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Proteins are complex macromolecules that perform vital biological functions in all living cell processes. Proteins consist of a sequence of building blocks called amino acids. The overall structure can be constituted of a sequence of amino acids (primary level), its folding in alpha helices or beta sheets in space (secondary level), the arrangement of the alpha helices and beta sheets in space (tertiary level) and the aggregation of several sequences (quaternary level). The protein’s function is strongly associated with its overall structure and can be affected by structural changes that may result from (i) genetic variations, (ii) alternatively spliced RNA transcripts or (iii) post-translational modifications (PTMs). Thus, any given protein may result (due to structural changes) in a complex mixture of different protein versions named proteoforms.

 

Protein measurement can be affected by the presence of different proteoforms of the protein and the measurement principles used, which in turn can lead to discrepancies in measurement results. Therefore, reliable and comparable measurements are needed as well as improved confidence in the data for proteins. This information is crucial for life science sectors such as food industry, food safety, health diagnosis, biopharma, and doping control. Given the complexity and heterogeneity of proteins and their structures, one of the main challenges is defining the measurand. This is further complicated by the different techniques used for protein measurement, as these rely on analytical targets that usually differ from the measurand.

 

The following are needed to address these challenges:

• development of accurate methods for the characterisation of the whole protein structure (primary to quaternary),
• robust evaluation of the impact of protein structure on measurement results,
• improved metrological developments focused on protein primary structure to higher order structure (HOS) (i.e. secondary, tertiary and quaternary structure is often collectively termed as HOS) protein analysis,
• tools to support better definition of the measurand(s) associated to specific proteoform(s).

Addressing these needs will require a multidisciplinary approach and collaboration between researchers, metrologists, and key stakeholders in the life science sectors in order to improve the accuracy and reliability of protein measurements.

Objectives

The project’s overall goal is to develop strategies to better understand the influence of protein structure on protein measurement and function and to establish a metrology framework with a better description of the protein measurand, the analytical targets and their influence on the measurement uncertainty.

The specific objectives are to:

1. To investigate the influence of primary sequence variants and PTMs of proteins, such as glycosylation, phosphorylation, deamidation, oxidation, or glycation on quantitative measurement results. Protein materials of increasing complexity will be characterised by expanding conventional metrological approaches based on mass spectrometry (MS) and nuclear magnetic resonance (NMR).
2. To investigate the influence of the primary sequence and PTMs variants considered in the Objective 1 on the secondary and tertiary structure of the protein and the influence of the presence of different structure variants (from primary to tertiary) on protein-protein interactions. This will be achieved by developing fit-for-purpose strategies using native and structural MS (e.g. ion mobility spectrometry-MS (IM‑MS), hydrogen deuterium exchange MS (HDX‑MS), chemical cross-linking MS (XL‑MS)) combined with other biophysical approaches (e.g. dynamic light scattering (DLS), cryoEM, and NMR) and existing computational techniques to characterise the higher-order structure (HOS) of the proteins to distinguish and quantify different structures and to explore the protein-protein interactions observed in biological systems (e.g. antibody-antigen interactions). Protocols to ensure traceability and estimation of the measurement uncertainties of the results will be developed.
3. To study the impact of structure heterogeneities from objective 1 and 2 on the measurement procedures using either purified peptides, recombinant proteins, extracted proteins or endogenous proteins in simplified buffer or matrix-matched solutions as calibrators. The influence of structure and interactions on isotope dilution-based reference measurement procedures (RMPs), and routine methods using proteomics approaches and immunoassays including sample preparation, calibration and incorporation of isotopically labelled analogues will be investigated.
4. To determine mathematical models based on the results of Objectives 1 to 3 to understand the influence of interferences, protein structure and protein-protein interactions on the different measurement procedures and evaluate their influence on protein function. Approaches to estimate the appropriate measurement uncertainty associated with procedures targeting entities that are not the intended measurand will be developed. Based on the measurement results and the model outcomes, guidelines will be developed for the definition of protein measurands (including appropriate units), of the analytical targets and the estimation of measurement uncertainty for the routine methods and the method developed within the project.
5. To facilitate the take up of the technology and measurement infrastructure developed in the project by the measurement supply chain (NMIs, DIs, research laboratories), research organisations (European Metrology Networks (EMNs) on Traceability in Laboratory Medicine and Safe and Sustainable Food), standards developing organisations (International Federation of Clinical Chemistry and Laboratory Medicine (IFCC), ISO and Codex Alimentarius.) and end users (biopharma, biomedicine, food producers, academic laboratories and clinical diagnostic laboratories).