Immunopeptidomics Sample Preparation

The isolation of peptides associated with MHC molecules and subsequent analysis using mass spectrometry are valuable techniques for characterizing the immunopeptidome, however immunopeptidomics research comes with some unique challenges. Help ensure success in immunopeptidomics experiments by following these helpful tips.

Select the anti-MHC antibody

In humans, the MHC is known as the human leukocyte antigen (HLA) complex. There is a wide range of anti-MHC and anti-HLA antibodies available, offering recognition of entire MHC molecule families (anti-pan MHC/HLA) or targeting specific classes, individual alleles, or subtypes of MHC molecules. Important considerations for anti-MHC antibody selection also include choosing an antibody that reacts with the species of interest and is validated for immunoprecipitation.

Antibodies used for IP in immunopeptidomics experiments

Need larger amounts of anti-MHC antibody?

Select the purification support for the anti-MHC antibody

Purification supports for immunopeptidomics include magnetic and agarose beads with Protein A, G, or A/G on the surface. Protein A and G are immunoglobulin (Ig)-binding proteins that bind to the Fc portion of intact Ig antibodies, which helps enable coupling of the bead to the anti-MHC antibody. In general, agarose beads provide higher binding capacity, while magnetic beads allow faster and higher throughput immunoprecipitation experiments with automated magnetic purification systems such as the KingFisher sample purification instruments. Magnetic and agarose beads coated with streptavidin can also be used to attach biotinylated antibodies if the selected antibody class does not bind well to Protein A or G.

Common immunoaffinity purification resins and formats*

*These products have not all been validated for peptide purification in immunopeptidomics experiments. Consult published immunopeptidomics protocols for guidance with product selection and usage.
 

Need larger amounts of purification resin?

Crosslink the MHC antibody to the purification support

If desired, the antibody can be covalently crosslinked to the support to prevent its co-elution with the MHC-peptide complex, which can interfere with downstream Western blotting and mass spectrometry analysis. Crosslinkers such as DSS and DMP are commonly used to covalently attach antibodies to beads in immunoprecipitation experiments. Alternatively, for BSA-free antibodies, an activated protein immobilization support can be used to covalently couple the antibody to the beads without the need for a separate crosslinking reagent. It is recommended to test that the antibody retains its binding affinity with crosslinking in a small IP experiment or confirm if the antibody has been previously used with the crosslinking method selected.

Crosslinkers and covalent attachment methods for IP

Separate the peptides from the MHC molecules

Prior to mass spectrometry on the peptides, the MHC molecules are separated and removed to purify the released peptide antigens using either size exclusion chromatography or solid phase extraction. To sufficiently separate and prepare the peptides for MS analysis, two rounds of solid phase extraction are often required and can include an offline peptide desalting or reversed-phase high-performance liquid chromatography (RP-HPLC) followed by LC-MS/MS. MWCO filtration using 5–10 kDa cutoff filters can also be used to separate the smaller peptides (~8–11 amino acids for MHC Class I peptides and ~12–25 amino acids for MHC Class II peptides) from the larger MHC molecules (~30–46 kDa).