Projects

Project PM1 - Development of validated procedures and reference standards for the high confidence analysis of complex protein mixtures (2004-2007)

Robust, reproducible and validated ways to fractionate, identify and quantify individual proteins in a cellular proteome or in complex protein formulations, are still some way off. Widely used procedures quantify protein concentrations across 3-4 orders of magnitude. However, as cellular protein concentrations range over ~6 orders of magnitude, and serum proteins over ~10 orders, there is a formidable challenge in identifying and quantifying a significant proportion of a total proteome. Optimisation of each stage of analysis is therefore essential to maximise sample information.

Efficient and robust separation methods are particularly important for the analysis of complex protein mixtures. In 2004-2007, two separation methods dominated. The classical 2D-gel electrophoresis can provide relative quantification and can easily separate post-translationally modified proteins, but it is labour-intensive, making high throughput and automation difficult, and certain protein types are often under-represented.  Advances in coupling 2D-LC/MS (Liquid Chromatography Mass Spectrometry) yield an alternative approach that is very sensitive and potentially able to detect low-abundance proteins. It is also easy to automate, enabling high throughput, and allows several different strategies to obtain reliable relative quantification. There is a clear industrial demand for quantitative comparison of these technologies, and for development of reference material(s) to enable inter-laboratory assessment and process optimisation.

Mass spectrometry has become the technique of choice for high throughput identification of proteins. Success usually depends on matching the accurate mass of proteolytic peptides generated from a sample to those from a database. However database searching of protein digest mass spectra is too uncertain for the unambiguous identification of proteins and the variability associated with the measurements needs to be evaluated. An alternative approach, peptide sequencing by MSⁿ increases the complexity of the proteomic experiment, requiring further investment in computing power, instrumentation and operator expertise. Industry wants to understand the accuracy and precision of mass spectrometry, and to clarify the level of confidence of protein identification.

The accuracy of quantitation continues to be an issue in protein measurements. Methods of relative quantitation are evolving rapidly but are often highly selective and may introduce measurement bias. Analysis of more complex protein mixtures becomes progressively more challenging and plagued by misidentifications. Currently there are no appropriate standards for the absolute quantitation of proteins. The international metrology community has targeted the establishment of fully characterised traceable standards, tied to a reference method. Moreover, industry seeks traceable protein standards against which to calibrate in–house proteins.

This project represented an integrated approach to the development and validation of procedures and reference samples for the optimised separation, high confidence identification and high accuracy quantification of proteins from complex mixtures. It helped realise an international biometrology strategy for national measurement institutes (NMIs) and ensure wide relevance to biomeasurement community users.

The key consortia members for this project are concerned with the separation, and high confidence identification and quantification of complex protein samples, and in metrology. This consortium consisted of LGC, University of York, and relevant instrument manufacturers and pharmaceutical companies. The consortium aimed to collaborate widely with other key NMIs (NIST, PTB, AIST) to secure global acceptance of its findings.

The key successes of this project included:

• Production of a poster describing absolute traceable protein quantification;
• Production of a report on the comparison study of 2D gel and multi-dimensional LCMS separation methods for simple and complex proteomes;
• Production of a guide on the use of MS methods for the identification of relevant proteomes;
• Development of an absolute traceable method for protein quantification from amino acids to peptides;
• Production of quantified peptide reference controls;
• Characterisation of protein reference materials.

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