Smart capture phases for proteomics, glycomics and biomarker assays

Informations projet

BioCapture

N° de convention de subvention: 722171

DOI

10.3030/722171

Projet clôturé

Date de signature de la CE 31 Août 2016

Date de début 1 Novembre 2016

Date de fin 31 Octobre 2021

Financé au titre de

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Coût total

€ 2 982 872,07

Contribution de l’UE

€ 2 982 872,07

Coordonné par

MALMO UNIVERSITET
Sweden

Periodic Reporting for period 2 - BioCapture (Smart capture phases for proteomics, glycomics and biomarker assays)

Période du rapport: 2018-11-01 au 2021-10-31

Résumé du contexte et des objectifs généraux du projet

Cancer medicine experiences currently a strong need for sensitive, robust and affordable diagnostic tools that can sense cellular states early during disease progression. In BioCapture we have addressed this need while harnessing novel molecular capture technology targeting tumor specific markers. Molecularly imprinted polymers (MIPs) and “plastic antibodies” and other smart materials have been developed and used to detect previously inaccessible tumor markers and to discover novel disease biomarkers. The project engaged 11 ESRs full time within a time frame of four years. The research performed was highly interdisciplinary comprising molecular imprinting, materials chemistry, optical sensor technology, proteomics and diagnostics.

The objectives of Bioacapture were to:
• prepare 11 young researchers for a succesfull career in industry and academia.
• develop proteomics capture phases for unstable post-translational modifications (PTMs) and PTM peptide sequence motifs.
• develop imprinted capture phases for lung cancer biomarkers
• develop saccharide specific capture phases.
• develop addressable fluorescent MIPs.
• develop high throughput multiplex assays.
• validate the novel diagnostic tools using clinical samples
• establish long lasting collaborations among polymer/materials, proteomics and clinical researchers.

Main conclusions:
We have progressed beyond expectation towards our goals to develop affinity based tools and reagents. Robust peptide- and PTM- specific capture materials compatible with proteomics work flows and MS have been used to develop assays with a potential diagnostic value. We have also advanced well on a fundamental materials, analytical and cell biology science level. A detailed macroscopic and molecular level characterisation of the imprinted receptors have increased our understanding of the nature of the binding sites and how they are formed. Finally, genetically engineered cell libraries has provided a perfect model system for imitating cancer cells and for probing receptor binding affinity and specificity.

Travail effectué depuis le début du projet jusqu’à la fin de la période considérée dans le rapport et principaux résultats atteints jusqu’à présent

The purpose of the BioCapture project was to develop a novel robust and cost-effective platform for streamlined assays of proteinaceous and glycosylated biomarkers. The key results listed below show that most of the original objectives have been achieved:

1. The first phosphopeptide sequence specific MIPs and its use for highly specific phosphopeptide enrichment from native whole cell lysates.

2. A MIP based mild approach for enrichment of proteins/peptides phosphorylated at histidine.

3. Saccharide MIPs capable of discriminating between glycosidic linkage isomers, a new potential lectin complement.

4. High affinity sialic acid MIPs displaying strong sialic acid retentivity of sialylated peptides.

5. Molecular level characterization of phospho- and sialo- MIPs revealing new insights into recognition mechanisms and the buildup of imprinting memory.

6. A method drastically increasing the production yield of high affinity “plastic antibodies”.

7. A "plastic antibody" based magnetic fluorescent displacement assay.

8. A versatile approach to magnetic MIPs for direct peptide enrichments.

9. The first on-line duplex assay for low-abundant cancer biomarkers.

10. Cell libraries genetically engineered for controlled presentation of human mucine O-glycans or 2,3- versus 2,6- linked sialic acids.

11. Core/shell type fluorescent MIPs displaying a pronounced and specific light up in response to a phosphorylated peptide.

12. A MIP microfluidic based detection system demonstrated for a phosphorylated peptide.

The above results were reported by the Biocapture partners in 27 publications and 11 manuscripts in preparation or submitted. These were in part published in high ranking journals comprising Angewandte Chemie, JACS, Nature Communications and PNAS. Results 1-4, 6-9 are currently being exploited for commercial use by Biocapture partners. Patents are either part of background IP or will be filed.

Progrès au-delà de l’état des connaissances et impact potentiel prévu (y compris l’impact socio-économique et les conséquences sociétales plus larges du projet jusqu’à présent)

Proteins constitute the most abundant and versatile biomolecules present in living cells, properties dictated by the genetic code. Their diverse structure and function is responsible for key cellular processes including metabolism and cell movement and this is to large part controlled by their post translational modifications (PTMs) such as glycosylations and phosphorylations. Proteins are also implicated in many diseases whether genetic, multi-factorial or degenerative. Studying the function and shape of proteins and their PTMs is central for understanding disease and for the discovery of new drugs. In Biocapture we have progressed beyond expectation towards our goals to develop affinity based tools and reagents enabling more efficient detection and quantification of both proteins and their PTMs in the form of phosphorylations and glycosylations. In brief the main achievements are as follows:

- Phosphopeptide sequence specific MIPs. This is a main breakthrough and has now been demonstrated on native cell lysates for two key sequences involved in early T-cell receptor signaling with immediate practical relevance in cancer research, drug discovery and testing and potentially in companion diagnostics.

- A MIP based approach for enrichment of proteins containing labile PTMs. This addresses a current lack of efficient tools for this purpose.

- Development and molecular level characterisation of MIPs engineered to recognize tumor specific glycans. A potential new substitute for lectins in glycobiology research.

- A method drastically increasing the production yield of high affinity “plastic antibodies”. This advance constitutes a breakthrough and relieves the bottle neck in producing plastic antibodies for essentially any given target.

- A "plastic antibody" based magnetic fluorescent displacement assay for ultrasensitive analyte detection. This is an entirely new approach to design fast label free assays.

- Genetically engineered cell libraries presenting glycan structures as found on cancer cells. These unique tailored cells are crucial for understanding and validating the glycan binders developed in the project.

- Core/shell type fluorescent MIPs displaying light up in response to a phosphorylated peptide. This is the first step towards the dual MS/fluorescence readout idea.

The above results demonstrate significant progress beyond the state of the art. The novel capture phases can pave the way for robust PTM-peptide assays of clinical relevance. The advances made in material science are also significant. We have overcome one main bottleneck for producing practical quantities of soft nano gels as plastic antibodies. This is likely to be widely adopted by the scientific community and industry. We also foresee a broader impact of MIP based specific enrichments of proteotypic peptides. We expect this approach to be applicable to essentially any MS detectable peptide biomarker and to enable MS based bottom up protein diagnostics on a large scale, significantly impacting healthcare and disease management. Finally, the potential use of the robust tools in home-based tests addresses a crucial patient need and key environmental sustainability goals.

Research highlights 2

Principle of Molecularly Imprinted Polymer (MIP) based biomarker capture and enrichment

Affinity based analytical tools

Training highlight 1 - Polymer and monolith workshop

Research highlights 1

Training highlight 2 - Biomarker, optical sensors and business development workshop

Principle of molecular imprinting

Training highlight 1 - Polymer and monolith workshop

Research approach

Training highlight 3 - Team building course