scholarly journals Mass Spectrometry versus Conventional Techniques of Protein Detection: Zika Virus NS3 Protease Activity towards Cellular Proteins

Molecules ◽  
2021 ◽  
Vol 26 (12) ◽  
pp. 3732
Author(s):  
Agnieszka Dabrowska ◽  
Aleksandra Milewska ◽  
Joanna Ner-Kluza ◽  
Piotr Suder ◽  
Krzysztof Pyrc
Keyword(s):  
Mass Spectrometry ◽  
Zika Virus ◽  
Viral Infections ◽  
Protein Detection ◽  
Extensive Discussion ◽  
Protein Targets ◽  
Highly Sensitive ◽  
Infected Cells ◽  
Ns3 Protease ◽  
To Receive

Mass spectrometry (MS) used in proteomic approaches is able to detect hundreds of proteins in a single assay. Although undeniable high analytical power of MS, data acquired sometimes lead to confusing results, especially during a search of very selective, unique interactions in complex biological matrices. Here, we would like to show an example of such confusing data, providing an extensive discussion on the observed phenomenon. Our investigations focus on the interaction between the Zika virus NS3 protease, which is essential for virus replication. This enzyme is known for helping to remodel the microenvironment of the infected cells. Several reports show that this protease can process cellular substrates and thereby modify cellular pathways that are important for the virus. Herein, we explored some of the targets of NS3, clearly shown by proteomic techniques, as processed during infection. Unfortunately, we could not confirm the biological relevance of protein targets for viral infections detected by MS. Thus, although mass spectrometry is highly sensitive and useful in many instances, also being able to show directions where cell/virus interaction occurs, we believe that deep recognition of their biological role is essential to receive complete insight into the investigated process.

scholarly journals Zika virus NS3 protease and its cellular substrates

2020 ◽  
Author(s):  
Agnieszka Dabrowska ◽  
Aleksandra Milewska ◽  
Joanna Ner-Kluza ◽  
Piotr Suder ◽  
Krzysztof Pyrc
Keyword(s):  
Mass Spectrometry ◽  
Virus Infection ◽  
Zika Virus ◽  
Brain Disorders ◽  
Biological Validation ◽  
Ns3 Protein ◽  
Highly Sensitive ◽  
Virus Interaction ◽  
Cellular Pathways ◽  
Ns3 Protease

AbstractZika virus is a flavivirus discovered in 1947, but the association between Zika virus infection and brain disorders was not demonstrated until 2015 in Brazil. Infection mostly poses a threat to women during pregnancy, since it may cause microcephaly and other neurological dysfunctions in the developing fetus. However, infection is also associated with Guillain-Barré syndrome. The nonstructural NS3 protein is essential for virus replication because it helps to remodel the cellular microenvironment. Several reports show that this protease can process cellular substrates and thereby modify cellular pathways that are important for the virus. Herein, we explored some of the targets of NS3, but we could not confirm the biological relevance of its protease activity. Thus, although mass spectrometry is highly sensitive and useful in many instances, being also able to show directions, where cell/virus interaction occurs, we believe that biological validation of the observed results is essential.

scholarly journals Highly sensitive and multiplexed in situ protein profiling with cleavable fluorescent streptavidin

2019 ◽  
Author(s):  
Renjie Liao ◽  
Diego Mastroeni ◽  
Paul D. Coleman ◽  
Jia Guo
Keyword(s):  
Signal Amplification ◽  
Individual Cell ◽  
Protein Detection ◽  
Protein Profiling ◽  
Detection Sensitivity ◽  
Layer By Layer ◽  
Protein Targets ◽  
Highly Sensitive ◽  
Wide Range

AbstractThe ability to perform highly sensitive and multiplexed in situ protein analysis is crucial to advance our understanding of normal physiology and disease pathogenesis. To achieve this goal, here we develop an approach using cleavable biotin conjugated antibodies and cleavable fluorescent streptavidin (CFS). In this approach, protein targets are first recognized by the cleavable biotin labeled antibodies. Subsequently, CFS is applied to stain the protein targets. Though layer-by-layer signal amplification using cleavable biotin conjugated orthogonal antibodies and CSF, the protein detection sensitivity can be enhanced by at least 10 fold, compared with the existing methods. After imaging, the fluorophores and the biotins unbound to streptavidin are removed by chemical cleavage. The leftover streptavidin is blocked by biotin. Upon reiterative analysis cycles, a large number of different proteins with a wide range of expression levels can be unambiguously detected in individual cell in situ.

Highly sensitive protein detection by combination of atomic force microscopy fishing with charge generation and mass spectrometry analysis

FEBS Journal ◽  
2014 ◽  
Vol 281 (20) ◽  
pp. 4705-4717 ◽  
Author(s):  
Yuri D. Ivanov ◽  
Tatyana Pleshakova ◽  
Krystina Malsagova ◽  
Andrey Kozlov ◽  
Anna Kaysheva ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Atomic Force Microscopy ◽  
Protein Detection ◽  
Mass Spectrometry Analysis ◽  
Charge Generation ◽  
Spectrometry Analysis ◽  
Force Microscopy ◽  
Atomic Force ◽  
Highly Sensitive

scholarly journals Modulation of acyl-carnitines, the broad mechanism behind Wolbachia-mediated inhibition of medically important flaviviruses in Aedes aegypti

2020 ◽  
Vol 117 (39) ◽  
pp. 24475-24483 ◽  
Author(s):  
Gayathri Manokaran ◽  
Heather A. Flores ◽  
Conor T. Dickson ◽  
Vinod K. Narayana ◽  
Komal Kanojia ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Aedes Aegypti ◽  
Zika Virus ◽  
Liquid Chromatography Mass Spectrometry ◽  
Comprehensive Picture ◽  
Infected Cells ◽  
Virus Blocking ◽  
Flavivirus Infection

Wolbachia-infected mosquitoes are refractory to flavivirus infections, but the role of lipids in Wolbachia-mediated virus blocking remains to be elucidated. Here, we use liquid chromatography mass spectrometry to provide a comprehensive picture of the lipidome of Aedes aegypti (Aag2) cells infected with Wolbachia only, either dengue or Zika virus only, and Wolbachia-infected Aag2 cells superinfected with either dengue or Zika virus. This approach identifies a class of lipids, acyl-carnitines, as being down-regulated during Wolbachia infection. Furthermore, treatment with an acyl-carnitine inhibitor assigns a crucial role for acyl-carnitines in the replication of dengue and Zika viruses. In contrast, depletion of acyl-carnitines increases Wolbachia density while addition of commercially available acyl-carnitines impairs Wolbachia production. Finally, we show an increase in flavivirus infection of Wolbachia-infected cells with the addition of acyl-carnitines. This study uncovers a previously unknown role for acyl-carnitines in this tripartite interaction that suggests an important and broad mechanism that underpins Wolbachia-mediated pathogen blocking.

scholarly journals Highly Sensitive and Multiplexed In-Situ Protein Profiling with Cleavable Fluorescent Streptavidin

Cells ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 852 ◽  
Author(s):  
Renjie Liao ◽  
Thai Pham ◽  
Diego Mastroeni ◽  
Paul D. Coleman ◽  
Joshua Labaer ◽  
...  
Keyword(s):  
Optical Resolution ◽  
Protein Detection ◽  
Protein Analysis ◽  
Protein Profiling ◽  
Detection Sensitivity ◽  
Layer By Layer ◽  
Protein Targets ◽  
Highly Sensitive ◽  
Wide Range

The ability to perform highly sensitive and multiplexed in-situ protein analysis is crucial to advance our understanding of normal physiology and disease pathogenesis. To achieve this goal, we here develop an approach using cleavable biotin-conjugated antibodies and cleavable fluorescent streptavidin (CFS). In this approach, protein targets are first recognized by the cleavable biotin-labeled antibodies. Subsequently, CFS is applied to stain the protein targets. Though layer-by-layer signal amplification using cleavable biotin-conjugated orthogonal antibodies and CSF, the protein detection sensitivity can be enhanced at least 10-fold, compared with the current in-situ proteomics methods. After imaging, the fluorophore and the biotin unbound to streptavidin are removed by chemical cleavage. The leftover streptavidin is blocked by biotin. Upon reiterative analysis cycles, a large number of different proteins with a wide range of expression levels can be profiled in individual cells at the optical resolution. Applying this approach, we have demonstrated that multiple proteins are unambiguously detected in the same set of cells, regardless of the protein analysis order. We have also shown that this method can be successfully applied to quantify proteins in formalin-fixed paraffin-embedded (FFPE) tissues.

scholarly journals The inflammasome components NLRP3 and ASC act in concert with IRGM to rearrange the Golgi during viral infections

2020 ◽  
Author(s):  
Coralie F. Daussy ◽  
Sarah C. Monard ◽  
Coralie Guy ◽  
Sara Muñoz-González ◽  
Maxime Chazal ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Zika Virus ◽  
Viral Infections ◽  
Hcv Infection ◽  
Structural Remodeling ◽  
Infected Cells ◽  
Golgi Fragmentation ◽  
Golgi Structure

AbstractHepatitis C virus (HCV) infection triggers Golgi fragmentation through the Golgi-resident protein immunity-related GTPase M (IRGM). Here, we report the role of NLRP3 and ASC, two inflammasome components, in the initial events leading to this fragmentation. We show that ASC resides at the Golgi with IRGM at homeostasis. Upon infection, ASC dissociates from both IRGM and Golgi and associates with HCV-induced NLRP3. NLRP3 silencing inhibits Golgi fragmentation. ASC silencing disrupts the Golgi structure in both control and infected cells and reduces the localization of IRGM at the Golgi. Silencing IRGM cannot totally restore the Golgi structure. These data highlight a role for ASC, upstream of the formation of the inflammasome, in regulating IRGM through its control on the Golgi. A similar mechanism occurs in response to Nigericin or infection with Zika virus (ZIKV). We propose a model for a newly ascribed function of the inflammasome components in Golgi structural remodeling.

Dicarbonyl derived post-translational modifications: chemistry bridging biology and aging-related disease

2020 ◽  
Vol 64 (1) ◽  
pp. 97-110
Author(s):  
Christian Sibbersen ◽  
Mogens Johannsen
Keyword(s):  
Mass Spectrometry ◽  
Future Research ◽  
Amino Acid Residues ◽  
Post Translational Modification ◽  
Dicarbonyl Compounds ◽  
Protein Targets ◽  
Post Translational Modifications ◽  
Reactive Protein ◽  
Glycation End Products ◽  
Direct Mass Spectrometry

Abstract In living systems, nucleophilic amino acid residues are prone to non-enzymatic post-translational modification by electrophiles. α-Dicarbonyl compounds are a special type of electrophiles that can react irreversibly with lysine, arginine, and cysteine residues via complex mechanisms to form post-translational modifications known as advanced glycation end-products (AGEs). Glyoxal, methylglyoxal, and 3-deoxyglucosone are the major endogenous dicarbonyls, with methylglyoxal being the most well-studied. There are several routes that lead to the formation of dicarbonyl compounds, most originating from glucose and glucose metabolism, such as the non-enzymatic decomposition of glycolytic intermediates and fructosyl amines. Although dicarbonyls are removed continuously mainly via the glyoxalase system, several conditions lead to an increase in dicarbonyl concentration and thereby AGE formation. AGEs have been implicated in diabetes and aging-related diseases, and for this reason the elucidation of their structure as well as protein targets is of great interest. Though the dicarbonyls and reactive protein side chains are of relatively simple nature, the structures of the adducts as well as their mechanism of formation are not that trivial. Furthermore, detection of sites of modification can be demanding and current best practices rely on either direct mass spectrometry or various methods of enrichment based on antibodies or click chemistry followed by mass spectrometry. Future research into the structure of these adducts and protein targets of dicarbonyl compounds may improve the understanding of how the mechanisms of diabetes and aging-related physiological damage occur.

A Novel Simultaneous Estimation of Sofosbuvir and Velpatasvir in Human Plasma by Liquid Chromatography Tandem-Mass Spectrometry after Protein Precipitation Method

2019 ◽  
Vol 15 (7) ◽  
pp. 710-715
Author(s):  
S.T. Narenderan ◽  
Basuvan Babu ◽  
T. Gokul ◽  
Subramania Nainar Meyyanathan
Keyword(s):  
Mass Spectrometry ◽  
Tandem Mass Spectrometry ◽  
Human Plasma ◽  
Multiple Reaction Monitoring ◽  
Simultaneous Estimation ◽  
Pharmacokinetic Studies ◽  
Tandem Mass ◽  
Mass Spectrometric Method ◽  
Highly Sensitive ◽  
Tandem Mass Spectrometric

Objective: The aim of the present work is to achieve a novel highly sensitive chromatographic method for the simultaneous determination of hepatitis C agents, sofosbuvir and velpatasvir from human plasma using ritonavir as an internal standard. Methods: Chromatographic separation was achieved using Hypersil C18 column (50mm x 4.6mm, 3μm) with an isocratic elution mode using the mobile phase composition 10 mM ammonium formate buffer (pH 5.0): acetonitrile (20:80 v/v) pumped at a flow rate of 0.5 ml/min. The detection was carried out by tandem mass spectrometry using Multiple Reaction Monitoring (MRM) positive Electrospray Ionization (ESI) with proton adducts at m/z 530.10 > 243.10, 883.40 > 114.0 and 721.25 > 197.0. Results: The method validated as per USFDA guidelines with respect to linearity, accuracy, and precision was found to be acceptable over the concentration range of 0.2–2000 ng/ml and 5-2000 ng/ml for sofosbuvir and velpatasvir respectively and the method was found to be highly sensitive and selective. Conclusion: The developed tandem mass spectrometric method is robust and can be applied for the monitoring of plasma levels of the analyzed drug in preclinical and clinical pharmacokinetic studies.

Sign in / Sign up

Export Citation Format

Share Document