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Services description

The Plateforme Proteome is supported by a research team specialized in the analysis of biomolecules and provides services based on the separation and the analysis of proteins. The Plateforme Proteome also makes available to the scientific instruments covering a range of important methodology.

Request

New projects should be discussed with Caroline Tokarski. Other requests may be made directly with operators A-M. Lomenech, J-W. Dupuy or S. Claverol.
Analysis request forms are completed by users (Green parts) before being sunmitted to the platform. Platform competent staff study the analysis feasibility. If necessary, an advanced discussion or meeting between the partners is planned to complete the application analysis. The manager assigns a member of the staff in the Platform as the "privileged" contact for users. The user is finally aked to valid the form (specifications, timing ...).

Inquiries regarding the availability of the equipment are to be asked to J-W. Dupuy.

Sample reception

"Tips and precautions about sample preparation" are given at the end of this page. We strongly encourage users to follw up those simple rules so as to otimize conditions for getting better results.

  • Receipt of the samples (solution, gel, band / spot) on site (business hours) or via mail (-20 ° C for samples in solution, gel piece at room temperature in 1% acetic acid).
  • Immediate storage of samples at the appropriate temperature (-20 ° C for samples in solution, 4 ° C for gels and spots / bands). Possibility of storage at -80 ° C on request.
  • Sample and records follow-up according to a precise nomenclature.

The Plateforme Proteome accepts to deal with gel pieces stained with silver nitrate. In this case, the Plateforme Proteome can not be held responsible in case of failure of the analysis.
In case of samples in solution, the user will avoid the presence in his samples of salts, glycerol, detergents or polymers. Otherwise, the user will indicate the Platform presence and concentration of these contaminants.
For mass spectrometry, the only accepted containers are colorless polypropylene tubes or microplates.
The user shall ensure the tightness of his plates/tubes.

Service realisation

Sample preparation, electrophoresis

Electrophoretic separation tailored to the user.

 

Bradford assay

A standard curve will be performed at each assay.

 

1-dimension Electrophoresis SDS-PAGE, BN-PAGE, 16-BAC-PAGE, IEF

Molecular weight markers are placed on each gel for ensuring the quality of the electrophoretic separation and subsequent staining efficiency.

 

2-dimension Electrophoresis BN/SDS-PAGE, 16-BAC/SDS-PAGE, IEF/SDS-PAGE Small (System Invitrogen), IEF/SDS-PAGE Large (System Amersham)

 

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Staining Coomassie Blue, Colloidal Blue, Silver (MS Compatible), Pro Q Diamond, Ruthenium II, Sypro, Deep Purple

Molecular weight markers are placed on each gel for ensuring the quality of the electrophoretic separation and subsequent staining efficiency.

 

Image analysis

The gels are scanned on the scanner adapted to the staining protocol. Gel images are sent by default in .Jpeg format. Raw data are saved and are sent on request to the format of the user's choice.

 

Liquid vein isoelectrofocalisation Rotofor large cuve, Rotofor large cuve, Offgel 12 fractions, Offgel 24 fractions

A pI curve is made ??from all collected fractions.

 

Western

Primary Ab are sent by the user.

 

Sample preparation for Mass Spectrometry analysis

Biweekly proteolysis campaign in a controlled environment to limit potential  "keratin" contaminations. Trypsin is used as default endoprotease but the choice of the endoprotease can be adapted to the protein or application desired by the user and is made ??in consultation with him.

 

LC-MS/MS (normal gradient ) on  LTQ Orbitrap XL (low and medium-complexity samples)

 

LC-MS/MS (long gradient ) on  LTQ Orbitrap XL (high-complexity samples)

 

Identification of proteins by nanoLC-ESI-MS/MS coupled to LTQ Orbitrap XL or Q-Exactive. The duration of the chromatographic gradient is matched to the complexity of the sample. The data is queried via Proteome Discoverer 1.4 against the database that best fits the sample. The choice of the length of the gradient and of the database is done in consultation with users.

 

In the case of complex mixtures, a false positive rate is measured and transmitted to the user. The results include the proteins identified on the basis of single peptide.

 

In the case of less complex mixtures, reports include only proteins identified by a  minimum of two peptides.

 

LC-MS systems are qualified weekly in terms of chomatographic performances and mass spectrometry sensitivity.

 

MALDI (+8 MS/MS)

 

Protein identification by MALDI-TOF MS / MS: Analysis by peptide mass fingerprint is associated with a MS / MS confirmation of proteins identified in the first approach. The data is queried using Mascot (local license) against the database that best fits the sample. The choice of bank is performed in consultation with users.

 

iTRAQ 4-Plex or 8-Plex labelling (with OffGel fractionation)

 

 

Lable Free Analyses

Label-Free quantitative analyzes are conducted on a LTQ Orbitrap XL or Q-Exactive with gradients of 105 minutes long.

 

LC-MS/MS data are aligned using the software ProGenesis QI (NonLinear Dynamics). The detected peaks are integrated and quantitative data were statistically validated. Only peptides with ANOVA below 5% are selected and taken into account in the calculation of the intensity of the protein (the sum of the intensity of the peptides). A new statistical validation is applied at the protein level to retain only proteins with ANOVA below 5%. Finally, only the proteins quantified on the basis of two peptides (and more) are retained.

 

Reports are given in excel format.

 

TiO2 phosphopeptides enrichment

 

Phosphorylation search (MS3, MS² ETD)

 

The choice of the methodology is performed in consultation with users.

 

de novo sequencing

 

Protein molecular weight measurment  

 

Results restitution

Reports are sent to users .xlsx format.

Reports indicating the complete treatment of the samples and the drawn conclusion of the staff of the platform are sent in .docx.

Documents posted on the website of the Platform can help understanding the results, but the staff of the platform remains at the disposal of users to answer all the questions raised and assist them for results exploitation steps.

The full results are deposited on the ftp server of the University Segalen Bordeaux for a period of 10 days. In this case, the download is protected by a password sent to the user by email. Upon request, the results can be communicated to the user on the medium of choice.

The platform considers that, without response within 10 days of the client, the folder is accepted and closed.

The Platform will not disclose any information about the project other than the user's name and title of the project. Upon request, the project can also be confidential.

The Platform Proteome only provides sample storage for the duration of performance of the service. The sample storage conditions are specified by the customer in the Analysis Form. Otherwise, the biological samples are stored at -20 ° C. The gels are usually stored at 4 ° C. The client can also specify if he wishes to recover the remaining samples. Otherwise, samples will be eliminated.

The Platform Proteome only ensures the conservation of raw data for the period of performance of the service. The client, if he wishes to get raw data, must specify in the Analysis Form.

After sales service
The platform and its project members propose a meeting to present the results and the follow-up. The project leader can apply for this final meeting with his contact at the Proteome Platform.

Provided that the raw data is always available, the PF may re-examine the raw data. Applications for re-search will be motivated and addressed to the responsable of the structure directly.

The Plateform Proteome can write the corresponding material and methods and can assist project leaders in the exploitation of the results.