Services

Protein identification and quantification

We can identify proteins from SDS-PAGE bands, 2D spots or immunopurified samples. Samples should be provided without buffer or detergent when possible. We will proceed with reduction/alkylation and trypsin digestion (see protocols). LC-MS/MS data will be processed using Mascot, PEAKS or Maxquant. Protein identification results will be provided as a Scaffold file to facilitate data review (http://www.proteomesoftware.com/).

Phosphopeptide enrichment

Cell lysates should be provided dry. We are performing large-scale phosphopeptide enrichment typically from 1 mg of starting material with Titansphere Ti-O kit from GL Sciences. Data processing is performed by either PEAKS or Maxquant. Phosphosite assignment will be confirmed with A-Score (Zhai B, et al. J Proteome Res. 2008 Apr;7(4):1675-82) also available with Scaffold PTM.

SUMO peptide enrichment

For in vivo SUMO site identification the desired cell line must express our custom SUMO1, SUMO2 or SUMO3 variant in a stable or transient fashion (Galisson et al., Mol Cell Proteomics, , 10, M110.004796, 2011; Lamoliatte et al., Nature Commun. 5, 5409, 2014). Plasmid will be provided upon request. To identify >100 SUMO sites, we require at least 20 million cells, which correspond to ~ 8 mg of total protein. SUMO expressing cells are lysed under strongly denaturing conditions to inactivate SUMO proteases and the SUMOylated proteins enriched on Ni-NTA beads. Proteins are subsequently digested with trypsin on the Ni-NTA beads and the released SUMOylated peptides purified using our custom antibody that recognizes the SUMO remnant left upon tryptic digestion. In HEK293 SUMO3 cells, this approach yields more than 500 SUMO sites with >50% of the peptides being SUMOylated in the final sample.

Ubiquitin peptide enrichment

For the identification of ubiquitin sites in the proteome, samples can be submitted as cell pellets, protein extracts or as peptides. For cell pellets, we lyse the cells under highly denaturing conditions and follow up with an in solution digestion, typically with trypsin. The peptides are then desalted and the ubiquitin modified peptides are enriched using the commercially available anti-GG antibody from Cell Signaling. The number of ubiquitin sites identified is highly dependent on the nature and amount of sample. Typically, at least 5 mg of protein is required for the identification several hundred sites.

MHC peptide enrichment

The minimum number of cell required is 500 millions. MHC peptides are isolated from the surface of the cell with an acid elution (see Fortier et al., J. Exp. Med., 205, 595, 2008; Granados et al, Nature Commun. 5, 3600. doi: 10.1038, 2014). After desalting and molecular cut-off at 3kDa, the MHC sample is analyzed by LC-MS/MS using high resolution in MS and MS/MS modes. Data processing is performed with PEAKS and the binding score to MHC class-I molecule is predicted with NetMHCcons.

Acetylated peptide enrichment

For the identification of acetylation sites in the proteome, samples can be submitted as cell pellets, protein extracts or as peptides. For cell pellets, we lyse the cells under highly denaturing conditions and follow up with an in solution digestion, typically with trypsin. The peptides are then desalted and the acetylated peptides are enriched using the commercially available antibody from Cell Signaling. The number of acetylation sites identified is highly dependent on the nature and amount of sample. Typically, at least 5 mg of protein is required for the identification several hundred sites.

Identification of Protein-Protein Interaction by BioID

Mass spectrometry is the ideal method to identify partner proteins that interact with your protein of interest due to its non-targeted nature. To identify protein-protein interaction from BioID experiments simply provide the final streptavidin bead bound material that has been washed 5 times or more with milliQ water. Avoid the use of detergents. Inquire if you are uncertain if your sample is compatible with MS.

Identification of Protein-Protein Interaction by Immuno-purifications

Mass spectrometry is the ideal method to identify partner proteins that interact with your protein of interest due to its non-targeted nature. Samples from immune-purifications can be submitted in one of two ways. The first method, which is the method of choice, consists of submitting the eluted material, where the immune-complex has been removed from the beads. The second method, is submitting the bead with the immuno-complex still attached in as little volume of buffer as possible. The later method is less specific and generally does not work as well as the former. In both instances avoid the use of detergents. Inquire if you are uncertain if your sample is compatible with MS.