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Molecular & Cellular Proteomics: MCP

Sibylle Pfammatter, Eric Bonneil, Francis P McManus, Satendra Prasad, Derek J Bailey, Michael Belford, Jean-Jacques Dunyach, Pierre Thibault
The depth of proteomic analyses is often limited by the overwhelming proportion of confounding background ions that compromise the identification and quantification of low abundance peptides. To alleviate these limitations, we present a new high field asymmetric waveform ion mobility spectrometry (FAIMS) interface that can be coupled to the Orbitrap Tribrid mass spectrometers. The interface provides several advantages over previous generations of FAIMS devices including ease of operation, robustness, and high ion transmission...
July 14, 2018: Molecular & Cellular Proteomics: MCP
Chuan-Chih Hsu, Yingfang Zhu, Justine V Arrington, J Sebastian Paez, Pengcheng Wang, Peipei Zhu, I-Hsuan Chen, Jian-Kang Zhu, Weiguo Andy Tao
Phosphorylation-mediated signaling transduction plays a crucial role in the regulation of plant defense mechanisms against environmental stresses. To address the high complexity and dynamic range of plant proteomes and phosphoproteomes, we present a universal sample preparation procedure that facilitates plant phosphoproteomic profiling. This advanced workflow significantly improves phosphopeptide identifications, enabling deep insight into plant phosphoproteomes. We then applied the workflow to study the phosphorylation events involved in tomato cold tolerance mechanisms...
July 13, 2018: Molecular & Cellular Proteomics: MCP
Chun Yip Chan, Alex Cheow Khoon Soh, Dorinda Yan Qin Kioh, Jianguo Li, Chandra Verma, Siew Kwan Koh, Roger Wilmer Beuerman, Lei Zhou, Eric Chun Yong Chan
Although covalent protein binding is established as the pivotal event underpinning acetaminophen (APAP) toxicity, its mechanistic details remain unclear. In this study, we demonstrated that APAP induces widespread protein glutathionylation in a time-, dose- and bioactivation-dependent manner in HepaRG cells. Proteo-metabonomic mapping provided evidence that APAP-induced glutathionylation resulted in functional deficits in energy metabolism, elevations in oxidative stress and cytosolic calcium, as well as mitochondrial dysfunction that correlate strongly with the well-established toxicity features of APAP...
July 13, 2018: Molecular & Cellular Proteomics: MCP
Dongsic Choi, Laura Montermini, Dae-Kyum Kim, Brian Meehan, Frederick P Roth, Janusz Rak
Glioblastoma multiforme (GBM) is a highly aggressive and heterogeneous form of primary brain tumors, driven by a complex repertoire of oncogenic alterations, including the constitutively active epidermal growth factor receptor (EGFRvIII). EGFRvIII impacts both cell-intrinsic and non-cell autonomous aspects of GBM progression, including cell invasion, angiogenesis and modulation of the tumor microenvironment. This is, at least in part, attributable to the release and intercellular trafficking of extracellular vesicles (EVs), heterogeneous membrane structures containing multiple bioactive macromolecules...
July 13, 2018: Molecular & Cellular Proteomics: MCP
Fumei Chen, Qiang Fu, Liping Pu, Pengfei Zhang, Yulin Huang, Zhen Hou, Zhuangzhuang Xu, Dongrong Chen, Fengling Huang, Tingxian Deng, Xianwei Liang, Yangqing Lu, Ming Zhang
Maternal-effect genes are especially critical for early embryonic development after fertilization and until massive activation of the embryonic genome occurs. By applying a tandem mass tag (TMT)-labeled quantitative proteomics combined with RNA sequencing approach, the proteome of the buffalo was quantitatively analyzed during parthenogenesis of mature oocytes and the two-cell stage embryo. Of 1,908 quantified proteins, 123 differed significantly. The transcriptome was analyzed eight stages (GV, MII, 2-cell, 4-cell, 8-cell, 16-cell, morula, blastocyst) of Buffalo using the RNA sequencing approach , and a total of 3567 unique genes were identified to be differently expressed between all consecutive stages of pre-implantation development...
July 12, 2018: Molecular & Cellular Proteomics: MCP
Kévin Jacquet, Sara L Banerjee, François J M Chartier, Sabine Elowe, Nicolas Bisson
Signals from cell surface receptors are often relayed via adaptor proteins. NCK1 and NCK2 are Src-Homology (SH) 2 and 3 domain adaptors that regulate processes requiring a remodelling of the actin cytoskeleton. Evidence from gene inactivation in mouse suggests that NCK1 and NCK2 are functionally redundant, although recent reports support the idea of unique functions for NCK1 and NCK2. We sought to examine this question further by delineating NCK1- and NCK2-specific signalling networks. We used both affinity purification-mass spectrometry and BioID proximity labelling to identify NCK1/2 signalling networks comprised of 98 proteins...
July 12, 2018: Molecular & Cellular Proteomics: MCP
Rahul Sureka, Rashi Wadhwa, Suman S Thakur, Rashmi Upadhyay Pathak, Rakesh K Mishra
Chromatin condenses several folds to form mitotic chromosomes during cell division and decondenses post-mitotically to re-occupy their nuclear territory and re-gain their specific transcriptional profile in a precisely lineage specific manner.  This necessitates that the features of nuclear architecture and DNA topology persist through mitosis. We compared the proteome of  nuclease and high salt resistant fraction of interphase nucleus known as nuclear matrix (NuMat) and an equivalent biochemical fraction in the mitotic chromosome known as mitotic chromosome scaffold (MiCS)...
July 10, 2018: Molecular & Cellular Proteomics: MCP
Payman Samavarchi-Tehrani, Hala Abdouni, Reuben Samson, Anne-Claude Gingras
Proximity-dependent biotinylation strategies have emerged as powerful tools to characterize the subcellular context of proteins in living cells.  The popular BioID approach employs an abortive E. coli biotin ligase mutant (R118G; denoted as BirA*), which when fused to a bait protein enables the covalent biotinylation of endogenous proximal polypeptides. This approach has been mainly applied to the study of protein proximity in immortalized mammalian cell lines.  To expand the application space of BioID, here we describe a set of lentiviral vectors that enable the inducible expression of BirA*-tagged bait fusion proteins for performing proximity-dependent biotinylation in diverse experimental systems...
July 10, 2018: Molecular & Cellular Proteomics: MCP
Weixuan Wang, Yadong Hu, Changmei Yang, Songbiao Zhu, Xiaofei Wang, Zhenyu Zhang, Haiteng Deng
Nicotinamide adenine dinucleotide (NAD) plays an essential role in all aspects of human life. NAD levels decrease as humans age, and supplementation with NAD precursors plays a protective role against aging and associated disease. Less is known about the effects of decreased NAD on cellular processes, which is the basis for understanding the relationship between cellular NAD levels and aging-associated disease. In the present study, cellular NAD levels were decreased by overexpression of CD38, a NAD hydrolase, or by treating cells with FK866, an inhibitor of nicotinamide phosphoribosyltransferase (NAMPT)...
July 6, 2018: Molecular & Cellular Proteomics: MCP
Andrea C Becker, Monique Gannage, Sebastian Giese, Zehan Hu, Shadi Abou-Eid, Carole Roubaty, Petra Paul, Lea Katharina Bühler, Christine Gretzmeier, Veronica I Dumit, Stephanie Kaeser-Pebernard, Martin Schwemmle, Christian Münz, Joern Dengjel
Seasonal epidemics of influenza A virus are a major cause of severe illness and are of high socio-economic relevance. For the design of effective anti-viral therapies, a detailed knowledge of pathways perturbed by virus infection is critical. We performed comprehensive expression and organellar proteomics experiments to study the cellular consequences of influenza A virus infection using three human epithelial cell lines derived from human lung carcinomas: A549, Calu-1 and NCI-H1299. As a common response, the type I interferon pathway was upregulated upon infection...
July 6, 2018: Molecular & Cellular Proteomics: MCP
Anna Ressa, Evert Bosdriesz, Joep de Ligt, Sara Mainardi, Gianluca Maddalo, Anirudh Prahallad, Myrthe Jager, Lisanne de la Fonteijne, Martin Fitzpatrick, Stijn Groten, A F Maarten Altelaar, René Bernards, Edwin Cuppen, Lodewyk Wessels, Albert J R Heck
Intrinsic and/or acquired resistance represents one of the great challenges in targeted cancer therapy. A deeper understanding of the molecular biology of cancer has resulted in more efficient strategies, where one or multiple drugs are adopted in novel therapies to tackle resistance. This beneficial effect of using combination treatments has also been observed in colorectal cancer patients harboring the BRAF(V600E) mutation, whereby dual inhibition of BRAF(V600E) and EGFR increases antitumor activity. Notwithstanding this success, it is not clear whether this combination treatment is the only or most effective treatment to block intrinsic resistance to BRAF inhibitors...
July 3, 2018: Molecular & Cellular Proteomics: MCP
Miquel Rosas-Salvans, Tommaso Cavazza, Guadalupe Espadas, Eduard Sabido, Isabelle Vernos
Microtubules (MTs) and associated proteins can self-organize into complex structures such as the bipolar spindle, a process in which RanGTP plays a major role. Addition of RanGTP to M-phase Xenopus egg extracts promotes the nucleation and self-organization of MTs into asters and bipolar-like structures in the absence of centrosomes or chromosomes. We show here that the complex proteome of these RanGTP-induced MT assemblies is similar to that of mitotic spindles. Using proteomic profiling we show that MT self-organization in the M-phase cytoplasm involves the non-linear and non-stoichiometric recruitment of proteins from specific functional groups...
July 3, 2018: Molecular & Cellular Proteomics: MCP
Jung-Eun Choi, Jae-Jin Lee, Wonmo Kang, Hyun Jung Kim, Jin-Hwan Cho, Pyung-Lim Han, Kong-Joo Lee
Chronic physical restraint stress increases oxidative stress in the brain, and dysregulation of oxidative stress can be one of the causes of major depressive disorder. In order to understand the underlying mechanisms, we undertook a systematic proteomic analysis of hippocampus in a chronic restraint stress mouse model of depression. Combining two-dimensional gel electrophoresis (2D-PAGE) for protein separation with nanoUPLC-ESI-q-TOF tandem mass spectrometry, we identified sixty-three protein spots that changed in the hippocampus of mice subjected to chronic restraint stress...
June 29, 2018: Molecular & Cellular Proteomics: MCP
Hyebin Lee, Kwangsoo Kim, Jongmin Woo, Joonho Park, Hyeyoon Kim, Kyung Eun Lee, Hyeyeon Kim, Youngsoo Kim, Kyung Chul Moon, Ji Young Kim, In Ae Park, Bo Bae Shim, Ji Hye Moon, Dohyun Han, Han Suk Ryu
Cytological examination of urine is the most widely used noninvasive pathologic screen for bladder urothelial carcinoma (BLCA); however, inadequate diagnostic accuracy remains a major challenge. We performed mass spectrometry-based proteomic analysis of urine samples of ten patients with BLCA and ten paired patients with benign urothelial lesion (BUL) to identify ancillary proteomic markers for use in liquid-based cytology (LBC). A total of 4,839 proteins were identified and 112 proteins were confirmed as expressed at significantly different levels between the two groups...
June 27, 2018: Molecular & Cellular Proteomics: MCP
Evan Graehl Williams, Yibo Wu, Dongryeol Ryu, Jun Yong Kim, Jiayi Lan, Moaraj Hasan, Witold Wolski, Pooja Jha, Christian Halter, Johan Auwerx, Ruedi Aebersold
We have used SWATH mass spectrometry to quantify 3648 proteins across 76 proteomes collected from genetically-diverse BXD mouse strains in two fractions (mitochondria and total cell) from five tissues: liver, quadriceps, heart, brain, and brown adipose (BAT). Across tissues, expression covariation between genes' proteins and transcripts-measured in the same individuals-broadly aligned. Covariation was however far stronger in certain subsets than others: only 8% of transcripts in the lowest expression and variance quintile covaried with their protein, in contrast to 65% of transcripts in the highest quintiles...
June 26, 2018: Molecular & Cellular Proteomics: MCP
Ying Zhu, Maowei Dou, Paul D Piehowski, Yiran Liang, Fangjun Wang, Rosalie K Chu, Will Chrisler, Jordan N Smith, Kaitlynn C Schwarz, Yufeng Shen, Anil K Shukla, Ronald J Moore, Richard D Smith, Wei-Jun Qian, Ryan T Kelly
Current mass spectrometry (MS)-based proteomics approaches are ineffective for mapping protein expression in tissue sections with high spatial resolution due to the limited overall sensitivity of conventional workflows. Here we report an integrated and automated method to advance spatially resolved proteomics by seamlessly coupling laser capture microdissection (LCM) with a recently developed nanoliter-scale sample preparation system termed nanoPOTS (Nanodroplet Processing in One pot for Trace Samples). The workflow is enabled by prepopulating nanowells with DMSO, which serves as a sacrificial capture liquid for microdissected tissues...
June 24, 2018: Molecular & Cellular Proteomics: MCP
Rekha Raghunathan, Nicole K Polinski, Joshua A Klein, John D Hogan, Chun Shao, Kshitij Khatri, Deborah R Leon, Mark E McComb, Fredric P Manfredsson, Caryl E Sortwell, Joseph Zaia
Parkinson's disease (PD) is a neurological disorder characterized by the progressive loss of functional dopaminergic neurons in the nigrostriatal pathway in the brain.  While current treatments provide only symptomatic relief, gene therapy has the potential to slow or halt the degeneration of nigrostriatal dopamine neurons in PD patients. Adeno-associated viruses (AAV) are vectors of choice in gene therapy due to their well-characterized safety and efficacy profiles; however, while gene therapy has been successful in preclinical models of the disease, clinical trials in humans have failed to demonstrate efficacy...
June 18, 2018: Molecular & Cellular Proteomics: MCP
Dean E Hammond, J Dinesh Kumar, Lorna Raymond, Deborah M Simpson, Robert J Beynon, Graham J Dockray, Andrea Varro
Analysis of secretomes critically underpins the capacity to understand the mechanisms determining interactions between cells and between cells and their environment. In the context of cancer cell micro-environments, the relevant interactions are recognised to be an important determinant of tumor progression. Global proteomic analyses of secretomes are often performed at a single time point and frequently identify both classical secreted proteins (possessing an N-terminal signal sequence), as well as many intracellular proteins, the release of which is of uncertain biological significance...
June 18, 2018: Molecular & Cellular Proteomics: MCP
Jean-David Morel, Anja O Paatero, Jiajie Wei, Jonathan W Yewdell, Laure Guenin-Macé, Delphi Van Haver, Francis Impens, Natalia Pietrosemoli, Ville O Paavilainen, Caroline Demangel
Mycolactone is a bacteria-derived macrolide that blocks the biogenesis of a large array of secretory and integral transmembrane proteins (TMP) through potent inhibition of the Sec61 translocon. Here, we used quantitative proteomics to delineate the direct and indirect effects of mycolactone-mediated Sec61 blockade in living cells. In T lymphocytes, dendritic cells and sensory neurons, Sec61 substrates downregulated by mycolactone were in order of incidence: secretory proteins (with a signal peptide but no transmembrane domain), TMPs with a signal peptide (Type I) and TMPs without signal peptide and a cytosolic N-terminus (Type II)...
June 18, 2018: Molecular & Cellular Proteomics: MCP
Auriane Corbière, Marie-Laure Walet-Balieu, Philippe Chan, Magali Basille-Dugay, Julie Hardouin, David Vaudry
The cerebellum is a brain structure involved in motor and cognitive functions. The development of the cerebellar cortex (the external part of the cerebellum) is under the control of numerous factors. Among these factors, neuropeptides including PACAP or somatostatin modulate the survival, migration and/or differentiation of cerebellar granule cells. Interestingly, such peptides contributing to cerebellar ontogenesis usually exhibit a specific transient expression profile with a low abundance at birth, a high expression level during the developmental processes, which take place within the first two postnatal weeks in rodents, and a gradual decline toward adulthood...
June 12, 2018: Molecular & Cellular Proteomics: MCP
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