Description of the PhD project
Oxidation plays a central role in cellular processes. Its physiological role, its involvement in cancer or aging are little understood. If oxidative stress can disrupt biological functions, the reactions of oxidation-reduction (redox) in a cell are often very finely regulated. A growing number of comments indicate that oxidation of the Cys should be regarded as a posttranslational modification involved in the regulation of proteins. Methionine residues are also very reactive and have been reported to induce a crosstalk between oxidation and glycosylation of proteins. The redox protein status often has an impact on their catalytic activity, the conformation or the metal/protein interaction.
We developed new strategies to access the redox index of a protein and its level of expression. These approaches should provide the closest snapshot of the redox state as possible. However, they are difficult to downscale, because they involve several steps incompatible with miniaturization, like protein precipitation. We developped a device for protein proteolysis using the Filter-Assisted Sample proteolysis strategy with a reactor total volume of 10µL instead of the commercial 200µL setups. We have shown that it is possible increase the overall coverage of the sample. The constraints are simple: work with a minimum of pressure drop, in a relatively small volume, and however avoiding the aggregation of our compounds which would plug our membrane. It must also avoid any component polymer incompatible with mass spectrometry. But we still lack the next steps of the workflows for specific analysis of the correlation of the redox state and the glycosylation state of the proteins.
Microfluidics is well suited to a comprehensive study of proteomes, with approaches on a broad spectrum of samples reproducibly prepared in small quantities. The developments of the microfluidics bricks will be carried out separately so that they can then be used on other biochemistry of proteins applications, the main constraint is to stay compatible with proteomic approaches.
Several aspects of the project:
- Work on confined microfluidic systems to overcome redox phenomena induced by the presence of surrounding oxidizing species.
- Develop a lysis of cells in situ that will split the proteins according to their subcellular compartmentation under protective atmosphere.
- Build microreactors using immobilized reagents. The diffusion time is proportional to the square of the characteristic length of the system and can be drastically reduced by miniaturization. The increase of the ratio surface/volume can be optimized with an increased catalytic surface due to the dispersion on functionalized beads., as it has been shown in glycoproteomics in the lab.
The group will strongly support this central project which will benefit from the unique infrastructures for proteomics (ESPCI facility) and microfluidics (IPGG facility).
Description of the research Unit/subunit
USR3249 Biological Mass Spectrometry & Proteomics
Our research unit was created in 2009 and provides proteomics solutions and performs constant technological developments. We have been particularly involved in sensitive multidimensional strategies for protein quantification, in differential analysis, including SILAC, and in the characterization of post-translational modifications by mass spectrometry. In particular, we have been lately combining analytical chemistry, biochemistry and protein chemistry to study oxidation and glycosylation of proteins. We recently developed a specific strategy to identify of biotinylated proteins which will be implemented in this project. We host a full equipped proteomics technological platform. Microfluidics devices will be produced in the microfab platform at the IPGG Institute.
The project will involve a multidisciplinary strategy using mass spectrometry, microfluidics, microfabrication, proteomics, and bioinformatics. It is of upmost importance for industrial application since there is a stron need for post-translational modifications studies, either for quality control of proction and storage, or for point-of-care diagnostic.
The project is an interdisplinary project at the interface between protein chemistry, physical chemistry, microfluidics and biology. It requires a strong involvement of the candidate who will have to be trained on different era, from nanochromatography to microfabrication and device designs, together with the biology and proteomics.
If successfull the redoxomics strategy will be applied to projects in collaboration with italian groups on thryroid cancer. More genrally, there is no strategy available to address redox and glycosylation of protein simultaneaously, and once validated the protocol will be provided to the ESPCI prteomics facility (more than 100 users/year)
Further information on the following link : https://www.upto.paris/Why-apply.html
The eligibility conditions can be found here: https://www.upto.paris/Eligibility-conditions.html
Details on the selection process can be found here: https://www.upto.paris/Evaluation-of-applications.html
The timeline of the 3rd call is available here: https://www.upto.paris/Timeline.html