Applications and Services

CAPA has extensive experience in identifying and quantitating proteins at different expression levels from an assortment of biological matrices, including cell culture and tissues. The four generic workflows listed below are typically used for protein identification and quantitation. Customized workflows can be developed upon request. Please specify workflow type and refer to pricing when ordering.

We can identify proteins from SDS-PAGE excised gel bands, 2D gel spots, purified fractions, cell lysates, tissue, paraffin embedded tissue or immunopurified samples. Samples should be provided without buffer or detergent when possible. We will proceed with reduction/alkylation and trypsin digestion or another enzyme upon request (see protocols section). LC-MS/MS raw data will be processed using Mascot, PEAKS or Maxquant. Protein identification results will be provided as a Scaffold file to facilitate data review; Scaffold software can be downloaded as a free demo viewer from proteomesoftware.com.

The analysis of major histocompatibility class I (MHC I) peptides from antigen presenting cells using LC-MS was one of the original applications developed by Dr. Pierre Thibault's group at the IRIC. In close collaboration with Dr. Claude Perreault, CAPA has developed a unique expertise with profiling MHC I peptides from normal and cancer cells. Using our combined instrumental and bioinformatic workflows, high throughput sequencing and quantitation of MHC I peptides can result in unique insights in the generation of the MHC peptide repertoire in normal and neoplastic cells. The study of the immunopeptidome has shown to have great potential in the discovery of more effective cancer immunotherapeutic approaches.

• 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 CAPA.
• The cell extract is further prepared by ultra filtration with MW cutoff filters, and desalting using solid phase extraction. Analysis is performed using high resolution LC-MS/MS. The acquired RAW files are processed using PEAKS studio, and the binding score to MHC class-I molecule is predicted with NetMHCcons

The interaction of proteins is essential in all cellular processes and regulation. Characterizing exact topological contacts between proteins is an important step to further understand biological function, as well as to ascertain mechanism of action (MoA) in therapeutic drug development. At CAPA, we have developed three mass spectrometry-based approaches to help researchers better understand the nature of PPI in their samples. The three workflows are summarized below:

Post-translational modifications (PTMs) of proteins increase both the diversity and complexity of the proteome. CAPA has developed specialized protocols and workflows to identify and quantity PTMs over a wide dynamic range. Our current methodologies for PTM enrichment include phosphorylation, acetylation, ubiquitination, and sumoylation. Please inquire for other PTMs and/or customized protocols to fit your application needs.

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, CAPA 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.

We use up to 8 different proteases with complementary specificities to perform separate or sequential digestion of the antibody. The digests are then analyzed by LC-MS-MS. We conduct de novo sequencing and peptide assembly using the Peaks Ab software. We also perform glycan analysis to decipher the glycan structure and its position on the heavy chain. We validate the sequence of the light and heavy chains after deglycosylation with PNGase F, digestion with IdeS (FabRICATOR) and reduction with TECP. Epitope mapping is performed by crosslinking between the antigen and the antibody. After digestion with chymotrypsin and LC-MS/MS analysis, the data are analyzed with MaxLynx (Maxquant) or XLinkX (Proteome Discoverer) or pLink to assess on which CDR the antigen is crosslinked.