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phosphoproteomics rat

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
Jaime Santo-Domingo, Isabelle Chareyron, Loïc Dayon, Antonio Núñez Galindo, Ornella Cominetti, María Pilar Giner Giménez, Umberto De Marchi, Carles Canto, Martin Kussmann, Andreas Wiederkehr
Mitochondria play a central role in pancreatic β-cell nutrient sensing by coupling their metabolism to plasma membrane excitability and insulin granule exocytosis. Whether non-nutrient secretagogues stimulate mitochondria as part of the molecular mechanism to promote insulin secretion is not known. Here, we show that PKC signaling, which is employed by many non-nutrient secretagogues, augments mitochondrial respiration in INS-1E (rat insulinoma cell line clone 1E) and human pancreatic β-cells. The phorbol ester, phorbol 12-myristate 13-acetate, accelerates mitochondrial respiration at both resting and stimulatory glucose concentrations...
December 7, 2016: FASEB Journal: Official Publication of the Federation of American Societies for Experimental Biology
Shaoling Jiang, Hanchang He, Lishan Tan, Liangliang Wang, Zhengxiu Su, Yufeng Liu, Hongguo Zhu, Menghuan Zhang, Fan Fan Hou, Aiqing Li
Salt plays an essential role in the progression of chronic kidney disease and hypertension. However, the mechanisms underlying pathogenesis of salt-induced kidney damage remain largely unknown. Here, Sprague-Dawley rats, that underwent 5/6 nephrectomy (5/6Nx, a model of advanced kidney damage) or sham operation, were treated for 2 weeks with a normal or high-salt diet. We employed aTiO2 enrichment, iTRAQ labeling and liquid-chromatography tandem mass spectrometry strategy for proteomic and phosphoproteomic profiling of the renal cortex...
October 24, 2016: Scientific Reports
Matthew T Rich, Thomas B Abbott, Lisa Chung, Erol E Gulcicek, Kathryn L Stone, Christopher M Colangelo, TuKiet T Lam, Angus C Nairn, Jane R Taylor, Mary M Torregrossa
UNLABELLED: Successful addiction treatment depends on maintaining long-term abstinence, making relapse prevention an essential therapeutic goal. However, exposure to environmental cues associated with drug use often thwarts abstinence efforts by triggering drug using memories that drive craving and relapse. We sought to develop a dual approach for weakening cocaine memories through phosphoproteomic identification of targets regulated in opposite directions by memory extinction compared with reconsolidation in male Sprague-Dawley rats that had been trained to self-administer cocaine paired with an audiovisual cue...
July 20, 2016: Journal of Neuroscience: the Official Journal of the Society for Neuroscience
Mahdokht Kohansal-Nodehi, John Je Chua, Henning Urlaub, Reinhard Jahn, Dominika Czernik
Neurotransmitter release is mediated by the fast, calcium-triggered fusion of synaptic vesicles with the presynaptic plasma membrane, followed by endocytosis and recycling of the membrane of synaptic vesicles. While many of the proteins governing these processes are known, their regulation is only beginning to be understood. Here we have applied quantitative phosphoproteomics to identify changes in phosphorylation status of presynaptic proteins in resting and stimulated nerve terminals isolated from the brains of Wistar rats...
2016: ELife
Shuxin Wang, Zheyi Li, Hongyan Shen, Zhong Zhang, Yuxin Yin, Qingsong Wang, Xuyang Zhao, Jianguo Ji
Protein phosphorylation is central to the understanding of multiple cellular signaling pathways responsible for regulating the self-renewal and differentiation of neural stem cells (NSCs). Here we performed a large-scale phosphoproteomic analysis of rat fetal NSCs using strong cation exchange chromatography prefractionation and citric acid-assisted two-step enrichment with TiO2 strategy followed by nanoLC-MS/MS analysis. Totally we identified 32,546 phosphosites on 5,091 phosphoproteins, among which 23,945 were class I phosphosites, and quantified 16,000 sites during NSC differentiation...
August 2016: Stem Cells
Liyuan Zhang, Zhen Liang, Lihua Zhang, Yukui Zhang, Shujuan Shao
Despite recent advances in phosphoproteome research, detection and characterization of multi-phosphopeptides have remained a challenge. Here we present a novel IMAC strategy for effective extracting multi-phosphopeptides from complex samples, through Ga(3+) chelation to the adenosine tri-phosphate (ATP)-functionalized magnetic nanoparticles (Ga(3+)-ATP-MNPs). The high specificity of Ga(3+)-ATP-MNPs was demonstrated by efficient enriching multi-phosphopeptides from the digest mixture of β-casein and BSA with molar ratio as low as 1:5000...
November 5, 2015: Analytica Chimica Acta
Jiaming Li, Qingrun Li, Jiashu Tang, Fangying Xia, Jiarui Wu, Rong Zeng
As central tissue of glucose homeostasis, islet has been an important focus of diabetes research. Phosphorylation plays pivotal roles in islet function, especially in islet glucose-stimulated insulin secretion. A systematic view on how phosphorylation networks were coordinately regulated in this process remains lacking, partially due to the limited amount of islets from an individual for a phosphoproteomic analysis. Here we optimized the in-tip and best-ratio phosphopeptide enrichment strategy and a SILAC-based workflow for processing rat islet samples...
November 6, 2015: Journal of Proteome Research
Rima Chaudhuri, Arash Sadrieh, Nolan J Hoffman, Benjamin L Parker, Sean J Humphrey, Jacqueline Stöckli, Adam P Hill, David E James, Jean Yee Hwa Yang
BACKGROUND: Most biological processes are influenced by protein post-translational modifications (PTMs). Identifying novel PTM sites in different organisms, including humans and model organisms, has expedited our understanding of key signal transduction mechanisms. However, with increasing availability of deep, quantitative datasets in diverse species, there is a growing need for tools to facilitate cross-species comparison of PTM data. This is particularly important because functionally important modification sites are more likely to be evolutionarily conserved; yet cross-species comparison of PTMs is difficult since they often lie in structurally disordered protein domains...
2015: BMC Genomics
Diego M Assis, Luiz Juliano, Thaysa Paschoalin, Maria Kouyoumdjian, Joao B Calixto, Robson A S Santos, Thelma A Pertinhez, Francis Gauthier, Thierry Moreau, Michael Blaber, Maria A Juliano
Phosphorylated kininogen and some of its fragments containing serine phosphorylated bradykinin ([pS(6)]-Bk) were identified in human serum and plasma by a phosphoproteomic approach. We report the kininogenase ability of human tissue and plasma kallikreins and tryptase to generate [pS(6)]-Bk or Lys-[pS(6)]-Bk having as substrate the synthetic human kininogen fluorescent fragment Abz-MISLMKRPPGF[pS(386)]PFRSSRI-NH2. The pharmacological assays of [pS(6)]-Bk showed it as a full B2 bradykinin receptor agonist in smooth muscle, it produces a portal liver hypertensive response in rat and mouse paw edema that lasts longer than Bk...
September 15, 2015: Biochemical Pharmacology
Xiao-Mei He, Xi Chen, Gang-Tian Zhu, Qian Wang, Bi-Feng Yuan, Yu-Qi Feng
Sample preparation methods with high selectivity, efficiency, and matrix resistance are essential for phosphoproteomic analysis. In this study, carboxyl cotton chelator-titanium(IV) (CCC-Ti4+) fibers, a novel CCC-based fibrous sorbent with excellent biocompatibility, were successfully synthesized on the basis of the coordination effect between double carboxyl groups on CCC and Ti4+. The synthesis of CCC-Ti4+ fibers was easy, and the incorporated titanium content was high. On the basis of immobilized metal ion affinity chromatography (IMAC), CCC-Ti4+ fibers were used for specific capture of phosphopeptides using a lab-in-syringe solid-phase extraction (SPE) from multiple biological samples, including standard protein digests, nonfat milk digests, human serum, and animal tissue...
August 12, 2015: ACS Applied Materials & Interfaces
Enwu Xu, Juan Chen, Yu Wang, Zhiyong Ke, Shenqiu Luo, Zhipeng Zou
The reprogrammed lipopolysaccharide (LPS) pathway has been reported to render patients more susceptible to the development of post-traumatic multiple organ dysfunction syndrome (MODS). To facilitate thorough understanding of this mechanism, a phosphoproteomic study was utilized to screen the potential signaling molecules. Interestingly, a truncated form of Src homology 2-domain-containing tyrosine phosphatase 1 (shp-1) emerged in human THP-1 macrophages sequentially treated with H2O2 and LPS and not with either of the treatments alone...
October 14, 2015: Journal of Proteomics
Luis Carretero, Pablo Llavona, Alejandro López-Hernández, Pedro Casado, Pedro R Cutillas, Pilar de la Peña, Francisco Barros, Pedro Domínguez
The transduction pathway mediating the inhibitory effect that TRH exerts on r-ERG channels has been thoroughly studied in GH3 rat pituitary cells but some elements have yet to be discovered, including those involved in a phosphorylation event(s). Using a quantitative phosphoproteomic approach we studied the changes in phosphorylation caused by treatment with 1μM TRH for 5min in GH3 cells. The activating residues of Erk2 and Erk1 undergo phosphorylation increases of 5.26 and 4.87 fold, respectively, in agreement with previous reports of ERK activation by TRH in GH3 cells...
September 2015: Cellular Signalling
Carine Poussin, Carole Mathis, Leonidas G Alexopoulos, Dimitris E Messinis, Rémi H J Dulize, Vincenzo Belcastro, Ioannis N Melas, Theodore Sakellaropoulos, Kahn Rhrissorrakrai, Erhan Bilal, Pablo Meyer, Marja Talikka, Stéphanie Boué, Raquel Norel, John J Rice, Gustavo Stolovitzky, Nikolai V Ivanov, Manuel C Peitsch, Julia Hoeng
The biological responses to external cues such as drugs, chemicals, viruses and hormones, is an essential question in biomedicine and in the field of toxicology, and cannot be easily studied in humans. Thus, biomedical research has continuously relied on animal models for studying the impact of these compounds and attempted to 'translate' the results to humans. In this context, the SBV IMPROVER (Systems Biology Verification for Industrial Methodology for PROcess VErification in Research) collaborative initiative, which uses crowd-sourcing techniques to address fundamental questions in systems biology, invited scientists to deploy their own computational methodologies to make predictions on species translatability...
2014: Scientific Data
Judy C Triplett, Aaron Swomley, Jessime Kirk, Katilyn Lewis, Miranda Orr, Karl Rodriguez, Jian Cai, Jon B Klein, Rochelle Buffenstein, D Allan Butterfield
Naked mole-rats (NMRs) are the oldest-living rodent species. Living underground in a thermally stable ecological niche, NMRs have evolved certain exceptional traits, resulting in sustained health spans, negligible cognitive decline, and a pronounced resistance to age-related disease. Uncovering insights into mechanisms underlying these extraordinary traits involved in successful aging may conceivably provide crucial clues to extend the human life span and health span. One of the most fundamental processes inside the cell is the production of ATP, which is an essential fuel in driving all other energy-requiring cellular activities...
August 2015: Journal of Neurochemistry
D B McClatchy, J N Savas, S Martínez-Bartolomé, S K Park, P Maher, S B Powell, J R Yates
Prepulse inhibition (PPI) is an example of sensorimotor gating and deficits in PPI have been demonstrated in schizophrenia patients. Phencyclidine (PCP) suppression of PPI in animals has been studied to elucidate the pathological elements of schizophrenia. However, the molecular mechanisms underlying PCP treatment or PPI in the brain are still poorly understood. In this study, quantitative phosphoproteomic analysis was performed on the prefrontal cortex from rats that were subjected to PPI after being systemically injected with PCP or saline...
February 2016: Molecular Psychiatry
Aswin Pyakurel, Claudia Savoia, Daniel Hess, Luca Scorrano
Controlled changes in mitochondrial morphology participate in cellular signaling cascades. However, the molecular mechanisms modifying mitochondrial shape are largely unknown. Here we show that the mitogen-activated protein (MAP) kinase cascade member extracellular-signal-regulated kinase (ERK) phosphorylates the pro-fusion protein mitofusin (MFN) 1, modulating its participation in apoptosis and mitochondrial fusion. Phosphoproteomic and biochemical analyses revealed that MFN1 is phosphorylated at an atypical ERK site in its heptad repeat (HR) 1 domain...
April 16, 2015: Molecular Cell
Tiina Öhman, Sandra Söderholm, Maruthibabu Paidikondala, Niina Lietzén, Sampsa Matikainen, Tuula A Nyman
Sendai virus (SeV) is a common respiratory pathogen in mice, rats, and hamsters. Host cell recognition of SeV is mediated by pathogen recognition receptors, which recognize viral components and induce intracellular signal transduction pathways that activate the antiviral innate immune response. Viruses use host proteins to control the activities of signaling proteins and their downstream targets, and one of the most important host protein modifications regulated by viral infection is phosphorylation. In this study, we used phosphoproteomics combined with bioinformatics to get a global view of the signaling pathways activated during SeV infection in human lung epithelial cells...
June 2015: Proteomics
Francesca Zappacosta, Gilbert F Scott, Michael J Huddleston, Roland S Annan
While analysis of the phosphoproteome has become an important component of understanding how cells function, it remains a nontrivial task in terms of the number of sample preparation steps and instrument time needed to achieve sufficient depth of coverage to produce meaningful results. We previously described a multidimensional method that uses hydrophilic interaction chromatography (HILIC) followed by Fe(3+) immobilized metal affinity chromatography (IMAC) to reduce complexity, improve selectivity, and increase phosphopeptide identifications...
February 6, 2015: Journal of Proteome Research
Maxime Rotival, Jeong-Hun Ko, Prashant K Srivastava, Audrey Kerloc'h, Alex Montoya, Claudio Mauro, Peter Faull, Pedro R Cutillas, Enrico Petretto, Jacques Behmoaras
Macrophage multinucleation (MM) is essential for various biological processes such as osteoclast-mediated bone resorption and multinucleated giant cell-associated inflammatory reactions. Here we study the molecular pathways underlying multinucleation in the rat through an integrative approach combining MS-based quantitative phosphoproteomics (LC-MS/MS) and transcriptome (high-throughput RNA-sequencing) to identify new regulators of MM. We show that a strong metabolic shift toward HIF1-mediated glycolysis occurs at transcriptomic level during MM, together with modifications in phosphorylation of over 50 proteins including several ARF GTPase activators and polyphosphate inositol phosphatases...
March 2015: Molecular & Cellular Proteomics: MCP
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