scholarly journals DIA-based systems biology approach unveils E3 ubiquitin ligase-dependent responses to a metabolic shift

2020 ◽  
Vol 117 (51) ◽  
pp. 32806-32815
Author(s):  
Ozge Karayel ◽  
André C. Michaelis ◽  
Matthias Mann ◽  
Brenda A. Schulman ◽  
Christine R. Langlois
Keyword(s):  
Mass Spectrometry ◽  
Systems Biology ◽  
Large Scale ◽  
E3 Ligase ◽  
Cellular Systems ◽  
Mass Spectrometry Analysis ◽  
High Throughput Analysis ◽  
Metabolic Switch ◽  
Spectrometry Analysis ◽  
Yeast Proteome

The yeastSaccharomyces cerevisiaeis a powerful model system for systems-wide biology screens and large-scale proteomics methods. Nearly complete proteomics coverage has been achieved owing to advances in mass spectrometry. However, it remains challenging to scale this technology for rapid and high-throughput analysis of the yeast proteome to investigate biological pathways on a global scale. Here we describe a systems biology workflow employing plate-based sample preparation and rapid, single-run, data-independent mass spectrometry analysis (DIA). Our approach is straightforward, easy to implement, and enables quantitative profiling and comparisons of hundreds of nearly complete yeast proteomes in only a few days. We evaluate its capability by characterizing changes in the yeast proteome in response to environmental perturbations, identifying distinct responses to each of them and providing a comprehensive resource of these responses. Apart from rapidly recapitulating previously observed responses, we characterized carbon source-dependent regulation of the GID E3 ligase, an important regulator of cellular metabolism during the switch between gluconeogenic and glycolytic growth conditions. This unveiled regulatory targets of the GID ligase during a metabolic switch. Our comprehensive yeast system readout pinpointed effects of a single deletion or point mutation in the GID complex on the global proteome, allowing the identification and validation of targets of the GID E3 ligase. Moreover, this approach allowed the identification of targets from multiple cellular pathways that display distinct patterns of regulation. Although developed in yeast, rapid whole-proteome–based readouts can serve as comprehensive systems-level assays in all cellular systems.

scholarly journals DIA-based systems biology approach unveils novel E3-dependent responses to a metabolic shift

2020 ◽  
Author(s):  
Ozge Karayel ◽  
André C. Michaelis ◽  
Matthias Mann ◽  
Brenda A. Schulman ◽  
Christine R. Langlois
Keyword(s):  
Mass Spectrometry ◽  
Systems Biology ◽  
Large Scale ◽  
E3 Ligase ◽  
Cellular Systems ◽  
Mass Spectrometry Analysis ◽  
High Throughput Analysis ◽  
Metabolic Switch ◽  
Spectrometry Analysis ◽  
Yeast Proteome

ABSTRACTYeast Saccharomyces cerevisiae is a powerful model system for systems-wide biology screens and large-scale proteomics methods. Nearly complete proteomics coverage has been achieved owing to advances in mass spectrometry. However, it remains challenging to scale this technology for rapid and high-throughput analysis of the yeast proteome to investigate biological pathways on a global scale. Here we describe a systems biology workflow employing plate-based sample preparation and rapid, single-run data independent mass spectrometry analysis (DIA). Our approach is straightforward, easy to implement and enables quantitative profiling and comparisons of hundreds of nearly complete yeast proteomes in only a few days. We evaluate its capability by characterizing changes in the yeast proteome in response to environmental perturbations, identifying distinct responses to each of them, and providing a comprehensive resource of these responses. Apart from rapidly recapitulating previously observed responses, we characterized carbon source dependent regulation of the GID E3 ligase, an important regulator of cellular metabolism during the switch between gluconeogenic and glycolytic growth conditions. This unveiled new regulatory targets of the GID ligase during a metabolic switch. Our comprehensive yeast system read-out pinpointed effects of a single deletion or point mutation in the GID complex on the global proteome, allowing the identification and validation novel targets of the GID E3 ligase. Moreover, our approach allowed the identification of targets from multiple cellular pathways that display distinct patterns of regulation. Although developed in yeast, rapid whole proteome-based readouts can serve as comprehensive systems-level assay in all cellular systems.

Acoustic Ejection/Full-Scan Mass Spectrometry Analysis for High-Throughput Compound Quality Control

2020 ◽  
pp. 247263032096762
Author(s):  
Jun Zhang ◽  
Yong Zhang ◽  
Chang Liu ◽  
Tom Covey ◽  
Julia Nielsen ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Quality Control ◽  
Data Processing ◽  
High Throughput ◽  
Data Extraction ◽  
Mass Spectrometry Analysis ◽  
High Throughput Analysis ◽  
Spectrometry Analysis ◽  
Microtiter Plates ◽  
Full Scan

High-throughput analysis of compound dissolved in DMSO and arrayed in multiwell plates for quality control (QC) purposes has widespread utility in drug discovery, ranging from the QC of assay-ready plates dispatched by compound management, to compound integrity check in the screening collection, to reaction monitoring of chemical syntheses in microtiter plates. Due to the large number of samples (thousands per batch) involved, these workflows can put a significant burden on the liquid chromatography–mass spectrometry (LC-MS) platform typically used. To achieve the required speed of seconds per sample, several chromatography-free MS approaches have previously been used with mixed results. In this study, we demonstrated the feasibility of acoustic ejection–mass spectrometry (AE-MS) in full-scan mode for high-throughput compound QC in miniaturized formats, featuring direct, contactless liquid sampling, minimal sample consumption, and ultrafast analytical speed. The sample consumption and analysis time by AE-MS represent, respectively, a 1000-fold and 30-fold reduction compared with LC-MS. In qualitative QC, AE-MS generated comparable results to conventional LC-MS in identifying the presence and absence of expected compounds. AE-MS also demonstrated its utility in relative quantifications of the same compound in serial dilution plates, or substrate in chemical synthesis. To facilitate the processing of a large amount of data generated by AE-MS, we have developed a data processing platform using commercially available tools. The platform demonstrated fast and straightforward data extraction, reviewing, and reporting, thus eliminating the need for the development of custom data processing tools. The overall AE-MS workflow has effectively eliminated the analytical bottleneck in the high-throughput compound QC work stream.

scholarly journals Alternative proteins are functional regulators in cell reprogramming by PKA activation

2020 ◽  
Vol 48 (14) ◽  
pp. 7864-7882 ◽  
Author(s):  
Tristan Cardon ◽  
Julien Franck ◽  
Etienne Coyaud ◽  
Estelle M N Laurent ◽  
Marina Damato ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Large Scale ◽  
Epithelial Mesenchymal Transition ◽  
Gene Annotation ◽  
Mass Spectrometry Analysis ◽  
Cell Reprogramming ◽  
Pathway Enrichment Analysis ◽  
Mesenchymal Transition ◽  
Spectrometry Analysis ◽  
Biological Studies

Abstract It has been recently shown that many proteins are lacking from reference databases used in mass spectrometry analysis, due to their translation templated on alternative open reading frames. This questions our current understanding of gene annotation and drastically expands the theoretical proteome complexity. The functions of these alternative proteins (AltProts) still remain largely unknown. We have developed a large-scale and unsupervised approach based on cross-linking mass spectrometry (XL-MS) followed by shotgun proteomics to gather information on the functional role of AltProts by mapping them back into known signalling pathways through the identification of their reference protein (RefProt) interactors. We have identified and profiled AltProts in a cancer cell reprogramming system: NCH82 human glioma cells after 0, 16, 24 and 48 h Forskolin stimulation. Forskolin is a protein kinase A activator inducing cell differentiation and epithelial–mesenchymal transition. Our data show that AltMAP2, AltTRNAU1AP and AltEPHA5 interactions with tropomyosin 4 are downregulated under Forskolin treatment. In a wider perspective, Gene Ontology and pathway enrichment analysis (STRING) revealed that RefProts associated with AltProts are enriched in cellular mobility and transfer RNA regulation. This study strongly suggests novel roles of AltProts in multiple essential cellular functions and supports the importance of considering them in future biological studies.

Integrated plant proteomics — putting the green genomes to work

2003 ◽  
Vol 30 (5) ◽  
pp. 471 ◽  
Author(s):  
Joshua L. Heazlewood ◽  
A. Harvey Millar
Keyword(s):  
Mass Spectrometry ◽  
Data Storage ◽  
Large Scale ◽  
Protein Identification ◽  
Mass Spectrometry Analysis ◽  
Plant Science ◽  
Genomic Information ◽  
Plant Proteomics ◽  
Spectrometry Analysis ◽  
Complete Set

Protein analysis has been at the heart of plant science for many years, but with new questions emerging from an abundance of genomic information and further improvements in technology, there are now new opportunities to undertake large-scale analyses and to move to more complex systems than has been possible previously. This explosion of interest and data is often referred to simply as proteomics, which is the study of the complete set of proteins expressed at a given time and place, the proteome. As its name suggests proteomics is intricately linked to allied technologies such as genomics, transcriptomics and metabolomics. In this review of plant proteomics we outline a series of issues that face the practical user, particularly the largest problem that currently faces researchers, the myriad of options to choose from. The choices, problems and pitfalls of entering into gel-based and non-gel-based arraying techniques are discussed together with advances in pre-fractionation of samples, liquid chromatography separations and subcellular analyses. Issues relating to mass spectrometry analysis and the eventual protein identification are outlined, and the dilemmas of data storage and analysis are highlighted. During this tour we provide a series of references to the literature — experimental, theoretical and technical — to illustrate the breadth of current investigations using these techniques.

scholarly journals A New Drug Discovery Approach Based on Thermal Proteome Profiling to Develop More Effective Drugs

2021 ◽  
Vol 8 (2) ◽  
Author(s):  
Elham Gholizadeh ◽  
Mostafa Rezaei-Tavirani ◽  
Alireza Emadi ◽  
Reza Karbalaei ◽  
Ali Khaleghian
Keyword(s):  
Mass Spectrometry ◽  
Protein Interactions ◽  
Large Scale ◽  
Protein Extraction ◽  
Mass Spectrometry Analysis ◽  
Structural Stabilization ◽  
Proteome Profiling ◽  
Spectrometry Analysis ◽  
Drug Concentrations ◽  
Low Efficiency

: The search for disease-related targets and studying drug-protein and protein-protein interactions are central issues that would accelerate the clinical approval of a drug. Also, by developing an accurate method in this regard, time and resource consumption will significantly decrease. The low efficiency of some drugs in humans is a grave issue leading to a low rate of FDA approval after spending billions of dollars and decades of research. Several strategies and methods have been expanded to fill this gap, such as drug affinity responsive target stability (DARTS), stability of proteins from rates of oxidation (SPROX), cellular thermal shift assay (CETSA), and finally, thermal proteome profiling (TPP). The TPP is based on the combination of CETSA and quantitative mass spectrometry. Among recently introduced proteomics technologies, TPP demonstrates the ability to offer detailed proteomic profiles for the large-scale analysis of protein-ligand interactions, including endogenous ligands and proteins like cofactors and metabolites. TPP facilitates the identification of the markers governing drug efficacy and toxicity and provides an unbiased measure for estimating the rate of drug-target engagement. At a glance at TPP steps, after protein extraction, the molecule is exposed to different temperatures and drug concentrations. After discarding solubilized and stabilized proteins, the protein’s identity is investigated by mass spectrometry analysis. As a result of the protein’s structural stabilization after binding to its substrate, TTP helps to accurately identify target proteins with high throughput. In this study, we aimed to introduce the basics of this method and review most recent studies on this technique.

scholarly journals A dry method for preserving tear protein samples

2017 ◽  
Author(s):  
Weiwei Qin ◽  
Chan Zhao ◽  
Linpei Zhang ◽  
Ting Wang ◽  
Youhe Gao
Keyword(s):  
Mass Spectrometry ◽  
Large Scale ◽  
Low Cost ◽  
Mass Spectrometry Analysis ◽  
Protein Patterns ◽  
Spectrometry Analysis ◽  
Disease Biomarkers ◽  
Tear Protein ◽  
Significant Difference ◽  
Biomarker Research

AbstractTears covering the ocular surface is an important bio-fluid containing thousands of molecules, including proteins, lipids, metabolites, nucleic acids, and electrolytes. Tears are valuable resources for biomarker research of ocular and even systemic diseases. For application in biomarker studies, tear samples should ideally be stored using a simple, low-cost, and efficient method along with the patient’s medical records. For this purpose, we developed a novel Schirmer’s strip-based dry method that allows for storage of tear samples in vacuum bags at room temperature. Using this method, tear protein patterns can also be preserved. Liquid chromatography-mass spectrometry/mass spectrometry analysis of proteins recovered from the dry method and traditional wet method showed no significant difference. Some tissue/organ enriched proteins were identified in tear, thus tear might be a good window for monitoring the change of these tissues or organs. This dry method facilitates sample transportation and enables the storage of tear samples on a large scale, increasing the availability of samples for studying disease biomarkers in tears.

Application of Mass Spectrometry Methods to the Analysis of Lipid Composition of the Human Brain

2021 ◽  
Vol 37 (5) ◽  
pp. 80-87
Author(s):  
A.I. Tkachev ◽  
M.S. Osetrova ◽  
D.N. Smirnov ◽  
O.I. Efimova ◽  
E.E. Khrameev
Keyword(s):  
Mass Spectrometry ◽  
Human Brain ◽  
Lipid Composition ◽  
Large Scale ◽  
Lipid Extraction ◽  
Simultaneous Detection ◽  
Molecular Organization ◽  
Mass Spectrometry Analysis ◽  
Spectrometry Analysis ◽  
The Brain

Abstract-Lipids make up more than half of the dry matter of the human brain and play a key role in its functioning. However, the lipid composition of the brain anatomical structures remains poorly understood. The first such studies were carried out more than 50 years ago, but since then, a small number of works have been published describing the concentration of lipids in only a few areas of the human brain. A fundamentally new step towards understanding the molecular organization of the brain and identifying the molecular basis of human cognitive abilities should be a detailed large-scale study of the brain lipidome. However, there is no description in the literature of methods optimized for studying the lipid composition of the human brain. In this work, we develop and present methods for lipid extraction and mass spectrometry analysis, which ensure simultaneous detection of the maximum amount of different lipid classes and individual substances in the human brain, as well as the approaches to bioinformatics analysis of the obtained data. Their use makes it possible to create a comprehensive picture of the molecular organization of the human brain, which has no analogues in the world in terms of its completeness. Key words: lipidome, brain, mass spectrometry, high-performance liquid chromatography, bioinformatics The reported study was funded by the Russian Foundation for Basic Research, project no. 20-34-70077.

scholarly journals Stability of the human pregnane X receptor is regulated by E3 ligase UBR5 and serine/threonine kinase DYRK2

2014 ◽  
Vol 459 (1) ◽  
pp. 193-203 ◽  
Author(s):  
Su Sien Ong ◽  
Asli N. Goktug ◽  
Ayesha Elias ◽  
Jing Wu ◽  
Darren Saunders ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
E3 Ligase ◽  
Pregnane X Receptor ◽  
Mass Spectrometry Analysis ◽  
Serine Threonine Kinase ◽  
Sirna Screen ◽  
Spectrometry Analysis ◽  
Threonine Kinase ◽  
Major Chemical

Mass spectrometry analysis and a kinome-wide siRNA screen revealed that phosphorylation of pregnane X receptor, a major chemical toxin sensor, by the serine/threonine kinase DYRK2 facilitates its subsequent ubiquitination by the E3 ligase UBR5.

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