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Novel strategies for biomarker discovery and quantification

Final Report Summary - BIOMAH (Novel strategies for biomarker discovery and quantification)

Novel strategies for biomarker discovery and quantification

This project was designed to develop the skills of a Marie Curie Research fellow in the area of quantitative proteomics. The overall aims were to apply these skills to specific projects that would ensure the supply of sufficient normal and pathological samples to gain a clear insight into the protein changes associated with pathology. The examples would be chosen from the medical and veterinary fields, reflecting the prior training of the Fellow.

The first phase of the programme was devoted to building advanced skills in quantitative proteomics, based on two types of instruments, a matrix assisted desorption ionisation time of flight mass spectrometer and an electrospray ionisation ion trap mass spectrometer. However, during the project, the host laboratory enjoyed a period of unprecedented expansion and acquisition of new instrumentation, coincident with a laboratory move to state-of-the-art space (www.liv.ac.uk/pfg). Thus, it was possible to enhance the training to include two flagship instruments, an Orbitrap Velos mass spectrometer and a Synapt G2 instrument. The Fellow was given full access to these instruments and as a consequence, the training was more advanced and the quality of the scientific data were commensurately improved.

The quality of the work programme was always going to be dependent upon the availability of high quality sample streams. In the intervening period between project approval and commencement, access to the clinical samples originally specified was less certain. According we decided to shift focus to samples for which we had guaranteed access in sufficient numbers. The change was without effect on the training, and increased the changes that high quality publications would ensure, an essential outcome for the fellow. Two main sample streams were developed. The first was a detailed profiling of the bovine urinary proteome. Urine poses particular challenges for proteomics studies, and an important aspect of this work was the development of methodology for sample clean up. The Fellow explored several approaches and has developed a simple yet remarkably effective protocol for the preparation of proteomics-friendly urinary protein preparations. As part of this programme, the Fellow has shown that specific antimicrobial proteins can be detected in urine samples, which might have value as generic biomarkers for infection status.

The second sample stream emphasised a rather more challenging problem, that of bacterial meningitis in children in the developing world (specifically Malawi). In collaboration with clinical colleagues, the fellow analysed cerebrospinal full samples using high resolution instrumentation. This programme was highly successful, reflecting the remarkable skill set acquire by the fellow in the early stages of the project. The protein profile in infected children was highly altered, pointing to several high confidence biomarkers of infection status. Additionally the ability to detect proteins specific to the infectious bacterium allows for more informed selection of antibiotic regimen, a scarce resource that should be used optimally. This aspect would require development into a field test, based on the development of antibody methodology.

To conclude, the main outcomes of the programme were:

* An exceptionally high level of skills in proteomics acquired and used by the Research Fellow.
* Three papers, currently in preparation, that will be submitted to high quality journals. In all three papers, the fellow will be first author.*
* Clear candidate biomarkers for bovine infectious disease that may be developed for herd health profiling.*
* A detailed understanding of the CSF protein changes in bacterial meningitis.
* Clear candidate biomarkers for intensity of infection and infectious organisms.*

* subject to examination for intellectual property potential, currently underway, which has caused a short delay in submission