scholarly journals TurboID-mediated proximity labelling of cytoophidium proteome in Drosophila

2019 ◽  
Author(s):  
Bo Zhang ◽  
Yuanbing Zhang ◽  
Ji-Long Liu
Keyword(s):  
Mass Spectrometry ◽  
Developmental Stages ◽  
Metabolic Enzyme ◽  
Wide Range ◽  
Powerful Approach ◽  
Ctp Synthase ◽  
Subcellular Compartmentation ◽  
Cell Type Specific ◽  
Living Organisms

AbstractProximity-based biotinylation combined with mass spectrometry has emerged as a powerful approach to study protein interaction networks and protein subcellular compartmentation. However, low kinetics and the requirement of toxic chemicals limit the broad utilisation of current proximity labelling methods in living organisms. TurboID, the newly engineered promiscuous ligase, has been reported to label bait proteins effectively in various species. Here, we systematically demonstrated the application of TurboID-mediated biotinylation in a wide range of developmental stages and tissues, and we also verified the feasibility of TurboID-mediated labelling in desired cells via cell-type-specific GAL4 driver in Drosophila. Furthermore, using TurboID-mediated biotinylation coupled with mass spectrometry, we characterized the proximate proteome of the cytoophidium, a newly identified filamentous structure containing the metabolic enzyme CTP synthase (CTPS) in Drosophila. Our study demonstrates a referable tool and resource for research in subcellular compartments of metabolic enzymes in vivo.

scholarly journals Highly effective proximate labeling in Drosophila

2021 ◽  
Author(s):  
Bo Zhang ◽  
Yuanbing Zhang ◽  
Ji-Long Liu
Keyword(s):  
Mass Spectrometry ◽  
Developmental Stages ◽  
Cell Types ◽  
Good Resolution ◽  
Spatiotemporal Resolution ◽  
Protein Protein Interaction ◽  
Ppi Networks ◽  
Wide Range

Abstract The protein-protein interaction (PPI) is a basic strategy for life to operate. The analysis of PPIs in multicellular organisms is very important but extremely challenging because PPIs are particularly dynamic and variable among different development stages, tissues, cells, and even organelles. Therefore, understanding PPI needs a good resolution of time and space. More importantly, understanding in vivo PPI needs to be realized in situ. Proximity-based biotinylation combined with mass spectrometry has emerged as a powerful approach to study PPI networks and protein subcellular compartmentation. TurboID, the newly engineered promiscuous ligase, has been reported to label bait proteins effectively in various species. In Drosophila, we systematically apply TurboID-mediated biotinylation in a wide range of developmental stages and tissues, and demonstrate the feasibility of TurboID-mediated labeling system in desired cell types. For a proof-of-principle, we use the TurboID-mediated biotinylation coupled with mass spectrometry to distinguish CTP synthase with or without the ability to form filamentous cytoophidia, retrieving two distinct sets of proximate proteomes. Therefore, this makes it possible to map PPIs in vivo and in situ at a defined spatiotemporal resolution, and demonstrates a referable resource for cytoophidium proteome in Drosophila.

scholarly journals In vivo measurement of synthesis rate of multiple plasma proteins in humans

2006 ◽  
Vol 291 (1) ◽  
pp. E190-E197 ◽  
Author(s):  
Abdul Jaleel ◽  
Vandana Nehra ◽  
Xuan-Mai T. Persson ◽  
Yves Boirie ◽  
Maureen Bigelow ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Synthesis ◽  
Stable Isotope ◽  
Plasma Proteins ◽  
Large Scale ◽  
Protein Quantification ◽  
Gas Chromatography Mass Spectrometry ◽  
Synthesis Rate ◽  
Wide Range

Advances in quantitative proteomics have facilitated the measurement of large-scale protein quantification, which represents net changes in protein synthesis and breakdown. However, measuring the rate of protein synthesis is the only way to determine the translational rate of gene transcripts. Here, we report a technique to measure the rate of incorporation of amino acids from ingested protein labeled with stable isotope into individual plasma proteins. This approach involves three steps: 1) production of stable isotope-labeled milk whey protein, oral administration of this intrinsically labeled protein, and subsequent collection of blood samples; 2) fractionation of the plasma and separation of the individual plasma proteins by a combination of anion exchange high-pressure liquid chromatography and gel electrophoresis; and 3) identification of individual plasma proteins by tandem mass spectrometry and measurement of stable isotopic enrichment of these proteins by gas chromatography-mass spectrometry. This method allowed the measurement of the fractional synthesis rate (FSR) of 29 different plasma proteins by using the same precursor pool. We noted a 30-fold difference in FSR of different plasma proteins with a wide range of physiological functions. This approach offers a tremendous opportunity to study the regulation of plasma proteins in humans in many physiological and pathological states.

Antimicrobial Peptides: An Approach to Combat Resilient Infections

2020 ◽  
Vol 17 (4) ◽  
pp. 542-552 ◽  
Author(s):  
Debaprasad Parai ◽  
Pia Dey ◽  
Samir K. Mukherjee
Keyword(s):  
Antimicrobial Peptides ◽  
Developmental Stages ◽  
Rapid Development ◽  
Resistance Mechanisms ◽  
New Drugs ◽  
Bacterial Strains ◽  
Common People ◽  
Wide Range ◽  
Microbial Infections ◽  
Living Organisms

Background:It was apparent by the end of 1980s that the success against the threats of bacterial pathogens on public health was an illusion, with the rapid development of resistant strains more than the discovery of new drugs. As a consequence, the remedial services were in the backfoot position of being on the losing side of this never-ending evolutionary war. The quest for new antibiotics to overcome resistance problems has long been a top research priority for the researchers and the pharmaceutical industry. However, the resistance problems remain unresolved due to the abrupt misuse of antibiotics by common people, which has immensely worsened the scenario by disseminating antibiotic-resistant bacterial strains around the world.Objective:Thus, immediate action is needed to measure emerging and re-emerging microbial diseases having new resistance mechanisms and to manage their rapid spread among the common public by means of novel alternative metabolites.Conclusion:Antimicrobial Peptides (AMPs) are short, cationic peptides evolved in a wide range of living organisms and serve as the essential part of the host innate immunity. For humans, these effector molecules either can directly kill the foreign microbes or modulate the host immune systems so that the human body could develop some resistance against the microbial infections. In this review, we discuss their history, structural classifications, modes of action, and explain their biological roles as anti-infective agents. We also scrutinize their clinical potentiality, current limitations in various developmental stages and strategies to overcome for their successful clinical applications.

scholarly journals Direct Visualization of Live Zebrafish Glycan via Single-step Metabolic Labeling with Fluorophore-tagged Nucleotide Sugars

2019 ◽  
Author(s):  
Senlian Hong ◽  
Pankaj Sahai-Hernandez ◽  
David Traver ◽  
Peng Wu
Keyword(s):  
Developmental Stages ◽  
Single Step ◽  
Zebrafish Embryos ◽  
Metabolic Labeling ◽  
Cell Stage ◽  
Imaging Probes ◽  
Nucleotide Sugars ◽  
Living Organisms ◽  
The One

ABSTRACTDynamic turnover of cell-surface glycans is involved in a myriad of biological events, making this process an attractive target for in vivo molecular imaging. The metabolic glycan labeling coupled with ‘bioorthogonal chemistry’ has paved the way for visulizing glycans in living organisms. However, a two-step labeling sequence is required, which is prone to tissue penetration difficulties of the imaging probes. Here, by exploring the substrate promiscuity of endogenous glycosyltransferases, we developed a single-step fluorescent glycan labeling strategy by using fluorophore-tagged analogs of nucleotide sugars directly. Injecting the fluorophore-tagged sialic acid and fucose into the yolk of zebrafish embryos at the one-cell stage enables a systematic imaging of sialylation and fucosylation in live zebrafish embryos at various developmental stages. From these studies, we obtained insights into the role of sialylated and fucosylated glycans in zebrafish hematopoiesis.

scholarly journals Mass Spectrometry-Based Zebrafish Toxicometabolomics: A Review of Analytical and Data Quality Challenges

Metabolites ◽  
2021 ◽  
Vol 11 (9) ◽  
pp. 635
Author(s):  
Katyeny Manuela da Silva ◽  
Elias Iturrospe ◽  
Chloe Bars ◽  
Dries Knapen ◽  
Steven Van Cruchten ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Chemical Exposure ◽  
Research Field ◽  
In Vivo Studies ◽  
In Vitro Assays ◽  
Special Focus ◽  
Zebrafish Model ◽  
Wide Range

Metabolomics has achieved great progress over the last 20 years, and it is currently considered a mature research field. As a result, the number of applications in toxicology, biomarker, and drug discovery has also increased. Toxicometabolomics has emerged as a powerful strategy to provide complementary information to study molecular-level toxic effects, which can be combined with a wide range of toxicological assessments and models. The zebrafish model has gained importance in recent decades as a bridging tool between in vitro assays and mammalian in vivo studies in the field of toxicology. Furthermore, as this vertebrate model is a low-cost system and features highly conserved metabolic pathways found in humans and mammalian models, it is a promising tool for toxicometabolomics. This short review aims to introduce zebrafish researchers interested in understanding the effects of chemical exposure using metabolomics to the challenges and possibilities of the field, with a special focus on toxicometabolomics-based mass spectrometry. The overall goal is to provide insights into analytical strategies to generate and identify high-quality metabolomic experiments focusing on quality management systems (QMS) and the importance of data reporting and sharing.

scholarly journals DNA-Encoded Multivalent Display of Protein Tetramers on Phage: Synthesis and In Vivo Aplications

2021 ◽  
Author(s):  
Guilherme M. Lima ◽  
Alexey Atrazhev ◽  
Susmita Sarkar ◽  
Mirat Sojitra ◽  
Revathi Reddy ◽  
...  
Keyword(s):  
Coat Protein ◽  
Biological Properties ◽  
Universal Method ◽  
Phagocytic Cells ◽  
Clearance Mechanism ◽  
Powerful Approach ◽  
Multiple Copies ◽  
Multivalent Display ◽  
Living Organisms

AbstractPhage display links phenotype of displayed polypeptides with DNA sequence in phage genome and offers a universal method for discovery of proteins with novel properties. Injection of phage-displayed libraries in living organisms further provides a unique and powerful approach to optimize biochemical, pharmacological and biological properties of the displayed peptides, antibodies and other proteins in vivo. However, over 60% of the proteome is comprised of multi-domain proteins, and display of large multi-subunit proteins on phages remains a challenge. Majority of protein display systems are based on monovalent phagemid constructs but methods for robust display of multiple copies of large proteins are scarce. Here, we describe a DNA-encoded display of a ∼200 kDa tetrameric protein tetrameric L-asparaginase on M13 phage produced by ligation of SpyCatcher-Asparaginase fusion (ScA) to prospectively barcoded phage clones displaying SpyTag peptide. Starting from the SpyTag display on p3 minor coat protein or p8 major coat protein yielded constructs with five copies of ScA displayed on p3 (ScA5-phage) and 50 copies of ScA on p8 protein (ScA50-phage). ScA remained active after conjugation. It could be easily produced directly from lysates of bacteria that express ScA. Display constructs of different valency can be injected into mice and analyzed by deep-sequencing of the DNA barcodes associated phage clones. In these multiplexed studies, we observed a density-dependent clearance rate in vivo. A known clearance mechanism of L-asparaginase is endocytosis by phagocytic cells. Our observations, thus, link the increase in density of the displayed protein with the increased rate of the endocytosis by cells in vivo. In conclusion, we demonstrate that a multivalent display of L-asparaginase on phage could be used to study the circulation life of this protein in vivo and such approach opens the possibility to use DNA sequencing to investigate multiplexed libraries of other multi-subunit proteins in vivo.Abstract Graphic

scholarly journals 1D “Spikelet” Projections from Heteronuclear 2D NMR Data—Permitting 1D Chemometrics While Preserving 2D Dispersion

Metabolites ◽  
2019 ◽  
Vol 9 (1) ◽  
pp. 16 ◽  
Author(s):  
Maryam Tabatabaei Anaraki ◽  
Wolfgang Bermel ◽  
Rudraksha Dutta Majumdar ◽  
Ronald Soong ◽  
Myrna Simpson ◽  
...  
Keyword(s):  
1H Nmr ◽  
13C Nmr ◽  
2D Nmr ◽  
Complex Samples ◽  
Software Packages ◽  
Nmr Data ◽  
Wide Range ◽  
Spectral Dispersion ◽  
Living Organisms

Nuclear magnetic resonance (NMR) spectroscopy is a powerful tool for the non-targeted metabolomics of intact biofluids and even living organisms. However, spectral overlap can limit the information that can be obtained from 1D 1H NMR. For example, magnetic susceptibility broadening in living organisms prevents any metabolic information being extracted from solution-state 1D 1H NMR. Conversely, the additional spectral dispersion afforded by 2D 1H-13C NMR allows a wide range of metabolites to be assigned in-vivo in 13C enriched organisms, as well as a greater depth of information for biofluids in general. As such, 2D 1H-13C NMR is becoming more and more popular for routine metabolic screening of very complex samples. Despite this, there are only a very limited number of statistical software packages that can handle 2D NMR datasets for chemometric analysis. In comparison, a wide range of commercial and free tools are available for analysis of 1D NMR datasets. Overtime, it is likely more software solutions will evolve that can handle 2D NMR directly. In the meantime, this application note offers a simple alternative solution that converts 2D 1H-13C Heteronuclear Single Quantum Correlation (HSQC) data into a 1D “spikelet” format that preserves not only the 2D spectral information, but also the 2D dispersion. The approach allows 2D NMR data to be converted into a standard 1D Bruker format that can be read by software packages that can only handle 1D NMR data. This application note uses data from Daphnia magna (water fleas) in-vivo to demonstrate how to generate and interpret the converted 1D spikelet data from 2D datasets, including the code to perform the conversion on Bruker spectrometers.

scholarly journals Soil-Associated Bacillus Species: A Reservoir of Bioactive Compounds with Potential Therapeutic Activity against Human Pathogens

2021 ◽  
Vol 9 (6) ◽  
pp. 1131
Author(s):  
Galal Yahya ◽  
Asmaa Ebada ◽  
Eman M. Khalaf ◽  
Basem Mansour ◽  
Nehal A. Nouh ◽  
...  
Keyword(s):  
Bioactive Compounds ◽  
Therapeutic Potential ◽  
Antibiotic Production ◽  
Molecular Techniques ◽  
Bacillus Species ◽  
Rrna Gene ◽  
Human Pathogens ◽  
Wide Range ◽  
Living Organisms

Soil hosts myriads of living organisms with the extensive potential to produce bioactive compounds. Bacteria are the major soil inhabitants that represent a rich reservoir for antibiotic production along with their role in recycling nutrients and maintenance of the soil ecosystem. Here, from 55 tested soil samples, we isolated and identified a novel antibiotic-producing bacterial strain with a phylogenetically closest match to Bacillus subtilis sp. based on BLASTN search of GenBank for the 16S rRNA gene sequence. We characterized this novel strain through microscopic, biochemical, and molecular techniques, combined with testing its potential antimicrobial activity. Chemical studies revealed that the antibiotic produced by this strain is a glycopeptide. It exhibited profound activity against both methicillin-resistant Staphylococcus aureus (MRSA) and Candida albicans. The antibiotic is optimally produced at 37 °C after 28 h of growth. The biocompatibility of the extracted antibiotic was tested over a wide range of factors including temperature, pH, surfactants, and metal salts. To confirm its therapeutic potential, a sterile solution of the antibiotic was tested in vivo against bacteria-induced keratitis in rats where significant healing activity was recorded. Hence, this soil Bacillus strain may lead to the development of novel antibiotics for the treatment of human pathogens.

The metabolism of [1- 13 C]-glucose by Paracoccus denitrificans , investigated by gas chromatography-mass spectrometry

1984 ◽  
Vol 220 (1221) ◽  
pp. 423-437 ◽  
Keyword(s):  
Mass Spectrometry ◽  
Metabolic Profiling ◽  
Sole Carbon Source ◽  
Paracoccus Denitrificans ◽  
Growth Conditions ◽  
Pentose Phosphate ◽  
Gas Chromatography Mass Spectrometry ◽  
Wide Range ◽  
Pentose Phosphate Pathways

The metabolism of Paracoccus denitrificans , grown on [1- 13 C]-glucose as the sole carbon source, was investigated by using gas chromatographymass spectrometry and metabolic profiling. The distribution of label in a wide range of metabolites (42 in total) showed that the EmbdenMeyerhoff pathway did not operate in vivo whereas both the EntnerDoudoroff and pentose phosphate pathways were operative under the specified growth conditions. It was calculated that 31% of the [1- 13 C]-glucose was metabolized by the Entner-Doudoroff pathway and 69% by the pentose phosphate pathway.

Sign in / Sign up

Export Citation Format

Share Document