Electrochemically-enabled high-throughput peptidomics for next-generation precision medicine

Project Information

ElectroMed

Grant agreement ID: 862539

DOI

10.3030/862539

Project closed

EC signature date 3 September 2019

Start date 1 January 2020

End date 31 December 2023

Funded under

• EXCELLENT SCIENCE - Future and Emerging Technologies (FET)

Total cost

€ 2 999 167,50

EU contribution

€ 2 999 167,50

Coordinated by

LUXEMBOURG INSTITUTE OF SCIENCE AND TECHNOLOGY
Luxembourg

Periodic Reporting for period 3 - ElectroMed (Electrochemically-enabled high-throughput peptidomics for next-generation precision medicine)

Reporting period: 2022-07-01 to 2023-12-31

Summary of the context and overall objectives of the project

Over the last decade, precision (or personalized) medicines have emerged as a viable treatment option for diseases with high prevalence, such as cancer and infections.
Recent scientific advances have demonstrated the efficacy of immunological treatments in the remission of specific cancer types by screening neoepitopes against immune system proteins. In silico methods based on computer algorithms can identify peptide candidates that allow for an effective immune system response in the fight against cancer. However, developing personalized therapies remains a costly and time-consuming process, since methods based on prediction models generate a large number of false positive results. Furthermore, current instruments are limited in their ability to fully characterize the interactions between these molecules and the immune system to assess the safety of the treatments.
ElectroMed was designed to provide a peptide screening platform for sequences that can be programmed onto sensors detecting interactions with proteins such as antibodies or MHC class II molecules. These last ones can detect neopitpoes and stimulate an efficient immune response.
ElectroMed brings three innovations:
-The synthesis is carried out using a novel solid phase peptide synthesis strategy that involves protecting the N-terminal of amino acids with acid labile groups, which are deprotected using electrochemically generated acid in spots with high acid contrast, enabling the use of acid labile groups employed in organic synthesis to produce high-purity crude sequences.
-Using electrochemically generated acid reduces the use of harsh chemicals, enabling the miniaturisation of a microfluidic management system for the automation of the peptide synthesis, using laminar flows that eliminate diffusion limited processes. This fastens the time process to shorten the time to results.
-Our microarray detects using FET technology, which enables real-time charge monitoring and quality control during the synthesis.
-Furthermore, continuous monitoring of protein binding with label-free sensors such as our FET sensors provides more information, such as the affinity of peptide protein interactions, providing higher quality information than traditional end-point assays.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

UCPH first investigated and assessed the relevance of linear peptide sequences that can interact with the immune system. The Cytomegalovirus (HHV-5) proteome contains peptides that interact with human antibodies and MHC Class II molecules, resembling cancer-related neopitopes. Furthermore, in collaboration with University Hospital Copenhagen, UCPH collected serum samples from COVID-19 and non-COVID-19 patients. Novel peptide epitope targets were identified and selected for single residue mapping; tested for inhibition of neutralizing anti-SARS-CoV2 antibodies; and used to investigate the presence of peptide-specific antibodies in a cohort of Covid-19 patients.
ElectroMed scientists, led by ELV, developed a functional prototype for sequential injection of multiple solutions using an Ibidi chip. The system has overcome the challenges of working with organic chemistry to miniaturize liquid management, resulting in a fully atomized system capable of delivering parallel peptide synthesis at a rate of one amino acid every five minutes, with the potential to reduce the synthesis time to less than a minute for each amino acid addition. Also, liquid volumes have been reduced, and in our current protocol, we use half ml with 0.3M concentrations, with the possibility of further reducing concentrations and dead volumes.
The synthesis takes place in a microfluidic chamber designed by LIST, where electrochemically generated acid is produced in addressable FET sensors that can monitor the synthesis process. We demonstrated the electrochemical production of acid quantities equivalent to traditional synthesis, which can be used to deprotect acid labile groups such as BOC in each addressable sensor. Furthermore, we completed the synthesis of the first peptide, resulting in a three-amino acid sequence detected by an antibody interaction, which was validated throughout our project.
UPF developed a protocol for coupling standard amino acids on surfaces similar to those used in sensors. This involved using APTES functionalized porous glass beads with adequate yield for HPLC-MS. The protocol was validated in collaboration with LIST and ELV in planar surfaces using mass spectrometry and SPR, allowing for continuous monitoring of the synthesis process.
The response of our FET sensors was modelled through a collaboration between LIST and UoT, led by UoG. We developed a surface interface model that can be used to predict the sensor response to peptide protein interactions as well as to provide a chemical characterisation of the sensor interface that can be used to monitor the synthesis and provide non-destructive quality control of each spot, which is a unique feature of our technology. In addition, we discovered a method for detecting amino acid and peptide fingerprints using the sensor signal from our FET.
UoT created FET sensors based on planar junctionless transistors, which can provide greater reliability. Our initial studies demonstrated that we can detect the chemical fingerprints of changes occurring during peptide synthesis using the detection of Boc protection and deprotection. To accomplish this, UoT created a control system that can accurately control pH levels.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

-ElectroMed's approach involves synthesizing peptides using electrochemically produced acid. For the first time, a peptide was synthesized and identified using electrochemically generated acid, due to our innovative reactors that combine nanolitter chambers with high surface area innert electrodes to produce acidity without the usage of additional chemicals.
-Solid phase peptide synthesis employs acid labile protection of N-peptide terminals and base labile protection of side chains to produce a high quality crude in situ peptide synthesis, reducing the use of harsh reagents. We obtained more than 90% yield from decapeptide sequences employing all standard amino acids.
-Custom microfluidic liquid management based on laminar flow without diffusion for rapid synthesis. In multiplexed synthesis, we accomplished rapid peptide synthesis with 5 minutes per amino acid addition while using 0.5ml of reagents at 0.3M resulting in significant chemical savings.
- FET detection of charges during peptide synthesis allows for monitoring of protection and deprotection processes, providing non-destructive quality control for each spot. We showed the signal providing from protection and deprotection steps.
-The same FET sensors are used to detect peptide-protein interactions in real time, providing information about the kinetics of protein absorption from which the affinity of the peptide-protein interaction can be inferred, providing more information than end point assays such as fluorescence.
Electromed Technology provides thus the following advantages respect to other peptide protein screening platforms:
-High-quality information on peptide-protein interactions obtained by detecting kinetics during charge absorption.
-Acid-labile chemistries and chemical synthesis monitoring provide superior chemical reliability.
-Speed to discovery thanks to microfluidic hips with laminar flow, the elimination of diffusion-limited processes, and the chip's massive multiplexing capabilities.

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