TY  - JOUR
AB  - Highly specialized cells are fundamental for proper functioning of complex organs. Variations in cell-type specific gene expression and protein composition have been linked to a variety of diseases. Investigation of the distinctive molecular makeup of these cells within tissues is therefore critical in biomedical research. Although several technologies have emerged as valuable tools to address this cellular heterogeneity, most workflows lack sufficient in situ resolution and are associated with high cost and extremely long analysis times. Here, we present a combination of experimental and computational approaches that allows a more comprehensive investigation of molecular heterogeneity within tissues than by either shotgun LC-MS/MS or MALDI imaging alone. We applied our pipeline on mouse brain, which contains a wide variety of cell types that not only perform unique functions but also exhibit varying sensitivities to insults. We explored the distinct neuronal populations within the hippocampus, a brain region crucial for learning and memory that is involved in various neurological disorders. As an example, we identified the groups of proteins distinguishing the neuronal populations of dentate gyrus (DG) and the cornu ammonis (CA) in the same brain section. Most of the annotated proteins matched the regional enrichment of their transcripts, thereby validating the method. As the method is highly reproducible, the identification of individual masses through the combination of MALDI-IMS and LC-MS/MS methods can be used for the much faster and more precise interpretation of MALDI-IMS measurements only. This greatly speeds up spatial proteomic analyses and allows the detection of local protein variations within the same population of cells. The method's general applicability has the potential to be used to investigate different biological conditions and tissues and a much higher throughput than other techniques making it a promising approach for clinical routine applications.
AU  - Schäfer, F.
AU  - Tomar, A.
AU  - Sato, S.*
AU  - Teperino, R.
AU  - Imhof, A.*
AU  - Lahiri, S.*
C1  - 71133
C2  - 55878
CY  - Radarweg 29, 1043 Nx Amsterdam, Netherlands
TI  - Enhanced in situ spatial proteomics by effective combination of MALDI imaging and LC-MS/MS.
JO  - Mol. Cell. Proteomics
VL  - 23
IS  - 8
PB  - Elsevier
PY  - 2024
SN  - 1535-9476
ER  - 

TY  - JOUR
AB  - Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancers and known for its extensive genetic heterogeneity, high therapeutic resistance, and strong variation in intrinsic radiosensitivity. To understand the molecular mechanisms underlying radioresistance, we screened the phenotypic response of 38 PDAC cell lines to ionizing radiation. Subsequent phosphoproteomic analysis of two representative sensitive and resistant lines led to the reproducible identification of 7,800 proteins and 13,000 phosphorylation sites (p-sites). Approximately 700 p-sites on 400 proteins showed abundance changes after radiation in all cell lines regardless of their phenotypic sensitivity. Apart from recapitulating known radiation response phosphorylation markers such as on proteins involved in DNA damage repair, the analysis uncovered many novel members of a radiation-responsive signaling network that was apparent only at the level of protein phosphorylation. These regulated p-sites were enriched in potential ATM substrates and in vitro kinase assays corroborated 10 of these. Comparing the proteomes and phosphoproteomes of radiosensitive and -resistant cells pointed to additional tractable radioresistance mechanisms involving apoptotic proteins. For instance, elevated NADPH quinine oxidoreductase 1 (NQO1) expression in radioresistant cells may aid in clearing harmful reactive oxygen species. Resistant cells also showed elevated phosphorylation levels of proteins involved in cytoskeleton organization including actin dynamics and focal adhesion kinase (FAK) activity and one resistant cell line showed a strong migration phenotype. Pharmacological inhibition of the kinases FAK by Defactinib and of CHEK1 by Rabusertib showed a statistically significant sensitization to radiation in radioresistant PDAC cells. Together, the presented data map a comprehensive molecular network of radiation-induced signaling, improves the understanding of radioresistance and provides avenues for developing radiotherapeutic strategies.
AU  - Wiechmann, S.*
AU  - Saupp, E.*
AU  - Schilling, D.
AU  - Heinzlmeir, S.*
AU  - Schneider, G.*
AU  - Schmid, R.M.*
AU  - Combs, S.E.
AU  - Kuster, B.*
AU  - Dobiasch, S.
C1  - 60327
C2  - 49256
CY  - 11200 Rockville Pike, Suite 302, Rockville, Md, United States
SP  - 1649-1663
TI  - Radiosensitization by kinase inhibition revealed by phosphoproteomic analysis of pancreatic cancer cells.
JO  - Mol. Cell. Proteomics
VL  - 19
IS  - 10
PB  - Amer Soc Biochemistry Molecular Biology Inc
PY  - 2020
SN  - 1535-9476
ER  - 

TY  - JOUR
AB  - The adipose organ, including white and brown adipose tissues, is an important player in systemic energy homeostasis, storing excess energy in form of lipids while releasing energy upon various energy demands. Recent studies have demonstrated that white and brown adipocytes also function as endocrine cells and regulate systemic metabolism by secreting factors that act locally and systemically. However, a comparative proteomic analysis of secreted factors from white and brown adipocytes and their responsiveness to adrenergic stimulation has not been reported yet. Therefore, we studied and compared the secretome of white and brown adipocytes, with and without norepinephrine (NE) stimulation. Our results reveal that carbohydrate-metabolism-regulating proteins are preferably secreted from white adipocytes, while brown adipocytes predominantly secrete a large variety of proteins. Upon NE stimulation, an increased secretion of known adipokines is favored by white adipocytes while brown adipocytes secreted higher amounts of novel adipokines. Furthermore, the secretory response between NE-stimulated and basal state was multifaceted addressing lipid and glucose metabolism, adipogenesis, and antioxidative reactions. Intriguingly, NE stimulation drastically changed the secretome in brown adipocytes. In conclusion, our study provides a comprehensive catalogue of novel adipokine candidates secreted from white and brown adipocytes with many of them responsive to NE. Given the beneficial effects of brown adipose tissue activation on its endocrine function and systemic metabolism, this study provides an archive of novel batokine candidates and biomarkers for activated brown adipose tissue.
AU  - Ali Khan, A.
AU  - Hansson, J.*
AU  - Weber, P.
AU  - Foehr, S.*
AU  - Krijgsveld, J.*
AU  - Herzig, S.
AU  - Scheideler, M.
C1  - 54181
C2  - 45433
CY  - 9650 Rockville Pike, Bethesda, Md 20814-3996 Usa
SP  - 2358-2370
TI  - Comparative secretome analyses of primary murine white and brown adipocytes reveal novel adipokines.
JO  - Mol. Cell. Proteomics
VL  - 17
IS  - 12
PB  - Amer Soc Biochemistry Molecular Biology Inc
PY  - 2018
SN  - 1535-9476
ER  - 

TY  - JOUR
AB  - The cell surface proteome is dynamic and has fundamental roles in cell signaling. Many surface membrane proteins are proteolytically released into a cell's secretome, where they can have additional functions in cell-cell-communication. Yet, it remains challenging to determine the surface proteome and to compare it to the cell secretome, under serum-containing cell culture conditions. Here, we set up and evaluated the 'surface-spanning protein enrichment with click sugars' (SUSPECS) method for cell surface membrane glycoprotein biotinylation, enrichment and label-free quantitative mass spectrometry. SUSPECS is based on click chemistry-mediated labeling of glycoproteins, is compatible with labeling of living cells and can be combined with secretome analyses in the same experiment. Immunofluorescence-based confocal microscopy demonstrated that SUSPECS selectively labeled cell surface proteins. Nearly 700 transmembrane glycoproteins were consistently identified at the surface of primary neurons. To demonstrate the utility of SUSPECS, we applied it to the protease BACE1, which is a key drug target in Alzheimer's disease. Pharmacological BACE1-inhibition selectively remodeled the neuronal surface glycoproteome, resulting in up to 7-fold increased abundance of the BACE1 substrates APP, APLP1, SEZ6, SEZ6L, CNTN2, and CHL1, whereas other substrates were not or only mildly affected. Interestingly, protein changes at the cell surface only partly correlated with changes in the secretome. Several altered proteins were validated by immunoblots in neurons and mouse brains. Apparent nonsubstrates, such as TSPAN6, were also increased, indicating that BACE1-inhibition may lead to unexpected secondary effects. In summary, SUSPECS is broadly useful for determination of the surface glycoproteome and its correlation with the secretome.
AU  - Herber, J.*
AU  - Njavro, J.*
AU  - Feederle, R.
AU  - Schepers, U.*
AU  - Müller, U.C.*
AU  - Bräse, S.*
AU  - Müller, S.A.*
AU  - Lichtenthaler, S.F.*
C1  - 54113
C2  - 45312
CY  - 9650 Rockville Pike, Bethesda, Md 20814-3996 Usa
SP  - 1487-1501
TI  - Click chemistry-mediated biotinylation reveals a function for the protease BACE1 in modulating the neuronal surface glycoproteome.
JO  - Mol. Cell. Proteomics
VL  - 17
IS  - 8
PB  - Amer Soc Biochemistry Molecular Biology Inc
PY  - 2018
SN  - 1535-9476
ER  - 

TY  - JOUR
AB  - Liquid chromatography-mass spectrometry (LC-MS)-based proteomics studies of large sample cohorts can easily require from months to years to complete. Acquiring consistent, high-quality data in such large-scale studies is challenging because of normal variations in instrumentation performance over time, as well as artifacts introduced by the samples themselves, such as those because of collection, storage and processing. Existing quality control methods for proteomics data primarily focus on post-hoc analysis to remove low-quality data that would degrade downstream statistics; they are not designed to evaluate the data in near real-time, which would allow for interventions as soon as deviations in data quality are detected. In addition to flagging analyses that demonstrate outlier behavior, evaluating how the data structure changes over time can aide in understanding typical instrument performance or identify issues such as a degradation in data quality because of the need for instrument cleaning and/or re-calibration. To address this gap for proteomics, we developed Quality Control Analysis in Real-Time (QC-ART), a tool for evaluating data as they are acquired to dynamically flag potential issues with instrument performance or sample quality. QC-ART has similar accuracy as standard post-hoc analysis methods with the additional benefit of real-time analysis. We demonstrate the utility and performance of QC-ART in identifying deviations in data quality because of both instrument and sample issues in near real-time for LC-MS-based plasma proteomics analyses of a sample subset of The Environmental Determinants of Diabetes in the Young cohort. We also present a case where QC-ART facilitated the identification of oxidative modifications, which are often underappreciated in proteomic experiments.
AU  - Stanfill, B.A.*
AU  - Nakayasu, E.S.*
AU  - Bramer, L.M.*
AU  - Thompson, A.M.*
AU  - Ansong, C.K.*
AU  - Clauss, T.R.*
AU  - Gritsenko, M.A.*
AU  - Monroe, M.E.*
AU  - Moore, R.J.*
AU  - Orton, D.J.*
AU  - Piehowski, P.D.*
AU  - Schepmoes, A.A.*
AU  - Smith, R.D.*
AU  - Webb-Robertson, B.M.*
AU  - Metz, T.O.*
AU  - TEDDY Study Group (Ziegler, A.-G.
AU  - Beyerlein, A.
AU  - Hummel, M.
AU  - Hummel, S.
AU  - Knopff, A.
AU  - Roth, R.
AU  - Scholz, M.
AU  - Stock, J.
AU  - Warncke, K.
AU  - Wendel, L.
AU  - Winkler, C.)
C1  - 55463
C2  - 46169
SP  - 1824-1836
TI  - Quality control analysis in real-time (QC-ART): A tool for real-time quality control assessment of mass spectrometry-based proteomics data.
JO  - Mol. Cell. Proteomics
VL  - 17
IS  - 9
PY  - 2018
SN  - 1535-9476
ER  - 

TY  - JOUR
AB  - Identification of interactors is a major goal in cell biology. Not only protein-protein but also protein-carbohydrate interactions are of high relevance for signal transduction in biological systems. Here we aim to identify novel interacting binding partners for the β-galactoside-binding proteins Galectin-1 (Gal-1) and Galectin-3 (Gal-3) relevant in the context of the eye disease proliferative vitreoretinopathy (PVR). PVR is one of the most common failures after retinal detachment surgeries and is characterized by the migration, adhesion and epithelial-to-mesenchymal transition (EMT) of retinal pigment epithelial cells (RPE) and the subsequent formation of sub- and epiretinal fibrocellular membranes. Gal-1 and Gal-3 bind in a dose- and carbohydrate-dependent manner to mesenchymal RPE cells and inhibit cellular processes like attachment and spreading. Yet knowledge about glycan-dependent interactors of Gal-1 and Gal-3 on RPE cells is very limited, although this is a prerequisite for unravelling the influence of galectins on distinct cellular processes in RPE cells. We identify here 131 Gal-3 and 15 Gal-1 interactors by galectin pull-down experiments combined with quantitative proteomics. They mainly play a role in multiple binding processes and are mostly membrane proteins. We focused on two novel identified interactors of Gal-1 and Gal-3 in the context of PVR: the low-density lipoprotein receptor LRP1 and the platelet-derived growth factor receptor beta PDGFRB. Addition of exogenous Gal-1 and Gal-3 induced crosslinking with LRP1/PDGFRB and Integrin-β1 (ITGB1) on the cell surface of human RPE cells and induced ERK/MAPK and Akt signaling. Treatment with Kifunensine, an inhibitor of complex-type-N-glycosylation, weakened the binding of Gal-1 and Gal-3 to these interactors and prevented lattice formation. In conclusion, the identified specific glycoprotein ligands shed light into the highly specific binding of galectins to dedifferentiated RPE cells and the resulting prevention of PVR-associated cellular events.
AU  - Obermann, J.
AU  - Priglinger, C.S.*
AU  - Merl-Pham, J.
AU  - Geerlof, A.
AU  - Priglinger, S.G.*
AU  - Götz, M.
AU  - Hauck, S.M.
C1  - 51236
C2  - 42860
CY  - Bethesda
SP  - 1528-1546
TI  - Proteome-wide identification of glycosylation-dependent interactors of Galectin-1 and Galectin-3 on mesenchymal retinal pigment epithelial cells.
JO  - Mol. Cell. Proteomics
VL  - 16
IS  - 8
PB  - Amer Soc Biochemistry Molecular Biology Inc
PY  - 2017
SN  - 1535-9476
ER  - 

TY  - JOUR
AB  - Atherosclerosis leads to vascular lesions that involve major rearrangements of the vascular proteome, especially of the extracellular matrix (ECM). Using single aortas from ApoE knock out mice, we quantified formation of plaques by single-run, high-resolution mass spectrometry (MS)based proteomics. To probe localization on a proteome-wide scale we employed quantitative detergent solubility profiling. This compartment- and time-resolved resource of atherogenesis comprised 5117 proteins, 182 of which changed their expression status in response to vessel maturation and atherosclerotic plaque development. In the insoluble ECM proteome, 65 proteins significantly changed, including relevant collagens, matrix metalloproteinases and macrophage derived proteins. Among novel factors in atherosclerosis, we identified matrilin-2, the collagen IV crosslinking enzyme peroxidasin as well as the poorly characterized MAM-domain containing 2 (Mamdc2) protein as being up-regulated in the ECM during atherogenesis. Intriguingly, three subunits of the osteoclast specific V-ATPase complex were strongly increased in mature plaques with an enrichment in macrophages thus implying an active de-mineralization function.
AU  - Wierer, M.*
AU  - Prestel, M.*
AU  - Schiller, H. B.
AU  - Yan, G.*
AU  - Schaab, C.*
AU  - Azghandi, S.*
AU  - Werner, J.*
AU  - Kessler, T.*
AU  - Malik, R.*
AU  - Murgia, M.*
AU  - Aherrahrou, Z.*
AU  - Schunkert, H.*
AU  - Dichgans, M.*
AU  - Mann, M.*
C1  - 52496
C2  - 44021
CY  - Bethesda
SP  - 321-334
TI  - Compartment-resolved proteomic analysis of mouse aorta during atherosclerotic plaque formation reveals osteoclast-specific protein expression.
JO  - Mol. Cell. Proteomics
VL  - 17
IS  - 2
PB  - Amer Soc Biochemistry Molecular Biology Inc
PY  - 2017
SN  - 1535-9476
ER  - 

TY  - JOUR
AB  - To date, the proteomic profiling of Muller cells, the dominant macroglia of the retina, has been hampered due to the absence of suitable enrichment methods. We established a novel protocol to isolate native, intact Muller cells from adult murine retinae at excellent purity which retain in situ morphology and are well suited for proteomic analyses. Two different strategies of sample preparation, an in StageTips (iST) and a subcellular fractionation approach including cell surface protein profiling were used for quantitative LC MSMS comparing Muller cell enriched to depleted neuronal fractions. Pathway enrichment analyses on both datasets enabled us to identify Muller cell specific functions which included focal adhesion kinase signaling, signal transduction mediated by calcium as second messenger, transmembrane neurotransmitter transport and antioxidant activity. Pathways associated with RNA processing, cellular respiration and phototransduction were enriched in the neuronal subpopulation. Proteomic results were validated for selected Muller cell genes by quantitative RT-PCR, confirming the high expression levels of numerous members of the angiogenic and anti-inflammatory annexins and antioxidant enzymes (e.g. paraoxonase 2, peroxiredoxin 1, 4 and 6). Finally, the significant enrichment of antioxidant proteins in Muller cells was confirmed by measurements on vital retinal cells using the oxidative stress indicator CM-H2DCFDA. In contrast to photoreceptors or bipolar cells, Muller cells were most efficiently protected against H2O2-induced reactive oxygen species formation, which is in line with the protein repertoire identified in the proteomic profiling. Our novel approach to isolate intact glial cells from adult retina in combination with proteomic profiling enabled the identification of novel Muller glia specific proteins, which were validated as markers and for their functional impact in glial physiology. This provides the basis to allow the discovery of novel glial specializations and will enable us to elucidate the role of Muller cells in retinal pathologies, a topic still controversially discussed.
AU  - Grosche, A.*
AU  - Hauser, A.*
AU  - Lepper, M.F.
AU  - Mayo, R.*
AU  - von Toerne, C.
AU  - Merl-Pham, J.
AU  - Hauck, S.M.
C1  - 46696
C2  - 37726
CY  - Bethesda
SP  - 462-480
TI  - The proteome of native adult Muller glial cells from murine retina.
JO  - Mol. Cell. Proteomics
VL  - 15
IS  - 2
PB  - Amer Soc Biochemistry Molecular Biology Inc
PY  - 2016
SN  - 1535-9476
ER  - 

TY  - JOUR
AB  - Naive CD4+ T cells are the common precursors of multiple effector and memory T cell subsets and possess a high plasticity in terms of differentiation potential. This stem-cell like character is important for cell therapies aiming at regeneration of specific immunity. Cell surface proteins are crucial for recognition and response to signals mediated by other cells or environmental changes. Knowledge of cell surface proteins of human naive CD4+ T cells and their changes during the early phase of T cell activation is urgently needed for a guided differentiation of naive T cells and may support the selection of pluripotent cells for cell therapy. Periodate oxidation and aniline-catalyzed oxime ligation (PAL) technology was applied with subsequent quantitative LC-MS/MS (PAL-qLC-MS/MS) to generate a dataset describing the surface proteome of primary human naive CD4+ T cells and to monitor dynamic changes during the early phase of activation. This led to the identification of 173 N-glycosylated surface proteins. To independently confirm the proteomic dataset and to analyse the cell surface by an alternative technique a systematic phenotypic expression analysis of surface antigens via flow cytometry was performed. This screening expanded the previous dataset, resulting in 229 surface proteins, which were expressed on naive unstimulated and activated CD4+ T cells. Furthermore, we generated a surface expression atlas based on transcriptome data, experimental annotation and predicted subcellular localization, and correlated the proteomics result with this transcriptional dataset. This extensive surface atlas provides an overall naive CD4+ T cell surface resource and will enable future studies aiming at a deeper understanding of mechanisms of T cell biology allowing the identification of novel immune targets usable for the development of therapeutic treatments.
AU  - Graessel, A.
AU  - Hauck, S.M.
AU  - von Toerne, C.
AU  - Kloppmann, E.*
AU  - Goldberg, T.*
AU  - Koppensteiner, H.
AU  - Schindler, M.
AU  - Knapp, B.
AU  - Krause, L.
AU  - Dietz, K.
AU  - Schmidt-Weber, C.B.
AU  - Suttner, K.
C1  - 45063
C2  - 37186
CY  - Bethesda
SP  - 2085-2102
TI  - A combined omics approach to generate the surface atlas of human naive CD4+ T cells during early TCR activation.
JO  - Mol. Cell. Proteomics
VL  - 14
IS  - 8
PB  - Amer Soc Biochemistry Molecular Biology Inc
PY  - 2015
SN  - 1535-9476
ER  - 

TY  - JOUR
AB  - While it is widely accepted that ectopic lipid accumulation in the liver is associated with hepatic insulin resistance, the underlying molecular mechanisms have not been well characterized. Here we employed time resolved quantitative proteomic profiling of mice fed a high fat diet to determine which pathways were affected during the transition of the liver to an insulin-resistant state. We identified several metabolic pathways underlying altered protein expression. In order to test the functional impact of a critical subset of these alterations, we focused on the epoxyeicosatrienoic acid (EET) eicosanoid pathway, whose deregulation coincided with the onset of hepatic insulin resistance. These results suggested that EETs may be positive modulators of hepatic insulin signaling. Analyzing EET activity in primary hepatocytes, we found that EETs enhance insulin signaling on the level of Akt. In contrast, EETs did not influence insulin receptor or insulin receptor substrate-1 phosphorylation. This effect was mediated through the eicosanoids, as overexpression of the deregulated enzymes in absence of arachidonic acid had no impact on insulin signaling. The stimulation of insulin signaling by EETs and depression of the pathway in insulin resistant liver suggest a likely role in hepatic insulin resistance. Our findings support therapeutic potential for inhibiting EET degradation.
AU  - Schäfer, A.
AU  - Neschen, S.
AU  - Kahle-Stephan, M.
AU  - Sarioglu, H.
AU  - Gaisbauer, T.
AU  - Imhof, A.*
AU  - Adamski, J.
AU  - Hauck, S.M.
AU  - Ueffing, M.
C1  - 45193
C2  - 37250
SP  - 2764-2774
TI  - The epoxyeicosatrienoic acid pathway enhances hepatic insulin signaling and is repressed in insulin-resistant mouse liver.
JO  - Mol. Cell. Proteomics
VL  - 14
IS  - 10
PY  - 2015
SN  - 1535-9476
ER  - 

TY  - JOUR
AB  - Hepatitis C virus (HCV) is a global health problem and one of the main reasons for chronic liver disease like cirrhosis and hepatocellular carcinoma. The HCV genome is translated into a polyprotein which is proteolytically processed into ten viral proteins. The interactome of the HCV proteins with the host cell has been worked out, however it remains unclear how viral proteins interact with each other. We aimed to generate the interaction network of these ten HCV proteins by a flow cytometry based FRET assay established in our laboratory (Banning et al, 2010 PLoS ONE 5(2): e9344). HCV proteins were constructed as fusions with the chromophores CFP and YFP. All HCV fusions were expressed and localized to specific subcellular compartments, indicating that they are functional. FACS-FRET measurements identified a total of 20 interactions. 13 of these were previously described and are now confirmed by our method in living cells. Among the seven novel protein binding pairs HCV p7 plays a pivotal role. It binds to the HCV capsid protein Core and the two glycoproteins E1 and E2. These interplays were further demonstrated in the relevant context of Huh7.5 liver cells expressing infectious HCV. Our work demonstrates the feasibility to rapidly generate small interaction networks by FACS-FRET and defines the network of intra HCV protein interactions. Furthermore, our data supports an important role of p7 in HCV assembly.
AU  - Hagen, N.*
AU  - Bayer, K.
AU  - Roesch, K.*
AU  - Schindler, M.
C1  - 31299
C2  - 34312
CY  - Bethesda
SP  - 1676-1689
TI  - The intra viral protein interaction network of hepatitis C virus.
JO  - Mol. Cell. Proteomics
VL  - 13
IS  - 7
PB  - Amer Soc Biochemistry Molecular Biology Inc
PY  - 2014
SN  - 1535-9476
ER  - 

TY  - JOUR
AB  - Liquid chromatography coupled to mass spectrometry (LC-MS) has become a standard technology in metabolomics. In particular, label-free quantification based on LC-MS is easily amenable to large-scale studies and thus well suited to clinical metabolomics. Large-scale studies, however, require automated processing of the large and complex LC-MS datasets. We present a novel algorithm for the detection of mass traces and their aggregation into features (i.e. all signals caused by the same analyte species) that is computationally efficient and sensitive and that leads to reproducible quantification results. The algorithm is based on a sensitive detection of mass traces, which are then assembled into features based on mass-to-charge spacing, co-elution information, and a support vector machine-based classifier able to identify potential metabolite isotope patterns. The algorithm is not limited to metabolites but is applicable to a wide range of small molecules (e.g. lipidomics, peptidomics), as well as to other separation technologies. We assessed the algorithm's robustness with regard to varying noise levels on synthetic data and then validated the approach on experimental data investigating human plasma samples. We obtained excellent results in a fully automated data-processing pipeline with respect to both accuracy and reproducibility. Relative to state-of-the art algorithms, ours demonstrated increased precision and recall of the method. The algorithm is available as part of the open-source software package OpenMS and runs on all major operating systems.
AU  - Kenar, E.*
AU  - Franken, H.*
AU  - Forcisi, S.
AU  - Wörmann, K.
AU  - Häring, H.-U.
AU  - Lehmann, R.
AU  - Schmitt-Kopplin, P.
AU  - Zell, A.*
AU  - Kohlbacher, O.*
C1  - 29234
C2  - 33785
SP  - 348-359
TI  - Automated label-free quantification of metabolites from liquid chromatography-mass spectrometry data.
JO  - Mol. Cell. Proteomics
VL  - 13
IS  - 1
PB  - Amer Soc Biochemistry Molecular Biology Inc
PY  - 2014
SN  - 1535-9476
ER  - 

TY  - JOUR
AB  - A complex and still not comprehensively resolved panel of transmembrane proteins regulates the outgrowth and the subsequent morphological and functional development of neuronal processes. In order to gain a more detailed description of these events at the molecular level, we have developed a cell surface biotinylation assay to isolate, detect, and quantify neuronal membrane proteins. When we applied our assay to investigate neuron maturation in vitro, we identified 439 differentially expressed proteins, including 20 members of the immunoglobulin superfamily. Among these candidates, we focused on Negr1, a poorly described cell adhesion molecule. We demonstrated that Negr1 controls the development of neurite arborization in vitro and in vivo. Given the tight correlation existing among synaptic cell adhesion molecules, neuron maturation, and a number of neurological disorders, our assay results are a useful tool that can be used to support the understanding of the molecular bases of physiological and pathological brain function.
AU  - Pischedda, F.*
AU  - Szczurkowska, J.*
AU  - Cirnaru, M.D.*
AU  - Giesert, F.
AU  - Vezzoli, E.*
AU  - Ueffing, M.
AU  - Sala, C.*
AU  - Francolini, M.*
AU  - Hauck, S.M.
AU  - Cancedda, L.*
AU  - Piccoli, G.*
C1  - 30729
C2  - 33800
CY  - Bethesda
SP  - 733-748
TI  - A cell surface biotinylation assay to reveal membrane-associated neuronal cues: Negr1 regulates dendritic arborization.
JO  - Mol. Cell. Proteomics
VL  - 13
IS  - 3
PB  - Amer Soc Biochemistry Molecular Biology Inc
PY  - 2014
SN  - 1535-9476
ER  - 

TY  - JOUR
AB  - Analyzing the molecular architecture of native multiprotein complexes via biochemical methods has so far been difficult and error prone. Protein complex isolation by affinity purification can define the protein repertoire of a given complex, yet, it remains difficult to gain knowledge of its substructure or modular composition. Here, we introduce SDS concentration gradient induced decomposition of protein complexes coupled to quantitative mass spectrometry and in silico elution profile distance analysis. By applying this new method to a cellular transport module, the IFT/lebercilin complex, we demonstrate its ability to determine modular composition as well as sensitively detect known and novel complex components. We show that the IFT/lebercilin complex can be separated into at least five submodules, the IFT complex A, the IFT complex B, the 14-3-3 protein complex and the CTLH complex, as well as the dynein light chain complex. Furthermore, we identify the protein TULP3 as a potential new member of the IFT complex A and showed that several proteins, classified as IFT complex B-associated, are integral parts of this complex. To further demonstrate EPASIS general applicability, we analyzed the modular substructure of two additional complexes, that of B-RAF and of 14-3-3-epsilon. The results show, that EPASIS provides a robust as well as sensitive strategy to dissect the substructure of large multiprotein complexes in a highly time- as well as cost-effective manner.
AU  - Texier, Y.
AU  - Toedt, G.*
AU  - Gorza, M.
AU  - Mans, D.A.*
AU  - van Reeuwijk, J.*
AU  - Horn, N.*
AU  - Willer, J.*
AU  - Katsanis, N.*
AU  - Roepman, R.*
AU  - Gibson, T.J.*
AU  - Ueffing, M.
AU  - Boldt, K.*
C1  - 31653
C2  - 34611
CY  - Bethesda
SP  - 1382-1391
TI  - Elution profile analysis of SDS-induced subcomplexes by quantitative mass spectrometry.
JO  - Mol. Cell. Proteomics
VL  - 13
IS  - 5
PB  - Amer Soc Biochemistry Molecular Biology Inc
PY  - 2014
SN  - 1535-9476
ER  - 

TY  - JOUR
AB  - Retinal Muller glial cells (RMG) have a primary role in maintaining the homeostasis of the retina. In pathological situations, RMG execute protective and regenerative effects, but can also contribute to neurodegeneration. Cultured primary RMG have recently been recognized to secrete pro-survival factors for retinal neurons for up to two weeks in culture, but this ability is lost when RMG are cultivated for longer durations. In our study, we investigated RMG supernatants for novel neuroprotective factors using a quantitative proteomic approach. Stable isotope labeling by amino acids in cell culture (SILAC) was used on primary porcine RMG. Supernatants of RMG cultivated for two weeks were compared to supernatants from cells which had already lost their protective capacity. Using this approach, we detected established neurotrophic factors such as transferrin, osteopontin (SPP1), and leukemia inhibitory factor (LIF), and identified C-X-C motif chemokine 10 (CXCL10) as a novel candidate neuroprotective factor. All factors prolonged photoreceptor survival in vitro. Ex-vivo treatment of retinal explants with LIF or CXCL10 demonstrated a neuroprotective effect on photoreceptors (PR). Western blots on CXCL10 and LIF stimulated explanted retina and PR lysates indicated activation of pro-survival Signal Transducer and Activator of Transcription (STAT) signaling and B-cell lymphoma (BCL) pathways. These findings suggest that CXCL10 contributes to the supportive potential of RMG towards retinal neurons.
AU  - von Toerne, C.
AU  - Menzler, J.
AU  - Ly, A.
AU  - Senninger, N.
AU  - Ueffing, M.
AU  - Hauck, S.M.
C1  - 31606
C2  - 34630
CY  - Bethesda
SP  - 2371-2381
TI  - Identification of a novel neurotrophic factor from primary retinal Müller cells using SILAC.
JO  - Mol. Cell. Proteomics
VL  - 13
IS  - 9
PB  - Amer Soc Biochemistry Molecular Biology Inc
PY  - 2014
SN  - 1535-9476
ER  - 

TY  - JOUR
AB  - MALDI imaging mass spectrometry (MALDI IMS) is a powerful tool for the visualization of proteins in tissues and has demonstrated considerable diagnostic and prognostic value. One main challenge is that the molecular identity of such potential biomarkers mostly remains unknown. We introduce a generic method that removes this issue by systematically identifying the proteins embedded in the MALDI matrix using a combination of bottom-up and top-down proteomics. The analyses of ten human tissues lead to the identification of 1,400 abundant and soluble proteins constituting the set of proteins detectable by MALDI IMS including >90% of all IMS biomarkers reported in the literature. Top-down analysis of the matrix proteome identified 124 mostly N- and C-terminally fragmented proteins indicating considerable protein processing activity in tissues. All protein identification data from this study as well as the IMS literature has been deposited into MaTisse, a new publically available database which we anticipate will become a valuable resource for the IMS community.
AU  - Maier, S.K.
AU  - Hahne, H.*
AU  - Moghaddas Gholami, A.*
AU  - Balluff, B.
AU  - Meding, S.
AU  - Schoene, C.
AU  - Walch, A.K.
AU  - Kuster, B.*
C1  - 24957
C2  - 31734
CY  - Bethesda
SP  - 2901-2910
TI  - Comprehensive identification of proteins from MALDI imaging.
JO  - Mol. Cell. Proteomics
VL  - 12
IS  - 10
PB  - Amer. Soc. Biochemistry Molecular Biology Inc.
PY  - 2013
SN  - 1535-9476
ER  - 

TY  - JOUR
AB  - High confidence definition of protein interactions is an important objective towards biological systems understanding. Isotope labeling in combination with affinity-based isolation of protein complexes has increased in accuracy and reproducibility, yet, larger organisms - including humans - are hardly accessible to metabolic labeling and thus, a major limitation has been its restriction to small animals, cell lines and yeast. As composition as well as stoichiometries of protein complexes can significantly differ in primary tissues, there is a great demand for methods capable to combine the selectivity of affinity-based isolation as well as the accuracy and reproducibility of isotope-based labeling with its application towards analysis of protein interactions from intact tissue. Towards this goal, we combined isotope coded protein labeling (ICPL) with immunoprecipitation (IP) and quantitative mass spectrometry (MS). ICPL-IP allows sensitive and accurate analysis of protein interactions from primary tissue. We applied ICPL-IP to immuno-isolate protein complexes from bovine retinal tissue. Protein complexes of immunoprecipitated β-tubulin, a highly abundant protein with known interactors as well as the lowly expressed small GTPase RhoA were analyzed. The results of both analyses demonstrate sensitive and selective identification of known as well as new protein interactions by our method
AU  - Vogt, A.*
AU  - Fuerholzner, B.*
AU  - Kinkl, N.*
AU  - Boldt, K.*
AU  - Ueffing, M.
C1  - 22923
C2  - 30959
SP  - 1395-1406
TI  - Isotope Coded Protein Labeling Coupled Immunoprecipitation (ICPL-IP): A novel approach for quantitative protein complex analysis from native tissue.
JO  - Mol. Cell. Proteomics
VL  - 12
IS  - 5
PB  - American Society of Biochemistry and Molecular Biology Inc.
PY  - 2013
SN  - 1535-9476
ER  - 

TY  - JOUR
AB  - Argonaute (Ago) proteins interact with small regulatory RNAs such as microRNAs (miRNAs) and facilitate gene-silencing processes. miRNAs guide Ago proteins to specific mRNAs leading to translational silencing or mRNA decay. In order to understand the mechanistic details of miRNA function, it is important to characterize Ago protein interactors. Although several proteomic studies have been performed, it is not clear how the Ago interactome changes on miRNA or mRNA binding. Here, we report the analysis of Ago protein interactions in miRNA-containing and miRNA-depleted cells. Using stable isotope labeling in cell culture in conjunction with Dicer knock out mouse embryonic fibroblasts, we identify proteins that interact with Ago2 in the presence or the absence of Dicer. In contrast to our current view, we find that Ago-mRNA interactions can also take place in the absence of miRNAs. Our proteomics approach provides a rich resource for further functional studies on the cellular roles of Ago proteins.
AU  - Frohn, A.*
AU  - Eberl, H.C.*
AU  - Stöhr, J.*
AU  - Glasmacher, E.
AU  - Rüdel, S.*
AU  - Heissmeyer, V.
AU  - Mann, M.*
AU  - Meister, G.*
C1  - 11274
C2  - 30575
SP  - 1442-1456
TI  - Dicer-dependent and -independent Argonaute2 protein interaction networks in mammalian cells.
JO  - Mol. Cell. Proteomics
VL  - 11
IS  - 11
PB  - American Society of Biochemistry and Molecular Biology Inc.
PY  - 2012
SN  - 1535-9476
ER  - 

TY  - JOUR
AB  - Matrix-assisted laser desorption/ionization (MALDI) molecular imaging technology attracts increasing attention in the field of biomarker discovery. The unambiguous correlation between histopathology and MALDI images is a key feature for success. MALDI imaging mass spectrometry (IMS) at high definition thus calls for technological developments that were established by a number of small steps. This included tissue and matrix preparation steps, dedicated lasers for MALDI imaging, increase of the robustness against cell debris and matrix sublimation, software for precision matching of molecular and microscopic images, and the analysis of MALDI imaging data using multivariate statistical methods. The goal of these developments is to approach single cell resolution with IMS. Currently a performance level of 20 µm image resolution was achieved with an unmodified and commercially available instrument for proteins detected in the 2-16 kDa range. The rat testis was used as a relevant model for validating and optimizing our technological developments. Indeed, testicular anatomy is amongst the most complex found in mammalian bodies. In the present study, we were able to visualize, at 20 µm image resolution level, different stages of germ cell development in testicular seminiferous tubules, to provide a molecular correlate for its well-established stage-specific classification, to identify proteins of interest using a top-down approach and superimpose molecular and immunohistochemistry images.
AU  - Lagarrigue, M.*
AU  - Becker, M.*
AU  - Lavigne, R.*
AU  - Deininger, S.O.*
AU  - Walch, A.K.
AU  - Aubry, F.*
AU  - Suckau, D.*
AU  - Pineau, C.*
C1  - 6139
C2  - 28151
TI  - Revisiting rat spermatogenesis with MALDI imaging at 20- μm resolution.
JO  - Mol. Cell. Proteomics
VL  - 10
IS  - 3
PB  - American Society for Biochemistry and Molecular Biology, Inc.
PY  - 2011
SN  - 1535-9476
ER  - 

TY  - JOUR
AB  - Mutations in human leucine-rich repeat kinase 2 (Lrrk2), a protein of yet unknown function, are linked to Parkinson's disease caused by degeneration of midbrain dopaminergic neurons. The protein comprises several domains including a GTPase- and a kinase domain both affected by several pathogenic mutations. To elucidate the molecular interaction network of endogenous Lrrk2 under stoichiometric constraints, we applied QUICK (quantitative immunoprecipitation combined with knockdown) in NIH3T3 cells. The identified interactome reveals actin isoforms as well as actin-associated proteins involved in actin filament assembly, organization, rearrangement and maintenance, suggesting that the biological function of Lrrk2 is linked to cytoskeletal dynamics. In fact, we demonstrate Lrrk2 de novo binding to F-actin and its ability to modulate its assembly in vitro. When tested in intact cells, knockdown of Lrrk2 causes morphological alterations in NIH3T3 cells. In developing dopaminergic midbrain primary neurons, Lrrk2 knockdown results in shortened neurite processes, indicating a physiological role of Lrrk2 in cytoskeletal organization and dynamics of dopaminergic neurons. Hence, our results demonstrate that molecular interactions as well as the physiological function of Lrrk2 are closely related to the organization of the actin-based cytoskeleton, a crucial feature of neuronal development and neuron function.
AU  - Meixner, A.
AU  - Boldt, K.
AU  - van Troys, M.*
AU  - Askenazi, M.*
AU  - Gloeckner, C.J.
AU  - Bauer, M.
AU  - Marto, J.A.*
AU  - Ampe, C.*
AU  - Kinkl, N.
AU  - Ueffing, M.
C1  - 4974
C2  - 27750
TI  - A QUICK screen for Lrrk2 interaction partners - leucine-rich repeat kinase 2 is involved in actin cytoskeleton dynamics.
JO  - Mol. Cell. Proteomics
VL  - 10
IS  - 1
PB  - American Society for Biochemistry and Molecular Biology
PY  - 2011
SN  - 1535-9476
ER  - 

TY  - JOUR
AB  - Tumors of the head and neck represent a molecularly diverse set of human cancers, but relatively few proteins have actually been shown to drive the disease at the molecular level. In order to identify new targets for individualized diagnosis or therapeutic intervention, we performed a kinase centric chemical proteomics screen and quantified 146 kinases across 34 head and neck squamous cell carcinoma (HNSCC) cell lines using intensity-based label-free mass spectrometry. Statistical analysis of the profiles revealed significant inter-cell line differences for 42 kinases (p<0.05), and loss of function experiments using siRNA in high- and low- expressing cell lines identified kinases including EGFR, NEK9, LYN, JAK1, WEE1 and EPHA2involved in cell survival and proliferation. EGFR inhibition by the small molecule inhibitors lapatinib, gefitinib and erlotinib as well as siRNA led to strong reduction of viability in high- but not low- expressing lines confirming EGFR as a drug target in 10-20% of HNSCC cell lines. Similarly, high, but not low EPHA2-expressing cells showed strongly reduced viability concomitant with down-regulation of AKT and ERK signaling following EPHA2 siRNA treatment or EPHA1-Fc ligand exposure, suggesting that EPHA2 is a novel drug target in HNSCC. This notion is underscored by immunohistochemical analyses showing that high EPHA2 expression is detected in a subset of HNSCC tissues and is associated with poor prognosis. Given that the approved pan-SRC family kinase inhibitor, dasatinib is also a very potent inhibitor of EPHA2, our findings may lead to new therapeutic options for HNSCC patients. Importantly, the strategy employed in this study is generic and therefore also of more general utility for the identification of novel drug targets and molecular pathway markers in tumors. This may ultimately lead to a more rational approach to individualized cancer diagnosis and therapy.
AU  - Wu, Z.*
AU  - Doondeea, J.B.*
AU  - Moghaddas Gholami, A.*
AU  - Janning, M.C.*
AU  - Lemeer, S.*
AU  - Kramer, K.*
AU  - Eccles, S.A.*
AU  - Gollin, S.M.*
AU  - Grenman, R.*
AU  - Walch, A.K.
AU  - Feller, S.M.*
AU  - Kuster, B.*
C1  - 6723
C2  - 29153
TI  - Quantitative chemical proteomics reveals new potential drug targets in head and neck cancer.
JO  - Mol. Cell. Proteomics
VL  - 10
IS  - 12
PB  - American Society for Biochemistry and Molecular Biolog
PY  - 2011
SN  - 1535-9476
ER  - 

TY  - JOUR
AB  - Protein affinity reagents (PARs), most commonly antibodies, are essential reagents for protein characterization in basic research, biotechnology, and diagnostics as well as the fastest growing class of therapeutics. Large numbers of PARs are available commercially; however, their quality is often uncertain. In addition, currently available PARs cover only a fraction of the human proteome, and their cost is prohibitive for proteome scale applications. This situation has triggered several initiatives involving large scale generation and validation of antibodies, for example the Swedish Human Protein Atlas and the German Antibody Factory. Antibodies targeting specific subproteomes are being pursued by members of Human Proteome Organisation (plasma and liver proteome projects) and the United States National Cancer Institute (cancer-associated antigens). ProteomeBinders, a European consortium, aims to set up a resource of consistently quality-controlled protein-binding reagents for the whole human proteome. An ultimate PAR database resource would allow consumers to visit one on-line warehouse and find all available affinity reagents from different providers together with documentation that facilitates easy comparison of their cost and quality. However, in contrast to, for example, nucleotide databases among which data are synchronized between the major data providers, current PAR producers, quality control centers, and commercial companies all use incompatible formats, hindering data exchange. Here we propose Proteomics Standards Initiative (PSI)-PAR as a global community standard format for the representation and exchange of protein affinity reagent data. The PSI-PAR format is maintained by the Human Proteome Organisation PSI and was developed within the context of ProteomeBinders by building on a mature proteomics standard format, PSI-molecular interaction, which is a widely accepted and established community standard for molecular interaction data. Further information and documentation are available on the PSI-PAR web site.
AU  - Gloriam, D.E.*
AU  - Orchard, S.*
AU  - Bertinetti, D.*
AU  - Björling, E.*
AU  - Bongcam-Rudloff, E.*
AU  - Borrebaeck, C.A.K.*
AU  - Bourbeillon, J.*
AU  - Bradbury, A.R.M.*
AU  - de Daruvar, A.*
AU  - Dübel, S.*
AU  - Frank, R.*
AU  - Gibson, T.J.*
AU  - Gold, L.*
AU  - Haslam, N.*
AU  - Herberg, F.W.*
AU  - Hiltke, T.*
AU  - Hoheisel, J.D.*
AU  - Kerrien, S.*
AU  - Koegl, M.*
AU  - Konthur, Z.*
AU  - Korn, B.*
AU  - Landegren, U.*
AU  - Montecchi-Palazzi, L.*
AU  - Palcy, S.*
AU  - Rodriguez, H.*
AU  - Schweinsberg, S.*
AU  - Sievert, V.*
AU  - Stoevesandt, O.*
AU  - Taussig, M.J.*
AU  - Ueffing, M.
AU  - Uhlén, M.*
AU  - van der Maarel, S.*
AU  - Wingren, C.*
AU  - Woollard, P.*
AU  - Sherman, D.J.*
AU  - Hermjakob, H.*
C1  - 5984
C2  - 27766
SP  - 1-10
TI  - A community standard format for the representation of protein affinity reagents.
JO  - Mol. Cell. Proteomics
VL  - 9
IS  - 1
PB  - Amer Soc Biochemistry Molecular Biology Inc.
PY  - 2010
SN  - 1535-9476
ER  - 

TY  - JOUR
AB  - Because of its availability, ease of collection, and correlation with physiology and pathology, urine is an attractive source for clinical proteomics/peptidomics. However, the lack of comparable data sets from large cohorts has greatly hindered the development of clinical proteomics. Here, we report the establishment of a reproducible, high resolution method for peptidome analysis of naturally occurring human urinary peptides and proteins, ranging from 800 to 17,000 Da, using samples from 3,600 individuals analyzed by capillary electrophoresis coupled to MS. All processed data were deposited in an Structured Query Language (SQL) database. This database currently contains 5,010 relevant unique urinary peptides that serve as a pool of potential classifiers for diagnosis and monitoring of various diseases. As an example, by using this source of information, we were able to define urinary peptide biomarkers for chronic kidney diseases, allowing diagnosis of these diseases with high accuracy. Application of the chronic kidney disease-specific biomarker set to an independent test cohort in the subsequent replication phase resulted in 85.5% sensitivity and 100% specificity. These results indicate the potential usefulness of capillary electrophoresis coupled to MS for clinical applications in the analysis of naturally occurring urinary peptides.
AU  - Good, D.M.*
AU  - Zürbig, P.*
AU  - Argilés, A.*
AU  - Bauer, H.W.*
AU  - Behrens, G.*
AU  - Coon, J.J.*
AU  - Dakna, M.*
AU  - Decramer, S.*
AU  - Delles, C.*
AU  - Dominiczak, A.F.*
AU  - Ehrich, J.H.*
AU  - Eitner, F.*
AU  - Fliser, D.*
AU  - Frommberger, M.
AU  - Ganser, A.*
AU  - Girolami, M.A.*
AU  - Golovko, I.*
AU  - Gwinner, W.*
AU  - Haubitz, M.*
AU  - Herget-Rosenthal, S.*
AU  - Jankowski, J.*
AU  - Jahn, H.*
AU  - Jerums, G.*
AU  - Julian, B.A.*
AU  - Kellmann, M.*
AU  - Kliem, V.*
AU  - Kolch, W.*
AU  - Krolewski, A.S.*
AU  - Luppi, M.*
AU  - Massy, Z.*
AU  - Melter, M.*
AU  - Neusüss, C.*
AU  - Novak, J.*
AU  - Peter, K.*
AU  - Rossing, K.*
AU  - Rupprecht, H.*
AU  - Schanstra, J.P.*
AU  - Schiffer, E.*
AU  - Stolzenburg, J.U.*
AU  - Tarnow, L.*
AU  - Theodorescu, D.*
AU  - Thongboonkerd, V.*
AU  - Vanholder, R.*
AU  - Weissinger, E.M.*
AU  - Mischak, H.*
AU  - Schmitt-Kopplin, P.
C1  - 4556
C2  - 27695
SP  - 2424-2437
TI  - Naturally occurring human urinary peptides for use in diagnosis of chronic kidney disease.
JO  - Mol. Cell. Proteomics
VL  - 9
IS  - 11
PB  - American Society for Biochemistry and Molecular Biology, Inc.
PY  - 2010
SN  - 1535-9476
ER  - 

TY  - JOUR
AB  - Autoimmune uveitis is a blinding disease presenting with autoantibodies against eye-specific proteins as well as autoagressive T cells invading and attacking the immune-privileged target tissue retina. The molecular events enabling T cells to invade and attack the tissue have remained elusive. Changes in membrane protein expression patterns between diseased and healthy stages are especially interesting because initiating events of disease will most likely occur at membranes. Since disease progression is accompanied with a break-down of the blood-retinal barrier, serum-derived proteins mask the potential target tissue-related changes. To overcome this limitation, we used membrane-enriched fractions derived from retinas of the only available spontaneous animal model for the disease equine recurrent uveitis, and compared expression levels by a label-free LC-MSMS-based strategy to healthy control samples. We could readily identify a total of 893 equine proteins with 57% attributed to the Gene Ontology project term "membrane." Of these, 179 proteins were found differentially expressed in equine recurrent uveitis tissue. Pathway enrichment analyses indicated an increase in proteins related to antigen processing and presentation, TNF receptor signaling, integrin cell surface interactions and focal adhesions. Additionally, loss of retina-specific proteins reflecting decrease of vision was observed as well as an increase in Müller glial cell-specific proteins indicating glial reactivity. Selected protein candidates (caveolin 1, integrin alpha 1 and focal adhesion kinase) were validated by immunohistochemistry and tissue staining pattern pointed to a significant increase of these proteins at the level of the outer limiting membrane which is part of the outer blood-retinal barrier. Taken together, the membrane enrichment in combination with LC-MSMS-based label-free quantification greatly increased the sensitivity of the comparative tissue profiling and resulted in detection of novel molecular pathways related to equine recurrent uveitis.
AU  - Hauck, S.M.
AU  - Dietter, J.
AU  - Kramer, R.L.*
AU  - Hofmaier, F.*
AU  - Zipplies, J.K.*
AU  - Amann, B.*
AU  - Feuchtinger, A.
AU  - Deeg, C.A.*
AU  - Ueffing, M.
C1  - 5478
C2  - 27567
SP  - 2292-2305
TI  - Deciphering membrane-associated molecular processes in target tissue of autoimmune uveitis by label-free quantitative mass spectrometry.
JO  - Mol. Cell. Proteomics
VL  - 9
IS  - 10
PB  - American Society for Biochemistry and Molecular Biology, Inc.
PY  - 2010
SN  - 1535-9476
ER  - 

TY  - JOUR
AB  - Glial cells support neuronal survival and function by secreting neurotrophic cytokines. Retinal Mueller glial cells (RMGs) support retinal neurons, especially photoreceptors. These highly light-sensitive sensory neurons receive vision, and their death results in blinding diseases. It has been proposed that RMGs release factors that support photoreceptor survival, but the nature of these factors remains to be elucidated. To discover such neurotrophic factors, we developed an integrated work flow toward systematic identification of neuroprotective proteins, which are, like most cytokines, expressed only in minute amounts. This strategy can be generally applied to identify secreted bioactive molecules from any body fluid once a recipient cell for this activity is known. Toward this goal we first isolated conditioned medium (CM) from primary porcine RMGs cultured in vitro and tested for survival-promoting activity using primary photoreceptors. We then developed a large scale, microplate-based cellular high content assay that allows rapid assessment of primary photoreceptor survival concomitant with biological activity in vitro. The enrichment strategy of bioactive proteins toward their identification consists of several fractionation steps combined with tests for biological function. Here we combined 1) size fractionation, 2) ion exchange chromatography, 3) reverse phase liquid chromatography, and 4) mass spectrometry (Q-TOF MS/MS or MALDI MS/MS) for protein identification. As a result of this integrated work flow, the insulin-like growth factor-binding proteins IGFBP5 and IGFBP7 and connective tissue growth factor (CTGF) were identified as likely candidates. Cloning and stable expression of these three candidate factors in HEK293 cells produced conditioned medium enriched for either one of the factors. IGFBP5 and CTGF, but not IGFBP7, significantly increased photoreceptor survival when secreted from HEK293 cells and when added to the original RMG-CM. This indicates that the survival-promoting activity in RMG-CM is multifactorial with IGFBP5 and CTGF as an integral part of this activity.
AU  - Hauck, S.M.
AU  - Gloeckner, C.J.
AU  - Harley, M.E.
AU  - Schoeffmann, S.
AU  - Boldt, K.
AU  - Ekström, P.A.*
AU  - Ueffing, M.
C1  - 4347
C2  - 25774
SP  - 1349-1361
TI  - Identification of paracrine neuroprotective candidate proteins by a functional assay-driven proteomics approach.
JO  - Mol. Cell. Proteomics
VL  - 7
IS  - 7
PB  - American Soc. of Biochemistry and Molecular Biology
PY  - 2008
SN  - 1535-9476
ER  - 

TY  - JOUR
AB  - Green fluorescent proteins (GFPs) and variants thereof are widely used to study protein localization and dynamics. We engineered a specific binder for fluorescent proteins based on a 13-kDa GFP binding fragment derived from a llama single chain antibody. This GFP-binding protein (GBP) can easily be produced in bacteria and coupled to a monovalent matrix. The GBP allows a fast and efficient (one-step) isolation of GFP fusion proteins and their interacting factors for biochemical analyses including mass spectroscopy and enzyme activity measurements. Moreover GBP is also suitable for chromatin immunoprecipitations from cells expressing fluorescent DNA-binding proteins. Most importantly, GBP can be fused with cellular proteins to ectopically recruit GFP fusion proteins allowing targeted manipulation of cellular structures and processes in living cells. Because of the high affinity capture of GFP fusion proteins in vitro and in vivo and a size in the lower nanometer range we refer to the immobilized GFP-binding protein as GFP-nanotrap. This versatile GFP-nanotrap enables a unique combination of microscopic, biochemical, and functional analyses with one and the same protein.
AU  - Rothbauer, U.*
AU  - Zolghadr, K.*
AU  - Muyldermans, S.*
AU  - Schepers, A.
AU  - Cardoso, M.C.*
AU  - Leonhardt, H.*
C1  - 742
C2  - 25412
SP  - 282-289
TI  - A versatile nanotrap for biochemical and functional studies with fluorescent fusion proteins.
JO  - Mol. Cell. Proteomics
VL  - 7
IS  - 2
PB  - American Soc. of Biochemistry and Molecular Biology
PY  - 2008
SN  - 1535-9476
ER  - 

TY  - JOUR
AB  - As part of a comprehensive survey of the impact of the environmental pollutant and hepatocarcinogen 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the proteome of hepatic cells, we have performed a high resolution two-dimensional gel electrophoresis study on the rat hepatoma cell line 5L. 78 protein species corresponding to 73 different proteins were identified as up- or down-regulated following exposure of the cells to 1 nm TCDD for 8 h. There was an overlap of only nine proteins with those detected as altered by TCDD in our recent study using the non-gel-based isotope-coded protein label method (Sarioglu, H., Brandner, S., Jacobsen, C., Meindl, T., Schmidt, A., Kellermann, J., Lottspeich, F., and Andrae, U. (2006) Quantitative analysis of 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced proteome alterations in 5L rat hepatoma cells using isotope-coded protein labels. Proteomics 6, 2407-2421) indicating a strong complementarity of the two approaches. For the majority of the altered proteins, an effect of TCDD on their abundance or posttranslational modifications had not been known before. Several observations suggest that a sizable fraction of the proteins with altered abundance was induced as an adaptive response to TCDD-induced oxidative stress that was demonstrated using the fluorescent probe dihydrorhodamine 123. A prominent group of these proteins comprised various enzymes for which there is evidence that their expression is regulated via the Keap1/Nrf2/antioxidant response element pathway. Other proteins included several involved in the maintenance of mitochondrial energy production and the regulation of the mitochondrial apoptotic pathway. A particularly intriguing finding was the up-regulation of the mitochondrial outer membrane pore protein, voltage-dependent anion channel-selective protein 2 (VDAC2), which was dependent on the presence of a functional aryl hydrocarbon receptor. The regulatability of VDAC2 protein abundance has not been described previously. In view of the recently discovered central role of VDAC2 as an inhibitor of the activation of the proapoptotic protein BAK and the mitochondrial apoptotic pathway, the present data point to a hitherto unrecognized mechanism by which TCDD may affect cellular homeostasis and survival.
AU  - Sarioglu, H.
AU  - Brandner, S.
AU  - Haberger, M.
AU  - Jacobsen, C.
AU  - Lichtmannegger, J.
AU  - Wormke, M.
AU  - Andrae, U.
C1  - 4028
C2  - 25135
SP  - 394-410
TI  - Analysis of 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced proteome changes in 5L rat hepatoma cells reveals novel targets of dioxin action including the mitochondrial apoptosis regulator VDAC2.
JO  - Mol. Cell. Proteomics
VL  - 7
IS  - 2
PB  - American Soc. of Biochemistry and Molecular Biology
PY  - 2008
SN  - 1535-9476
ER  - 

TY  - JOUR
AB  - The development, progression, and recurrence of autoimmune diseases are frequently driven by a group of participatory autoantigens. We identified and characterized novel autoantigens by analyzing the autoantibody binding pattern from horses affected by spontaneous equine recurrent uveitis to the retinal proteome. Cellular retinaldehyde-binding protein (cRALBP) had not been described previously as autoantigen, but subsequent characterization in equine recurrent uveitis horses revealed B and T cell autoreactivity to this protein and established a link to epitope spreading. We further immunized healthy rats and horses with cRALBP and observed uveitis in both species with typical tissue lesions at cRALBP expression sites. The autoantibody profiling outlined here could be used in various autoimmune diseases to detect autoantigens involved in the dynamic spreading cascade or serve as predictive markers. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.
AU  - Deeg, C.A.*
AU  - Pompetzki, D.*
AU  - Raith, A.J.*
AU  - Hauck, S.M.
AU  - Amann, B.*
AU  - Suppmann, S.
AU  - Goebel, T.W.*
AU  - Olazabal, U.
AU  - Gerhards, H.*
AU  - Reese, S.*
AU  - Stangassinger, M.*
AU  - Kaspers, B.*
AU  - Ueffing, M.
C1  - 2844
C2  - 24061
SP  - 1462-1470
TI  - Identification and functional validation of novel autoantigens in equine uveitis.
JO  - Mol. Cell. Proteomics
VL  - 5
IS  - 8
PY  - 2006
SN  - 1535-9476
ER  - 

TY  - JOUR
AU  - Hauck, S.M.
AU  - Ekström, P.A.R.*
AU  - Ahuja-Jensen, P.*
AU  - Suppmann, S.
AU  - Paquet-Durand, F.*
AU  - van Veen, T.*
AU  - Ueffing, M.
C1  - 2608
C2  - 23674
SP  - 324-336
TI  - Differential modification of phosducin protein in degenerating rd1 retina is associated with constitutively active Ca2+/calmodulin kinase II in rod outer segments.
JO  - Mol. Cell. Proteomics
VL  - 5
PY  - 2006
SN  - 1535-9476
ER  - 

TY  - JOUR
AU  - Zischka, H.
AU  - Braun, R.J.
AU  - Marantidis, E.P.
AU  - Büringer, D.*
AU  - Bornhövd, C.*
AU  - Hauck, S.M.
AU  - Demmer, O.
AU  - Gloeckner, C.J.
AU  - Reichert, A.S.*
AU  - Madeo, F.*
AU  - Ueffing, M.
C1  - 3922
C2  - 24204
SP  - 2185-2200
TI  - Differential analysis of Saccharomyces cerevisiae mitochondria by free flow electrophoresis.
JO  - Mol. Cell. Proteomics
VL  - 5
PY  - 2006
SN  - 1535-9476
ER  -