Publications by authors named "Helmut E Meyer"

281 Publications

Differential Proteome Analysis Using 2D-DIGE.

Methods Mol Biol 2021 ;2228:77-84

Medizinisches Proteom-Center (MPC), Medical Faculty, Ruhr-University Bochum, Bochum, Germany.

Classical 2D-PAGE allows comparison and quantitation of proteomes by visualization of protein patterns using gel stains and comparative image analysis. The introduction of fluorescent reagents for protein labeling (difference in-gel electrophoresis or DIGE) has brought substantial improvement in this field. It provides multiplexing of up to three samples in one gel, higher sensitivity compared to normal protein staining methods, and a higher linear range for quantitation. This article gives detailed protocols for 2D-DIGE, including both minimal and saturation labeling.
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http://dx.doi.org/10.1007/978-1-0716-1024-4_7DOI Listing
January 2021

Proteome Analysis with Classical 2D-PAGE.

Methods Mol Biol 2021 ;2228:53-62

Medizinisches Proteom-Center (MPC), Medical Faculty, Ruhr-University Bochum, Bochum, Germany.

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is based on the combination of two orthogonal separation techniques. In the first dimension, proteins are separated by their isoelectric point, a technique known as isoelectric focusing (IEF). There are two important variants of IEF, which are carrier-ampholine (CA)-based IEF and immobilized pH-gradient (IPG)-based IEF. In the second dimension, proteins are further separated by their electrophoretic mobility using SDS-PAGE. Finally, proteins can be visualized and quantified by different staining procedures such as Coomassie, silver staining, or fluorescence labeling. This article gives detailed protocols for 2D-PAGE, using both CA- and IPG-based separation in the first dimension.
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http://dx.doi.org/10.1007/978-1-0716-1024-4_5DOI Listing
January 2021

A combinatorial native MS and LC-MS/MS approach reveals high intrinsic phosphorylation of human Tau but minimal levels of other key modifications.

J Biol Chem 2020 12 26;295(52):18213-18225. Epub 2020 Oct 26.

DZNE (German Center for Neurodegenerative Diseases), Bonn, Germany; CAESAR Research Center, Bonn, Germany. Electronic address:

Abnormal changes of neuronal Tau protein, such as phosphorylation and aggregation, are considered hallmarks of cognitive deficits in Alzheimer's disease. Abnormal phosphorylation is thought to precede aggregation and therefore to promote aggregation, but the nature and extent of phosphorylation remain ill-defined. Tau contains ∼85 potential phosphorylation sites, which can be phosphorylated by various kinases because the unfolded structure of Tau makes them accessible. However, methodological limitations ( in MS of phosphopeptides, or antibodies against phosphoepitopes) led to conflicting results regarding the extent of Tau phosphorylation in cells. Here we present results from a new approach based on native MS of intact Tau expressed in eukaryotic cells (Sf9). The extent of phosphorylation is heterogeneous, up to ∼20 phosphates per molecule distributed over 51 sites. The medium phosphorylated fraction P showed overall occupancies of ∼8 P (± 5) with a bell-shaped distribution; the highly phosphorylated fraction P had 14 P (± 6). The distribution of sites was highly asymmetric (with 71% of all P-sites in the C-terminal half of Tau). All sites were on Ser or Thr residues, but none were on Tyr. Other known posttranslational modifications were near or below our detection limit ( acetylation, ubiquitination). These findings suggest that normal cellular Tau shows a remarkably high extent of phosphorylation, whereas other modifications are nearly absent. This implies that abnormal phosphorylations at certain sites may not affect the extent of phosphorylation significantly and do not represent hyperphosphorylation. By implication, the pathological aggregation of Tau is not likely a consequence of high phosphorylation.
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http://dx.doi.org/10.1074/jbc.RA120.015882DOI Listing
December 2020

Intricate Crosstalk Between Lipopolysaccharide, Phospholipid and Fatty Acid Metabolism in Modulates Proteolysis of LpxC.

Front Microbiol 2018 14;9:3285. Epub 2019 Jan 14.

Microbial Biology, Ruhr University Bochum, Bochum, Germany.

Lipopolysaccharides (LPS) in the outer membrane of Gram-negative bacteria provide the first line of defense against antibiotics and other harmful compounds. LPS biosynthesis critically depends on LpxC catalyzing the first committed enzyme in this process. In , the cellular concentration of LpxC is adjusted in a growth rate-dependent manner by the FtsH protease making sure that LPS biosynthesis is coordinated with the cellular demand. As a result, LpxC is stable in fast-growing cells and prone to degradation in slow-growing cells. One of the factors involved in this process is the alarmone guanosine tetraphosphate (ppGpp) but previous studies suggested the involvement of yet unknown factors in LpxC degradation. We established a quantitative proteomics approach aiming at the identification of proteins that are associated with LpxC and/or FtsH at high or low growth rates. The identification of known LpxC and FtsH interactors validated our approach. A number of proteins involved in fatty acid biosynthesis and degradation, including the central regulator FadR, were found in the LpxC and/or FtsH interactomes. Another protein associated with LpxC and FtsH was WaaH, a LPS-modifying enzyme. When overproduced, several members of the LpxC/FtsH interactomes were able to modulate LpxC proteolysis. Our results go beyond the previously established link between LPS and phospholipid biosynthesis and uncover a far-reaching network that controls LPS production by involving multiple enzymes in fatty acid metabolism, phospholipid biosynthesis and LPS modification.
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http://dx.doi.org/10.3389/fmicb.2018.03285DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6339880PMC
January 2019

Direct-acting antivirals-based therapy decreases hepatic fibrosis serum biomarker microfibrillar-associated protein 4 in hepatitis C patients.

Clin Mol Hepatol 2019 03 19;25(1):42-51. Epub 2018 Nov 19.

Medizinisches Proteom-Center, Ruhr University Bochum, Bochum, Germany.

Background/aims: An estimated 80 million people worldwide are infected with viremic hepatitis C virus (HCV). Even after eradication of HCV with direct acting antivirals (DAAs), hepatic fibrosis remains a risk factor for hepatocarcinogenesis. Recently, we confirmed the applicability of microfibrillar-associated protein 4 (MFAP4) as a serum biomarker for the assessment of hepatic fibrosis. The aim of the present study was to assess the usefulness of MFAP4 as a biomarker of liver fibrosis after HCV eliminating therapy with DAAs.

Methods: MFAP4 was measured using an immunoassay in 50 hepatitis C patients at baseline (BL), the end-of-therapy (EoT), and the 12-week follow-up visit (FU). Changes in MFAP4 from BL to FU and their association with laboratory parameters including alanine aminotransferase (ALT), aspartate aminotransferase (AST), platelets, the AST to platelet ratio index (APRI), fibrosis-4 score (FIB-4), and albumin were analyzed.

Results: MFAP4 serum levels were representative of the severity of hepatic fibrosis at BL and correlated well with laboratory parameters, especially APRI (Spearman correlation, R²=0.80). Laboratory parameters decreased significantly from BL to EoT. MFAP4 serum levels were found to decrease from BL and EoT to FU with high statistical significance (Wilcoxon P<0.001 for both).

Conclusion: Our findings indicate that viral eradication resulted in reduced MFAP4 serum levels, presumably representing a decrease in hepatic fibrogenesis or fibrosis. Hence, MFAP4 may be a useful tool for risk assessment in hepatitis C patients with advanced fibrosis after eradication of the virus.
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http://dx.doi.org/10.3350/cmh.2018.0029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6435967PMC
March 2019

The deubiquitination of the PTS1-import receptor Pex5p is required for peroxisomal matrix protein import.

Biochim Biophys Acta Mol Cell Res 2019 02 6;1866(2):199-213. Epub 2018 Nov 6.

Biochemie Intrazellulärer Transportprozesse, Ruhr-Universität Bochum, 44801 Bochum, Germany. Electronic address:

Peroxisomal biogenesis depends on the correct import of matrix proteins into the lumen of the organelle. Most peroxisomal matrix proteins harbor the peroxisomal targeting-type 1 (PTS1), which is recognized by the soluble PTS1-receptor Pex5p in the cytosol. Pex5p ferries the PTS1-proteins to the peroxisomal membrane and releases them into the lumen. Finally, the PTS1-receptor is monoubiquitinated on the conserved cysteine 6 in Saccharomyces cerevisiae. The monoubiquitinated Pex5p is recognized by the peroxisomal export machinery and is retrotranslocated into the cytosol for further rounds of protein import. However, the functional relevance of deubiquitination has not yet been addressed. In this study, we have analyzed a Pex5p-truncation lacking Cys6 [(Δ6)Pex5p], a construct with a ubiquitin-moiety genetically fused to the truncation [Ub-(Δ6)Pex5p], as well as a construct with a reduced susceptibility to deubiquitination [Ub(G75/76A)-(Δ6)Pex5p]. While the (Δ6)Pex5p-truncation is not functional, the Ub-(Δ6)Pex5p chimeric protein can facilitate matrix protein import. In contrast, the Ub(G75/76A)-(Δ6)Pex5p chimera exhibits a complete PTS1-import defect. The data show for the first time that not only ubiquitination but also deubiquitination rates are tightly regulated and that efficient deubiquitination of Pex5p is essential for peroxisomal biogenesis.
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http://dx.doi.org/10.1016/j.bbamcr.2018.11.002DOI Listing
February 2019

Precipitation with polyethylene glycol followed by washing and pelleting by ultracentrifugation enriches extracellular vesicles from tissue culture supernatants in small and large scales.

J Extracell Vesicles 2018 17;7(1):1528109. Epub 2018 Oct 17.

Institute for Transfusion Medicine, University Hospital Essen, University of Duisburg-Essen, Essen, Germany.

Extracellular vesicles (EVs) provide a complex means of intercellular signalling between cells at local and distant sites, both within and between different organs. According to their cell-type specific signatures, EVs can function as a novel class of biomarkers for a variety of diseases, and can be used as drug-delivery vehicles. Furthermore, EVs from certain cell types exert beneficial effects in regenerative medicine and for immune modulation. Several techniques are available to harvest EVs from various body fluids or cell culture supernatants. Classically, differential centrifugation, density gradient centrifugation, size-exclusion chromatography and immunocapturing-based methods are used to harvest EVs from EV-containing liquids. Owing to limitations in the scalability of any of these methods, we designed and optimised a polyethylene glycol (PEG)-based precipitation method to enrich EVs from cell culture supernatants. We demonstrate the reproducibility and scalability of this method and compared its efficacy with more classical EV-harvesting methods. We show that washing of the PEG pellet and the re-precipitation by ultracentrifugation remove a huge proportion of PEG co-precipitated molecules such as bovine serum albumine (BSA). However, supported by the results of the size exclusion chromatography, which revealed a higher purity in terms of particles per milligram protein of the obtained EV samples, PEG-prepared EV samples most likely still contain a certain percentage of other non-EV associated molecules. Since PEG-enriched EVs revealed the same therapeutic activity in an ischemic stroke model than corresponding cells, it is unlikely that such co-purified molecules negatively affect the functional properties of obtained EV samples. In summary, maybe not being the purification method of choice if molecular profiling of pure EV samples is intended, the optimised PEG protocol is a scalable and reproducible method, which can easily be adopted by laboratories equipped with an ultracentrifuge to enrich for functional active EVs.
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http://dx.doi.org/10.1080/20013078.2018.1528109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6197019PMC
October 2018

Next-Generation Trapping of Protease Substrates by Label-Free Proteomics.

Methods Mol Biol 2018 ;1841:189-206

Lehrstuhl für Biologie der Mikroorganismen, Ruhr-Universität Bochum, Bochum, Germany.

AAA proteases (ATPases associated with various cellular activities) shape the cellular protein pool in response to environmental conditions. A prerequisite for understanding the underlying recognition and degradation principles is the identification of as many protease substrates as possible. Most previous studies made use of inactive protease variants to trap substrates, which were identified by 2D-gel based proteomics. Since this method is known for limitations in the identification of low-abundant proteins or proteins with many transmembrane domains, we established a trapping approach that overcomes these limitations. We used a proteolytically inactive FtsH variant (FtsH) of Escherichia coli (E. coli) that is still able to bind and translocate substrates into the proteolytic chamber but no longer able to degrade proteins. Proteins associated with FtsH or FtsH (proteolytically active FtsH) were purified, concentrated by an 1D-short gel, and identified by LC-coupled mass spectrometry (LC-MS) followed by label-free quantification. The identification of four known FtsH substrates validated this approach and suggests that it is generally applicable to AAA proteases.
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http://dx.doi.org/10.1007/978-1-4939-8695-8_14DOI Listing
April 2019

An Integrated Proteomic Approach Uncovers Novel Substrates and Functions of the Lon Protease in Escherichia coli.

Proteomics 2018 07 6;18(13):e1800080. Epub 2018 Jun 6.

Department of Microbial Biology, Ruhr University Bochum, Universitätsstraße 150, D-44801, Bochum, Germany.

Controlling the cellular abundance and proper function of proteins by proteolysis is a universal process in all living organisms. In Escherichia coli, the ATP-dependent Lon protease is crucial for protein quality control and regulatory processes. To understand how diverse substrates are selected and degraded, unbiased global approaches are needed. We employed a quantitative Super-SILAC (stable isotope labeling with amino acids in cell culture) mass spectrometry approach and compared the proteomes of a lon mutant and a strain producing the protease to discover Lon-dependent physiological functions. To identify Lon substrates, we took advantage of a Lon trapping variant, which is able to translocate substrates but unable to degrade them. Lon-associated proteins were identified by label-free LC-MS/MS. The combination of both approaches revealed a total of 14 novel Lon substrates. Besides the identification of known pathways affected by Lon, for example, the superoxide stress response, our cumulative data suggests previously unrecognized fundamental functions of Lon in sulfur assimilation, nucleotide biosynthesis, amino acid and central energy metabolism.
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http://dx.doi.org/10.1002/pmic.201800080DOI Listing
July 2018

Identification of new channels by systematic analysis of the mitochondrial outer membrane.

J Cell Biol 2017 11 15;216(11):3485-3495. Epub 2017 Sep 15.

Institute of Biochemistry and Molecular Biology, ZBMZ, Faculty of Medicine, University of Freiburg, Freiburg, Germany.

The mitochondrial outer membrane is essential for communication between mitochondria and the rest of the cell and facilitates the transport of metabolites, ions, and proteins. All mitochondrial outer membrane channels known to date are β-barrel membrane proteins, including the abundant voltage-dependent anion channel and the cation-preferring protein-conducting channels Tom40, Sam50, and Mdm10. We analyzed outer membrane fractions of yeast mitochondria and identified four new channel activities: two anion-preferring channels and two cation-preferring channels. We characterized the cation-preferring channels at the molecular level. The mitochondrial import component Mim1 forms a channel that is predicted to have an α-helical structure for protein import. The short-chain dehydrogenase-related protein Ayr1 forms an NADPH-regulated channel. We conclude that the mitochondrial outer membrane contains a considerably larger variety of channel-forming proteins than assumed thus far. These findings challenge the traditional view of the outer membrane as an unspecific molecular sieve and indicate a higher degree of selectivity and regulation of metabolite fluxes at the mitochondrial boundary.
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http://dx.doi.org/10.1083/jcb.201706043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5674900PMC
November 2017

Altered mitochondrial and peroxisomal integrity in lipocalin-2-deficient mice with hepatic steatosis.

Biochim Biophys Acta Mol Basis Dis 2017 09 7;1863(9):2093-2110. Epub 2017 Apr 7.

Institute of Molecular Pathobiochemistry, Experimental Gene Therapy and Clinical Chemistry, RWTH University Hospital Aachen, Aachen, Germany. Electronic address:

Lipocalin-2 (LCN2) is a secreted adipokine that transports small hydrophobic molecules such as fatty acids and steroids. LCN2 limits bacterial growth by sequestering iron-containing siderophores and in mammalian liver protects against inflammation, infection, injury and other stressors. Because LCN2 modulates hepatic fat metabolism and homeostasis, we performed a comparative profiling of proteins and lipids of wild type (WT) and Lcn2-deficient mice fed either standard chow or a methionine- and choline-deficient (MCD) diet. Label-free proteomics and 2D-DIGE protein expression profiling revealed differential expression of BRIT1/MCPH1, FABP5, HMGB1, HBB2, and L-FABP, results confirmed by Western blotting. Gene ontology enrichment analysis identified enrichment for genes associated with mitochondrial membrane permeabilization and metabolic processes involving carboxylic acid. Measurements of mitochondrial membrane potential, mitochondrial chelatable iron pool, intracellular lipid peroxidation, and peroxisome numbers in primary hepatocytes confirmed that LCN2 regulates mitochondrial and peroxisomal integrity. Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight (MALDI-TOF) mass spectrometry imaging identified significant changes to sphingomyelins, triglycerides, and glycerophospholipids in livers of mice fed an MCD diet regardless of LCN2 status. However, two arachidonic acid-containing glycerophospholipids were increased in Lcn2-deficient livers. Thus, LCN2 influences peroxisomal and mitochondrial biology in the liver to maintain triglyceride balance, handle oxidative stress, and control apoptosis.
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http://dx.doi.org/10.1016/j.bbadis.2017.04.006DOI Listing
September 2017

LMD proteomics provides evidence for hippocampus field-specific motor protein abundance changes with relevance to Alzheimer's disease.

Biochim Biophys Acta Proteins Proteom 2017 Jun 2;1865(6):703-714. Epub 2017 Apr 2.

Leibniz-Institut für Analytische Wissenschaften -ISAS- e. V., Dortmund, Germany.

Background: Human hippocampal area Cornu Ammonis (CA) 1 is one of the first fields in the human telencephalon showing Alzheimer disease (AD)-specific neuropathological changes. In contrast, CA2 and CA3 are far later affected pointing to functional differences, which may be accompanied by differences in proteome endowment and changes.

Methods: Human pyramidal cell layers of hippocampal areas CA1, CA2, and CA3 from neurologically unaffected individuals were excised using laser microdissection. The proteome of each individual sample was analyzed and differentially abundant proteins were validated by immuno-histochemistry.

Results: Comparison of CA1 to CA2 revealed 223, CA1 to CA3 197 proteins with differential abundance, among them we found motor proteins MYO5A and DYNC1H1. Extension of the study to human hippocampus slices from AD patients revealed extensive depletion of these proteins in CA1 area compared to unaffected controls.

Conclusion: High abundance of motor proteins in pyramidal cell layers CA1 compared to CA2 and CA3 points the specific vulnerability of this hippocampal area to transport-associated changes based on microtubule dysfunction and destabilization in AD.
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http://dx.doi.org/10.1016/j.bbapap.2017.03.013DOI Listing
June 2017

Strategies in relative and absolute quantitative mass spectrometry based proteomics.

Biol Chem 2017 05;398(5-6):687-699

Ruhr-University Bochum, Medizinisches Proteom-Center, Universitätsstraße 150, D-44801 Bochum.

Quantitative mass spectrometry approaches are used for absolute and relative quantification in global proteome studies. To date, relative and absolute quantification techniques are available that differ in quantification accuracy, proteome coverage, complexity and robustness. This review focuses on most common relative or absolute quantification strategies exemplified by three experimental studies. A label-free relative quantification approach was performed for the investigation of the membrane proteome of sensory cilia to the depth of olfactory receptors in Mus musculus. A SILAC-based relative quantification approach was successfully applied for the identification of core components and transient interactors of the peroxisomal importomer in Saccharomyces cerevisiae. Furthermore, AQUA using stable isotopes was exemplified to unraveling the prenylome influenced by novel prenyltransferase inhibitors. Characteristic enrichment and fragmentation strategies for a robust quantification of the prenylome are also summarized.
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http://dx.doi.org/10.1515/hsz-2017-0104DOI Listing
May 2017

Glyoxalase 1-knockdown in human aortic endothelial cells - effect on the proteome and endothelial function estimates.

Sci Rep 2016 11 29;6:37737. Epub 2016 Nov 29.

Medical Proteom-Center, Ruhr-Universität Bochum, Bochum, Germany.

Methylglyoxal (MG), an arginine-directed glycating agent, is implicated in diabetic late complications. MG is detoxified by glyoxalase 1 (GLO1) of the cytosolic glyoxalase system. The aim was to investigate the effects of MG accumulation by GLO1-knockdown under hyperglycaemic conditions in human aortic endothelial cells (HAECs) hypothesizing that the accumulation of MG accounts for the deleterious effects on vascular function. SiRNA-mediated knockdown of GLO1 was performed and MG concentrations were determined. The impact of MG on the cell proteome and targets of MG glycation was analysed, and confirmed by Western blotting. Markers of endothelial function and apoptosis were assessed. Collagen content was assayed in cell culture supernatant. GLO1-knockdown increased MG concentration in cells and culture medium. This was associated with a differential abundance of cytoskeleton stabilisation proteins, intermediate filaments and proteins involved in posttranslational modification of collagen. An increase in fibrillar collagens 1 and 5 was detected. The extracellular concentration of endothelin-1 was increased following GLO1-knockdown, whereas the phosphorylation and amount of eNOS was not influenced by GLO1-knockdown. The expression of ICAM-1, VCAM-1 and of MCP-1 was elevated and apoptosis was increased. MG accumulation by GLO1-knockdown provoked collagen expression, endothelial inflammation and dysfunction and apoptosis which might contribute to vascular damage.
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http://dx.doi.org/10.1038/srep37737DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5127188PMC
November 2016

Molecular profiles of thyroid cancer subtypes: Classification based on features of tissue revealed by mass spectrometry imaging.

Biochim Biophys Acta Proteins Proteom 2017 Jul 17;1865(7):837-845. Epub 2016 Oct 17.

Center for Translational Research and Molecular Biology of Cancer, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology Gliwice Branch, ul. Wybrzeze Armii Krajowej 15, 44101 Gliwice, Poland. Electronic address:

Determination of the specific type of thyroid cancer is crucial for the prognosis and selection of treatment of this malignancy. However, in some cases appropriate classification is not possible based on histopathological features only, and it might be supported by molecular biomarkers. Here we aimed to characterize molecular profiles of different thyroid malignancies using mass spectrometry imaging (MSI) which enables the direct annotation of molecular features with morphological pictures of an analyzed tissue. Fifteen formalin-fixed paraffin-embedded tissue specimens corresponding to five major types of thyroid cancer were analyzed by MALDI-MSI after in-situ trypsin digestion, and the possibility of classification based on the results of unsupervised segmentation of MALDI images was tested. Novel method of semi-supervised detection of the cancer region of interest (ROI) was implemented. We found strong separation of medullary cancer from malignancies derived from thyroid epithelium, and separation of anaplastic cancer from differentiated cancers. Reliable classification of medullary and anaplastic cancers using an approach based on automated detection of cancer ROI was validated with independent samples. Moreover, extraction of spectra from tumor areas allowed the detection of molecular components that differentiated follicular cancer and two variants of papillary cancer (classical and follicular). We concluded that MALDI-MSI approach is a promising strategy in the search for biomarkers supporting classification of thyroid malignant tumors. This article is part of a Special Issue entitled: MALDI Imaging, edited by Dr. Corinna Henkel and Prof. Peter Hoffmann.
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http://dx.doi.org/10.1016/j.bbapap.2016.10.006DOI Listing
July 2017

Quantitative proteome analysis reveals the correlation between endocytosis-associated proteins and hepatocellular carcinoma dedifferentiation.

Biochim Biophys Acta 2016 11 9;1864(11):1579-85. Epub 2016 Aug 9.

Medizinisches Proteom-Center, Ruhr-Universität Bochum, 44801 Bochum, Germany. Electronic address:

The majority of poorly differentiated hepatocellular carcinomas (HCCs) develop from well-differentiated tumors. Endocytosis is a cellular function which is likely to take part in this development due to its important role in regulating the abundances of vital signaling receptors. Here, we aimed to investigate the abundance of endocytosis-associated proteins in HCCs with various differentiation grades. Therefore, we analyzed 36 tissue specimens from HCC patients via LC-MS/MS-based label-free quantitative proteomics including 19 HCC tissue samples with different degrees of histological grades and corresponding non-tumorous tissue controls. As a result, 277 proteins were differentially regulated between well-differentiated tumors and controls. In moderately and poorly differentiated tumors, 278 and 1181 proteins, respectively, were significantly differentially regulated compared to non-tumorous tissue. We explored the regulated proteins based on their functions and identified thirty endocytosis-associated proteins, mostly overexpressed in poorly differentiated tumors. These included proteins that have been shown to be up-regulated in HCC like clathrin heavy chain-1 (CLTC) as well as unknown proteins, such as secretory carrier-associated membrane protein 3 (SCAMP3). The abundances of SCAMP3 and CLTC were immunohistochemically examined in tissue sections of 84 HCC patients. We demonstrate the novel association of several endocytosis-associated proteins, in particular, SCAMP3 with HCC progression.
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http://dx.doi.org/10.1016/j.bbapap.2016.08.005DOI Listing
November 2016

Tissue-based quantitative proteome analysis of human hepatocellular carcinoma using tandem mass tags.

Biomarkers 2017 Mar 28;22(2):113-122. Epub 2016 Jul 28.

a Medizinisches Proteom-Center, Ruhr-Universität Bochum , Germany.

Context And Objective: Human hepatocellular carcinoma (HCC) is a severe malignant disease, and accurate and reliable diagnostic markers are still needed. This study was aimed for the discovery of novel marker candidates by quantitative proteomics.

Methods And Results: Proteomic differences between HCC and nontumorous liver tissue were studied by mass spectrometry. Among several significantly upregulated proteins, translocator protein 18 (TSPO) and Ras-related protein Rab-1A (RAB1A) were selected for verification by immunohistochemistry in an independent cohort. For RAB1A, a high accuracy for the discrimination of HCC and nontumorous liver tissue was observed.

Conclusion: RAB1A was verified to be a potent biomarker candidate for HCC.
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http://dx.doi.org/10.1080/1354750X.2016.1210678DOI Listing
March 2017

Evaluation of the biomarker candidate MFAP4 for non-invasive assessment of hepatic fibrosis in hepatitis C patients.

J Transl Med 2016 07 4;14(1):201. Epub 2016 Jul 4.

Medizinisches Proteom-Center, Ruhr-Universität Bochum, 44801, Bochum, Germany.

Background: The human microfibrillar-associated protein 4 (MFAP4) is located to extracellular matrix fibers and plays a role in disease-related tissue remodeling. Previously, we identified MFAP4 as a serum biomarker candidate for hepatic fibrosis and cirrhosis in hepatitis C patients. The aim of the present study was to elucidate the potential of MFAP4 as biomarker for hepatic fibrosis with a focus on the differentiation of no to moderate (F0-F2) and severe fibrosis stages and cirrhosis (F3 and F4, Desmet-Scheuer scoring system).

Methods: MFAP4 levels were measured using an AlphaLISA immunoassay in a retrospective study including n = 542 hepatitis C patients. We applied a univariate logistic regression model based on MFAP4 serum levels and furthermore derived a multivariate model including also age and gender. Youden-optimal cutoffs for binary classification were determined for both models without restrictions and considering a lower limit of 80 % sensitivity (correct classification of F3 and F4), respectively. To assess the generalization error, leave-one-out cross validation (LOOCV) was performed.

Results: MFAP4 levels were shown to differ between no to moderate fibrosis stages F0-F2 and severe stages (F3 and F4) with high statistical significance (t test on log scale, p value <2.2·10(-16)). In the LOOCV, the univariate classification resulted in 85.8 % sensitivity and 54.9 % specificity while the multivariate model yielded 81.3 % sensitivity and 61.5 % specificity (restricted approaches).

Conclusions: We confirmed the applicability of MFAP4 as a novel serum biomarker for assessment of hepatic fibrosis and identification of high-risk patients with severe fibrosis stages in hepatitis C. The combination of MFAP4 with existing tests might lead to a more accurate non-invasive diagnosis of hepatic fibrosis and allow a cost-effective disease management in the era of new direct acting antivirals.
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http://dx.doi.org/10.1186/s12967-016-0952-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4932744PMC
July 2016

Minichromosome Maintenance Complex Is a Critical Node in the miR-183 Signaling Network of MYCN-Amplified Neuroblastoma Cells.

J Proteome Res 2016 07 9;15(7):2178-86. Epub 2016 Jun 9.

Department of Pediatric Hematology/Oncology/Stem Cell Transplantation Charité - Universitätsmedizin Berlin , Campus Virchow-Klinikum, Augustenburger Platz 1, 13353 Berlin, Germany.

MYCN and HDAC2 jointly repress the transcription of tumor suppressive miR-183 in neuroblastoma. Enforced miR-183 expression induces neuroblastoma cell death and inhibits xenograft growth in mice. Here we aimed to focus more closely on the miR-183 signaling network using a label-free mass spectrometric approach. Analysis of neuroblastoma cells transfected with either control or miR-183 expression vectors identified 85 differentially expressed proteins. All six members of the minichromosome maintenance (MCM) complex, which is indispensable for initiation and elongation during DNA replication and transcriptionally activated by MYCN in neuroblastoma, emerged to be down-regulated by miR-183. Subsequent annotation category enrichment analysis revealed a ∼14-fold enrichment in the "MCM" protein module category, which highlighted this complex as a critical node in the miR-183 signaling network. Down-regulation was confirmed by Western blotting. MCMs 2-5 were predicted by in silico methods as direct miR-183 targets. Dual-luciferase reporter gene assays with 3'-UTR constructs of the randomly selected MCMs 3 and 5 experimentally confirmed them as direct targets of miR-183. Our results reveal the MCM complex to be a critical and directly regulated node within the miR-183 signaling network in MYCN-amplified neuroblastoma cells.
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http://dx.doi.org/10.1021/acs.jproteome.6b00134DOI Listing
July 2016

MFAP4: a candidate biomarker for hepatic and pulmonary fibrosis?

Sarcoidosis Vasc Diffuse Lung Dis 2016 Mar 29;33(1):41-50. Epub 2016 Mar 29.

Department of Gastroenterology and Hepatology, Berufsgenossenschaftliches Universitätsklinikum Bergmannsheil, Bürkle-de-la-Camp-Platz 1, 44789 Bochum, Germany.

Background: Several comparable mechanisms have been identified for hepatic and pulmonary fibrosis. The human microfibrillar associated glycoprotein 4 (MFAP4), produced by activated myofibroblasts, is a ubiquitous protein playing a potential role in extracellular matrix (ECM) turnover and was recently identified as biomarker for hepatic fibrosis in hepatitis C patients. The current study aimed to evaluate serum levels of MFAP4 in patients with pulmonary fibrosis in order to test its potential as biomarker in clinical practice. A further aim was to determine whether MFAP4 deficiency in mice affects the formation of pulmonary fibrosis in the bleomycin model of lung fibrosis.

Methods: 91 patients with idiopathic pulmonary fibrosis (IPF), 23 with hypersensitivity pneumonitis (HP) and 31 healthy subjects were studied. In the mouse model, C57BL/6 Mfap4+/+ and Mfap4-/- mice between 6-8 weeks of age were studied. Serum levels of MFAP4 were measured by ELISA in patients and in mice. Surfactant protein D (SP-D) and LDH were measured as comparison biomarkers in patients with pulmonary fibrosis. Morphometric assessment and the Sircol kit were used to determine the amount of collagen in the lung tissue in the mouse model.

Results: Serum levels of MFAP4 were not elevated in lung fibrosis - neither in the patients with IPF or HP nor in the animal model. Furthermore no significant correlations with pulmonary function tests of IPF patients could be found for MFAP4. MFAP4 levels were increased in BAL of bleomycin treated mice with pulmonary fibrosis.

Conclusions: MFAP4 is not elevated in sera of patients with pulmonary fibrosis or bleomycin treated mice with pulmonary fibrosis. This may be due to different pathogenic mechanisms of liver and lung fibrogenesis. MFAP4 seems to be useful as serum biomarker for hepatic but not for lung fibrosis.
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March 2016

Key players in neurodegenerative disorders in focus-New insights into the proteomic profile of Alzheimer's disease, schizophrenia, ALS, and multiple sclerosis-24th HUPO BPP Workshop: September 29, 2015, Vancouver, Canada.

Proteomics 2016 Apr;16(7):1047-50

University of São Paulo Medical School, Department of Pathology, Brazil.

The HUPO Brain Proteome Project (HUPO BPP) held its 24th workshop in Vancouver, Canada, September 29, 2015. The focus of the autumn workshop was on new insights into the proteomic profile of Alzheimer's disease, schizophrenia, ALS and multiple sclerosis.
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http://dx.doi.org/10.1002/pmic.201600047DOI Listing
April 2016

Tissue MALDI Mass Spectrometry Imaging (MALDI MSI) of Peptides.

Methods Mol Biol 2016 ;1394:129-150

Leibniz-Institut für Analytische Wissenschaften-ISAS-e.V., Dortmund, Germany.

Matrix assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) is a technique to visualize molecular features of tissues based on mass detection. This chapter focuses on MALDI MSI of peptides and provides detailed operational instructions for sample preparation of cryoconserved and formalin-fixed paraffin-embedded (FFPE) tissue. Besides sample preparation we provide protocols for the MALDI measurement, tissue staining, and data analysis. On-tissue digestion and matrix application are described for two different commercially available and commonly used spraying devices: the SunCollect (SunChrom) and the ImagePrep (Bruker Daltonik GmbH).
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http://dx.doi.org/10.1007/978-1-4939-3341-9_10DOI Listing
October 2016

Immunohistochemical Markers Distinguishing Cholangiocellular Carcinoma (CCC) from Pancreatic Ductal Adenocarcinoma (PDAC) Discovered by Proteomic Analysis of Microdissected Cells.

Mol Cell Proteomics 2016 Mar 7;15(3):1072-82. Epub 2015 Dec 7.

From the ‡Medizinisches Proteom-Center, Ruhr-Universität Bochum, Germany;

Cholangiocellular carcinoma (CCC) and pancreatic ductal adenocarcinoma (PDAC) are two highly aggressive cancer types that arise from epithelial cells of the pancreatobiliary system. Owing to their histological and morphological similarity, differential diagnosis between CCC and metastasis of PDAC located in the liver frequently proves an unsolvable issue for pathologists. The detection of biomarkers with high specificity and sensitivity for the differentiation of these tumor types would therefore be a valuable tool. Here, we address this problem by comparing microdissected CCC and PDAC tumor cells from nine and eleven cancer patients, respectively, in a label-free proteomics approach. The novel biomarker candidates were subsequently verified by immunohistochemical staining of 73 CCC, 78 primary, and 18 metastatic PDAC tissue sections. In the proteome analysis, we found 180 proteins with a significantly differential expression between CCC and PDAC cells (p value < 0.05, absolute fold change > 2). Nine candidate proteins were chosen for an immunohistochemical verification out of which three showed very promising results. These were the annexins ANXA1, ANXA10, and ANXA13. For the correct classification of PDAC, ANXA1 showed a sensitivity of 84% and a specificity of 85% and ANXA10 a sensitivity of 90% at a specificity of 66%. ANXA13 was higher abundant in CCC. It presented a sensitivity of 84% at a specificity of 55%. In metastatic PDAC tissue ANXA1 and ANXA10 showed similar staining behavior as in the primary PDAC tumors (13/18 and 17/18 positive, respectively). ANXA13, however, presented positive staining in eight out of eighteen secondary PDAC tumors and was therefore not suitable for the differentiation of these from CCC. We conclude that ANXA1 and ANXA10 are promising biomarker candidates with high diagnostic values for the differential diagnosis of intrahepatic CCC and metastatic liver tumors deriving from PDAC.
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http://dx.doi.org/10.1074/mcp.M115.054585DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4813689PMC
March 2016

Quantitative Tissue Proteomics Analysis Reveals Versican as Potential Biomarker for Early-Stage Hepatocellular Carcinoma.

J Proteome Res 2016 Jan 18;15(1):38-47. Epub 2015 Dec 18.

Medizinisches Proteom-Center, Ruhr-Universität Bochum , 44801 Bochum, Germany.

Hepatocellular carcinoma (HCC) is one of the most aggressive tumors, and the treatment outcome of this disease is improved when the cancer is diagnosed at an early stage. This requires biomarkers allowing an accurate and early tumor diagnosis. To identify potential markers for such applications, we analyzed a patient cohort consisting of 50 patients (50 HCC and 50 adjacent nontumorous tissue samples as controls) using two independent proteomics approaches. We performed label-free discovery analysis on 19 HCC and corresponding tissue samples. The data were analyzed considering events known to take place in early events of HCC development, such as abnormal regulation of Wnt/b-catenin and activation of receptor tyrosine kinases (RTKs). 31 proteins were selected for verification experiments. For this analysis, the second set of the patient cohort (31 HCC and corresponding tissue samples) was analyzed using selected (multiple) reaction monitoring (SRM/MRM). We present the overexpression of ATP-dependent RNA helicase (DDX39), Fibulin-5 (FBLN5), myristoylated alanine-rich C-kinase substrate (MARCKS), and Serpin H1 (SERPINH1) in HCC for the first time. We demonstrate Versican core protein (VCAN) to be significantly associated with well differentiated and low-stage HCC. We revealed for the first time the evidence of VCAN as a potential biomarker for early-HCC diagnosis.
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http://dx.doi.org/10.1021/acs.jproteome.5b00420DOI Listing
January 2016

A Long Journey from Childhood to Senility: The 23rd HUPO BPP Workshop: 16-17 April 2015, São Paulo, Brazil.

Proteomics 2015 Sep;15(17):2895-7

Department of Pathology, University of São Paulo Medical School, Brazil.

The HUPO Brain Proteome Project (HUPO BPP) held its 23rd workshop in São Paulo, Brazil, April 16-17, 2015. The focus of the spring workshop was on strategies and predictive therapies concerning neurodegenerative diseases.
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http://dx.doi.org/10.1002/pmic.201570154DOI Listing
September 2015

Differential proteomic and tissue expression analyses identify valuable diagnostic biomarkers of hepatocellular differentiation and hepatoid adenocarcinomas.

Pathology 2015 Oct;47(6):543-50

1Institute of Pathology, University Hospital of Essen, University of Duisburg-Essen, Essen 2Medizinisches Proteom-Center, Ruhr-Universität Bochum, Bochum 3Institute for Medical Informatics, Biometry and Epidemiology, University Hospital of Essen, University of Duisburg-Essen, Essen 4Institute of Pathology, Klinikum Dortmund gGmbH, Dortmund 5Department of General, Visceral and Transplantation Surgery, University Hospital of Essen, University of Duisburg-Essen, Essen 6West German Cancer Centre Essen, University Hospital of Essen, University of Duisburg-Essen, Essen 7Department of Gastroenterology and Hepatology, University Hospital of Essen, University of Duisburg-Essen, Essen 8Leibniz-Institut für Analytische Wissenschaften - ISAS - e.V., Dortmund, Germany *contributed equally as senior authors.

The exact discrimination of lesions with true hepatocellular differentiation from secondary tumours and neoplasms with hepatocellular histomorphology like hepatoid adenocarcinomas (HAC) is crucial. Therefore, we aimed to identify ancillary protein biomarkers by using complementary proteomic techniques (2D-DIGE, label-free MS). The identified candidates were immunohistochemically validated in 14 paired samples of hepatocellular carcinoma (HCC) and non-tumourous liver tissue (NT). The candidates and HepPar1/Arginase1 were afterwards tested for consistency in a large cohort of hepatocellular lesions and NT (n = 290), non-hepatocellular malignancies (n = 383) and HAC (n = 13). Eight non-redundant, differentially expressed proteins were suitable for further immunohistochemical validation and four (ABAT, BHMT, FABP1, HAOX1) for further evaluation. Sensitivity and specificity rates for HCC/HAC were as follows: HepPar1 80.2%, 94.3% / 80.2%, 46.2%; Arginase1 82%, 99.4% / 82%, 69.2%; BHMT 61.4%, 93.8% / 61.4%, 100%; ABAT 84.4%, 33.7% / 84.4%, 30.8%; FABP1 87.2%, 95% / 87.2%, 69.2%; HAOX1 95.5%, 36.3% / 95.5%, 46.2%. The best 2×/3× biomarker panels for the diagnosis of HCC consisted of Arginase1/HAOX1 and BHMT/Arginase1/HAOX1 and for HAC consisted of Arginase1/FABP1 and BHMT/Arginase1/FABP1. In summary, we successfully identified, validated and benchmarked protein biomarker candidates of hepatocellular differentiation. BHMT in particular exhibited superior diagnostic characteristics in hepatocellular lesions and specifically in HAC. BHMT is therefore a promising (panel based) biomarker candidate in the differential diagnostic process of lesions with hepatocellular aspect.
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http://dx.doi.org/10.1097/PAT.0000000000000298DOI Listing
October 2015

Characterization of Regenerative Phenotype of Unrestricted Somatic Stem Cells (USSC) from Human Umbilical Cord Blood (hUCB) by Functional Secretome Analysis.

Mol Cell Proteomics 2015 Oct 16;14(10):2630-43. Epub 2015 Jul 16.

From the ‡Molecular Proteomics Laboratory (MPL), Institute for Molecular Medicine, Heinrich Heine University, Universitätsstr. 1, 40225 Düsseldorf, Germany; **Biologisch-Medizinisches Forschungszentrum (BMFZ), Heinrich Heine University, Universitätsstr. 1, 40225 Düsseldorf, Germany

Stem cell transplantation is a promising therapeutic strategy to enhance axonal regeneration after spinal cord injury. Unrestricted somatic stem cells (USSC) isolated from human umbilical cord blood is an attractive stem cell population available at GMP grade without any ethical concerns. It has been shown that USSC transplantation into acute injured rat spinal cords leads to axonal regrowth and significant locomotor recovery, yet lacking cell replacement. Instead, USSC secrete trophic factors enhancing neurite growth of primary cortical neurons in vitro. Here, we applied a functional secretome approach characterizing proteins secreted by USSC for the first time and validated candidate neurite growth promoting factors using primary cortical neurons in vitro. By mass spectrometric analysis and exhaustive bioinformatic interrogation we identified 1156 proteins representing the secretome of USSC. Using Gene Ontology we revealed that USSC secretome contains proteins involved in a number of relevant biological processes of nerve regeneration such as cell adhesion, cell motion, blood vessel formation, cytoskeleton organization and extracellular matrix organization. We found for instance that 31 well-known neurite growth promoting factors like, e.g. neuronal growth regulator 1, NDNF, SPARC, and PEDF span the whole abundance range of USSC secretome. By the means of primary cortical neurons in vitro assays we verified SPARC and PEDF as significantly involved in USSC mediated neurite growth and therewith underline their role in improved locomotor recovery after transplantation. From our data we are convinced that USSC are a valuable tool in regenerative medicine as USSC's secretome contains a comprehensive network of trophic factors supporting nerve regeneration not only by a single process but also maintained its regenerative phenotype by a multitude of relevant biological processes.
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http://dx.doi.org/10.1074/mcp.M115.049312DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4597141PMC
October 2015

ProCon - PROteomics CONversion tool.

J Proteomics 2015 Nov 13;129:56-62. Epub 2015 Jul 13.

Medizinisches Proteom Center (MPC), Ruhr-Universität Bochum, D-44801 Bochum, Germany. Electronic address:

With the growing amount of experimental data produced in proteomics experiments and the requirements/recommendations of journals in the proteomics field to publicly make available data described in papers, a need for long-term storage of proteomics data in public repositories arises. For such an upload one needs proteomics data in a standardized format. Therefore, it is desirable, that the proprietary vendor's software will integrate in the future such an export functionality using the standard formats for proteomics results defined by the HUPO-PSI group. Currently not all search engines and analysis tools support these standard formats. In the meantime there is a need to provide user-friendly free-to-use conversion tools that can convert the data into such standard formats in order to support wet-lab scientists in creating proteomics data files ready for upload into the public repositories. ProCon is such a conversion tool written in Java for conversion of proteomics identification data into standard formats mzIdentML and Pride XML. It allows the conversion of Sequest™/Comet .out files, of search results from the popular and often used ProteomeDiscoverer® 1.x (x=versions 1.1 to1.4) software and search results stored in the LIMS systems ProteinScape® 1.3 and 2.1 into mzIdentML and PRIDE XML. This article is part of a Special Issue entitled: Computational Proteomics.
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http://dx.doi.org/10.1016/j.jprot.2015.06.015DOI Listing
November 2015

The Canonical Immediate Early 3 Gene Product pIE611 of Mouse Cytomegalovirus Is Dispensable for Viral Replication but Mediates Transcriptional and Posttranscriptional Regulation of Viral Gene Products.

J Virol 2015 Aug 10;89(16):8590-8. Epub 2015 Jun 10.

Institut für Virologie, Universitätsklinikum Essen, Universität Duisburg-Essen, Essen, Germany

Unlabelled: Transcription of mouse cytomegalovirus (MCMV) immediate early ie1 and ie3 is controlled by the major immediate early promoter/enhancer (MIEP) and requires differential splicing. Based on complete loss of genome replication of an MCMV mutant carrying a deletion of the ie3-specific exon 5, the multifunctional IE3 protein (611 amino acids; pIE611) is considered essential for viral replication. Our analysis of ie3 transcription resulted in the identification of novel ie3 isoforms derived from alternatively spliced ie3 transcripts. Construction of an IE3-hemagglutinin (IE3-HA) virus by insertion of an in-frame HA epitope sequence allowed detection of the IE3 isoforms in infected cells, verifying that the newly identified transcripts code for proteins. This prompted the construction of an MCMV mutant lacking ie611 but retaining the coding capacity for the newly identified isoforms ie453 and ie310. Using Δie611 MCMV, we demonstrated the dispensability of the canonical ie3 gene product pIE611 for viral replication. To determine the role of pIE611 for viral gene expression during MCMV infection in an unbiased global approach, we used label-free quantitative mass spectrometry to delineate pIE611-dependent changes of the MCMV proteome. Interestingly, further analysis revealed transcriptional as well as posttranscriptional regulation of MCMV gene products by pIE611.

Importance: Cytomegaloviruses are pathogenic betaherpesviruses persisting in a lifelong latency from which reactivation can occur under conditions of immunosuppression, immunoimmaturity, or inflammation. The switch from latency to reactivation requires expression of immediate early genes. Therefore, understanding of immediate early gene regulation might add insights into viral pathogenesis. The mouse cytomegalovirus (MCMV) immediate early 3 protein (611 amino acids; pIE611) is considered essential for viral replication. The identification of novel protein isoforms derived from alternatively spliced ie3 transcripts prompted the construction of an MCMV mutant lacking ie611 but retaining the coding capacity for the newly identified isoforms ie453 and ie310. Using Δie611 MCMV, we demonstrated the dispensability of the canonical ie3 gene product pIE611 for viral replication and delineated pIE611-dependent changes of the MCMV proteome. Our findings have fundamental implications for the interpretation of earlier studies on pIE3 functions and highlight the complex orchestration of MCMV gene regulation.
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http://dx.doi.org/10.1128/JVI.01234-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4524224PMC
August 2015

PIA: An Intuitive Protein Inference Engine with a Web-Based User Interface.

J Proteome Res 2015 Jul 10;14(7):2988-97. Epub 2015 Jun 10.

Medizinisches Proteom-Center, Ruhr-Universität Bochum, 44801 Bochum, Germany.

Protein inference connects the peptide spectrum matches (PSMs) obtained from database search engines back to proteins, which are typically at the heart of most proteomics studies. Different search engines yield different PSMs and thus different protein lists. Analysis of results from one or multiple search engines is often hampered by different data exchange formats and lack of convenient and intuitive user interfaces. We present PIA, a flexible software suite for combining PSMs from different search engine runs and turning these into consistent results. PIA can be integrated into proteomics data analysis workflows in several ways. A user-friendly graphical user interface can be run either locally or (e.g., for larger core facilities) from a central server. For automated data processing, stand-alone tools are available. PIA implements several established protein inference algorithms and can combine results from different search engines seamlessly. On several benchmark data sets, we show that PIA can identify a larger number of proteins at the same protein FDR when compared to that using inference based on a single search engine. PIA supports the majority of established search engines and data in the mzIdentML standard format. It is implemented in Java and freely available at https://github.com/mpc-bioinformatics/pia.
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http://dx.doi.org/10.1021/acs.jproteome.5b00121DOI Listing
July 2015