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Zawartość zarchiwizowana w dniu 2024-06-18

Robust affinity materials for applications in proteomics and diagnostics

Final Report Summary - PEPMIP (Robust affinity materials for applications in proteomics and diagnostics)

SUMMARY OF PROJECT OBJECTIVES
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. Proteins are also implicated in many diseases whether genetic, multi-factorial or degenerative. Studying protein function and shape is central for understanding disease and for the discovery of new drugs.
The EU-funded ITN PEPMIP (Robust affinity materials for applications in proteomics and diagnostics) has engaged 12 partners in training 10 ESRs and 2 ERs with a focus on research tasks aiming at improving existing means of protein analysis through novel molecularly designed materials, at 12 host institutions or companies: Malmö University (MU), University of Strathclyde (STRATH), Umeå University (UME), University of Southern Denmark (SDU), Oslo University (UIO), Ruhr Universität Bochum (RUB), Universitätsklinikum Essen (UKE), Lund University (LU), MIP Technologies (MIPTech), GT Septech, Thermo Fisher Scientific (THERMO) and Linneaus University (LNU).
The objectives of PEPMIP were:
• Through a comprehensive training program to provide 12 young researchers with a well-balanced spectrum of scientific, business and entrepreneurial skills required for a succesfull career in industry or academia.
• To innovate and develop smart materials for molecular recognition, capture or sensing of biologically relevant proteins and peptides
• To develop and validate sensitive high throughput methods and platforms for the analysis of biologically relevant proteins and peptides, especially phosphorylated peptides and proteins, and biomarkers for cancer and neurodegenerative diseases.
• To strengthen European research through establishing and cementing long lasting interdisciplinary and intersectoral collaborations among polymer/materials, proteomics and clinical researchers.

TRAINING ACHIEVEMENTS
All ESRs were recruited during the first year of the project and enrolled in PhD programmes at their host universities. Personel career development plans outlining the research tasks and aims as well as educational aims and career perspectives were established for all researchers. The entire network wide training was carried according to plan. This comprised eight technical workshops covering topics overlapping with or related to the scientific research tasks, three business development seminars organized by the industrial partners and two open Graduate Student Symposia serving as an informal forum for the ESRs to present their results and to receive unbiased criticism. The three private sector partners were actively involved in the research and training activities. The technical training events were supplemented by university specific courses and various team-building activities and complementary skills training including a workshop in Science Communication. A total of 34 secondment periods ranging from 1 week to 2 months were registred. These involved all partners and all ESRs and ERs of the network. 11 secondments were between partners representing different sectors. One of the research fellows, Chuixiu Huang (ESR in the company GT Septech, Norway) has already been awarded her PhD degree from the University whereas the remaining ESRs are scheduled to pass their degrees during 2016.

SCIENTIFIC ACHIEVEMENTS

OBJECTIVE 1: TO CREATE ROBUST SEPARATION MATERIALS FOR PHOSPHORYLATED PEPTIDES AND PROTEINS
Mainly three of the partners, SDU, RUB and MU have been involved in addressing this objective in Work Package 1. Focusing on neutral phosphate receptors utilizing coordinative hydrogen bonding as the main affinity principle these partners have successfully addressed existing limitations of the current gold standard methods e.g. complicated workflows, large sample inputs, sequence bias, resulting in the following scientific highlights.
• A streamlined method for phosphopeptide enrichment was developed by RUB and MU which is compatible with extremely small sample sizes (10 µg scale) and is robust, fast and simple. The method was shown to enrich phosphopeptides more effectively than established approaches resulting in a peptide pool with amino acid distributions more closely resembling the native amino acid abundance.
• The first phosphotyrosine selective enrichment technique devoid of antibodies was developed by SDU and MU. The MIP strategy was found to be complementary to the gold standard reference method indicating that combined use of pY-MIPs and TiO2 for phosphoproteomics increase the entity coverage and pY-peptide signal intensity.

OBJECTIVE 2. TO CREATE ROBUST SEPARATION MATERIALS FOR SPECIFIC DISEASE BIOMARKERS
This objective was addressed in two work packages. Work package 2 aimed at developing novel capture materials and methods for biomarkers for neurodegenerative disease e.g. Alzheimers and Parkinson. The work involved mainly UKE, RUB, MU, STRATH and LU and aimed at comparing different materials formats of epitope imprinted receptors targeting the protein biomarkers beta-amyloid and alphasynuclein. Novel capture materials showing enhanced affinity for these biomarkers were developed and are currently undergoing testing using the analytical platforms available at RUB and LU.
Work package 3 on the other hand addressed this objective by targeting established biomarkers for Small Cell Lung Cancer. MU, STRATH and UIO developed an alternative enrichment strategy for targeted proteomics exploring MIP based capture of the SCLC biomarker ProGRP. This resulted in the following highlights:
• A bottom up targeted proteomics method allowing absolute quantification of the biomarker in fortified serum samples.
• A robust analytical platform for automated analysis of a ProGRP signal peptide in clinical samples. This represents the first fully automated method for the determination of ProGRP in patient samples.

OBJECTIVE 3: TO DEVELOP GENERIC AND SCALABLE PEPTIDE AND PROTEIN IMPRINTING TECHNIQUES
OBJECTIVE 4: TO DEVELOP IMPRINTED NANO- AND MICROBEADS AND EVALUATE SUCH BEADS IN PROTEIN ANALYSIS
Several surface imprinting approaches and bead polymerization techniques have been assessed by MU, STRATH and MIPTech to address these two objectives.
• Microbeads prepared as silica polymer composites, hierarchically imprinted polymers, grafted thin film composites, precipitation polymers and controlled pore size beads have been compared and are currently used in trap columns to capture the SCLC biomarker. These formats display enhanced peptide retentivity and have also been shown to retain beta-amyloid with enhanced affinity under denaturing conditions. This satisfies Objective 4.
• A scalable method to produce controlled pore size polymer beads using nanoparticle templates were developed by MIPTech. The method developed provides a simple and effective way of controlling the pore size in highly cross-linked polymers. This satisfies Objective 3.

OBJECTIVE 5: TO DEVELOP MONOLITHIC SEPARATION MEDIA FOR MINIATURIZED FORMATS
• A novel simple approach to prepare monolithic separation columns was developed by UME and SDU. Monolithic materials were prepared by polymerization of urea and formaldehyde in the presence of an organophosphate compound. This resulted in a surprisingly strong selective retention for phosphopeptides. The columns feature a pronounced imprint related phosphopeptide selectivity when tested on line in simple model systems.

OBJECTIVE 6: TO DEVELOP NOVEL CHIP-BASED MICROARRAYS FOR MULTIPLEX ANALYSIS
The consortium has made a concerted effort to develop novel microanalytical systems, to demonstrate these in real world applications and to couple them with the smart capture materials.
• The integration of MIPs in lab on chip devices has been carried out by LU with promising results. The platform has been assessed both for potential multiplex analysis of biomarkers and has been proven for fast parallel optimization of binding conditions for solid phase extraction.

OBJECTIVE 7: TO DEVELOP CAPILLARY BASED NANO-LIQUID CHROMATOGRAPHY-MASS-SPECTROMETRY SYSTEMS
• Using standard miniaturized trap columns slurry-packed with MIP particles we have demonstrated effective on-line enrichments of proteotypic peptides and phosphopeptides. Spherical microparticles prepared by template polymerization or precipitation polymerization have been particularly amenable to this application. This format will be of interest in the context of high throughput omics applications.

OBJECTIVE 8: TO DEVELOP ELECTRO MEMBRANE EXTRACTIONS (EME) AS A MILD SERUM CLEAN-UP METHOD
• GT Septech has developed a novel sample pretreatment method based on electrically driven membrane extractions. This provides effective clean-up and potential enrichment of the target. The system has shown very promising performance and has been applied to the biomarkers of the project.

DISSEMINATION
The PEPMIP fellows gave 22 oral presentations and 21 poster presentations at different national and international conferences (excluding the network wide training events). Within the network training program each researcher presented their achievements resulting in totally on the average 10 oral presentations per researcher. The research fellows have 14 scientific manuscripts published or under revision and in addition 9 manuscripts in preparation or ready for submission. In total 12 publications are joint productions involving at least 2 partners of the network and the researchers are first authors on 21 of the papers published or planned. The consortium has organized 9 technical workshops open to external researchers and 2 International Symposia dedicated to graduate students working in the field of imprinting science. All ESR have actively communicated their science to the public.
The project website (www.pepmip.eu) has provided a summary of the project, its achievements and schedule of events and has served as a forum for exchange of documents, knowledge and tips among the researchers. The latter has been suplemented by the projects Facebook (https://www.facebook.com/Pepmip-278077505657349/?ref=ts&fref=ts) and Twitter sites (https://twitter.com/pepmip). The projects achievements has been highlighted in ca 7 press releases.

POTENTIAL IMPACT AND USE OF RESULTS
In PEPMIP we have developed and implemented MIPs targeting the phosphorylated peptides, protein biomarkers and specific proteotypic peptide sequence motifs. Given the programmable nature of the imprinting technique, materials targeting other phosphorylation motifs can be generated relatively easily, thereby extending the method to encompass other relevant amino acid side chains. Indeed, imprinted polymers targeting the pY motif are currently being implemented for the sensitive extraction of low-abundance pY-peptides by leading proteomics institutes outside the PEPMIP consortium. Indeed the publication by RUB and MU in Nature Scientific Reports generated significant response among leading groups in the field as well as proteomic and diagnostic companies. In view of the enhanced sequence coverage using the MIP based approach, it is also reasonable to assume that new phosphorylation sites will be discovered as a result of these efforts. Due to their link to cancer this can in turn result in the identification of new biomarkers of clinical diagnostic relevance.
The advances made in material science are also significant. We have established thin film composite materials prepared by RAFT mediated grafting as well as hierarchically imprinted materials as robust and reproducible formats for specific peptide capture. The new templating approach to produce hierarchically imprinted materials represents an interesting scalable technique to produce mesoporous materials with a very narrow pore size distribution. Finally the monoliths prepared by step-growth polymerization is first of the kind and we expect broad applications in separation science as a whole.
We also foresee a broader impact of our recent proof of concept demonstration 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.