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low-throughput proteomics

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...
October 8, 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 Foldýnová 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 co-immunoprecipitation 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...
August 30, 2018: Journal of Biological Chemistry
Christopher D Chouinard, Gabe Nagy, Ian K Webb, Tujin Shi, Erin S Baker, Spencer A Prost, Tao Liu, Yehia M Ibrahim, Richard D Smith
Phosphoproteomics greatly augments proteomics and holds tremendous potential for insights into the modulation of biological systems for various disease states. However, numerous challenges hinder conventional methods in terms of measurement sensitivity, throughput, quantification, and capabilities for confident phosphopeptide and phosphosite identification. In this work, we report the first example of integrating structures for lossless ion manipulations ion mobility-mass spectrometry (SLIM IM-MS) with online reversed-phase liquid chromatography (LC) to evaluate its potential for addressing the aforementioned challenges...
September 18, 2018: Analytical Chemistry
Nicolai Bache, Philipp Emanuel Geyer, Dorte B Bekker-Jensen, Ole Hoerning, Lasse Falkenby, Peter V Treit, Sophia Doll, Igor Paron, Johannes Bruno Müller, Florian Meier, Jesper V Olsen, Ole Vorm, Matthias Mann
To further integrate mass spectrometry (MS)-based proteomics into biomedical research and especially into clinical settings, high throughput and robustness are essential requirements. They are largely met in high-flow rate chromatographic systems for small molecules but these are not sufficiently sensitive for proteomics applications. Here we describe a new concept that delivers on these requirements while maintaining the sensitivity of current nano-flow LC systems. Low-pressure pumps elute the sample from a disposable trap column, simultaneously forming a chromatographic gradient that is stored in a long storage loop...
August 13, 2018: Molecular & Cellular Proteomics: MCP
Cassandra M Modahl, Seth Frietze, Stephen P Mackessy
High-throughput technologies were used to identify venom gland toxin expression and to characterize the venom proteomes of two rear-fanged snakes, Ahaetulla prasina (Asian Green Vine Snake) and Borikenophis portoricensis (Puerto Rican Racer). Sixty-nine complete toxin-coding transcripts from 12 venom protein superfamilies (A. prasina) and 50 complete coding transcripts from 11 venom protein superfamilies (B. portoricensis) were identified in the venom glands. However, only 18% (A. prasina) and 32% (B. portoricensis) of the translated protein isoforms were detected in the proteome of these venoms...
September 15, 2018: Journal of Proteomics
Shichen Shen, Bo An, Xue Wang, Shannon P Hilchey, Jun Li, Jin Cao, Yu Tian, Chenqi Hu, Liang Jin, Andrew Ng, Chengjian Tu, Miao Qu, Martin S Zand, Jun Qu
For quantitative proteomics, efficient, robust, and reproducible sample preparation with high throughput is critical yet challenging, especially when large cohorts are involved, as is often required by clinical/pharmaceutical studies. We describe a rapid and straightforward surfactant cocktail-aided extraction/precipitation/on-pellet digestion (SEPOD) strategy to address this need. Prior to organic solvent precipitation and on-pellet digestion, SEPOD treats samples with a surfactant cocktail (SC) containing multiple nonionic/anionic surfactants, which achieves (i) exhaustive/reproducible protein extraction, including membrane-bound proteins; (ii) effective removal of detrimental nonprotein matrix components (e...
September 4, 2018: Analytical Chemistry
Viviana Greco, Cristian Piras, Luisa Pieroni, Maurizio Ronci, Lorenza Putignani, Paola Roncada, Andrea Urbani
The development of precision medicine requires advanced technologies to address the multifactorial disease stratification and to support personalized treatments. Among omics techniques, proteomics based on Mass Spectrometry (MS) is becoming increasingly relevant in clinical practice allowing a phenotypic characterization of the dynamic functional status of the organism. From this perspective, Matrix Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF) MS is a suitable platform for providing a high-throughput support to clinics...
August 2018: Expert Review of Proteomics
Ryszard A Zielke
Combining high-throughput mass spectrometry with isobaric tags for relative and absolute quantification (iTRAQ) allows for the identification and relative quantification of proteins from multiple samples. Furthermore, low-abundance proteins that are usually not detected can be enriched by using only the relevant fraction of the proteome, e.g., cytoplasmic, membrane proteins, or secreted proteins. Described here is a workflow for isolation and enrichment of secreted and membrane proteins that is compatible with mass spectrometry...
2018: Methods in Molecular Biology
John W Thompson, Alexander W Sorum, Linda C Hsieh-Wilson
The dynamic posttranslational modification O-linked β- N-acetylglucosamine glycosylation (O-GlcNAcylation) is present on thousands of intracellular proteins in the brain. Like phosphorylation, O-GlcNAcylation is inducible and plays important functional roles in both physiology and disease. Recent advances in mass spectrometry (MS) and bioconjugation methods are now enabling the mapping of O-GlcNAcylation events to individual sites in proteins. However, our understanding of which glycosylation events are necessary for regulating protein function and controlling specific processes, phenotypes, or diseases remains in its infancy...
July 10, 2018: Biochemistry
Ryohei Narumi, Keiko Masuda, Takeshi Tomonaga, Jun Adachi, Hiroki R Ueda, Yoshihiro Shimizu
High-sensitivity mass spectrometry approaches using selected reaction monitoring (SRM) or multiple reaction monitoring (MRM) methods are powerful tools for targeted quantitative proteomics-based investigation of dynamics in specific biological systems. Both high-sensitivity detection of low-abundance proteins and their quantification using this technique employ stable isotope-labeled peptide internal standards. Currently, there are various ways for preparing standards, including chemical peptide synthesis, cellular protein expression, and cell-free protein or peptide synthesis...
June 2018: Synthetic and Systems Biotechnology
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