochemical analysis of reversible protein phosphorylation in the past years has led to the development of several new techniques to isolate or enrich phosphopeptides, particularly in large-scale analyses. This chapter deals with three such examples.

Kinesin motors play crucial roles in the delivery of membranous cargo to its destination and thus for the establishment and maintenance of cellular polarization. Recently, calsyntenin-1 was identified as a cargo-docking protein for Kinesin-1-mediated axonal transport of tubulovesicular organelles along axons of central nervous system neurons. To further define the function of calsyntenin-1, we immunoisolated calsyntenin-1 organelles from murine brain homogenates and determined their proteome by MS. We found that calsyntenin-1 organelles are endowed with components of the endosomal trafficking machinery and contained the β-amyloid precursor protein (APP). Detailed biochemical analyses of calsyntenin-1 immunoisolates in conjunction with immunocytochemical colocalization studies with cultured hippocampal neurons, using endosomal marker proteins for distinct subcompartments of the endosomal pathways, indicated that neuronal axons contain at least two distinct, nonoverlapping calsyntenin-1-containing transport packages: one characterized as early-endosomal, APP positive, the other as recycling-endosomal, APP negative. We postulate that calsyntenin-1 acts as a general mediator of anterograde axonal transportation of endosomal vesicles. In this role, calsyntenin-1 may actively contribute to axonal growth and pathfinding in the developing as well as to the maintenance of neuronal polarity in the adult nervous system; further, it may actively contribute to the stabilization of APP during its anterograde axonal trajectory.

Background— Marfan syndrome (MFS) is a heritable disorder of connective tissue, affecting principally skeletal, ocular, and cardiovascular systems. The most life-threatening manifestations are aortic aneurysm and dissection. We investigated changes in the proteome of aortic media in patients with and without MFS to gain insight into molecular mechanisms leading to aortic dilatation. Methods and Results— Aortic samples were collected from 46 patients. Twenty-two patients suffered from MFS, 9 patients had bicuspid aortic valve, and 15 patients without connective tissue disorder served as controls. Aortic media was isolated and its proteome was analyzed in 12 patients with the use of 2-dimensional difference gel electrophoresis and mass spectrometry. We found higher amounts of filamin A C-terminal fragment, calponin 1, vinculin, microfibril-associated glycoprotein 4, and myosin-10 heavy chain in aortic media of MFS aneurysm samples than in controls. Regulation of filamin A C-terminal fragmentation was validated in all patient samples by immunoblotting. Cleavage of filamin A and the calpain substrate spectrin was increased in the MFS and bicuspid aortic valve groups. Extent of cleavage correlated positively with calpain 2 expression and negatively with the expression of its endogenous inhibitor calpastatin. Conclusions— Our observation demonstrates for the first time upregulation of the C-terminal fragment of filamin A in dilated aortic media of MFS and bicuspid aortic valve patients. In addition, our results present evidence that the cleavage of filamin A is highly likely the result of the protease calpain. Increased calpain activity might explain, at least in part, histological alterations in dilated aorta.

The target of rapamycin complex 1 (TORC1) is an essential multiprotein complex conserved from yeast to humans. Under favorable growth conditions, and in the absence of the macrolide rapamycin, TORC1 is active, and influences virtually all aspects of cell growth. Although two direct effectors of yeast TORC1 have been reported (Tap42, a regulator of PP2A phosphatases and Sch9, an AGC family kinase), the signaling pathways that couple TORC1 to its distal effectors were not well understood. To elucidate these pathways we developed and employed a quantitative, label-free mass spectrometry approach. Analyses of the rapamycin-sensitive phosphoproteomes in various genetic backgrounds revealed both documented and novel TORC1 effectors and allowed us to partition phosphorylation events between Tap42 and Sch9. Follow-up detailed characterization shows that Sch9 regulates RNA polymerases I and III, the latter via Maf1, in addition to translation initiation and the expression of ribosomal protein and ribosome biogenesis genes. This demonstrates that Sch9 is a master regulator of protein synthesis.

Protein modifications play a major role for most biological processes in living organisms. Amino-terminal acetylation of proteins is a common modification found throughout the tree of life: the N-terminus of a nascent polypeptide chain becomes co-translationally acetylated, often after the removal of the initiating methionine residue. While the enzymes and protein complexes involved in these processes have been extensively studied, only little is known about the biological function of such N-terminal modification events. To identify common principles of N-terminal acetylation, we analyzed the amino-terminal peptides from proteins extracted from Drosophila Kc167 cells. We detected more than 1,200 mature protein N-termini and could show that N-terminal acetylation occurs in insects with a similar frequency as in humans. As the sole true determinant for N-terminal acetylation we could extract the (X)PX rule that indicates the prevention of acetylation under all circumstances. We could show that this rule can be used to genetically engineer a protein to study the biological relevance of the presence or absence of an acetyl group, thereby generating a generic assay to probe the functional importance of N-terminal acetylation. We applied the assay by expressing mutated proteins as transgenes in cell lines and in flies. Here, we present a straightforward strategy to systematically study the functional relevance of N-terminal acetylations in cells and whole organisms. Since the (X)PX rule seems to be of general validity in lower as well as higher eukaryotes, we propose that it can be used to study the function of N-terminal acetylation in all species. ISSN:1544-9173 ISSN:1545-7885

In plants the vacuolar functions are the cellular storage of soluble carbohydrates, organic acids, inorganic ions and toxic compounds. Transporters and channels located in the vacuolar membrane, the tonoplast, are modulated by PTMs to facilitate the optimal functioning of a large number of metabolic pathways. Here we present a phosphoproteomic approach for the identification of in vivo phosphorylation sites of tonoplast (vacuolar membrane) proteins. Highly purified tonoplast and tonoplast-enriched microsomes were isolated from photosynthetically induced barley (Hordeum vulgare) mesophyll protoplasts. Phosphopeptides were enriched by strong cation exchange (SCX) chromatography followed either by IMAC or titanium dioxide (TiO(2)) affinity chromatography and were subsequently analysed using LC-ESI-MS/MS. In total, 65 phosphopeptides of 27 known vacuolar membrane proteins were identified, including the two vacuolar proton pumps, aquaporins, CAX transporters, Na(+)/H(+) antiporters as well as other known vacuolar transporters mediating the transfer of potassium, sugars, sulphate and malate. The present study provides a novel source to further analyse the regulation of tonoplast proteins by protein phosphorylations, especially as most of the identified phosphorylation sites are highly conserved between Hordeum vulgare (Hv) and Arabidopsis thaliana.

Nogo-A has been extensively studied as a myelin-associated neurite outgrowth inhibitor in the lesioned adult central nervous system. However, its role in the intact central nervous system has not yet been clarified. Analysis of the intact adult nervous system of C57BL/6 Nogo-A knock-out (KO) versus wild-type (WT) mice by a combined two-dimensional gel electrophoresis and isotope-coded affinity tagging approach revealed regulation of cytoskeleton-, transport-, and signaling growth-related proteins, pointing to regulation of the actin cytoskeleton, the neuronal growth machinery, and in particular the Rho-GTPase/LIMK1/cofilin pathway. Nogo-A KO adult neurons showed enlarged, more motile growth cones compared with WT neurons. The phenotype was reproduced by acute in vitro neutralization of neuronal Nogo-A. LIMK1 phosphorylation was increased in Nogo-A KO growth cones, and its reduction caused the decrease of KO growth cone motility to WT levels. Our study suggests that in the unlesioned adult nervous system, neuronal Nogo-A can restrict neuronal growth through negative modulation of growth cone motility.

High-throughput quantitation of proteins is of essential importance for all systems biology approaches and provides complementary information on steady-state gene expression and perturbation-induced systems responses. This information is necessary because it is, e.g., difficult to predict protein concentrations from the level of mRNAs, since regulatory processes at the posttranscriptional level adjust protein concentrations to prevailing conditions. Despite its importance, quantitative proteomics is still a challenging task because of the high dynamic range of protein concentrations in the cell and the variation in the physical properties of proteins. In this chapter we review the current status of, and options for, protein quantification in high-throughput experiments and discuss the suitability and limitations of different existing methods.

In the present report, a method based on chip-based nanoelectrospray mass spectrometry (nanoESI-MS) is described to detect noncovalent ligand binding to the human estrogen receptor alpha ligand-binding domain (hERalpha LBD). This system represents an important environmental interest, because a wide variety of molecules, known as endocrine disruptors, can bind to the estrogen receptor (ER) and induce adverse health effects in wildlife and humans. Using proper experimental conditions, the nanoESI-MS approach allowed for the detection of specific ligand interactions with hERalpha LBD. The relative gas-phase stability of selected hERalpha LBD-ligand complexes did not mirror the binding affinity in solution, a result that demonstrates the prominent role of hydrophobic contacts for stabilizing ER-ligand complexes in solution. The best approach to evaluate relative solution-binding affinity by nanoESI-MS was to perform competitive binding experiments with 17beta-estradiol (E2) used as a reference ligand. Among the ligands tested, the relative binding affinity for hERalpha LBD measured by nanoESI-MS was 4-hydroxtamoxifen approximately diethylstilbestrol > E2 >> genistein >> bisphenol A, consistent with the order of the binding affinities in solution. The limited reproducibility of the bound to free protein ratio measured by nanoESI-MS for this system only allowed the binding constants (K(d)) to be estimated (low nanomolar range for E2). The specificity of nanoESI-MS combined with its speed (1 min/ligand), low sample consumption (90 pmol protein/ligand), and its sensitivity for ligand (30 ng/mL) demonstrates that this technique is a promising method for screening suspected endocrine disrupting compounds and to qualitatively evaluate their binding affinity.

High-throughput quantitation of proteins is of essential importance for all systems biology approaches and provides complementary information on steady-state gene expression and perturbation-induced systems responses. This information is necessary because it is, e.g., difficult to predict protein concentrations from the level of mRNAs, since regulatory processes at the posttranscriptional level adjust protein concentrations to prevailing conditions. Despite its importance, quantitative proteomics is still a challenging task because of the high dynamic range of protein concentrations in the cell and the variation in the physical properties of proteins. In this chapter we review the current status of, and options for, protein quantification in high-throughput experiments and discuss the suitability and limitations of different existing methods.

Organic aerosols are a major fraction, often more than 50%, of the total atmospheric aerosol mass. The chemical composition of the total organic aerosol mass is poorly understood, although hundreds of compounds have been identified in the literature. High molecular weight compounds have recently gained much attention because this class of compounds potentially represents a major fraction of the unexplained organic aerosol mass. Here we analyze secondary organic aerosols, generated in a smog chamber from alpha-pinene ozonolysis with ultra-high-resolution Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). About 450 compounds are detected in the mass range of m/z 200-700. The mass spectrum is clearly divided into a low molecular weight range (monomer) and a high molecular weight range, where dimers and trimers are distinguishable. Using the Kendrick mass analysis, the elemental composition of about 60% of all peaks could be determined throughout the whole mass range. Most compounds have high O:C ratios between 0.4 and 0.6. Small compounds (i.e., monomers) have a higher maximum O:C ratio than dimers and trimers, suggesting that condensation reactions with, for example, the loss of water are important in the oligomer formation process. A program developed in-house was used to determine exact mass differences between peaks in the monomer, dimer, and trimer mass range to identify potential monomer building blocks, which form the co-oligomers observed in the mass spectrum. A majority of the peaks measured in the low mass region of the spectrum (m/z < 300) is also found in the calculated results. For the first time the elemental composition of the majority of peaks over a wide mass range was determined using advanced data analysis methods for the analysis of ultra-high-resolution MS data. Possible oligomer formation mechanisms in secondary organic aerosols were investigated.

The ability to analyze and understand the mechanisms by which cells process information is a key question of systems biology research. Such mechanisms critically depend on reversible phosphorylation of cellular proteins, a process that is catalyzed by protein kinases and phosphatases. Here, we present PhosphoPep, a database containing more than 10 000 unique high‐confidence phosphorylation sites mapping to nearly 3500 gene models and 4600 distinct phosphoproteins of the Drosophila melanogaster Kc167 cell line. This constitutes the most comprehensive phosphorylation map of any single source to date. To enhance the utility of PhosphoPep, we also provide an array of software tools that allow users to browse through phosphorylation sites on single proteins or pathways, to easily integrate the data with other, external data types such as protein–protein interactions and to search the database via spectral matching. Finally, all data can be readily exported, for example, for targeted proteomics approaches and the data thus generated can be again validated using PhosphoPep, supporting iterative cycles of experimentation and analysis that are typical for systems biology research., Molecular Systems Biology, 3 (1), ISSN:1744-4292

Activity-dependent protein phosphorylation is a highly dynamic yet tightly regulated process essential for cellular signaling. Although recognized as critical for neuronal functions, the extent and stoichiometry of phosphorylation in brain cells remain undetermined. In this study, we resolved activity-dependent changes in phosphorylation stoichiometry at specific sites in distinct subcellular compartments of brain cells. Following highly sensitive phosphopeptide enrichment using immobilized metal affinity chromatography and mass spectrometry, we isolated and identified 974 unique phosphorylation sites on 499 proteins, many of which are novel. To further explore the significance of specific phosphorylation sites, we used isobaric peptide labels and determined the absolute quantity of both phosphorylated and non-phosphorylated peptides of candidate phosphoproteins and estimated phosphorylation stoichiometry. The analyses of phosphorylation dynamics using differentially stimulated synaptic terminal preparations revealed activity-dependent changes in phosphorylation stoichiometry of target proteins. Using this method, we were able to differentiate between distinct isoforms of Ca2+/calmodulin-dependent protein kinase (CaMKII) and identify a novel activity-regulated phosphorylation site on the glutamate receptor subunit GluR1. Together these data illustrate that mass spectrometry-based methods can be used to determine activity-dependent changes in phosphorylation stoichiometry on candidate phosphopeptides following large scale phosphoproteome analysis of brain tissue.

A reproducible high-resolution protein separation method is the basis for a successful differential proteome analysis. Of the techniques currently available, two-dimensional gel electrophoresis is most widely used, because of its robustness under various experimental conditions. With the introduction of narrow range immobilized pH gradient (IPG) strips (also referred to as ultra-zoom gels) in the first dimension, the depth of analysis, i.e. the number of proteins that can be resolved, has increased substantially. However, for poorly understood reasons isoelectric focusing on ultra-zoom gels in the alkaline region above pH 7 has suffered from problems with resolution and reproducibility. To tackle these difficulties we have optimized the separation of semipreparative amounts of proteins on alkaline IPG strips by focusing on two important phenomena: counteracting water transport during isoelectric focusing and migration of dithiothreitol (DTT) in alkaline pH gradients. The first problem was alleviated by the addition of glycerol and isopropanol to the focusing medium, leading to a significant improvement in the resolution above pH 7. Even better results were obtained by the introduction of excess of the reducing agent DTT at the cathode. With these adaptations together with an optimized composition of the IPG strip, separation efficiency in the pH 6.2-8.2 range is now comparable to the widely used acidic ultra-zoom gels. We further demonstrated the usefulness of these modifications up to pH 9.5, although further improvements are still needed in that range. Thus, by extending the range covered by conventional ultra-zoom gels, the depth of analysis of two-dimensional gel electrophoresis can be significantly increased, underlining the importance of this method in differential proteomics.

The phosphorylation and dephosphorylation of proteins by kinases and phosphatases constitute an essential regulatory network in eukaryotic cells. This network supports the flow of information from sensors through signaling systems to effector molecules, and ultimately drives the phenotype and function of cells, tissues, and organisms. Dysregulation of this process has severe consequences and is one of the main factors in the emergence and progression of diseases, including cancer. Thus, major efforts have been invested in developing specific inhibitors that modulate the activity of individual kinases or phosphatases; however, it has been difficult to assess how such pharmacological interventions would affect the cellular signaling network as a whole. Here, we used label-free, quantitative phosphoproteomics in a systematically perturbed model organism (Saccharomyces cerevisiae) to determine the relationships between 97 kinases, 27 phosphatases, and more than 1000 phosphoproteins. We identified 8814 regulated phosphorylation events, describing the first system-wide protein phosphorylation network in vivo. Our results show that, at steady state, inactivation of most kinases and phosphatases affected large parts of the phosphorylation-modulated signal transduction machinery, and not only the immediate downstream targets. The observed cellular growth phenotype was often well maintained despite the perturbations, arguing for considerable robustness in the system. Our results serve to constrain future models of cellular signaling and reinforce the idea that simple linear representations of signaling pathways might be insufficient for drug development and for describing organismal homeostasis.

The regulation of DNA double strand break (DSB) repair by phosphorylation dependent signaling pathways is crucial for the maintenance of genome stability; however remarkably little is known about the molecular mechanisms by which phosphoryla tion controls DSB repair. Here we show that PIN1 a phosphorylation specific prolyl isomerase inter acts with key DSB repair factors and affects the rela tive contributions of homologous recombination (HR) and nonhomologous end joining (NHEJ) to DSB repair. We find that PIN1 deficient cells display reduced NHEJ due to increased DNA end resection whereas resection and HR are compromised in PIN1 overexpressing cells. Moreover we identify CtIP as a substrate of PIN1 and show that DSBs become hyperresected in cells expressing a CtIP mutant refractory to PIN1 recognition. Mechanisti cally we provide evidence that PIN1 impinges on CtIP stability by promoting its ubiquitylation and sub sequent proteasomal degradation. Collectively these data uncover PIN1 mediated isomerization as a regulatory mechanism coordinating DSB repair.