Periodic Reporting for period 1 - ARIADNE (Redefining mass spectrometry – a breakthrough platform for real-time noninvasive breath analysis with single ion detection of intact viruses and bacteria and post-analysis molecular characterization)
Reporting period: 2021-06-01 to 2022-05-31
The objective of the ARIADNE project is to develop a breakthrough analytical platform based on mass spectrometry (MS) for detecting intact virus particles directly from breath. This is the main goal, upon many others listed as deliverables, which require an entire new range of highly disruptive technologies to be realized and applied successfully. These technologies relate to all the design aspects of the ARIADNE MS platform and include a novel sampling interface and complex aerosol processing methods, a new ionization source for supercharging particles and droplets, gas dynamically optimized transfer optics, advanced top down analytical workflows in the ultra-high mass range performed in the Omnitrap platform, and mass analyzer designs with integrated novel detection schemes. Successful implementation of the ideas currently being realized will have a significant impact in all aspects of instrumentation design and applications development based on MS.
WP1 - FIT identified the need to clean, concentrate and size-separate viral particles and the architecture was redefined, comprising: an atmospheric pressure aerosol concentrator, a virus-surfactant separation system and a high-resolution DMA size classifier. D1.3 now comprises of the aerosol concentrator and optionally the virus-surfactant separator, while D1.1 includes the DMA. FIT performed theoretical analyses of the concentrator. A first mechanical design was completed and a first unit was built and tested. A concentration factor of 9 is demonstrated, validating the concept of the architecture. A new design will be developed. A patent protecting this new architecture will be filled. FIT invented a new method to separate viruses and surfactants by inducing coulombic fissions. This requires supercharging the aerosols to a very high charge state. Theoretical models to understand the factors limiting ultra-high charging, and the design criteria that enhance aerosol super-charging were developed. A supercharging system that is embedded into a virus-surfactant separation system was designed. The mechanical components have been assembled, and the electronic controls completed. The system is being coupled to a high-resolution DMA. FIT will also produce a 'lung simulator'.
WP2 - FAST has designed and constructed a new PTR/cold plasma ionization source coupled to an oTOF MS for testing the idea of charging particles at low pressure. Although ionization with extremely high sensitivity was accomplished for small volatile species (<20ppt) based on charge-transfer and proton transfer ionization, interactions between the plasma and proteins produced by ESI were not observed. Results indicate that generating particles in higher charge states using this method is not a viable approach. A new alternative idea is thus being explored, combining traditional diffusion charging and FAIMS technology into a single device to increase the kinetic energy of protonated water molecules, and enhance the proton transfer kinetics in reactions with droplets. Advanced CFD modeling and several iterations to the mechanical design have been performed. Theoretical calculations are provided by partner FIT to evaluate the viability of this approach. Developing alternative approaches to supercharge aerosol particles is pivotal to the success of the ARIADNE project, since it will enable efficient transfer and detection by eTOF MS and/or CDMS. FAST has finalized the core mechanical design of the ARIADNE platform. Orders are currently underway and an approximate date for assembling core technologies of ARIADNE is June 2023.
WP3 - CNRS-Lyon designed, constructed and tested the new FT-CDMS using MDa ions. SIMION simulations were performed to optimize this unique geometry that would allow working with macroions with masses ranging across 6 orders of magnitude, from hudreds of kDa to hundreds of GDa. The new FT-CDMS was then coupled to an ESI source and operated in a stand-alone mode. New preamplifiers working at low temperature in vacuum and directly connected to the CDMS devices were implemented. Significant improvement in the limit of charge detection has been attained. The signal will be detected with an array of 8 identical charge detection devices (CDDs). Alternative approaches that yield the highest charge and frequency accuracy wil be explored by correlating the output signal to an expected output pattern. This task will be performed in collaboration with partner SPS based on advancements offered by the high performance data acquisition system architecture. Discussions with partner FAST to simulate ion injection in the FT-CDMS coupled to the ARIADNE platform are underway. SIMION modeling is employed to address the kinetic energy window of the ions for optimal trapping. SPS has developed the first prototype of a high-performance data acquisition system. The developed prototype was delivered to the CNRS partner and interfaced to the CDMS prototype.
WP4 - The delivery of the Omnitrap to partner USieg to upgrade the QE UHMR is now scheduled for Q2 2023. The delivery is delayed due to unforeseen problems during the final stages of the production (outgassing of materials - high background signals in ExD, stray magnetic fields defocusing electrons). Additional delays in the supply chain of electronics components have also interrupted the productization process. Two successful installations have already been performed at Institut Pasteur in Paris and at the School of Medicine, Boston University in Q2 2022. The top down capabilities of the Omnitrap systems currently being utilized in Athens and in Paris under the TopSpec FET project show exceptional performance compared to all other high-end platforms available on the market. In a preparation for the top-down analysis of viruses in the Omnitrap hyphenated with a Q Exactive UHMR instrument, SPS has performed simulations of data processing and data analysis for viruses above 1 MDa. The simulations were performed with the proprietary software tool, FTMS Simulator.
WP5 - KI decided to try and detect viruses or bacteria in single cells that we have analyzed so far. Briefly, the MS/MS datasets were first searched in human database, after which all hits were removed and the dataset was searched again, this time in the database containing viral and bacterial proteins. No non-human hits were found. Further work in single cell proteomics focused on interrogating human Jlat 10.6 cells infected with a modified Human Immunodeficiency Virus (HIV). In this experiment, we have used TMT-10plex labeling with a carrier proteome including 200 cells. With MS/MS we identified 3 peptides of the HIV gag-polyprotein. This result demonstrates that we can detect viral load in a single human cell. To enhance sensitivity of the TMT-based protein quantitation from single cells and reduce the number of missing values, partner SPS has advanced specifically the least-squares fitting (LSF) processing of the time-domain transients for detection of TMT reporter ions and TMT complementary (TMTc) reported ions.
WP2 - FAST has designed and constructed a new PTR/cold plasma ionization source coupled to an oTOF MS for testing the idea of charging particles at low pressure. Although ionization with extremely high sensitivity was accomplished for small volatile species (<20ppt) based on charge-transfer and proton transfer ionization, interactions between the plasma and proteins produced by ESI were not observed. Results indicate that generating particles in higher charge states using this method is not a viable approach. A new alternative idea is thus being explored, combining traditional diffusion charging and FAIMS technology into a single device to increase the kinetic energy of protonated water molecules, and enhance the proton transfer kinetics in reactions with droplets. Advanced CFD modeling and several iterations to the mechanical design have been performed. Theoretical calculations are provided by partner FIT to evaluate the viability of this approach. Developing alternative approaches to supercharge aerosol particles is pivotal to the success of the ARIADNE project, since it will enable efficient transfer and detection by eTOF MS and/or CDMS. FAST has finalized the core mechanical design of the ARIADNE platform. Orders are currently underway and an approximate date for assembling core technologies of ARIADNE is June 2023.
WP3 - CNRS-Lyon designed, constructed and tested the new FT-CDMS using MDa ions. SIMION simulations were performed to optimize this unique geometry that would allow working with macroions with masses ranging across 6 orders of magnitude, from hudreds of kDa to hundreds of GDa. The new FT-CDMS was then coupled to an ESI source and operated in a stand-alone mode. New preamplifiers working at low temperature in vacuum and directly connected to the CDMS devices were implemented. Significant improvement in the limit of charge detection has been attained. The signal will be detected with an array of 8 identical charge detection devices (CDDs). Alternative approaches that yield the highest charge and frequency accuracy wil be explored by correlating the output signal to an expected output pattern. This task will be performed in collaboration with partner SPS based on advancements offered by the high performance data acquisition system architecture. Discussions with partner FAST to simulate ion injection in the FT-CDMS coupled to the ARIADNE platform are underway. SIMION modeling is employed to address the kinetic energy window of the ions for optimal trapping. SPS has developed the first prototype of a high-performance data acquisition system. The developed prototype was delivered to the CNRS partner and interfaced to the CDMS prototype.
WP4 - The delivery of the Omnitrap to partner USieg to upgrade the QE UHMR is now scheduled for Q2 2023. The delivery is delayed due to unforeseen problems during the final stages of the production (outgassing of materials - high background signals in ExD, stray magnetic fields defocusing electrons). Additional delays in the supply chain of electronics components have also interrupted the productization process. Two successful installations have already been performed at Institut Pasteur in Paris and at the School of Medicine, Boston University in Q2 2022. The top down capabilities of the Omnitrap systems currently being utilized in Athens and in Paris under the TopSpec FET project show exceptional performance compared to all other high-end platforms available on the market. In a preparation for the top-down analysis of viruses in the Omnitrap hyphenated with a Q Exactive UHMR instrument, SPS has performed simulations of data processing and data analysis for viruses above 1 MDa. The simulations were performed with the proprietary software tool, FTMS Simulator.
WP5 - KI decided to try and detect viruses or bacteria in single cells that we have analyzed so far. Briefly, the MS/MS datasets were first searched in human database, after which all hits were removed and the dataset was searched again, this time in the database containing viral and bacterial proteins. No non-human hits were found. Further work in single cell proteomics focused on interrogating human Jlat 10.6 cells infected with a modified Human Immunodeficiency Virus (HIV). In this experiment, we have used TMT-10plex labeling with a carrier proteome including 200 cells. With MS/MS we identified 3 peptides of the HIV gag-polyprotein. This result demonstrates that we can detect viral load in a single human cell. To enhance sensitivity of the TMT-based protein quantitation from single cells and reduce the number of missing values, partner SPS has advanced specifically the least-squares fitting (LSF) processing of the time-domain transients for detection of TMT reporter ions and TMT complementary (TMTc) reported ions.
An outstanding list of new technologies is currently being developed within the framework of the project, and successful implementation of only a part of those ideas currently brought forward will be a great success. In this first stage of the project, different subsystems of the instrument are being developed independently by the partners.