Projects

Project PM2 - More comparable measurement of higher order structure and interactions of proteins (2004-2007)

This Protein Measurement Project addressed a series of issues associated with the analysis of protein structure, function and interactions. It covered a broad range of techniques used in protein analysis including mass spectrometry (MS), fluorescence labelling and micro-array, circular dichroism (CD) and surface plasmon resonance (SPR).

The measurement of the interactions between proteins, proteins and nucleic acids and proteins and small molecules is core to (bio)pharmaceutical drug discovery, lead optimisation, quality control, and medical diagnostics. Techniques range from the relatively low-throughput, such as isothermal calorimetry (ITC), SPR, and analytical ultra centrifugation (AUC), to higher-throughput, multiplexed approaches, such as ELISAs and protein micro-arrays. However, there is a current lack of comparability between platforms and of standardization within platforms - a major limitation in the exploitation of many methods. These projects addressed some of these issues through inter-platform comparison and development of coherent reference materials.

One factor controlling protein interactions, and protein structure/function is the post-translational modification (PTM) of the protein. Consequently the characterisation of PTMs is key to the exploitation of proteomics, for example in the development of therapeutic agents. The commonest PTMs of proteins are glycosylation and phosphorylation. Existing techniques tend to be labour-intensive and low-throughput, disadvantages in a process-linked QC regime. New approaches based on mass spectrometry, optical spectroscopy, and arrays show promise, but require development and validation. The BEACON project in MfB (Measurements for Biotechnology) 2001–2004 has made a promising start in addressing these problems. This was developed further under this project through an experimental review of emerging techniques for PTM analysis such as mass spectrometry, microarray fluorescence labelling and Liquid Chromatography (LC) separation.

The requirement for consistency and comparability between different laboratories is paramount to ensure confidence in techniques and efficient data transfer. Fluorescence and circular dichroism measurement are two techniques which have been identified as significantly important for International comparability - pilot studies under the Bioanalysis Working Group of CCQM were planned to determine the sources of error in these measurements and establish appropriate calibration techniques and controls to ensure wider uptake of the techniques and regulatory acceptance of the data. In addition, high throughput approaches to protein interaction measurement, such as protein microarrays, require the immobilization of target proteins on a substrate. This can have a major impact on the data obtained and consequently the validity of the interpretation. Often these methods rely on fluorescently labelling protein populations for visualisation and quantitation, which can introduce an additional bias to relative quantitation. The uncertainty associated with chip based fluorescence measurement is being fully investigated.

The key successes of this project included:

• Development of two sets of protein binding pairs as reference materials for the measurement of protein-protein interactions;
• Evaluation of comparability between three technology platforms routinely used by industry for biopharmaceutical drug development;
• Experimental comparison of different fluorescence labelling techniques to optimise fluorescent detection of proteins while retaining their biological activity;
• Preparation of guidance on best strategies for immobilising proteins on surfaces while retaining biological activity;
• Application of this guidance to multiplexed antibody assays with case studies on their application to protein arrays;
• Production of report: Characterisation and quantitation of post-translationally modified proteins;
• Production of report: Towards the development of protein-protein interaction standards to improve the comparability of protein interaction measurements;
• Production of report: Fluorescent labelling of protein analytes.

Further information can be obtained by contacting the NMS helpdesk at LGC.