General presentation

The Proteome Platform (PP) offers expertise and equipment for the separation, identification and characterization of proteins, quantification and differential analysis or the finding of protein partners.

The analysis of proteins is performed by the so-called "bottom-up" methodology using equipment combining nano-liquid chromatography with the latest generation of mass spectrometers equipped with Orbitrap type analysers. These new generations of analysers enable very high resolution analysis and very high scanning speed for optimal sensitivity, allowing the analysis of complex or even very complex samples.

Concerning quantitative/differential proteomics, the PP offers the possibility to analyze your samples by the label-free technique which allows to compare several samples without any protein/peptide labeling step. The choice of experimental design (label-free, SILAC, TMT, ...) is however guided by different factors including the type of samples (cell cultures, tissues ...), their complexity, the amount of material available ... Alternatively, isotope labelling methods are also available and allow to consider a protein or peptide fractionation with a minimum of experimental bias in the analysis.

The analysis of intact proteins (purified or in mixture) is possible by the so-called "top-down" approach, using methods implemented on the latest generations of instruments available in the PP.

For the study of non-sequenced species, the PP proposes its expertise in protein identification by de novo sequencing. Various software specific to this type of analysis are available.

Finally, the PP proposes different enrichment and/or bioinformatics analysis strategies for the characterization of co/post-translational modifications (i.e. phosphorylation, ubiquitination, glycosylation).

For protein separation, the PP proposes single (SDS-PAGE, BN-PAGE, 16-BAC-PAGE, IEF) or two-dimensional (BN/SDS-PAGE, 16-BAC/SDS-PAGE, IEF/SDS-PAGE) electrophoretic techniques. The gels can be stained with various visible and fluorescent dyes (Coomassie Blue, Colloidal Blue, Silver (MS Compatible), Pro Q Diamond, Ruthenium II, Sypro, Deep Purple). Membrane transfer of the gels is also possible.

The separation of proteins in liquid vein by electrofocusing (IEF) or by molecular weight can be performed on commercially available systems, respectively OffGel, Rotofor or GelFree.

As the PP is supported by the research team Mass Spectrometry of Biological Macromolecules, UMR 5248 CBMN, collaborative projects can be carried out on the following topics. For any new projects on the following themes, please contact the head of the research team Caroline Tokarski and the person in charge of the theme.

• Structural analysis of proteins by isotope exchange or cross-linking (responsible : Stéphane Chaignepain)
• Imaging by Mass Spectrometry (responsible : Nicolas Desbenoit).
• Analysis of N- and O-glycoproteins / glycopeptides (responsible : Katell Bathany).
• Lipid analysis (responsible : Corinne Buré).
• Tandem mass spectrometry analysis of intact proteins - top-down proteomics (responsible : Caroline Tokarski).
• Trace and ultra trace analysis in the field of cultural heritage (responsible: Caroline Tokarski).

 See the "Expertises" tab for more details on the services offered by the PP.

 

Terms of service and rates

Before submitting samples, it is advisable to contact the PP to establish a context-specific analysis strategy and to set up the experimental conditions (preparation and analysis).

New projects should be requested to Caroline Tokarski. Other requests can be made directly to the operators Anne-Marie Lomenech, Jean-William Dupuy or Stéphane Claverol. An analysis request form is available and must be completed in order to study the feasibility of the project (Analysis request). A preliminary meeting can be organized between the partners if necessary. The PP manager will designate a "contact" within the Platform who will become the user's privileged interlocutor. The user validates the analysis request (specifications, type of service, deadline, quotation, PP staff involved).

The services are provided either in the form of collaboration or as a research service:

• In the case of a collaboration, the laboratory in charge of the scientific project assumes the operating costs. In this case, the platform staff involved is co-owner of the results and co-author of any scientific publications resulting from the collaboration.

• In the case of a research service, the laboratory responsible for the scientific project assumes not only the operating costs but also the expertise costs. In this case, the laboratory carrying out the project remains the exclusive owner of the results.

A definition of the strict service framework as well as an quotation will be established for each request made to PP.

 

Sample delivery

Tips and precautions around sample preparation" are given for information on our website. We strongly advise users to follow these few rules in order to place in the optimal conditions for obtaining a relevant result.

• Reception of samples (solution, gel, strip/spot) on the spot (during working hours) or by mail (-20°C or dry ice for samples in solution, at room temperature in 1% acetic acid for gel pieces).
   
• Immediate storage of samples at the appropriate temperature: -20°C or -80°C on request for samples in solution, 4°C for gels and spots/strips).
   
• Tracking of samples and records according to a precise nomenclature.

The PP accepts to process silver nitrate stained gel pieces. In this case, the PP cannot be held responsible in case of failure of the analysis.

In the case of samples in solution, the user should avoid the presence of salts, glycerol, detergents or polymers in the samples. Otherwise, the user will indicate to the PP the presence and concentration of these contaminants.

For mass spectrometry, the only containers accepted are tubes or microplates made of non-coloured and untreated polypropylene. The user will ensure that the tubes or plate are leak-proof.

 

Performing the service

Services	Description of services
Preparation of samples	Sample preparation is performed in accordance with the technological strategies chosen in agreement with the project leader. The operating mode is communicated to the partner.
Protein assay	The protein assay is performed using the Bradford technique. A standard curve is established for each assay.
Electrophoretic methods and coloration of acrylamide gels	If the chosen electrophoretic technique allows it, uncoloured molecular weight markers are deposited on each gel to ensure the quality of the electrophoretic separation as well as the efficiency of the staining.
Image analysis	The gels are digitalized on the scanner adapted to the chosen staining method. The gel images are sent in .jpeg format by default. The raw data is stored and sent on request in the format of the user's choice.
Isoelectrofocusing in the liquid vein	For any isoelectrofocusing in liquid vein, whether it is performed on Rotofor system, Offgel system or GelFree system, a standard curve (pI, MW) is established from all the collected fractions.
Sample preparation for Mass Spectrometry	Weekly proteolysis campaign in a controlled environment to limit the possibility of "keratin" contamination. The endoprotease used is generally trypsin. The choice of endoprotease can be adapted specifically to a project.
LC-MS/MS analysis	Protein identification is performed by nanoLC-ESI-MS/MS orbitrap mass spectrometer. The length of the chromatographic gradient is adapted to the complexity of the sample. The data are queried via Proteome Discoverer / PEAKS against the most appropriate database defined in agreement with the project leader. The choice of gradient length and database is also made in agreement with the project leader. In the case of complex mixtures, a false positive rate is determined. The results include the proteins identified on the basis of a unique peptide. LC-MS/MS systems are calibrated and qualified weekly both in terms of chromatographic performance and the sensitivity of the mass spectrometer.
Label-Free Differential Expression Analysis	Label-Free quantitative analyses are performed on a Orbitrap Fusion Lumos mass spectrometer, which provides optimal sensitivity and scanning speed for the analysis of very complex samples. Raw LC-MS/MS data are then processed using Proteome Discoverer software to identify and quantify proteins.  The methods for calculating ratios and statistical tests are established in agreement with the project leader. The raw data are available for reprocessing by the project leader using the software of his choice. Raw LC-MS/MS results are then processed using Proteome Discoverer software for protein identification and quantification.  The methods for calculating ratios and statistical tests are established in agreement with the project leader and the raw data are available for reprocessing by the project leader using the software of his choice.  The results are given in excel format.

 

Reporting of results

The results will be sent electronically via email or ftp server. Upon request, the results can be communicated to the user on the media of his choice. The files are in *.zip, *.docx, *.xlsx or *.pptx format. The results are in *.xlsx format. The reports describing the complete processing of the samples and the conclusions formulated by the Platform staff are sent in .docx format.

Documents available on the Platform's website can help to understand the results (Description of the results obtained with Proteome Discoverer) but the Platform's staff remains at the full disposal of the users to answer any questions and to assist them in the steps of valorization of the results.

The PP considers that, if there is no response within 10 days from the project leader, the project will be considered accepted and closed.

PP engages itself not to disclose any information related to the project other than the user's name and the project title. Upon request, the title of the project may also be kept confidential.

PP will only retain samples for the duration of the service. The conditions for the storage of samples are specified by the customer in the analysis request. Otherwise, biological samples are stored at -20°C. Gels are systematically stored at 4°C. The customer may also specify whether he wishes to retrieve the remaining samples. If not, the samples will be eliminated within the biological waste elimination process.

PP only ensures the preservation of the raw data for the duration of the service. If the customer wishes to have access to the raw data, this must be specified in the analysis request.

 

Post-delivery service

PP and its members propose to the project leaders to organize a closing meeting to present the results and discuss the perspectives of the project. The project leader can request this closing meeting from his/her contact person and/or the head of the Platform.

Provided that the raw data are still available, the PP can interrogate the raw data again. Requests for re-interrogation should be motivated and addressed to the head of the Platform directly.

Within the framework of an evaluation of the results from PP, the staff undertakes to draft the corresponding material and method and to help the project leaders in the exploitation of the results.