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low-throughput proteomics

Yuanjie Pang, Michael V Holmes, Zhengming Chen, Christiana Kartsonaki
We aimed to review the epidemiologic literature examining lifestyle, metabolic risk factors, and blood-based biomarkers including multi-omics (genomics, proteomics, and metabolomics) and to discuss how these predictive markers can inform early diagnosis of pancreatic ductal adenocarcinoma (PDAC). A search of the PubMed database was conducted in June 2018 to review epidemiologic studies of (1) lifestyle and metabolic risk factors for PDAC, genome-wide association studies (GWAS), and risk prediction models incorporating these factors and (2) blood-based biomarkers for PDAC (conventional diagnostic markers, metabolomics, and proteomics)...
December 14, 2018: Journal of Gastroenterology and Hepatology
Kristian E Swearingen, Jimmy K Eng, David Shteynberg, Vladimir Vigdorovich, Timothy A Springer, Luis Mendoza, Noah D Sather, Eric W Deutsch, Stefan H I Kappe, Robert L Moritz
Thrombospondin type 1 repeats (TSRs), small adhesive protein domains with a wide range of functions, are usually modified with O-linked fucose, which may be extended to O-fucose-β1,3-glucose. Collision-induced dissociation (CID) spectra of O-fucosylated peptides cannot be sequenced by standard tandem mass spectrometry (MS/MS) sequence database search engines because O-linked glycans are highly labile in the gas phase and are effectively absent from the CID peptide fragment spectra, resulting in a large mass error...
December 7, 2018: Journal of Proteome Research
Ting Cao, Jing Lv, Lei Zhang, Guoquan Yan, Haojie Lu
The identification and quantification of low abundant proteins are always impeded by high abundant proteins in proteomic analysis because of the extreme complexity of peptide mixtures and wide dynamic range of protein abundances. Here, we developed a novel approach to enrich and quantify N-terminal glycine peptides through sortase A mediated ligation. This strategy was based on the formation of a covalent bond between the sortase A recognition motif LPXTG and a N-terminal glycine residue. And the quantification was achieved by introducing isotopically labelled threonine in the motif LPXTG...
November 30, 2018: Analytical Chemistry
Miljan Kuljanin, Ruth M Elgamal, Gillian I Bell, Dimetri Xenocostas, Anargyros Xenocostas, David A Hess, Gilles A Lajoie
Human multipotent stromal cells (hMSCs) are one of the most versatile cell types used in regenerative medicine due to their ability to respond to injury. In the context of diabetes, it has been previously shown that the regenerative capacity of hMSCs is donor specific after transplantation into streptozotocin (STZ)-treated immunodeficient mice. However, in vivo transplantation models to determine regenerative potency of hMSCs are lengthy, costly, and low throughput. Therefore, a high-throughput quantitative proteomics assay was developed to screen β cell regenerative potency of donor-derived hMSC lines...
November 27, 2018: Cell Reports
Anupam Guleria, Amit Kumar, Umesh Kumar, Ritu Raj, Dinesh Kumar
Metabolomics is an analytical approach to metabolism and involves quantitative and comparative analysis of low-molecular-weight metabolites in body fluids or cellular/tissues extracts. Owing to its ability to reveal disease-specific metabolic patterns or metabolic changes produced in response to a therapeutic intervention; it is gaining widespread applications virtually in all aspects of biomedical and pharmaceutical research pertaining to human healthcare management. It has also started playing a strategic role in pharmacological and toxicological research for evaluating therapeutic efficacy/safety of promising drug candidates either alone or in conjunction with other omics tools such as genomics, transcriptomics and proteomics...
November 20, 2018: Current Topics in Medicinal Chemistry
Zhicheng Jin, Timothy S Collier, Darlene L Y Dai, Virginia Chen, Zsuzsanna Hollander, Raymond T Ng, Bruce M McManus, Robert Balshaw, Sophia Apostolidou, Marc S Penn, Cory Bystrom
BACKGROUND: Cholesterol efflux capacity (CEC) is a measure of HDL function that, in cell-based studies, has demonstrated an inverse association with cardiovascular disease. The cell-based measure of CEC is complex and low-throughput. We hypothesized that assessment of the lipoprotein proteome would allow for precise, high-throughput CEC prediction. METHODS: After isolating lipoprotein particles from serum, we used LC-MS/MS to quantify 21 lipoprotein-associated proteins...
November 21, 2018: Clinical Chemistry
Magdalena Kuras, Lazaro Hiram Betancourt, Melinda Rezeli, Jimmy Rodriguez, Marcell Szasz, Qimin Zhou, Tasso Miliotis, Roland Andersson, György Marko-Varga
Large cohorts of carefully collected clinical tissue materials play a central role in acquiring sufficient depth and statistical power to discover disease-related mechanisms and biomarkers of clinical significance. Manual preparation of such large sample cohorts requires experienced laboratory personnel. This carries other possible downsides such as low throughput, high risk of errors and low reproducibility. In this work, three automated technologies for high-throughput proteomics of frozen sectioned tissues were compared...
November 21, 2018: Journal of Proteome Research
Andrew Yh Ng, Chengjian Tu, Shichen Shen, Ding Xu, Merry J Oursler, Jun Qu, Shuying Yang
Osteoclasts are bone-resorbing cells differentiated from macrophage/monocyte precursors in response to macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL). In vitro models are principally based on primary bone marrow macrophages (BMMs), but RAW 264.7 cells are frequently used because they are widely available, easy to culture, and more amenable to genetic manipulation than primary cells. Increasing evidence, however, has shown that the vastly different origins of these two cell types may have important effects on cell behavior...
November 2018: JBMR Plus
Evelína Kánová, Irene Jiménez-Munguía, Petra Majerová, Zuzana Tkáčová, Katarína Bhide, Patrícia Mertinková, Lucia Pulzová, Andrej Kováč, Mangesh Bhide
Neisseria meningitidis is able to translocate the blood-brain barrier and cause meningitis. Bacterial translocation is a crucial step in the onset of neuroinvasion that involves interactions between pathogen surface proteins and host cells receptors. In this study, we applied a systematic workflow to recover and identify proteins of N. meningitidis that may interact with human brain microvascular endothelial cells (hBMECs). Biotinylated proteome of N. meningitidis was incubated with hBMECs, interacting proteins were recovered by affinity purification and identified by SWATH-MS...
2018: Frontiers in Microbiology
Eva Borràs, Eduard Sabidó
Data-independent acquisition methods that acquire fragment ions from virtually any peptide in a sample have expanded the benefits of low-throughput targeted proteomics to proteome-wide analyses. While these methods have increased the reproducibility of peptide quantification across multiple samples, their sensitivity is still limited, and the quantification of complete proteomes remains a challenge. Here we present DIA+, a DIA method that combines signals from identical peptides with different charge states, resulting in improved signal-to-noise, additional number of fragments, and therefore in a higher number of identified and quantified peptides in complex samples...
November 6, 2018: Analytical Chemistry
Jingren Deng, Fumio Ikenishi, Nicole Smith, Iulia M Lazar
Modern high-throughput and high-content biological research is performed with advanced instrumentation and complex and time-consuming protocols, which, as a whole, pose a challenge for routine implementation in a research laboratory. In support of a "bioanalytical toolbox" with potential utility for exploring cellular functions mediated via protein phosphorylation-a post-translational modification (PTM) with essential regulatory roles in a variety of cellular processes-in this work, we describe the development of a simple, integrated microfluidic chip that can perform targeted, quantitative analysis of phosphopeptides involved in cancer-relevant signaling pathways...
September 14, 2018: Electrophoresis
Eliezer Keinan, Ayelet Chen Abraham, Aaron Cohen, Alexander I Alexandrov, Reshef Mintz, Merav Cohen, Dana Reichmann, Daniel Kaganovich, Yaakov Nahmias
Microfluidic sorting offers a unique ability to isolate large numbers of cells for bulk proteomic or metabolomics studies but is currently limited by low throughput and persistent clogging at low flow rates. Recently we uncovered the physical principles governing the inertial focusing of particles in high-Reynolds numbers. Here, we superimpose high Reynolds inertial focusing on Dean vortices, to rapidly isolate large quantities of young and adult yeast from mixed populations at a rate of 107 cells/min/channel...
September 13, 2018: Scientific Reports
Yong Zeng, Shiyan Wang, Shanshan Gao, Fraser Soares, Musadeqque Ahmed, Haiyang Guo, Miranda Wang, Junjie Tony Hua, Jiansheng Guan, Michael F Moran, Ming Sound Tsao, Housheng Hansen He
N6-Methyladenosine (m6A) accounts for approximately 0.2% to 0.6% of all adenosine in mammalian mRNA, representing the most abundant internal mRNA modifications. m6A RNA immunoprecipitation followed by high-throughput sequencing (MeRIP-seq) is a powerful technique to map the m6A location transcriptome-wide. However, this method typically requires 300 μg of total RNA, which limits its application to patient tumors. In this study, we present a refined m6A MeRIP-seq protocol and analysis pipeline that can be applied to profile low-input RNA samples from patient tumors...
September 2018: PLoS Biology
Bhupesh K Prusty, Nitish Gulve, Suvagata Roy Chowdhury, Michael Schuster, Sebastian Strempel, Vincent Descamps, Thomas Rudel
Human herpesvirus 6A and 6B frequently acquires latency. HHV-6 activation has been associated with various human diseases. Germ line inheritance of chromosomally integrated HHV-6 makes viral DNA-based analysis difficult for determination of early stages of viral activation. We characterized early stages of HHV-6 activation using high throughput transcriptomics studies and applied the results to understand virus activation under clinical conditions. Using a latent HHV-6A cell culture model in U2OS cells, we identified an early stage of viral reactivation, which we define as transactivation that is marked by transcription of several viral small non-coding RNAs (sncRNAs) in the absence of detectable increase in viral replication and proteome...
2018: NPJ Genomic Medicine
Nobuo Sekiguchi, Chiyomi Kubo, Ayako Takahashi, Kumiko Muraoka, Akira Takeiri, Shunsuke Ito, Mariko Yano, Futa Mimoto, Atsuhiko Maeda, Yuki Iwayanagi, Tetsuya Wakabayashi, Shotaro Takata, Naoaki Murao, Shuichi Chiba, Masaki Ishigai
Immunogenicity is a key factor capable of influencing the efficacy and safety of therapeutic antibodies. A recently developed method called MHC-associated peptide proteomics (MAPPs) uses liquid chromatography/mass spectrometry to identify the peptide sequences derived from a therapeutic protein that are presented by major histocompatibility complex class II (MHC II) on antigen-presenting cells, and therefore may induce immunogenicity. In this study, we developed a MAPPs technique (called Ab-MAPPs) that has high throughput and can efficiently identify the MHC II-presented peptides derived from therapeutic antibodies using magnetic nanoparticle beads coated with a hydrophilic polymer in the immunoprecipitation process...
September 10, 2018: MAbs
Divya Mohan, Daniel L Wansley, Brandon M Sie, Muhammad S Noon, Alan N Baer, Uri Laserson, H Benjamin Larman
The binding specificities of an individual's antibody repertoire contain a wealth of biological information. They harbor evidence of environmental exposures, allergies, ongoing or emerging autoimmune disease processes, and responses to immunomodulatory therapies, for example. Highly multiplexed methods to comprehensively interrogate antibody-binding specificities have therefore emerged in recent years as important molecular tools. Here, we provide a detailed protocol for performing 'phage immunoprecipitation sequencing' (PhIP-Seq), which is a powerful method for analyzing antibody-repertoire binding specificities with high throughput and at low cost...
September 2018: Nature Protocols
Stephen M Rappaport
Etiological studies of human exposures to environmental factors typically rely on low-throughput methods that target only a few hundred chemicals or mixtures. In this Perspectives article, I outline how environmental exposure can be defined by the blood exposome-the totality of chemicals circulating in blood. The blood exposome consists of chemicals derived from both endogenous and exogenous sources. Endogenous chemicals are represented by the human proteome and metabolome, which establish homeostatic networks of functional molecules...
2018: NPJ Systems Biology and Applications
Borjana Arsova, Michelle Watt, Björn Usadel
Protein post-translational modifications (PTMs) are among the fastest and earliest of plant responses to changes in the environment, making the mechanisms and dynamics of PTMs an important area of plant science. One of the most studied PTMs is protein phosphorylation. This review summarizes the use of targeted proteomics for the elucidation of the biological functioning of plant PTMs, and focuses primarily on phosphorylation. Since phosphorylated peptides have a low abundance, usually complex enrichment protocols are required for their research...
2018: Frontiers in Plant Science
Mikel Azkargorta, Iraide Escobes, Ibon Iloro, Nerea Osinalde, Blanca Corral, Jone Ibañez-Perez, Antonia Exposito, Begoña Prieto, Felix Elortza, Roberto Matorras
STUDY QUESTION: Is there any difference in the protein composition of the endometrial fluid aspirate (EFA) obtained the day of embryo transfer in in vitro fertilization (IVF) cycles achieving and not achieving pregnancy? SUMMARY ANSWER: Comparative analysis identified a differential protein expression pattern in 'implantative' and 'non-implantative' IVF cycles. WHAT IS KNOWN ALREADY: EFA allows non-invasive characterization of the endometrium, and may contain important information on its receptivity when performing (IVF) cycles...
October 1, 2018: Human Reproduction
Jakub Harnoš, Jan Ryneš, Pavlína Víšková, Silvie Foldynová-Trantírková, Lola Bajard-Ešner, Lukáš Trantírek, Vítězslav Bryja
Intrinsically disordered regions (IDRs) are protein regions that lack persistent secondary or tertiary structure under native conditions. IDRs represent >40% of the eukaryotic proteome and play a crucial role in protein-protein interactions. The classical approach for identification of these interaction interfaces is based on mutagenesis combined with biochemical techniques such as coimmunoprecipitation or yeast two-hybrid screening. This approach either provides information of low resolution (large deletions) or very laboriously tries to precisely define the binding epitope via single amino acid substitutions...
October 19, 2018: Journal of Biological Chemistry
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