scholarly journals Differential mitochondrial proteomic analysis of A549 cells infected with avian influenza virus subtypes H5 and H9

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Yuting Yang ◽  
Yun Zhang ◽  
Changcheng Yang ◽  
Fang Fang ◽  
Ying Wang ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Avian Influenza ◽  
Differential Expression ◽  
Gel Electrophoresis ◽  
A549 Cells ◽  
Infected Group ◽  
Differentially Expressed ◽  
Mitochondrial Proteins ◽  
Differentially Expressed Proteins ◽  
Pathogenic Avian Influenza

Abstract Background Both the highly pathogenic avian influenza (HPAI) H5N1 and low pathogenic avian influenza (LPAI) H9N2 viruses have been reported to cross species barriers to infect humans. H5N1 viruses can cause severe damage and are associated with a high mortality rate, but H9N2 viruses do not cause such outcomes. Our purpose was to use proteomics technology to study the differential expression of mitochondrial-related proteins related to H5N1 and H9N2 virus infections. Methods According to the determined viral infection titer, A549 cells were infected with 1 multiplicity of infection virus, and the mitochondria were extracted after 24 h of incubation. The protein from lysed mitochondria was analyzed by the BCA method to determine the protein concentration, as well as SDS-PAGE (preliminary analysis), two-dimensional gel electrophoresis, and mass spectrometry. Differential protein spots were selected, and Western blotting was performed to verify the proteomics results. The identified proteins were subjected to GO analysis for subcellular localization, KEGG analysis for functional classification and signaling pathways assessment, and STRING analysis for functional protein association network construction. Results In the 2-D gel electrophoresis analysis, 227 protein spots were detected in the H5N1-infected group, and 169 protein spots were detected in the H9N2-infected group. Protein spots were further subjected to mass spectrometry identification and removal of redundancy, and 32 differentially expressed proteins were identified. Compared with the H9N2 group, the H5N1-infected group had 16 upregulated mitochondrial proteins and 16 downregulated proteins. The differential expression of 70-kDa heat shock protein analogs, short-chain enoyl-CoA hydratase, malate dehydrogenase, and ATP synthase was verified by Western blot, and the results were consistent with the proteomics findings. Functional analysis indicated that these differentially expressed proteins were primarily involved in apoptosis and metabolism. Conclusions Compared with their expression in the H9N2 group, the differential expression of eight mitochondrial proteins in the H5N1 group led to host T cell activation, antigen presentation, stress response, ATP synthesis and cell apoptosis reduction, leading to higher pathogenicity of H5N1 than H9N2.

scholarly journals Differential mitochondrial proteomic analysis of A549 cells infected with avian influenza virus subtypes H5 and H9 

2020 ◽  
Author(s):  
Yuting Yang ◽  
Yun Zhang ◽  
Changcheng Yang ◽  
Fang Fang ◽  
Ying Wang ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Avian Influenza ◽  
Differential Expression ◽  
Gel Electrophoresis ◽  
A549 Cells ◽  
Infected Group ◽  
Differentially Expressed ◽  
Mitochondrial Proteins ◽  
Differentially Expressed Proteins ◽  
Pathogenic Avian Influenza

Abstract Background: Both the highly pathogenic avian influenza (HPAI) H5N1 and low pathogenic avian influenza (LPAI) H9N2 viruses have been reported to cross species barriers to infect humans. H5N1 viruses can cause severe damage and a high mortality rate, but H9N2 viruses cannot. Our purpose was to use proteomics technology to study the differential expression of mitochondrial-related proteins caused by H5N1 and H9N2 virus infections. Methods: According to the determined viral infection titer, A549 cells were infected with 1 MOI (multiplicity of infection) virus, and the mitochondria were extracted after 24 h of incubation. The protein from lysed mitochondria was analyzed by the BCA method to determine the protein concentration, SDS-PAGE preliminary analysis, two-dimensional gel electrophoresis, and mass spectrometry. Differential protein spots were selected,, and Western blotting was performed to verify the proteomics results. The identified proteins were subjected to GO analysis for subcellular localization, KEGG analysis for functional classification and signaling pathways assessment, STRING analysis for functional protein association network construction. Results: In the 2-D gel electrophoresis analysis, 227 protein spots were detected in the H5N1-infected group, and 169 protein spots were detected in the H9N2-infected group. Protein spots further mass spectrometry identification and removal of redundancy, 32 differentially expressed proteins were identified. Compared with the H9N2 group, the H5N1-infected group had 16 upregulated mitochondrial proteins and 16 downregulated proteins. The differential expression of 70-kDa heat shock protein analogs, short-chain enoyl-CoA hydratase, malate dehydrogenase, and ATP synthase was verified by Western blot, and the results were consistent with the proteomics findings. Functional analysis indicated that these differentially expressed proteins were involved mainly in apoptosis and metabolism. Conclusions: Compared with their expression in the H9N2 group, the differential expression of eight mitochondrial proteins in H5N1 group led to host T cell activation, antigen presentation, stress response, ATP synthesis and cell apoptosis reduction, leading to higher pathogenicity of H5N1 than H9N2.

scholarly journals Differential mitochondrial proteomic analysis of A549 cells infected with avian influenza virus subtypes H5 and H9

2021 ◽  
Author(s):  
Yuting Yang ◽  
Yun Zhang ◽  
Changcheng Yang ◽  
Fang Fang ◽  
Ying Wang ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Avian Influenza ◽  
Differential Expression ◽  
Gel Electrophoresis ◽  
A549 Cells ◽  
Infected Group ◽  
Differentially Expressed ◽  
Mitochondrial Proteins ◽  
Differentially Expressed Proteins ◽  
Pathogenic Avian Influenza

Abstract Background: Both the highly pathogenic avian influenza (HPAI) H5N1 and low pathogenic avian influenza (LPAI) H9N2 viruses have been reported to cross species barriers to infect humans. H5N1 viruses can cause severe damage and are associated with a high mortality rate, but H9N2 viruses do not cause such outcomes. Our purpose was to use proteomics technology to study the differential expression of mitochondrial-related proteins related to H5N1 and H9N2 virus infections.Methods: According to the determined viral infection titer, A549 cells were infected with 1 multiplicity of infection (MOI) virus, and the mitochondria were extracted after 24 h of incubation. The protein from lysed mitochondria was analyzed by the BCA method to determine the protein concentration, as well as SDS-PAGE (preliminary analysis), two-dimensional gel electrophoresis, and mass spectrometry. Differential protein spots were selected, and Western blotting was performed to verify the proteomics results. The identified proteins were subjected to GO analysis for subcellular localization, KEGG analysis for functional classification and signaling pathways assessment, and STRING analysis for functional protein association network construction.Results: In the 2-D gel electrophoresis analysis, 227 protein spots were detected in the H5N1-infected group, and 169 protein spots were detected in the H9N2-infected group. Protein spots were further subjected to mass spectrometry identification and removal of redundancy, and 32 differentially expressed proteins were identified. Compared with the H9N2 group, the H5N1-infected group had 16 upregulated mitochondrial proteins and 16 downregulated proteins. The differential expression of 70-kDa heat shock protein analogs, short-chain enoyl-CoA hydratase, malate dehydrogenase, and ATP synthase was verified by Western blot, and the results were consistent with the proteomics findings. Functional analysis indicated that these differentially expressed proteins were primarily involved in apoptosis and metabolism.Conclusions: Compared with their expression in the H9N2 group, the differential expression of eight mitochondrial proteins in the H5N1 group led to host T cell activation, antigen presentation, stress response, ATP synthesis and cell apoptosis reduction, leading to higher pathogenicity of H5N1 than H9N2.

scholarly journals Differential mitochondrial proteomic analysis of A549 cells infected with avian influenza virus subtypes H5 and H9

2020 ◽  
Author(s):  
Yuting Yang ◽  
Yun Zhang ◽  
Changcheng Yang ◽  
Fang Fang ◽  
Ying Wang ◽  
...  
Keyword(s):  
Avian Influenza ◽  
Western Blot ◽  
Differential Expression ◽  
Gel Electrophoresis ◽  
A549 Cells ◽  
Infected Group ◽  
Differentially Expressed ◽  
Mitochondrial Proteins ◽  
Differentially Expressed Proteins ◽  
Pathogenic Avian Influenza

Abstract Background In the past 20–30 years, both the highly pathogenic avian influenza (HPAI) H5N1 and low pathogenic avian influenza (LPAI) H9N2 viruses have been reported to cross species barriers to infect humans. However, H5N1 viruses could cause severe damage and a high death rate, but H9N2 viruses could not. In this study, we use H9N2 virus infection as a control to investigate the differential expression of mitochondrial-related proteins caused by H5N1 and H9N2 virus infections in A549 cells. Methods According to the determined viral infection titer, A549 cells were infected with 1 MOI (multiplicity of infection) virus, and the mitochondria were extracted after 24 hours of incubation. The lysed mitochondrial protein was analyzed by BCA method for protein concentration, SDS-PAGE preliminary analysis, two-dimensional gel electrophoresis, and mass spectrometry. Select different protein spots, perform Western Blot to verify the proteomics results, and then perform GO and KEGG analysis. Results In the 2-D gel electrophoresis analysis, 227 protein spots were detected in the H5N1-infected group, and 169 protein spots were detected in the H9N2-infected group. After further MS identification and removal of redundancy, 32 differentially expressed proteins were identified. Compared with the H9N2 group, the H5N1-infected group had 16 upregulated mitochondrial proteins and 16 downregulated proteins. The 70 kDa heat shock protein analogs, short-chain enoyl-CoA hydratase, malate dehydrogenase, and ATP synthase were verified by Western Blot, and the results were consistent with proteomics. Conclusions Functional analysis indicated that these differentially expressed proteins were involved mainly in apoptosis, metabolism and the cytoskeleton. The differential expression of eight mitochondrial proteins in H5N1-infected cells resulted in decreased T cell activation, decreased antigen presentation and stress response, reduced ATP synthesis, and decreased induction of apoptosis, resulting in the higher pathogenicity of H5N1 virus than H9N2 virus.These finding may provide a basis for analyzing the pathogenesis of influenza viruses with different virulence levels, identifying anti-influenza host targets and developing new influenza vaccines.

scholarly journals Differential mitochondrial proteomic analysis of A549 cells infected with avian influenza virus subtypes H5 and H9

2020 ◽  
Author(s):  
Yuting Yang ◽  
Yun Zhang ◽  
Changcheng Yang ◽  
Fang Fang ◽  
Ying Wang ◽  
...  
Keyword(s):  
Avian Influenza ◽  
Western Blot ◽  
Differential Expression ◽  
Gel Electrophoresis ◽  
A549 Cells ◽  
Infected Group ◽  
Differentially Expressed ◽  
Mitochondrial Proteins ◽  
Differentially Expressed Proteins ◽  
Pathogenic Avian Influenza

Abstract Background: Both the highly pathogenic avian influenza (HPAI) H5N1 and low pathogenic avian influenza (LPAI) H9N2 viruses have been reported to cross species barriers to infect humans. However, H5N1 viruses can cause severe damage and a high mortality rate, but H9N2 viruses can not. Our purpose was to use proteomics technology to study the differential expression of mitochondrial-related proteins caused by H5N1 and H9N2 virus infections.Methods: According to the determined viral infection titer, A549 cells were infected with 1 MOI (multiplicity of infection) virus, and the mitochondria were extracted after 24 hours of incubation. The lysed mitochondrial protein was analyzed by BCA method for protein concentration, SDS-PAGE preliminary analysis, two-dimensional gel electrophoresis, and mass spectrometry. Select different protein spots, perform Western Blot to verify the proteomics results. The identified proteins use GO analysis for subcellular localization, KEGG analysis for functional classification and signal pathways, STRING analysis for functional protein association networks.Results: In the 2-D gel electrophoresis analysis, 227 protein spots were detected in the H5N1-infected group, and 169 protein spots were detected in the H9N2-infected group. After further MS identification and removal of redundancy, 32 differentially expressed proteins were identified. Compared with the H9N2 group, the H5N1-infected group had 16 upregulated mitochondrial proteins and 16 downregulated proteins. The 70 kDa heat shock protein analogs, short-chain enoyl-CoA hydratase, malate dehydrogenase, and ATP synthase were verified by Western Blot, and the results were consistent with proteomics. Functional analysis indicated that these differentially expressed proteins were involved mainly in apoptosis, metabolism.Conclusions: Compared with H9N2 group, the differential expression of eight mitochondrial proteins in H5N1 group led to T cell activation, antigen presentation, stress response, ATP synthesis and cell apoptosis reduction, leading to higher pathogenicity of H5N1 than H9N2 virus.

scholarly journals In-Depth Proteomic Characterization of Lineage-Biased Hematopoietic Progenitor Cells in the Fetus and Adult

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3703-3703
Author(s):  
Maria Jassinskaja ◽  
Kristýna Pimková ◽  
Emil Johansson ◽  
Ewa Sitnicka Quinn ◽  
Jenny Hansson
Keyword(s):  
Mass Spectrometry ◽  
Progenitor Cells ◽  
Differential Expression ◽  
Normal Development ◽  
Differentially Expressed ◽  
Differential Sensitivity ◽  
Hematopoietic Progenitor Cells ◽  
Differentially Expressed Proteins ◽  
Hematopoietic Progenitor ◽  
The Difference

The process of hematopoiesis is subject to extensive ontogenic remodeling that is accompanied by alterations in cellular fate both during normal development and upon malignant transformation. Although the functional differences between fetal and adult hematopoiesis are well established, the responsible molecular mechanisms have long remained largely unexplored at the proteomic level. We hypothesize that an intrinsically programmed proteomic switch in hematopoietic stem and progenitor cells (HSPCs) during ontogeny regulates the outcome of hematopoiesis both during normal development and upon leukemia initiation, and that the proteomic makeup of the leukemia-initiating cell has an instructive role in determining the outcome of the resulting cancer. In our latest work, we utilized quantitative mass spectrometry-based proteomics to characterize and compare the proteomic makeup of fetal and adult Lin- Sca-1+ cKit+ (LSK) HSPCs (Jassinskaja et al., 2017, Cell Reports), representing all of the earliest stem and progenitors in fetal and adult hematopoiesis. We identified differences in several important cellular processes not previously described to play a role in hematopoiesis, highlighting the need for applying proteomic-centric approaches in the field. In order to further increase our understanding of normal and malignant hematopoiesis during ontogeny, we are now continuing this work by focusing on more stringently defined populations of lineage-biased hematopoietic progenitor cells (HPCs). Here, we have utilized encapsulated methods for preparation of microscale samples in combination with state-of-the-art mass spectrometry to gain deep coverage of the proteome of 100,000 fetal (E14.5) and adult lymphoid-primed multipotent progenitors (LMPPs), common lymphoid progenitors (CLPs) and granulocyte-macrophage progenitors (GMPs). Our analysis resulted in the identification and quantification of 4189 proteins, with over 200 proteins per cell type displaying differential expression between the fetus and the adult. Importantly, the differentially expressed proteins were enriched for a broad variety of biological processes. Similar to our previous findings in HSPCs, for all three cell types, proteins higher expressed in the fetus showed a strong enrichment for cell cycle- and translation-related processes, whereas those higher expressed in the adult were enriched for processes related to immune response and redox homeostasis. Our preliminary analysis of hematopoietic cell subset signatures associated with the differentially expressed proteins suggests a stronger lymphoid bias in fetal compared to adult LMPPs as well as CLPs. Surprisingly, the proteomic signature of fetal GMPs suggests a retained megakaryocyte-erythroid potential, which is corroborated by a significantly higher expression of megakaryocyte progenitor marker CD41 on the fetal cells. Upon analyzing expression of transcription factors (TFs) in fetal and adult HPCs, we could confirm differential expression of TFs known to have ontogeny-specific roles in hematopoiesis (e.g. Arid3a and Etv6). Importantly, we also identified several differentially expressed TFs that could represent novel regulators of fetal- and adult-specific features of hematopoiesis, such as Irf8, Btf3, Mndal and Pura. Furthermore, the difference in expression of Irf8 observed here could indicate a previously unknown ontogenic switch in the balance between neutrophil and monocyte production from myeloid-competent progenitors. Lastly, our data shows strong indications of a differential sensitivity towards Rho kinase inhibition between the fetal and the adult HPCs. Collectively, our work represents a significant advancement in the understanding of the molecular programs that govern ontogenic differences in hematopoiesis and provides a solid foundation for future investigation of which factors are responsible for the difference in susceptibility and outcome of different leukemias in infants and in adults. Disclosures No relevant conflicts of interest to declare.

Identification of differentially expressed proteins in retinoblastoma tumors using mass spectrometry-based comparative proteomic approach

2017 ◽  
Vol 159 ◽  
pp. 77-91 ◽  
Author(s):  
Jasmine Naru ◽  
Ritu Aggarwal ◽  
Ashok Kumar Mohanty ◽  
Usha Singh ◽  
Deepak Bansal ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Differentially Expressed ◽  
Differentially Expressed Proteins ◽  
Proteomic Approach

scholarly journals Bayesian identification of protein differential expression in multi-group isobaric labelled mass spectrometry data

2014 ◽  
Vol 13 (5) ◽  
Author(s):  
Howsun Jow ◽  
Richard J. Boys ◽  
Darren J. Wilkinson
Keyword(s):  
Mass Spectrometry ◽  
Simulated Data ◽  
Differentially Expressed ◽  
Mass Spectrometry Data ◽  
Control Group ◽  
Differentially Expressed Proteins ◽  
Statistical Methodology ◽  
Proteomic Data ◽  
Bayesian Identification ◽  
Multiple Groups

AbstractIn this paper we develop a Bayesian statistical inference approach to the unified analysis of isobaric labelled MS/MS proteomic data across multiple experiments. An explicit probabilistic model of the log-intensity of the isobaric labels’ reporter ions across multiple pre-defined groups and experiments is developed. This is then used to develop a full Bayesian statistical methodology for the identification of differentially expressed proteins, with respect to a control group, across multiple groups and experiments. This methodology is implemented and then evaluated on simulated data and on two model experimental datasets (for which the differentially expressed proteins are known) that use a TMT labelling protocol.

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