Towards harmonisation of proteomics methods. Implication for high resolution / accurate mass targeted methods. (Doctoral thesis)
- Proteomics of Cellular Signaling
Mass spectrometry plays a central role in proteomics studies which has allowed its expansion to biomedical research. In an effort to accelerate the understanding of the various aspects of protein biology, the comparison and integration of results across laboratories have gained importance. However, the variety of laboratory-specific protocols, instruments, and data processing methods limits the reliability and reproducibility of the proteomics datasets. The harmonization of LC-MS based proteomics experiments is thus urgently needed to ensure that the workflows used are suitable for the intended purpose of the experiments and that they generate consistent and reproducible results. In a first step towards this harmonization, the critical components of each step of the workflow must be identified. Consolidated sample preparation methods with defined recovery and qualified platforms along with systematic assessment of their performance have to be established. They should ultimately rely on well-defined recommendations and reference materials.
Towards these goals, the present project aimed to define, based on current proteomics practices and recent technologies, experimental protocols that will constitute reference methods for the community. The associated results will represent a baseline that can be used to benchmark workflows and platforms, and to conduct routine experiments. A quality control procedure was developed to routinely assess the uniformity of proteomics analyses. The combination of a simple protocol and the addition of reference materials at different stages of the workflow allowed a straightforward monitoring of both sample preparation and LC-MS performance. In addition, as high resolution/accurate mass instruments with fast scanning capabilities turned out to be particularly suited to targeted quantitative experiments, a significant part of the work has consisted in the evaluation of the capabilities offered by such mass spectrometers to promote good practice upon their inception. The methods developed based on these emerging technologies were compared to the existing workflows allowing recommendations to be made for their implementation for fit-for-purpose experiment