Protocoles:

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Identification et quantification des protéines
Identification et quantification des protéines
Chemoproteomics
Identifications des interactions protéine-protéine par BioID

PROID

PROIDL

PROIDEEP

PRMQUANT

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Identification et quantification des protéines

  • For MHC peptide identification and sequencing, a minimum starting material of 100-500 million cells is required. MHC peptides are isolated from the cell surface using either mild acid elution or immunoaffinity purification of MHC I complex protocols developed and validated at CAPCA.

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Identifications des interactions protéine-protéine par BioID

Identification et quantification des protéines
(BioID)

This workflow uses a mutant form of the biotin ligase enzyme (BirA) fused to the protein of interest, which will biotinylate proximal proteins in the surrounding micro-environment. The addition of biotin results in covalent linkages to be formed that can be further purified using streptavidin, and sequenced by high resolution LC-MS/MS. Peptide MS/MS spectra will provide information on the interacting partners and the location of interaction.

Identifications des interactions protéine-protéine par immuno-précipitations
(IP)

Using a FLAG tagged bait protein, the IP is performed using 1-5 mg of cell extract using commercially available anti-FLAG agarose beads. After basic elution of the protein complexes, tryptic peptides are analyzed and sequenced by high resolution LC-MS/MS. Peptide MS/MS spectra will provide information on the interacting partners and the location of interaction.


(CL-MS)

In this approach, a homo-bi-functional NHS-ester (i.e. DSS) is added to native proteins in solution to generate covalent links between neighbouring lysine residues. Once covalently bonded, transient interactions between proteins are stabilized. Tryptic peptides are then generated and analyzed by high resolution LC-MS/MS. Peptide MS/MS spectra will provide information on the interacting partners and the location of interaction within a distance constraint imposed by the cross-linking reagent.

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Détection de modifications post-traductionnelles

Phospho

Acetyl

Ubiquitin

SUMO

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Chemoproteomics

Different chemoproteomic approaches are available for early-stage target identification. These approaches are grouped into two categories:

  1. Cell-wide profiling of protein abundance and/or protein modification upon compound exposure
  2. Targeted chemoproteomics using activity- or affinity-based target profiling with chemical probes to enrich specific subsets of the cell proteome
To facilitate the identification of protein targets interacting with bioactive compound hits from high content screen (HCS) campaigns, and to independently validate candidate targets identified by chemogenomics, CAPCA offers two quantitative chemoproteomic strategies: affinity capture of target proteins and cellular thermal shift. These strategies build upon IRIC’s expertise in medicinal chemistry and quantitative proteomics to profile changes in protein abundance based on affinity capture of protein targets on functionalized beads or on ligand-induced changes in protein thermal stability.

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