Proteomics: Applications and Services

CAPCA provides an assortment of contract services for protein identification and quantification to support your research projects. Whether it is global protein profiling or targeting a subset of the proteome for quantification, our long established expertise can provide you with a solution for your biological challenges. Our services include the following established workflows; please inquire about customized workflows that can be designed according to your application needs.

Our main services include the following:

Protein identification and quantitation
MHC peptide identification and sequencing
Protein-Protein Interactions
PTM enrichment and analysis

Protein Identification and Quantitation (PROID and PRMQUANT)

CAPCA 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.

PROID

  • Standard LC-MS analysis of 70 minutes
  • Typical sample sizes and high sequence coverage expected

PROIDL

  • Longer LC-MS workflow of 120 minutes
  • Deeper proteome coverage for more complex samples

PROIDEEP

  • Extended LC-MS workflow of 240 minutes
  • Deepest proteome coverage possible, optimal for low abundant proteins and modifications

PRMQUANT

  • Targeted analysis of proteins of interest; high sensitivity and specificity
  • Inquire for more information about customizing a target panel of peptides for your samples

We can identify proteins from SDS-PAGE excised gel bands, 2D gel spots, HPLC purified fractions, or immuno-purified 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.

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MHC Peptide Identification and Quantitation

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, CAPCA 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 CAPCA.
  • 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

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Protein-Protein Interactions (PPI)

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 CAPCA, 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:

Proximity-dependent biotin identification
(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.

Protein-Protein Interactions by immuno-purifications
(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.

Protein-Protein Interactions by chemical cross-linking mass spectrometry
(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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Post-Translational Modifications (PTMs)

Post-translational modifications (PTMs) of proteins increase both the diversity and complexity of the proteome. CAPCA 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.

Phospho

Global or targeted LC-MS/MS approaches to detect and quantity phosphorylation on your proteins of interest. Cell lysates should be provided dry to the laboratory. We typically require about 1 mg of starting material for phosphopeptide enrichment prior to LC-MS/MS. Titanium dioxide (TiO2) microcolumns are used for sample enrichment prior to LC-MS/MS analysis.

Acetyl

At least 5 mg of starting material is required for the identification several hundred acetylation sites in the proteome. The number of acetylation sites identified is highly dependent on the nature and amount of starting material. Samples can be submitted as cell pellets, protein extracts or as peptides. Tryptic digests desalted and acetylated peptides are enriched with a commercially available antibody from Cell Signaling Technology for acetylome profiling.

Ubiquitin

At least 5 mg of starting material is required for the identification several hundred ubiquitination sites in the proteome. The number of ubiquitination sites identified is highly dependent on the nature and amount of starting material. Samples can be submitted as cell pellets, protein extracts or as peptides. Tryptic digests are desalted and ubiquitin modified peptides are enriched using the commercially available anti-GG antibody from Cell Signaling Technology

SUMO

To identify over 100 SUMOylated sites, we require at least 20 million cells, which corresponds to approximately 8 mg of starting material.
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 (see protocols section). Plasmids will be provided upon request. 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.

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