Projects

Project BA5 - Traceable Methods for the Characterisation Of Structurally Significant Proteins

Background

The fundamental issue for clinical, healthcare and metrology communities is the establishment of traceability for biologicals, in the same way as for simpler chemical entities by assigning values in SI. Complex biomolecules, such as proteins, have additional chemical heterogeneity (de-amidation, oxidation, isoforms) and plasma-based heterogeneity (complexing to other proteins) and the concept of ‘a unique, homogeneous chemical entity’ does not apply to proteins and other macromolecules. Furthermore few reference measurement methods exist and traceability cannot be determined readily.

The requirement for traceability in clinical measurement is addressed by ISO/EN 17511 ‘Measurement of quantities in samples of biological origin’ and in diagnostics, it is driven by the EU IVD Directive (98/79/EC). WHO international biological standards and reference reagents, based on arbitrarily assigned international units (IU), remain a pragmatic, ‘best-fit’ approach to comparisons of quantitative measurements of heterogeneous biologicals. However, this lack of traceability has been identified as an issue in achieving consistent measurement comparability by the laboratory medicine and industrial diagnostics community.

Building on previous research success in developing traceable methods for the quantification of proteins, this project will develop capability for metrological traceability of complex biomolecules, including their structural heterogeneity. This will be achieved by applying previously developed methods for the traceable determination of a proteins primary structure. These will be combined with mass spectrometry-based structural measurements, which will be investigated for the quantification of specific epitopes and/or structural motifs which infer biological activity.

Aims and objectives

Development of mass spectrometric approaches for the traceable quantification of protein structural confirmations. This will be achieved through a number of approaches including:

• The use of IDMS methodologies for the traceable quantification of amino acids and peptides
• Development of digestion protocols for the traceable quantification of proteins
• Preliminary investigations into the use of protein-ligand interaction hydrogen deuterium exchange for the determination of binding constants and protein structure