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Journal of Proteome Research

Ana Y Wang, Peter S Thuy-Boun, Gregory S Stupp, Andrew I Su, Dennis W Wolan
The lysis and extraction of soluble bacterial proteins from cells is a common practice for proteomics analyses, but insoluble bacterial biomasses are often left behind. Here, we show that with triflic acid treatment, the insoluble bacterial biomass of Gram- and Gram+ bacteria can be rendered soluble. We use LC-MS/MS shotgun proteomics to show that bacterial proteins in the soluble and insoluble post-lysis fractions differ significantly. Additionally, in the case of Gram- Pseudomonas aeruginosa, triflic acid treatment enriches for cell envelope-associated proteins...
July 18, 2018: Journal of Proteome Research
Anna Hakobyan, Werner Liesack, Timo Glatter
Methylocystis sp. strain SC2 is a representative of the alphaproteobacterial methane oxidizers or type IIa methanotrophs. These bacteria play a crucial role in methane cycling. Here, we developed an efficient analytical proteomics workflow for strain SC2. It tackles the major challenges related to the high amount of integral membrane proteins that need to be efficiently solubilized and digested for downstream analysis. Each step of the workflow, including cell lysis, protein solubilization and digestion, and MS peptide quantification, was assessed and optimized...
July 18, 2018: Journal of Proteome Research
Chengjian Tu, Michael V Fiandalo, Elena Pop, John J Stocking, Gissou Azabdaftari, Jun Li, Hua Wei, Danjun Ma, Jun Qu, James L Mohler, Li Tang, Yue Wu
Fetal bovine serum (FBS) is used commonly in cell culture. Charcoal-stripped FBS (CS-FBS) is used to study androgen responsiveness and androgen metabolism in cultured CaP cells. Switching CaP cells from FBS to CS-FBS may reduce activity of androgen receptor (AR), inhibit cell proliferation, or modulate intracellular androgen metabolism. Removal of proteins by charcoal stripping may cause changes in biological functions. Proteins in FBS and CS-FBS were profiled using an ion current-based quantitative platform consisting of reproducible surfactant-aided precipitation/on-pellet digestion, long-column nano-liquid chromatography (LC) separation, and ion current-based analysis (ICan)...
July 17, 2018: Journal of Proteome Research
Frank Klont, Marrit Hadderingh, Péter Horvatovich, Nick H T Ten Hacken, Rainer Bischoff
Antibodies are indispensable tools in biomedical research, but their size, complexity, and sometimes lack of reproducibility created a need for the development of alternative binders to overcome these limitations. Affimers are a novel class of affinity binders based on a structurally robust protease inhibitor scaffold (i.e. Cystatin A), which are selected by phage display and produced in a rapid and simple E. coli protein expression system. These binders have a defined amino acid sequence with defined binding regions and are versatile thereby allowing for easy engineering...
July 13, 2018: Journal of Proteome Research
Jasmin Schillinger, Katharina Severin, Farnusch Kaschani, Markus Kaiser, Michael Ehrmann
The HTRA1 gene encoding an evolutionary conserved protein quality-control factor can be epigenetically silenced or inactivated by mutation under pathologic conditions such as cancer. Recent evidence suggests that the loss of HTRA1 function causes multiple phenotypes, including the acceleration of cell growth, delayed onset of senescence, centrosome amplification, and polyploidy, suggesting an implication in the regulation of the cell cycle. To address this model, we performed a large-scale proteomics study to correlate the abundance of proteins and HTRA1 levels in various cell cycle phases using label-free-quantification mass spectrometry...
July 13, 2018: Journal of Proteome Research
Regine C Tölle, Cedric Gaggioli, Joern Dengjel
In vitro cell culture systems are an invaluable tool for cell biological research, to study molecular pathways and to characterize processes critical in human pathophysiology. However, the experimental conditions in two-dimensional (2D) cell cultures often differ substantially from the in vivo situation, which continuously raises concerns how reliable and how conferrable the obtained results are. Three-dimensional (3D) cell cultures have been shown to closer mimic in vivo conditions and are commonly employed, e...
July 10, 2018: Journal of Proteome Research
Lianyi Zhang, Paul D Veith, N Laila Huq, Yu-Yen Chen, Christine A Seers, Keith J Cross, Dhana G Gorasia, Eric C Reynolds
Porphyromonas gingivalis is a keystone periodontal pathogen that has been associated with autoimmune disorders. The cell surface proteases Lys-gingipain (Kgp) and Arg-gingipains (RgpA and RgpB) are major virulence factors and their proteolytic activity is enhanced by small peptides such as glycylglycine (GlyGly). The reaction kinetics suggested that GlyGly may function as an acceptor molecule for gingipain-catalysed transpeptidation. Purified gingipains and P. gingivalis whole cells were used to digest selected substrates including human haemoglobin in the presence or absence of peptide acceptors...
July 9, 2018: Journal of Proteome Research
Christian Trachsel, Christian Panse, Tobias Kockmann, Witold E Wolski, Jonas Grossmann, Ralph Schlapbach
Optimizing methods for liquid chromatography coupled to mass spectrometry (LC-MS) is a non-trivial task. Here we present rawDiag, a software tool supporting rational method optimization by providing MS operator-tailored diagnostic plots of scan level metadata. rawDiag is implemented as R package and can be executed on the command line, or through a graphical user interface (GUI) for less experienced users. The code runs platform independent and can process a hundred raw files in less than three minutes on current consumer hardware as we show by our benchmark...
July 6, 2018: Journal of Proteome Research
Luis Felipe Clemente, María Luisa Hernáez, Antonio Ramos-Fernández, Gertrudis Ligero, Concha Gil, Fernando José Corrales, Miguel Marcilla
Currently, 14% of the human proteome is made up of proteins whose existence is not confirmed by mass spectrometry. We performed a proteomic profiling of human mesenchymal stem cells derived from adipose tissue or umbilical cord (PRIDE accession number: PXD009893) and identified peptides derived from 13 of such missing proteins. Remarkably, we found compelling evidence of the expression of hyaluronan synthase 1 (NX_Q92839-1) and confirmed its identification by the fragmentation of four heavy-labeled peptides that coeluted with their endogenous light counterparts...
July 6, 2018: Journal of Proteome Research
Mengyang Xu, Fanyi Zhong, Richard S Bruno, Kevin D Ballard, Jing Zhang, Jiangjiang Zhu
Although higher intakes of dairy milk are associated with a lower risk of metabolic syndrome (MetS), the underlying protective mechanism remains unclear. This study investigated the dynamic metabolic profile shift following the ingestion of low-fat milk or an isocaloric volume of rice milk in obese individuals with metabolic syndrome (MetS). In a randomized, double-blind, crossover study, postprandial plasma samples (n = 266) were collected from 19 MetS participants. Plasma samples were analyzed by a targeted metabolomics platform which specifically detects 117 metabolites from 25 metabolic pathways...
July 5, 2018: Journal of Proteome Research
Michele Spiniello, Rachel A Knoener, Maisie I Steinbrink, Bing Yang, Anthony J Cesnik, Katherine E Buxton, Mark Scalf, David F Jarrard, Lloyd M Smith
RNA-protein interactions are integral to the regulation of gene expression. RNAs have diverse functions and the protein interactomes of individual RNAs vary temporally, spatially, and with physiological context. These factors make the global acquisition of individual RNA-protein interactomes an essential endeavor. Although techniques have been reported for discovery of the protein interactomes of specific RNAs they are largely laborious, costly, and accomplished singly in individual experiments. We developed HyPR-MS for the discovery and analysis of the protein interactomes of multiple RNAs in a single experiment while also reducing design time and improving efficiencies...
July 4, 2018: Journal of Proteome Research
Ryan Lillico, Courtney K Lawrence, Ted M Lakowski
Mixed lineage leukemia results from chromosomal rearrangements of the gene mixed lineage leukemia (MLL). MLL-AF9 is one such rearrangement that recruits the lysine methyltransferase, human disruptor of telomere silencing 1-like (DOT1L) and lysine specific demethylase 1 (LSD1), resulting in elevated expression of the Homeobox protein A9 (HOXA9), and leukemia. Inhibitors of LSD1 or DOT1L reduce HOXA9 expression, kill MLL-rearranged cells, and may treat leukemia. To quantify their effects on histone modifying enzyme activity and expression in MLL-rearranged leukemia, we tested inhibitors of DOT1L (EPZ-5676), LSD1 (GSK2879552), and HDAC (mocetinostat), in the MLL-AF9 cell line MOLM-13...
July 4, 2018: Journal of Proteome Research
Wei Yuan, Julius Benicky, Renhuizi Wei, Radoslav Goldman, Miloslav Sanda
Sex hormone binding globulin (SHBG) is a liver secreted glycoprotein and a major regulator of steroid distribution. It has been reported that serum concentration of SHBG changes in liver disease. To explore the involvement of SHBG in liver disease of different etiologies in greater detail, we developed a sensitive and selective LC-MS/PRM workflow to achieve quantitative analysis of SHBG glycosylation microheterogeneity. The method uses energy optimized "soft" fragmenation to extract informative Y-ions for maximal coverage of glycoforms and their quantitative comparisons...
July 4, 2018: Journal of Proteome Research
Ethan G Stoddard, Regan F Volk, James P Carson, Cecilia M Ljungberg, Taylor A Murphree, Jordan N Smith, Natalie C Sadler, Anil K Shukla, Charles Ansong, Aaron T Wright
Lung diseases and disorders are a leading cause of death among infants. Many of these diseases and disorders are caused by premature birth and underdeveloped lungs. In addition to developmentally-related disorders, the lungs are exposed to a variety of environmental contaminants and xenobiotics upon birth that can cause breathing issues and are progenitors of cancer. In order to gain a deeper understanding of the developing lung, we applied an activity-based chemoproteomics approach for the functional characterization of the xenometabolizing cytochrome P450 enzymes, active ATP and nucleotide binding enzymes, and serine hydrolases using a suite of activity-based probes (ABPs)...
July 4, 2018: Journal of Proteome Research
James R Anderson, Marie M Phelan, Peter D Clegg, Mandy J Peffers, Luis Rubio Martinez
Osteoarthritis (OA), osteochondrosis (OC) and synovial sepsis in horses cause loss of function and pain. Reliable biomarkers are required to achieve accurate and rapid diagnosis, with synovial fluid (SF) holding a unique source of biochemical information. Nuclear magnetic resonance (NMR) spectroscopy allows global metabolite analysis of a small volume of SF, with minimal sample pre-processing using a non-invasive and non-destructive method. Equine SF metabolic profiles from both non-septic joints (OA and OC) and septic joints were analysed using 1D 1 H NMR spectroscopy...
July 3, 2018: Journal of Proteome Research
Yu-Hsien Lin, Vojtech Franc, Albert J R Heck
Fetuin, also known as Alpha-2-Heremans Schmid Glycoprotein (AHSG), belongs to some of the most abundant glycoproteins secreted into the bloodstream. In blood, fetuins exhibit functions as carriers of metals and small molecules. Bovine fetuin, which harbors 3 N-glycosylation sites and a suggested half a dozen O-glycosylation sites, has been often used as a model glycoprotein to test novel analytical workflows in glycoproteomics. Here we characterize and compare fetuin in depth, using protein from three different biological sources; human serum, bovine serum and recombinant human fetuin expressed in HEK-293 cells, with the aim to elucidate similarities and differences between these proteins and the post-translational modifications they harbor...
July 2, 2018: Journal of Proteome Research
Balachandran Manavalan, Sathiyamoorthy Subramaniyam, Tae Hwan Shin, Myeong Ok Kim, Gwang Lee
Cell-penetrating peptides (CPPs) can enter cells as a variety of biologically active conjugates and have various biomedical applications. To offset the cost and effort of designing novel CPPs in laboratories, computational methods are necessitated to identify candidate CPPs before in vitro experimental studies. We developed a two-layer prediction framework called machine-learning-based prediction of cell-penetrating peptides (MLCPPs). The first-layer predicts whether a given peptide is a CPP or non-CPP, whereas the second-layer predicts the uptake efficiency of the predicted CPPs...
July 2, 2018: Journal of Proteome Research
Ming Chen, Min Zhang, Linhui Zhai, Hao Hu, Ping Liu, Minjia Tan
No abstract text is available yet for this article.
June 29, 2018: Journal of Proteome Research
Magali Palau-Rodriguez, Sara Tulipani, Anna Marco-Ramell, Antonio Miñarro, Olga Jauregui, Raul Gonzalez-Dominguez, Alex Sanchez-Pla, Bruno Ramos-Molina, Francisco J Tinahones, Cristina Andres-Lacueva
The exact impact of bariatric surgery in metabolically "healthy" (MH) or "unhealthy" (MU) phenotypes for the study of the metabolic improvement is still unknown. We applied an untargeted LC-ESI-TripleTOF-MS-driven metabolomics approach in serum samples from 39 patients with morbid obesity (MH and MU) 1, 3, and 6 months after bariatric surgery. Multiple factor analysis, along with correlation and enrichment analyses, was carried out to distinguish those metabolites associated with metabolic improvement...
June 29, 2018: Journal of Proteome Research
Enrique Almanza-Aguilera, Carl Brunius, M Rosa Bernal-Lopez, Mar Garcia-Aloy, Francisco Madrid-Gambin, Francisco J Tinahones, Ricardo Gómez-Huelgas, Rikard Landberg, Cristina Andres-Lacueva
Little is known regarding metabolic benefits of weight loss (WL) on the metabolically healthy obese (MHO) patients. We aimed to examine the impact of a lifestyle weight loss (LWL) treatment on the plasma metabolomic profile in MHO individuals. Plasma samples from 57 MHO women allocated to an intensive LWL treatment group (TG, hypocaloric Mediterranean diet and regular physical activity, n = 30) or to a control group (CG, general recommendations of a healthy diet and physical activity, n = 27) were analyzed using an untargeted 1 H NMR metabolomics approach at baseline, after 3 months (intervention), and 12 months (follow-up)...
June 28, 2018: Journal of Proteome Research
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