Project

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Investigations of protein expression during anti-viral response mechanisms based on innovative mass spectrometric and bioinformatic workflows

Applicant Hopfgartner Gérard
Number 136282
Funding scheme Sinergia
Research institution Laboratoire de spectrométrie de masse du vivant Université de Genève
Institution of higher education University of Geneva - GE
Main discipline Molecular Biology
Start/End 01.09.2011 - 31.08.2015
Approved amount 1'200'000.00
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All Disciplines (2)

Discipline
Molecular Biology
Information Technology

Keywords (8)

HIV-1; innate immunity; analytical proteomics; mass spectrometry; bioinformatics; protein-protein interactions; spectral library search; interferon-dependent pathway

Lay Summary (English)

Lead
Lay summary

Vaccination approaches capable of controlling viruses such as poliovirus and smallpox have not worked for HIV-1, and successful HIV-1 vaccination will more likely arise from basic research into the human immune response to retroviral infection. The effectiveness of immune defence mechanisms targeting microbial pathogens is largely determined by signals initiated by pattern recognition receptors (PRR) expressed in dendritic cells (DCs), which are the central coordinators of innate and acquired immune responses. Recent observations showed on the one hand that the HIV-1 restriction factor TRIM5 is a retroviral-specific PRR and on the other hand that the lentiviral accessory protein Vpx, is capable of effectively abrogating the capacity of DCs to resist HIV-1 infection. This latest view offers a unique handle on the molecular mechanisms underlying the capacity of DCs to function as key decision making sites along the pathway leading from initial challenge to full blown AIDS. Little is known about how DCs detect retroviruses and help mount an effective protective immunity against HIV-1. The aim of this project based on the complementary expertise on HIV-1 biology, analytical proteomics and bioinformatics is to utilize a systems biology approach to unravel the mechanisms by which DCs determine disease outcome in the first few hours post-infection and relies on an innovative integrated analytical proteomics/bioinformatics platform.

 

Direct link to Lay Summary Last update: 21.02.2013

Responsible applicant and co-applicants

Employees

Publications

Publication
Optimization by infusion of multiple reaction monitoring transitions for sensitive quantification of peptides by liquid chromatography/mass spectrometryOptimizing MRM transitions by FIA
Alghanem Bandar, Nikitin Frédéric, Stricker Thomas, Duchoslav Eva, Luban Jeremy, Strambio-De-Castillia Caterina, Muller Markus, Lisacek Frédérique, Varesio Emmanuel, Hopfgartner Gérard (2017), Optimization by infusion of multiple reaction monitoring transitions for sensitive quantification of peptides by liquid chromatography/mass spectrometryOptimizing MRM transitions by FIA, in Rapid Communications in Mass Spectrometry, 31(9), 753-761.
Processing Strategies and Software Solutions for Data-Independent Acquisition in Mass Spectrometry
Varesio Emmanuel, Luban Jeremy, Strambio-De-Castillia Caterina, Hopfgartner Gérard, Müller. Markus, Lisacek Frédérique (2015), Processing Strategies and Software Solutions for Data-Independent Acquisition in Mass Spectrometry, in Proteomics, 15(5-6), 964.
Optimization of human dendritic cell sample preparation for mass spectrometry-based proteomic studies
Zhang Ying, Bottinelli Dario, Lisacek Frédérique, Lisacek Frédérique, Luban Jeremy, Strambio-De-Castillia Caterina, Varesio Emmanuel, Hopfgartner Gérard (2015), Optimization of human dendritic cell sample preparation for mass spectrometry-based proteomic studies, in Analytical Biochemistry, 484, 40-50.
Ranking fragment ions based on outlier detection for improved label-free quantification in data-independent acquisition LC-MS/MS
Bilbao Aivett, Bilbao Aivett, Zhang Ying, Varesio Emmanuel, Luban Jeremy, Strambio-De-Castillia Caterina, Lisacek Frédérique, Lisacek Frédérique, Hopfgartner Gérard (2014), Ranking fragment ions based on outlier detection for improved label-free quantification in data-independent acquisition LC-MS/MS, in Journal of Proteome Research, 14(11), 4581-4593.
The Use of Variable Q1 Isolation Windows Improves Selectivity in LC-SWATH-MS Acquisition
Zhang Ying, Bilbao Aivett, Bilbao Aivett, Bruderer Tobias, Luban Jeremy, Strambio-De-Castillia Caterina, Lisacek Frédérique, Lisacek Frédérique, Hopfgartner Gérard, Varesio Emmanuel (2014), The Use of Variable Q1 Isolation Windows Improves Selectivity in LC-SWATH-MS Acquisition, in Journal of Proteome Research, 14(10), 4359-4371.

Collaboration

Group / person Country
Types of collaboration
SyBIT of SystemsX.ch Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Research Infrastructure

Scientific events

Active participation

Title Type of contribution Title of article or contribution Date Place Persons involved
IMSC2014 (20th International Mass Spectrometry Conference) Poster Towards Demulplexing of SWATH Spectra for Peptide Identification: Similarity Analysis of Fragment Ion Elution Profiles 24.08.2014 Geneva, Switzerland Bilbao Aivett;
IMSC2014 (20th International Mass Spectrometry Conference) Talk given at a conference Quantitative proteomic analysis by variable SWATH acquisition of differentially expressed proteins in monocyte-derived dendritic cells 24.08.2014 Geneva, Switzerland Zhang Ying;
61th ASMS Conference on Mass Spectrometry and Allied Topics Poster Gel-free sample preparation for LCMS/MS analysis based moDC proteomics studies 09.06.2013 Minneapolis, MN, United States of America Zhang Ying;
61th ASMS Conference on Mass Spectrometry and Allied Topics Poster Combining demultiplexing and label-free quantification analyses for high-resolution data-independent acquisition LC-MS/MS 09.06.2013 Minneapolis, MN, United States of America Bilbao Aivett;
IMSC2012 (19th International Mass Spectrometry Conference) Poster Comparing and validating protein expression in multiple experimental conditions of antiviral response using clustering techniques on label-free LC-MS/MS data 15.09.2012 Kyoto, Japan Bilbao Aivett;
ECCB’12 (11th European Conference on Computational Biology) Poster Comparing and validating protein expression in multiple experimental conditions of antiviral immune response using clustering techniques on label-free LC-MS/MS data. 09.09.2012 Basel, Switzerland Bilbao Aivett;


Abstract

Vaccination approaches capable of controlling viruses such as poliovirus and smallpox have not worked for HIV-1, and successful HIV-1 vaccination will be made more likely by basic research into the human immune response to retroviral infection. The effectiveness of immune defence mechanisms targeting microbial pathogens is largely determined by signals initiated by pattern recognition receptors (PRR) expressed in dendritic cells (DCs), which are the central coordinators of innate and acquired immune responses. Little is known about how DCs detect retroviruses and help mount an effective protective immunity against HIV-1. Recent observations from the Luban laboratory showing on the one hand that the HIV-1 restriction factor TRIM5 is a retroviral-specific PRR and on the other hand demonstrating that the lentiviral accessory protein Vpx, is capable of effectively abrogating the capacity of DCs to resist HIV-1 infection, offer a unique handle on the molecular mechanisms underlying the capacity of DCs to function as key decision making sites along the pathway leading from initial challenge to full blown AIDS. The aim of this project based on the complementary expertise on HIV-1 biology (Luban lab, group A), analytical proteomics (Hopfgartner lab, group B) and bioinformatics (Lisacek lab, group C) is to utilize a systems biology approach to unravel the mechanisms by which DCs determine disease outcome in the first few hours post-infection and relies on an innovative integrated analytical proteomics/bioinformatics platform. Large quantities of primary human DCs will be generated from peripheral blood monocytes of individual healthy donors (monocyte-derived DCs or moDCs). Exploiting experimental treatments developed in the lab of Group A (J. Luban), these cells will either elaborate a potent, innate, antiviral state that suppresses HIV-1 infection to undetectable levels or they will be rendered completely permissive for HIV-1 infection, even in the face of exogenous type 1 interferon (IFN). Group A will generate these moDCs; confirm the anti-viral or viral permissive states in samples subjected to proteomics analysis; will compare protein expression results emerging from the proteomics study with mRNA levels of expression; and will perform molecular and functional studies to characterize newly discovered pathways. In parallel to this, qualitative and quantitative (QUAL/QUAN) proteomic acquisition workflows will be developed in Group B (G. Hopfgartner) on the basis of novel fast MS acquisition (below 100 msec duty cycle) with resolution power above 30’000 and nano-ultra high performance liquid chromatography combined with tandem mass spectrometry (UHPLC - MS/MS) instrumentation using innovative unsupervised and supervised MS or MS/MS data acquisition and processing concepts. In addition, in order to generate relevant biological data of the required high-caliber, implementation of quality-control protocols from method development to data processing will be carried out in parallel and in continual synergy with Group C (F. Lisacek). In particular, Group C will develop iterative and integrated analytical proteomics/bioinformatics processes with a threefold aim in accordance with the three specific aims of the project: First, they will devise and perform the MS-based data analysis to dig deep into the mechanisms of the proteomic regulation of IFN-induced anti-viral states and of viral escape pathways. To this end, they will analyze the data coming from the three types of MS experiments in an iterative, result-driven way by focusing the MS analysis in successive replicates on not yet identified proteins. They will create spectrum libraries of all confident peptide identifications annotated with the quantitative profiles. Second, Group C will use these libraries to plan subsequent selected reaction monitoring (SRM) measurements. Third, they will support the interpretation of the quantitative results by means of dedicated pathway visualization and protein annotations methods.
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