Periodic Reporting for period 1 - TopSpec (Next generation precision antibody profiling - from science fiction to reality)
Reporting period: 2019-01-01 to 2019-12-31
MS: The complex hardware for the omnitrap are now generating high-quality top-down spectra of Abs. Effort is still required to optimize the versatility of the device. While many details of the Omnitrap have been finalized, developments of hyperthermal hydrogen atom bombardment (HHAB), IM separation of fragment ions and software continues. The original plan of producing two Omnitraps for the Q Exactive HF MS series has been revised. Instead, two platforms, the original one (Q Exactive) and one compatible with the new Exploris 480 MS, have been designed. This latter system will exploit enhanced sensitivity and mass resolution capabilities. Subsequent developments include testing the revised IM device and HHAB gun. The electron source design and investigating Coulomb explosion dissociation effects will also be undertaken.
PI-trap: Work to connect the pI-Trap system with electrospray ionization (ESI) is progressing well, with separation of protein mixtures. A small-molecule based ampholyte has been selected, providing improved detection. Efforts have been made to balance concentration of isoelectric focusing additives whilst maintaining a stable spray. The pI-Trap instrument design has been finalized and mechanical parts fabricated. The software is under development. The final configuration of the interface between pI-Trap Cell, buffer exchanger and ESI is in progress. The most recent implementation involves integration of a nanospray needle, electrode and buffer exchanger into a single assembly.
Data acquisition hardware/processing software: The first versions of hardware tools tailored for top-down analysis are ready to be integrated into the TopSpec platforms. 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. The core tools are ready for TopSpec data processing and analysis. Advanced processing modules will be added, providing bespoke data processing analysis capabilities, to augment the existing capability, once the performance envelope of the full Omnitrap/IMS/Orbitrap MS systems can be explored. Further hardware, method and software tools development are envisioned.
Ahead of project plan: Of note is on-going development of a method for processing Top-down protein datasets involving isotopically enriched or depleted samples. 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: A deuterium lamp has been integrated with the omnitrap and a series-simulations to investigate surface induced dissociation are finalized. We have also 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 are being developed. Progress in preparing growth media for expressing mAbs in monoisotopic media has been made. 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 outlined.
Impact and implications: If successful, TopSpec will provide a new tool that can be used to expand our knowledge of the immune system and have a dramatic impact on personalized, precision medicine. Another significant impact will be in the field of MS instrument design. Specific impacts: 1) Increase in the speed of diagnosis and drug development. 2) Increase knowledge on an individual´s Ab response to disease. 3) Expand scientific research around proteomics. 4) Create new business opportunities within and outside the project.