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Molecular & Cellular Proteomics: MCP

Tamar Ziv, Vered Chalifa-Caspi, Nadav Denekamp, Inbar Plaschkes, Sylwiya Sylwia Kierszniowska, Idit Blais, Arie Admon, Esther Lubzens
Numerous aquatic invertebrates remain dormant for decades in a hydrated state as encysted embryos. In search for functional pathways associated with this form of dormancy, we used label-free quantitative proteomics to compare the proteomes of hydrated encysted dormant embryos (resting eggs; RE) with non-dormant embryos (amictic eggs; AM) of the rotifer Brachionus plicatilis</p> <p>A total of 2,631 proteins were identified in rotifer eggs. About 62% proteins showed higher abundance in AM relative to RE (Fold Change>3; p=0...
July 20, 2017: Molecular & Cellular Proteomics: MCP
Johannes Leufken, Anna Niehues, Peter Sarin, Florian Wessel, Michael Hippler, Sebastian A Leidel, Christian Fufezan
Quantitative mass spectrometry (MS) is a key technique in many research areas (1), including proteomics, metabolomics, glycomics, and lipidomics. Because all of the corresponding molecules can be described by chemical formulas, universal quantification tools are highly desirable. Here we present pyQms, an open-source software for accurate quantification of all types of molecules measurable by MS. pyQms uses isotope pattern matching which offers an accurate quality assessment of all quantifications and the ability to directly incorporate mass spectrometer accuracy...
July 20, 2017: Molecular & Cellular Proteomics: MCP
Malika Chabi, Estelle Goulas, Celine Leclercq, Isabelle de Waele, Christophe Rihouey, Ugo Cenci, Arnaud Day, Anne-Sophie Blervacq, Godfrey Neutelings, Ludovic Duponchel, Patrice Lerouge, Jean-François Hausman, Jenny Renaut, Simon Hawkins
Experimentally-generated (nanoLC-MS/MS) proteomic analyses of four different flax organs/tissues (inner-stem, outer-stem, leaves and roots) enriched in proteins from 3 different sub-compartments (soluble-, membrane-, and cell wall-proteins) was combined with publically available data on flax seed and whole-stem proteins to generate a flax protein database containing 2996 non-redundant total proteins. Subsequent multiple analyses (MapMan, CAZy, WallProtDB and expert curation) of this database were then used to identify a flax cell wall proteome consisting of 456 non-redundant proteins localized in the cell wall and/or associated with cell wall biosynthesis, remodeling and other cell wall related processes...
July 13, 2017: Molecular & Cellular Proteomics: MCP
Tomáš Takáč, Olga Šamajová, Tibor Pechan, Ivan Luptovčiak, Jozef Šamaj
Microtubule organization and dynamics are critical for key developmental processes such as cell division, elongation and morphogenesis. Microtubule severing is an essential regulator of microtubules, and is exclusively executed by KATANIN 1 in Arabidopsis. In this study we comparatively studied the proteome-wide effects in two KATANIN 1 mutants. Thus, shot-gun proteomic analysis of roots and aerial parts of single nucleotide mutant fra2 and T-DNA insertion mutant ktn1-2 was carried out. We have detected 42 proteins differentially abundant in both fra2 and ktn1-2 KATANIN 1 dysfunction altered abundances of proteins involved in development, metabolism and stress responses...
July 13, 2017: Molecular & Cellular Proteomics: MCP
Ruijin Wang, Junbo Peng, Qing X Li, You-Liang Peng
Protein phosphorylation is known to regulate pathogenesis, mycelial growth, conidiation and stress response in Pyricularia oryzae However, phosphorylation mediated regulatory networks in the fungal pathogen remain largely to be uncovered. In this study, we identified 1621 phosphorylation sites of 799 proteins in mycelia of P. oryzae, including 899 new p-sites of 536 proteins and 47 new p-sites of 31 pathogenesis-relation proteins. From the sequences flanking the phosphorylation sites, 19 conserved phosphorylation motifs were identified...
July 13, 2017: Molecular & Cellular Proteomics: MCP
Ugljesa Djuric, Deivid C Rodrigues, Ihor Batruch, James Ellis, Patrick Shannon, Phedias Diamandis
Mass spectrometry (MS) analysis of human post-mortem central nervous system (CNS) tissue and induced pluripotent stem cell (iPSC)-based directed differentiations offer complementary avenues to define protein signatures of neurodevelopment. Methodological improvements of formalin-fixed, paraffin-embedded (FFPE) protein isolation now enable widespread proteomic analysis of well-annotated archival tissue samples in the context of development and disease. Here, we utilize a shotgun label-free quantification (LFQ) MS method to profile magnetically enriched human cortical neurons and neural progenitor cells (NPCs) derived from iPSCs...
July 7, 2017: Molecular & Cellular Proteomics: MCP
Romy D Zwittink, Diny van Zoeren-Grobben, Rocio Martin, Richard A van Lingen, Liesbeth J Groot Jebbink, Sjef Boeren, Ingrid B Renes, Ruurd M van Elburg, Clara Belzer, Jan Knol
Development of the gastrointestinal tract and immune system can be modulated by the gut microbiota. Establishment of the intestinal microbiota, in its turn, is affected by host- and environmental factors. As such, development of the gut microbiota is greatly impacted in preterm infants, who have an immature gut and are exposed to factors like hospitalisation, caesarean section, antibiotics, and respiratory support.</p> <p>Design: We analysed faecal microbiota composition and activity of ten preterm infants (gestational age 25-30 weeks; birthweight 630-1750 gram) during the first six postnatal weeks through metaproteomics (LC-MS/MS) and 16S-rRNA gene sequencing...
July 6, 2017: Molecular & Cellular Proteomics: MCP
Ineke Dhondt, Vladislav A Petyuk, Sophie Bauer, Heather M Brewer, Richard D Smith, Geert Depuydt, Bart P Braeckman
Protein turnover rates severely decline in aging organisms, including C. elegans However, limited information is available on turnover dynamics at the individual protein level during aging. We followed changes in protein turnover at one-day resolution using a multiple-pulse (15)N-labeling and accurate mass spectrometry approach. Forty percent of the proteome shows gradual slowdown in turnover with age, while only few proteins show increased turnover. Decrease in protein turnover was consistent for only a minority of functionally related protein subsets, including tubulins and vitellogenins, while randomly diverging turnover patterns with age were the norm...
July 5, 2017: Molecular & Cellular Proteomics: MCP
Jennifer Villers, Jérôme Savocco, Aleksandra Szopinska, Hervé Degand, Sylvain Nootens, Pierre Morsomme
Yeast cells, to be able to grow on a wide variety of nitrogen sources, regulate the set of nitrogen transporters present at their plasma membrane. Such regulation relies on both transcriptional and post-translational events. While microarray studies have identified most nitrogen-sensitive genes, nitrogen-induced post-translational regulation has only been studied for very few proteins among which the general amino acid permease Gap1. Adding a preferred nitrogen source to proline-grown cells triggers Gap1 endocytosis and vacuolar degradation in an Rsp5-Bul1/2-dependent manner...
July 5, 2017: Molecular & Cellular Proteomics: MCP
Edmund Wilkes, Pedro Casado, Vinothini Rajeeve, Pedro Rodriguez Cutillas
Cell survival is regulated by a signaling network driven by the activity of protein kinases; however, determining the contribution that each kinase in the network makes to such regulation remains challenging. Here, we report a computational approach that uses mass spectrometry-based phosphoproteomics data to rank protein kinases based on their contribution to cell regulation. We found that the scores returned by this algorithm, which we have termed Kinase Activity Ranking using Phosphoproteomics data (KARP), were a quantitative measure of the contribution that individual kinases make to the signaling output...
July 3, 2017: Molecular & Cellular Proteomics: MCP
Yanhua Liu, Qian Liu, Linlu Qi, Tao Ding, Zhen Wang, Jiaqi Fu, Mo Hu, Min Li, Jeongmin Song, Xiaoyun Liu
We previously examined Salmonella proteome within infected host cells and found differential expression of many proteins with defined functional roles such as metabolism or virulence. However, the precise roles of other altered proteins in Salmonella pathogenesis are largely unknown. A putative transcriptional regulator, YdcR, was highly induced intracellularly while barely expressed in vitro, implicating potential relevance to bacterial infection. To unveil its physiological functions, we exploited quantitative proteomics of intracellular Salmonella and found that genetic ablation of ydcR resulted in severe repression of SrfN, a known virulence factor...
July 3, 2017: Molecular & Cellular Proteomics: MCP
Joshua J Hamey, Beeke Wienert, Kate G R Quinlan, Marc R Wilkins
Lysine methylation is widespread on human proteins, however the enzymes that catalyse its addition remain largely unknown. This limits our capacity to study the function and regulation of this modification. Here we used the CRISPR/Cas9 system to knock out putative protein methyltransferases METTL21B and METTL23 in K562 cells, to determine if they methylate elongation factor eEF1A. The known eEF1A methyltransferase EEF1AKMT1 was also knocked out as a control. Targeted mass spectrometry revealed the loss of lysine 165 methylation upon knock out of METTL21B, and the expected loss of lysine 79 methylation on knock out of EEF1AKMT1...
June 29, 2017: Molecular & Cellular Proteomics: MCP
Stephanie Devaux, Dasa Cizkova, Khalil Mallah, Melodie-Anne Karnoub, Zahra Laouby, Firas Kobeissy, Juraj Blasko, Serge Nataf, Laurent Pays, Celine Meriaux, Isabelle Fournier, Michel Salzet
The therapeutic use of RhoA inhibitors (RhoAi) has been experimentally tested in spinal cord injury (SCI). In order to decipher the underlying molecular mechanisms involved in such a process, an in vitro neuroproteomic-systems biology platform was developed in which the pan-proteomic profile of the dorsal root ganglia (DRG) cell line ND7/23 DRG was assessed in a large array of culture conditions using RhoAi and/or conditioned media obtained from SCI ex-vivo derived spinal cord slices. A fine mapping of the spatio-temporal molecular events of the RhoAi treatment in SCI was performed...
June 28, 2017: Molecular & Cellular Proteomics: MCP
Gerhard Feil, Ralf Horres, Julia Schulte, Andreas F Mack, Svenja Petzoldt, Caroline Arnold, Chen Meng, Lukas Jost, Jochen Boxleitner, Nicole Kiessling-Wolf, Ender Serbest, Dominic Helm, Bernhard Kuster, Isabel Hartmann, Thomas Korff, Hannes Hahne
Preserving the native phenotype of primary cells in vitro is a complex challenge. Recently, hydrogel-based cellular matrices have evolved as alternatives to conventional cell culture techniques. We developed a bacterial cellulose-based aqueous gel-like biomaterial, dubbed Xellulin, which mimics a cellular microenvironment and seems to maintain the native phenotype of cultured and primary cells. When applied to human umbilical vein endothelial cells (HUVEC), it allowed the continuous cultivation of cell monolayers for more than one year without degradation or dedifferentiation...
June 21, 2017: Molecular & Cellular Proteomics: MCP
Ian Loke, Ole Østergaard, Niels H H Heegaard, Nicolle H Packer, Morten Thaysen-Andersen
Human neutrophil elastase (HNE) is an important N-glycosylated serine protease in the innate immune system, but the structure and immune-modulating functions of HNE N-glycosylation remain undescribed. Herein, LC-MS/MS-based glycan, glycopeptide and glycoprotein profiling were utilised to first determine the heterogeneous N-glycosylation of HNE purified from neutrophil lysates and then from isolated neutrophil granules of healthy individuals. The spatiotemporal expression of HNE during neutrophil activation and the biological importance of its N-glycosylation were also investigated using immunoblotting, cell surface capture, native MS, receptor interaction, protease inhibition, and bacteria growth assays...
June 19, 2017: Molecular & Cellular Proteomics: MCP
Joanna K Lempiäinen, Einari A Niskanen, Kaisa-Mari Vuoti, Riikka E Lampinen, Helka Göös, Markku Varjosalo, Jorma J Palvimo
Glucocorticoid receptor (GR) and androgen receptor (AR) are steroid-inducible transcription factors (TFs). GR and AR are central regulators of various metabolic, homeostatic and differentiation processes and hence important therapeutic targets, especially in inflammation and prostate cancer, respectively. Hormone binding to these steroid receptors (SRs) leads to DNA binding and activation or repression of their target genes with the aid of interacting proteins, coregulators. However, protein interactomes of these important drug targets have remained poorly defined...
June 13, 2017: Molecular & Cellular Proteomics: MCP
John D Lapek, Mary K Lewinski, Jacob M Wozniak, John Guatelli, David J Gonzalez
The mechanisms by which human immunodeficiency virus (HIV) circumvents and coopts cellular machinery to replicate and persist in cells are not fully understood. HIV accessory proteins play key roles in the HIV life cycle by altering host pathways that are often dependent on post-translational modifications (PTMs). Thus, the identification of HIV accessory protein host targets and their PTM status is critical to fully understand how HIV invades, avoids detection and replicates to spread infection. To date, a comprehensive characterization of HIV accessory protein host targets and modulation of their PTM status does not exist...
June 12, 2017: Molecular & Cellular Proteomics: MCP
Jara Obermann, Claudia S Priglinger, Juliane Merl-Pham, Arie Geerlof, Sigfried Priglinger, Magdalena Götz, Stefanie M Hauck
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...
June 2, 2017: Molecular & Cellular Proteomics: MCP
Jerrard M Hayes, Asa Frostell, Robert Karlsson, Steffen Müller, Silvia Millan-Martin, Martin Pauers, Franziska Reuss, Eoin Cosgrave, Cecilia Anneren, Gavin P Davey, Pauline M Rudd
Fc gamma receptors (FcyR) bind the Fc region of antibodies and therefore play a prominent role in antibody-dependent cell-based immune responses such as ADCC, CDC and ADCP. The immune effector cell activity is directly linked to a productive molecular engagement of FcyRs where both the protein and glycan moiety of antibody and receptor can affect the interaction and in the present study we focus on the role of the FcyR glycans in this interaction. We provide a complete description of the glycan composition of Chinese hamster ovary (CHO) expressed human Fcy receptors RI (CD64), RIIa Arg131/His131 (CD32a), RIIb (CD32b) and RIIIa Phe158/Val158 (CD16a) and analyze the role of the glycans in the binding mechanism with IgG...
June 2, 2017: Molecular & Cellular Proteomics: MCP
Virginia Sanchez-Quiles, Vyacheslav Akimov, Nerea Osinalde, Chiara Francavilla, Michele Puglia, Inigo Barrio-Hernandez, Irina Kratchmarova, Jesper V Olsen, Blagoy Blagoev
Cylindromatosis tumor suppressor protein (CYLD) is deubiquitinase, best known as an essential negative regulator of the NFkB pathway. Previous studies have suggested an involvement of CYLD in epidermal growth factor (EGF)-dependent signal transduction as well, as it was found enriched within the tyrosine-phosphorylated complexes in cells stimulated with the growth factor. EGF receptor (EGFR) signaling participates in central cellular processes and its tight regulation, partly through ubiquitination cascades, is decisive for a balanced cellular homeostasis...
June 1, 2017: Molecular & Cellular Proteomics: MCP
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