Periodic Reporting for period 2 - TopSpec (Next generation precision antibody profiling - from science fiction to reality)
Reporting period: 2020-01-01 to 2022-12-31
A growing challenge in EU health system is cost of drugs/targeted therapies. By reducing the time to develop therapies, costs are also reduced. To reduce development time, it is imperative to understand how we defend our self against diseases. In doing this, the biggest mystery to solve and challenge to address is the immune system. Specifically, we need to be able to characterize and understand the expressed sequence repertoire of antibodies (Abs) and identify their antigens. The aim of TopSpec is to address this challenge, thus opening extraordinary opportunities in medical research and drug development. We aim to create a technology that will revolutionize proteomics, and advance development of next generation Ab- and other protein-based therapeutics. Abs are the most sophisticated line of natural defense against disease. Knowing which Abs are produced in response to a given disease will enable us to understand how antibodies are optimized to recognize targets. This knowledge can lead us to next-generation cures via personalized therapeutic Abs. The current limitation iour inability s to efficiently and effectively sequence large protein molecules (e.g. Abs). In this project, experts in radical-assisted protein sequencing team up with the best developers in ultrahigh-resolution MS and top European scientists in protein separation and data processing. We will develop a ground-breaking TOP-down tandem mass SPECtrometry (MS/MS) platform designed to provide a significant improvement in our ability to sequence Abs, and thus to solve this current bottleneck problem. Achieving the seemingly impossible, we will create, test and validate a platform capitalizing on our ground-breaking innovations, enable scientists from academia and industry to access the Ab repertoire, enhancing our ability to explore new effective cures for global diseases.
The TopSpec platform includes a pI-trap device for protein separation by isoelectric point prior to the MS, a high-performance Orbitrap mass spectrometer, a versatile ion trapping device (omnitrap) for gas-phase ion activation, dissociation, manipulation and accumulation, an ion mobility (IM) drift cell for separating fragment ions, a high-performance data acquisition system and data processing and data analysis software tailored for Ab analysis (Figure).
PI-trap: The pI-Trap system was connected with electrospray ionization (ESI), with separation of protein mixtures online with MS analysis. A small-molecule based ampholyte has been selected, providing improved detection of protein molecules. Efforts have been made to balance concentration of isoelectric focusing additives whilst maintaining a stable spray. The pI-Trap instrument contains an autosampler, which can double as a fraction collector for off-line separation. The controlling software for that is developed. The pI Trap has successfully separated two Abs online with detected by MS and MS/MS.
MS: The complex hardware for the omnitrap is now generating high-quality top-down mass spectra of Abs. The omnitrap design has been finalized, including the high-energy electron gun for electron-based activation, hyperthermal source for hydrogen atom bombardment (HAB), IM and other parts of the TopSpec platform. The omnitrap can be connected with both Q Exactive (both Plus and HF), as well as with the new Exploris 480 MS. This latter system possesses enhanced sensitivity and mass resolution capabilities.
Data acquisition hardware/processing software: The hardware tool tailored for top-down analysis (FT Booster) is integrated into the TopSpec platform. The core software tools (data processing and analysis) have been developed and tested on reference data from Orbitrap MS-instruments equipped with ultra-high resolution, native MS, as well as HCD, ECD and UVPD MS/MS. Advanced processing modules have been added, providing bespoke data processing analysis capabilities, to augment the existing capability.
PI-trap: The pI-Trap system was connected with electrospray ionization (ESI), with separation of protein mixtures online with MS analysis. A small-molecule based ampholyte has been selected, providing improved detection of protein molecules. Efforts have been made to balance concentration of isoelectric focusing additives whilst maintaining a stable spray. The pI-Trap instrument contains an autosampler, which can double as a fraction collector for off-line separation. The controlling software for that is developed. The pI Trap has successfully separated two Abs online with detected by MS and MS/MS.
MS: The complex hardware for the omnitrap is now generating high-quality top-down mass spectra of Abs. The omnitrap design has been finalized, including the high-energy electron gun for electron-based activation, hyperthermal source for hydrogen atom bombardment (HAB), IM and other parts of the TopSpec platform. The omnitrap can be connected with both Q Exactive (both Plus and HF), as well as with the new Exploris 480 MS. This latter system possesses enhanced sensitivity and mass resolution capabilities.
Data acquisition hardware/processing software: The hardware tool tailored for top-down analysis (FT Booster) is integrated into the TopSpec platform. The core software tools (data processing and analysis) have been developed and tested on reference data from Orbitrap MS-instruments equipped with ultra-high resolution, native MS, as well as HCD, ECD and UVPD MS/MS. Advanced processing modules have been added, providing bespoke data processing analysis capabilities, to augment the existing capability.
Along project plan: Major achievements are superior performance of the omnitrap electron source and enhanced ExD ion activation capabilities. Ion-electron energy interactions are performed with high efficiency over an extended electron energy range, superior to commercially available instruments. Another innovation is the slow heating CID method, that produces unique and information rich, high mass Abs fragments. Means are implemented to utilize this benefit by generating and isolating Ab subunits, which offers superior performance in top down analysis of large molecular ions. A unique feature of TopSpec is IM separation performed to reduce the complexity of fragment ion populations. Its aim is to reduce spectral congestion and facilitate de novo sequencing of Abs. Additionally, a method for transient level deconvolution of top-down data using asymmetric apodization function has been developed. For validation, nanoLC-MS/MS middle- and top-down workflows on Orbitrap platforms to sequence reference Ab chains has been developed. The complementarity of MS/MS activation techniques on 25 and 50 kDa subunits as well as the effect of the number of selected precursors for MS/MS on sequence coverage have been evaluated. Furthermore, development of a novel algorithm for deconvolution of complex top-down spectra by deriving transient decay constant of individual peaks has been accomplished.
Beyond project plan: We have developed a method for charge state deconvolution of intact Abs, but still maintaining absorption mode type peak profiles - something that no other exiting method can offer. Software for accelerating manual processing of complex spectra with implementation of de novo sequencing algorithms is also developed. A novel method of determining which Ab binds to a given antigen without antigen mobilization and pull-down, but based on thermal profiling, has been developed (manuscript in preparation).
Impact and implications: TopSpec platform provides a new tool to expand our knowledge of the immune system. Broad application of this tool may have a dramatic impact on personalized, precision medicine. Another significant impact will be in the field of MS instrument design. Specific impacts: 1) An increase in the speed of diagnosis and drug development. 2) Increase dknowledge on an individual´s Ab response to disease. 3) Expanded scientific research using proteomics. 4) New business opportunities within and outside the project.
Beyond project plan: We have developed a method for charge state deconvolution of intact Abs, but still maintaining absorption mode type peak profiles - something that no other exiting method can offer. Software for accelerating manual processing of complex spectra with implementation of de novo sequencing algorithms is also developed. A novel method of determining which Ab binds to a given antigen without antigen mobilization and pull-down, but based on thermal profiling, has been developed (manuscript in preparation).
Impact and implications: TopSpec platform provides a new tool to expand our knowledge of the immune system. Broad application of this tool may have a dramatic impact on personalized, precision medicine. Another significant impact will be in the field of MS instrument design. Specific impacts: 1) An increase in the speed of diagnosis and drug development. 2) Increase dknowledge on an individual´s Ab response to disease. 3) Expanded scientific research using proteomics. 4) New business opportunities within and outside the project.