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Smart capture phases for proteomics, glycomics and biomarker assays

Periodic Reporting for period 1 - BioCapture (Smart capture phases for proteomics, glycomics and biomarker assays)

Reporting period: 2016-11-01 to 2018-10-31

To find new and better ways to diagnose and treat cancer is one of the most pressing tasks for researchers today. Projections for 2025 estimate an annual increase of 19.3 million new cancer cases world-wide whereof more than half occuring in low- and middle-income countries. The transformation from a normal cell into a malignant tumor cell is a multistage progression where cells become gradually less susceptible to treatment. Early diagnosis where the cancer is still curable is therefore crucial. This emphasizes the need for sensitive, robust and affordable (especially for low income countries) diagnostic tools that can sense the cellular state, commonly in the form of tumor specific protein markers, early in the process. In BioCapture we address this need while harnessing novel molecular capture technology targeting tumor specific markers. Molecularly imprinted polymers or “plastic antibodies” and other smart materials are being developed and used to detect previously inaccessible tumor markers or to discover novel disease biomarkers. By matching 6 polymer/materials research groups with 5 leading molecular biologists and diagnostics institutes and 4 SMEs we have created a highly interdisciplinary research-training network with ambitious scientific goals and an attractive training program for 11 ESRs.

The objectives of Bioacapture are:
• Through a comprehensive training program to provide 11 young researchers with a well-balanced spectrum of scientific, business and entrepreneurial skills required for a succesfull career in industry or academia.
• to develop and validate proteomics capture phases for unstable PTMs and pY sequence motifs.
• to develop imprinted capture phases for lung cancer biomarkers
• to develop saccharide specific capture phases for glycoprotein or glycopeptides.
• to develop addressable fluorescent MIPs for tumor biomarkers for assays or stains.
• to integrate capture phases in high throughput multiplex MS, fluorescence or ELISA assays.
• to validate the novel diagnostic tools using clinical samples
• To strengthen European research through establishing and cementing long lasting interdisciplinary and intersectoral collaborations among polymer/materials, proteomics and clinical researchers.
"The purpose of the BioCapture project is to develop a novel robust and cost-effective platform for streamlined assays of proteinaceous and glycosylated biomarkers. The research performed is highly interdisciplinary comprising molecular imprinting, materials chemistry, optical sensor technology, proteomics and diagnostics.

Summary of main results:

1. The first phosphopeptide sequence specific MIPs. The MIP targets the T-cell receptor signaling kinase Zap-70, a prognostic marker in chronic lymphocytic leukemia and SH2 domain targets. The MIPs display excellent target specificity in tests on complex peptide mixtures also when spiked at very low levels in a complex 12 protein digest

2. A MIP based chemo-affinity approach for enrichment of proteins/peptides phosphorylated at histidine. The method works under mild basic conditions and can enrich pHis peptides from spiked BSA digests. This offers a unique tool given the lack of affinity techniques compatible with basic conditions.

3. A method drastically increasing the production yield of high affinity “plastic antibodies”. The method has been demonstrated for the proteins trypsin and pepsin.

4. A ""plastic antibody"" based magnetic fluorescent displacement assay for ultrasensitive analyte detection.

5. An imprinted polymer displaying selectivity for a signature peptide for the ProGRP biomarker NSE.

6. Cell libraries genetically engineered to present homogeneous O-glycan structures (Core1, Tn-antigen and STn) as found on cancer cells.

7. HEK293 cells genetically engineered to alter their sialic acid capping capacity showing no, only 2,3- or only 2,6-linked sialic acids on a bi/tetra N-glycan.

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

9. A reproducible preparation scheme for micron-sized polymer core/silica shell/MIP shell (CSS) beads.

10. A MIP microfluidic flow cytometry based detection system demonstrated for a phosphorylated peptide.
"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 for two key sequences involved in early receptor signaling with immediate practical relevance in cancer research, drug discovery and testing and potentially in companion diagnostics. Manuscript is in preparation.

- A MIP based chemo-affinity approach for enrichment of proteins containing labile PTMs. This addresses a current lack of efficient tools for this purpose. Manuscript has been favorably reviewed for Anal. Chem. and is currently under revision.

- 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.

With the above tools in hand we foresee the discovery of new phosphorylation sites and the understanding of kinase expression level and activity. This is directly applicable to cancer research, drug discovery and testing and potentially in companion diagnostics. Moreover this can result in the identification of new biomarkers of clinical diagnostic 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."
Principle of Molecularly Imprinted Polymer (MIP) based biomarker capture and enrichment
Research highlights
Affinity based analytical tools
Training highlight 1 - Polymer and monolith workshop
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