Protein Characterization Unit
Purpose
The Protein Characterization Unit specializes in proteomics and intermolecular interaction analysis by surface plasmon resonance (SPR) and bio-layer interferometry (BLI).
- Proteomic :
-Identification of proteins after enzymatic digestion by coupling nanoLC-MS/MS
-Relative quantification of proteins
-characterization of post-translational modfications
-Molecular mass measurement
- BLItz to detect and quantify proteins and characterize biomolecular interactions
- Biacore T200 to study biomolecular interactions by surface plasmon resonance (SPR).
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Services
Proteome and biomolecular interaction analyses.
► MASS SPECTROMETRY
Additional information :
► MASS SPECTROMETRY
- Protein identification by mass spectrometry (LC-MS/MS) : peptides are obtained from a SDS-PAGE gel or solution following enzymatic treatment and are analyzed by nanoLC-MSMS. MS/MS spectra are compared with protein data from public or specific databases.
- Characterization of post-translational modifications (phosphorylation, acetylation, ...): post-translational modifications are identified in the MS/MS spectra and modification sites are validated by visual inspection of the MS/MS spectra.
- Relative quantification of proteins (to compare experimental conditions) : identification and relative quantification of proteins in a complex mixture by Label Free (LFQ), SILAC or TMT/iTRAQ labeling.
- Accurate mass measurement of small molecules, peptides, proteins,oligonucleotides and synthetic polymers : by MALDI/TOF or ESI/Orbitrap after deconvolution of multiply charged ions.
► ANALYSES OF BIOMOLECULAR INTERACTIONS by INTERFEROMETRY (BLItz system) and by Surface Plasmon Resonance (BIACORE T200 system)
For information :
Three types of services are available:
- Equipment: BLITZ and Biacore T200 and consumables (subject to access approval and training)
- Customized services
- Project Management
The BLItz system provides two types of analyses that are simple, quick and inexpensive:
- Detection and quantification of tagged proteins (His, GST, Strep…) or antibodies in a culture supernatant before checking the level of expression of the protein of interest,
- Detection and characterization of interactions (protein-protein or protein-small molecule, protein-carbohydrate, protein-DNA, protein-RNA) before advanced characterization on the Biacore T200.
The Biacore T200 provides several types of high-sensitive analyses:
- Characterization of interactions (protein-protein, protein-small molecule, protein-carbohydrate, protein-DNA and protein-RNA, protein-cell, protein-virus particle by determination of kinetic constants: association (ka) and dissociation (kd) constants, affinity (KD and KA) and thermodynamic properties of interactions (ΔG and ΔH; analyses at different temperatures)
- Characterization of antibodies: screening and selection of the best antibodies, determination of kinetic and affinity constants, identification of epitopes, immunization follow-up…
- Analysis of competitive process or multi-molecular assemblies: screening of drugs inhibiting interactions, characterization of sites of interaction by competition, analysis of complexes involving several partners…
- Determination of concentration of a biomolecule based on specific interactions with a high affinity binding partner
- And finally, capture, isolation and recovery of unknown binding partners and their identification by Mass Spectrometry.
Equipment
- 3 Mass spectrometers :
- Q Exactive HF Biopharma (Thermo Scientific) : nanoelectrospray source, quadripole-orbitrap, HCD fragmentation, nanoRSLC coupling (Thermo Scientific)
- VOYAGER De Pro (Sciex) : MALDI-TOF
- LTQ Velos (Thermo Scientific) : nanoelectrospray source, double trap analyser, fragmentations CID/ETD, nanoLC NCP U3000 (Thermo Scientific)
- 1 BLITZ (Pall ForteBio) for interferometry analyses
- 1 Biacore T200 (GE Healthcare) - SPR.
TRAINING
User fees
Information on user fees is available on intranet (équipes contributrices et équipes académiques) or upon demand.
Documentation
- Fiche échantillon à remplir pour la spectrométrie de masse.
- Biacore T100 Instrument Handbook
- Brochure Biacore T200
- BiacoreT200 System Data File
- Biacore Consommables Brochure et Catalogue
Links
- Réservation du Biacore (pour les utilisateurs déclarés – accès sécurisé)
- Réservation du BLITZ (pour les utilisateurs déclarés – accès sécurisé)