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https://www.readbyqxmd.com/read/28107008/robust-sensitive-and-automated-phosphopeptide-enrichment-optimized-for-low-sample-amounts-applied-to-primary-hippocampal-neurons
#1
Harm Post, Renske Penning, Martin A Fitzpatrick, Luc B Garrigues, W Wu, Harold D MacGillavry, Casper C Hoogenraad, Albert J R Heck, A F Maarten Altelaar
Because of the low stoichiometry of protein phosphorylation, targeted enrichment prior to LC-MS/MS analysis is still essential. The trend in phosphoproteome analysis is shifting toward an increasing number of biological replicates per experiment, ideally starting from very low sample amounts, placing new demands on enrichment protocols to make them less labor-intensive, more sensitive, and less prone to variability. Here we assessed an automated enrichment protocol using Fe(III)-IMAC cartridges on an AssayMAP Bravo platform to meet these demands...
December 6, 2016: Journal of Proteome Research
https://www.readbyqxmd.com/read/28106078/comparative-phosphoproteomics-reveals-an-important-role-of-mkk2-in-banana-musa-spp-cold-signal-network
#2
Jie Gao, Sheng Zhang, Wei-Di He, Xiu-Hong Shao, Chun-Yu Li, Yue-Rong Wei, Gui-Ming Deng, Rui-Bin Kuang, Chun-Hua Hu, Gan-Jun Yi, Qiao-Song Yang
Low temperature is one of the key environmental stresses, which greatly affects global banana production. However, little is known about the global phosphoproteomes in Musa spp. and their regulatory roles in response to cold stress. In this study, we conducted a comparative phosphoproteomic profiling of cold-sensitive Cavendish Banana and relatively cold tolerant Dajiao under cold stress. Phosphopeptide abundances of five phosphoproteins involved in MKK2 interaction network, including MKK2, HY5, CaSR, STN7 and kinesin-like protein, show a remarkable difference between Cavendish Banana and Dajiao in response to cold stress...
January 20, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28103766/long-term-exposure-to-imatinib-mesylate-downregulates-hippo-pathway-and-activates-yap-in-a-model-of-chronic-myelogenous-leukemia
#3
Anna Chorzalska, Javier Flores Kim, Karim Roder, Alexander Tepper, Nagib Ahsan, R Shyama Prasad Rao, Adam J Olszewski, Xiaoqing Yu, Dmitry Terentyev, John Morgan, Diana O Treaba, Ting Zhao, Olin Liang, Philip Gruppuso, Patrycja Dubielecka
Despite the success of tyrosine kinase inhibitor (TKI) therapy in chronic myelogenous leukemia (CML), leukemic stem/progenitor cells remain detectable even in the state of deep molecular remission. Mechanisms that allow them to persist despite continued kinase inhibition remain unclear. We have previously shown that prolonged exposure to imatinib mesylate results in dysregulation of Akt/Erk 1/2 signaling, upregulation of miR-181a, enhanced adhesiveness, and resistance to high imatinib mesylate. In order to characterize the molecular basis and reversibility of those effects, we applied gene and protein expression analysis, quantitative phosphoproteomic, and direct miR-181a inhibition to our cellular model of CML cells subjected to prolonged exposure to imatinib mesylate...
January 19, 2017: Stem Cells and Development
https://www.readbyqxmd.com/read/28103301/differences-in-beef-quality-between-angus-bos-taurus-taurus-and-nellore-bos-taurus-indicus-cattle-through-a-proteomic-and-phosphoproteomic-approach
#4
Rafael Torres de Souza Rodrigues, Mario Luiz Chizzotti, Camilo Elber Vital, Maria Cristina Baracat-Pereira, Edvaldo Barros, Karina Costa Busato, Rafael Aparecido Gomes, Márcio Machado Ladeira, Taiane da Silva Martins
Proteins are the major constituents of muscle and are key molecules regulating the metabolic changes during conversion of muscle to meat. Brazil is one of the largest exporters of beef and most Brazilian cattle are composed by zebu (Nellore) genotype. Bos indicus beef is generally leaner and tougher than Bos taurus such as Angus. The aim of this study was to compare the muscle proteomic and phosphoproteomic profile of Angus and Nellore. Seven animals of each breed previously subjected the same growth management were confined for 84 days...
2017: PloS One
https://www.readbyqxmd.com/read/28102545/-rice-calcium-dependent-protein-kinase-oscpk17-targets-plasma-membrane-intrinsic-protein-and-sucrose-phosphate-synthase-and-is-required-for-a-proper-cold-stress-response
#5
M Cecília Almadanim, Bruno M Alexandre, Margarida T G Rosa, Helena Sapeta, António E Leitão, José C Ramalho, TuKiet T Lam, Sónia Negrão, Isabel A Abreu, M Margarida Oliveira
Calcium-dependent protein kinases (CDPKs) are involved in plant tolerance mechanisms to abiotic stresses. Although CDPKs are recognized as key messengers in signal transduction, the specific role of most members of this family remains unknown. Here we test the hypothesis that OsCPK17 plays a role in rice cold stress response by analyzing OsCPK17 knockout, silencing, and overexpressing rice lines under low temperature. Altered OsCPK17 gene expression compromises cold tolerance performance, without affecting the expression of key cold stress-inducible genes...
January 19, 2017: Plant, Cell & Environment
https://www.readbyqxmd.com/read/28102081/deep-coverage-of-global-protein-expression-and-phosphorylation-in-breast-tumor-cell-lines-using-tmt-10-plex-isobaric-labeling
#6
Fang-Ke Huang, Guoan Zhang, Kevin Lawlor, Arpi Nazarian, John Philip, Paul Tempst, Noah Dephoure, Thomas A Neubert
Labeling peptides with isobaric tags is a popular strategy in quantitative bottom-up proteomics. In this study, we labeled six breast tumor cell lysates (1.34 mg proteins per channel) using 10-plex tandem mass tag reagents and analyzed the samples on a Q Exactive HF Quadrupole-Orbitrap mass spectrometer. We identified a total of 8706 proteins and 28186 phosphopeptides, including 7394 proteins and 23739 phosphosites common to all channels. The majority of technical replicates correlated with a R2 ≥ 0.98, indicating minimum variability was introduced after labeling...
January 19, 2017: Journal of Proteome Research
https://www.readbyqxmd.com/read/28098263/a-lab-on-a-chip-for-monolith-based-preconcentration-and-electrophoresis-separation-of-phosphopeptides
#7
Monica Araya-Farias, Szymon Dziomba, Benjamin Carbonnier, Mohamed Guerrouache, Ichraf Ayed, Nacera Aboud, Myriam Taverna, N Thuy Tran
A microdevice combining online preconcentration and separation of phosphopeptides was developed in a glass microchip. An ethylene glycol methacrylate phosphate (EGMP), acrylamide (AM) and bisacrylamide (BAA) based monolith was synthesized within microchannels through a photo-driven process. Morphological investigations revealed a homogeneous monolithic structure composed of uniform nodules (∼0.8 μm), with a large pore volume (0.62 cm(3) g(-1)) and sufficiently high specific surface area (34.1 m(2) g(-1))...
January 18, 2017: Analyst
https://www.readbyqxmd.com/read/28096466/a-phosphoproteomic-screen-identifies-a-guanine-nucleotide-exchange-factor-for-rab3a-as-a-map-kinase-phosphatase-5-regulated-map-kinase-target-in-il-6-secretion-and-myogenesis
#8
Hojin Lee, Kisuk Min, Jae-Sung Yi, Hao Shi, Woochul Chang, Leandra Jackson, Anton M Bennett
The mitogen-activated protein kinases (MAPKs) have been shown to regulate skeletal muscle function. Previously, we showed that MAPK phosphatase-5 (MKP-5) negatively regulates myogenesis and regeneration of skeletal muscle through inhibition of p38 MAPK and c-Jun N-terminal kinase (JNK). However, the identity and contribution of MKP-5-dependent MAPK targets in the regulation of skeletal muscle function and regenerative myogenesis has not been established. In order to identify MKP-5-dependent MAPK substrates in skeletal muscle, we performed a global differential phospho-MAPK substrate screen in regenerating skeletal muscles of wild type and MKP-5-deficient mice...
January 17, 2017: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/28092035/functionally-altered-sh2-domains-for-biochemical-studies-loss-of-function-mutant-and-domain-concatenation
#9
Mari Ogiue-Ikeda, Kazuya Machida
Recombinant modular protein domains have been a convenient proteomics tool for deciphering protein-protein interactions and elucidating the role of protein modifications in cell signaling. To obtain reliable experimental data, these protein domain probes require sufficient specificity and sensitivity. Since naturally evolved protein domains do not always have optimal biochemical characteristics for in vitro assays, functional alterations such as improved affinity are sometimes needed. In this chapter, we describe preparation of loss-of-function and concatenated (tandem) SH2 domains that should be widely applicable to both high- and low-throughput phosphoproteomics studies...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28087597/membrane-depolarizing-channel-blockers-induce-selective-glioma-cell-death-by-impairing-nutrient-transport-and-unfolded-protein-amino-acid-responses
#10
Mia Niklasson, Gianluca Maddalo, Zuzana Sramkova, Ercan Mutlu, Shimei Wee, Petra Sekyrova, Linnéa Schmidt, Nicolas Fritz, Ivar Dehnisch, Gregorios Kyriatzis, Michaela Krafcikova, Brittany B Carson, Jennifer Feenstra, Voichita D Marinescu, Anna Segerman, Martin Haraldsson, Anna-Lena Gustavsson, Lars Gj Hammarström, Annika Jenmalm-Jensen, Lene Uhrbom, A F Maarten Altelaar, Sten Linnarsson, Per Uhlén, Lukas Trantirek, C Theresa Vincent, Sven Nelander, Per Øyvind Enger, Michael Andäng
Glioma-initiating cells (GIC) are considered the underlying cause of recurrences of aggressive glioblastomas, replenishing the tumor population and undermining the efficacy of conventional chemotherapy. Here we report the discovery that inhibiting T-type voltage-gated Ca2+ and KCa channels can effectively induce selective cell death of GIC and increase host survival in an orthotopic mouse model of human glioma. At present, the precise cellular pathways affected by the drugs affecting these channels are unknown...
January 13, 2017: Cancer Research
https://www.readbyqxmd.com/read/28079298/from-phosphoproteins-to-phosphoproteomes-a-historical-account
#11
REVIEW
Andrea Venerando, Luca Cesaro, Lorenzo A Pinna
The first phosphoprotein (casein) was discovered in 1883, yet the enzyme responsible for its phosphorylation was identified only 130 years later, in 2012. During the very long time elapsed between these two events, with special reference to the last decades of the 1900s, it became evident that, far from being an oddity, phosphorylation affects the majority of eukaryotic proteins during their lifespan, and that this reaction is catalysed by the members of a large family of protein kinases, susceptible to a variety of stimuli controlling nearly every aspects of life and death...
January 12, 2017: FEBS Journal
https://www.readbyqxmd.com/read/28076760/enriching-the-circadian-proteome
#12
Joseph S Takahashi
Circadian clocks regulate most aspects of physiology and metabolism. Genome-wide approaches have uncovered widespread circadian rhythms in the transcriptome, cistrome, and epigenome of mice, and now two proteomics studies in this issue (Robles et al., 2016; Wang et al., 2016) reveal extensive circadian regulation of the nuclear and phosphoproteome.
January 10, 2017: Cell Metabolism
https://www.readbyqxmd.com/read/28073184/functional-lipidomics-palmitic-acid-impairs-hepatocellular-carcinoma-development-by-modulating-membrane-fluidity-and-glucose-metabolism
#13
Ling Lin, Ying Ding, Yi Wang, Zhenxin Wang, Xuefei Yin, Guoquan Yan, Lei Zhang, Pengyuan Yang, Huali Shen
: Lipids are essential cellular components and energy sources of living organisms, altered lipid composition is increasingly recognized as a signature of cancer. We performed the lipidomic analysis in a series of hepatocellular carcinoma (HCC) cells and identified over 1700 intact lipids originating from 3 major lipid categories. Comparative lipidomic screening revealed 93 significantly changed lipids and decreased palmitic acyl (C16:0)-containing glycerophospholipids (GPs) were positively associated with metastatic abilities of HCC cells...
January 10, 2017: Hepatology: Official Journal of the American Association for the Study of Liver Diseases
https://www.readbyqxmd.com/read/28072816/systematic-analysis-of-transcriptional-and-post-transcriptional-regulation-of-metabolism-in-yeast
#14
Emanuel Gonçalves, Zrinka Raguz Nakic, Mattia Zampieri, Omar Wagih, David Ochoa, Uwe Sauer, Pedro Beltrao, Julio Saez-Rodriguez
Cells react to extracellular perturbations with complex and intertwined responses. Systematic identification of the regulatory mechanisms that control these responses is still a challenge and requires tailored analyses integrating different types of molecular data. Here we acquired time-resolved metabolomics measurements in yeast under salt and pheromone stimulation and developed a machine learning approach to explore regulatory associations between metabolism and signal transduction. Existing phosphoproteomics measurements under the same conditions and kinase-substrate regulatory interactions were used to in silico estimate the enzymatic activity of signalling kinases...
January 2017: PLoS Computational Biology
https://www.readbyqxmd.com/read/28064214/hsp90-promotes-burkitt-lymphoma-cell-survival-by-maintaining-tonic-b-cell-receptor-signaling
#15
Roland Walter, Kuan-Ting Pan, Carmen Doebele, Federico Comoglio, Katarzyna Tomska, Hanibal Bohnenberger, Ryan M Young, Laura Jacobs, Ulrich Keller, Halvard Bönig, Michael Engelke, Andreas Rosenwald, Henning Urlaub, Louis M Staudt, Hubert Serve, Thorsten Zenz, Thomas Oellerich
Burkitt lymphoma (BL) is an aggressive B cell neoplasm that is currently treated by intensive chemotherapy in combination with anti-CD20 antibodies. Due to their toxicity, current treatment regimens are often not suitable for elderly patients or for patients in developing countries where BL is endemic. Targeted therapies for BL are therefore needed. In this study, we performed a compound screen in 17 BL cell lines to identify small molecule inhibitors affecting cell survival. We found that inhibitors of heat shock protein 90 (HSP90) induced apoptosis in BL cells in vitro at concentrations that did not affect normal B cells...
November 15, 2016: Blood
https://www.readbyqxmd.com/read/28062706/combinatorial-drug-screening-identifies-ewing-sarcoma-specific-sensitivities
#16
Branka Radic-Sarikas, Kalliopi P Tsafou, Kristina B Emdal, Theodore Papamarkou, Kilian V M Huber, Cornelia Mutz, Jeffrey A Toretsky, Keiryn L Bennett, Jesper V Olsen, Søren Brunak, Heinrich Kovar, Giulio Superti-Furga
Improvements in survival for Ewing sarcoma pediatric and adolescent patients have been modest over the past 20 years. Combinations of anticancer agents endure as an option to overcome resistance to single treatments caused by compensatory pathways. Moreover, combinations are thought to lessen any associated adverse side effects through reduced dosing, which is particularly important in childhood tumors. Using a parallel phenotypic combinatorial screening approach of cells derived from three pediatric tumor types, we identified Ewing sarcoma-specific interactions of a diverse set of targeted agents including approved drugs...
January 2017: Molecular Cancer Therapeutics
https://www.readbyqxmd.com/read/28060719/temporal-quantitative-phosphoproteomics-of-adp-stimulation-reveals-novel-central-nodes-in-platelet-activation-and-inhibition
#17
Florian Beck, Jörg Geiger, Stepan Gambaryan, Fiorella A Solari, Margherita Dell'Aica, Stefan Loroch, Nadine Mattheij, Igor Mindukshev, Oliver Pötz, Kerstin Jurk, Julia M Burkhart, Christian Fufezan, Johan W M Heemskerk, Ulrich Walter, René P Zahedi, Albert Sickmann
ADP enhances platelet activation by virtually any other stimulant to complete aggregation. It binds specifically to the G-protein coupled membrane receptors P2Y1 and P2Y12, stimulating intracellular signaling cascades leading to integrin αIIbβ3 activation, a process that is antagonized by endothelial prostacyclin. P2Y12 inhibitors are among the most successful anti-platelet drugs, however, show remarkable variability in efficacy. We reasoned whether a more detailed molecular understanding of ADP-induced protein phosphorylation could identify (i) critical hubs in platelet signaling towards aggregation, and (ii) novel molecular targets for anti-platelet treatment strategies...
November 9, 2016: Blood
https://www.readbyqxmd.com/read/28059163/phosphoproteomic-comparison-of-pik3ca-and-pten-signalling-identifies-the-nucleotidase-nt5c-as-a-novel-akt-substrate
#18
Larissa S Moniz, Silvia Surinova, Essam Ghazaly, Lorena Gonzalez Velasco, Syed Haider, Juan Carlos Rodríguez-Prados, Inma M Berenjeno, Claude Chelala, Bart Vanhaesebroeck
To identify novel effectors and processes regulated by PI3K pathway activation, we performed an unbiased phosphoproteomic screen comparing two common events of PI3K deregulation in cancer: oncogenic Pik3ca mutation (Pik3ca(H1047R)) and deletion of Pten. Using mouse embryonic fibroblast (MEF) models that generate inducible, low-level pathway activation as observed in cancer, we quantified 7566 unique phosphopeptides from 3279 proteins. A number of proteins were found to be differentially-regulated by Pik3ca(H1047R) and Pten loss, suggesting unique roles for these two events in processes such as vesicular trafficking, DNA damage repair and RNA splicing...
January 6, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28054942/a-comprehensive-proteomic-survey-of-aba-induced-protein-phosphorylation-in-rice-oryza-sativa-l
#19
Jiehua Qiu, Yuxuan Hou, Yifeng Wang, Zhiyong Li, Juan Zhao, Xiaohong Tong, Haiyan Lin, Xiangjin Wei, Hejun Ao, Jian Zhang
abscisic acid (ABA) is a key phytohormone regulating plant development and stress response. The signal transduction of ABA largely relies on protein phosphorylation. However; little is known about the phosphorylation events occurring during ABA signaling in rice thus far. By employing a label-free; MS (Mass Spectrometry)-based phosphoproteomic approach; we identified 2271 phosphosites of young rice seedlings and their intensity dynamics in response to ABA; during which 1060 proteins were found to be differentially phosphorylated...
January 3, 2017: International Journal of Molecular Sciences
https://www.readbyqxmd.com/read/28039027/the-map-kinase-mapklk1-is-essential-to-trypanosoma-brucei-proliferation-and-regulates-proteins-involved-in-mrna-metabolism
#20
Michel Batista, Fernanda G Kugeratski, Carla V de Paula Lima, Christian M Probst, Rafael L Kessler, Lyris M de Godoy, Marco A Krieger, Fabricio K Marchini
: Protein phosphorylation and dephosphorylation events regulate many cellular processes. The identification of all phosphorylation sites and their association to a respective protein kinase or phosphatase is a challenging and crucial step to have a deeper understanding of the effects of signaling networks on cells. Pathogenic trypanosomatids have a large number of protein kinases and phosphatases in comparison to other organisms, which reinforces the relevance of the phosphorylation process in these early eukaryotes, nevertheless little is known about protein phosphorylation in these protozoa...
December 27, 2016: Journal of Proteomics
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