First time ultra-sensitive and simultaneous quantification of proteins, interactions, and post-translational modifications in single cells to enable exponential growth in proteomics and interactomics

Informacje na temat projektu

PICO-NGS

Identyfikator umowy o grant: 190140802

DOI

10.3030/190140802

Realizacja projektu zakończyła się 8 Maja 2025

Data podpisania przez KE 11 Kwietnia 2023

Data rozpoczęcia 1 Sierpnia 2023

Data zakończenia 31 Lipca 2025

Finansowanie w ramach

• The European Innovation Council (EIC)

Koszt całkowity

€ 3 568 750,00

Wkład UE

€ 2 498 125,00

Koordynowany przez

ACTOME GMBH
Germany

Periodic Reporting for period 2 - PICO-NGS (First time ultra-sensitive and simultaneous quantification of proteins, interactions, and post-translational modifications in single cells to enable exponential growth in proteomics and interactomics)

Okres sprawozdawczy: 2024-08-01 do 2025-07-31

Podsumowanie kontekstu i ogólnych celów projektu

In a human cell there are about 20k protein coding genes (genome) that originate ~100k proteins (proteome). Proteins are involved in complex networks of protein-protein interactions (PPIs) influenced by post-translational modifications (PTMs), the interactome of living cells (~800,000 entities). The genome is mostly static throughout cell lifetime and can be sequenced today in <1h. The proteome and interactome are changing with other factors, such as environment and health, and not yet fully known. Today's methods for proteomics and interactomics are tedious, and lack sensitivity, quantitativity and parallelism.

Single-cell proteomics enables the study of protein expression in individual cells, revealing cellular heterogeneity that is often masked in bulk analyses. It is crucial for understanding disease mechanisms, such as identifying subpopulations of cells responsible for cancer progression or drug resistance. This approach advances precision medicine by providing patient-specific insights and helps in drug discovery by assessing individual cellular responses. Additionally, it contributes to fundamental biology by elucidating processes like cell differentiation and signaling pathways. Single-cell proteomics is revolutionizing research and clinical applications.

PICO-NGS tech is leading single-cell proteomics tool that combines ultra-high sensitivity (10 proteoform molecules per single-cell) with high parallelism measuring proteins, PPIs, and PTMs simultaneously (>50 targets) for hundreds of single cells. This will disrupt the global proteomics market (€29 B worth, 16% CAGR). This will fertilise ground-breaking academic research and accelerate developments in e.g. Pharma (drug discovery), Biotech (biomarkers/diagnosis), and Agrotech (crop sciences) industries.

The PICO-NGS project aimed to advance Actome’s single-cell proteomics capabilities by integrating digital PCR and NGS readouts with high-multiplex antibody panels. While the project was prematurely terminated due to strategic shifts and resource constraints, it achieved several significant scientific and technical milestones.

Prace wykonane od początku projektu do końca okresu sprawozdawczego oraz najważniejsze dotychczasowe rezultaty

A key success of the project was the validation of 41 out of the targeted 50 molecular markers across 9 of 10 biological pathways, laying the groundwork for a powerful multiplexed assay panel. The development of 1-million-compartment devices (QbMD and SbMD) demonstrated the technical feasibility of high-resolution proteomic analysis. These platforms supported multiplex assays and, in the case of SbMD, successful NGS library preparation.

Driven by emerging biological insights and the need for greater sensitivity, the project revised its original design to target 10 molecules per target per cell, requiring a shift from 1 million to 5 billion compartments per assay. Actome successfully developed the SPG cartridge to meet this need but encountered challenges in scaling its production and ensuring user-friendliness for untrained customers.

The project faced technical hurdles in finding a third-party manufacturer for the development of the 5-billion-compartment system, as the devices were not yet matured for external use. Combined with a strategic pivot toward the more commercially promising extracellular vesicle (EV) market, these factors led to the project's early termination.

Innowacyjność oraz oczekiwany potencjalny wpływ (w tym dotychczasowe znaczenie społeczno-gospodarcze i szersze implikacje społeczne projektu)

The PICO-NGS project has had a significant scientific impact by pushing the boundaries of multiplexed single-cell protein analysis and establishing key innovations that will inform future research and product development. One of the most notable achievements was the development and validation of high-performance antibody panels capable of detecting complex proteoform interactions at the single-cell level. These validated reagents have not only expanded Actome's internal antibody library but also provide a ready-to-use, high-quality resource for researchers, reducing the barrier to adoption and enabling a broader range of biological questions to be addressed with precision.

The project also contributed to a deeper understanding of the sensitivity requirements for functional proteomic analysis. The realization that biologically relevant interactions can occur at fewer than 100 molecules per cell—and in some cases as few as 10—has reshaped internal standards for detection thresholds. This insight challenges conventional assumptions and sets a new benchmark for what must be achieved to make single-cell protein interaction analysis biologically meaningful.

Furthermore, the project advanced methodological innovation in digital PCR and sequencing-based readouts. The development and evaluation of high-compartment prototypes, including the Qiacuity-based and Stilla-based 1-million compartment systems, demonstrated the feasibility of coupling digital protein assays with NGS workflows. This work has laid the groundwork for scalable, high-throughput single-cell proteomics and has the potential to bridge the gap between proteomic resolution and transcriptomic analysis.

Another scientific breakthrough was the correlation established between compartment size and detection sensitivity, which contributes to the theoretical and practical understanding of digital proteomics. This relationship provides a foundation for future assay optimization, enabling more precise quantification of low-abundance targets across diverse cell types and experimental conditions.

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