Steps for isolation and analysis of MHC-associated peptides
Immunopeptidomics involves the identification and characterization of peptides presented by major histocompatibility complex (MHC) Class I and II molecules on the cell surface. These peptides are recognized by T cell receptors and play a crucial role in immune recognition and antigen presentation.
Immunopeptidomics sample preparation includes several key steps to characterize and identify the immunopeptidome. Cells or tissues are collected and processed to isolate MHC-associated peptides (MAPs) using immunoprecipitation (also known as immunoaffinity purification or IP) with MHC-specific antibodies. Mass spectrometry (MS) analysis is performed for peptide characterization, comparing unknown peptides presented by the MHC against protein sequence databases to identify and quantify the peptide antigens.
Tips and challenges in immunopeptidomics research
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.
| Low abundance of MHC-associated peptides | Use large amounts of cell or tissue samples. Avoid peptide loss during the immunoprecipitation procedure:
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| Complexity and diversity of immunopeptides | MHC-associated peptides are not generated by tryptic cleavage of proteins:
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Step 1: Prepare the MHC immunoprecipitation column
Using an immunoaffinity purification column coupled with MHC-specific antibodies enables isolation of MHC-associated peptides from the cell or tissue lysate. To prepare the column, careful consideration must be given in selecting the anti-MHC antibody, purification resin and format that will effectively bind to the antibody of choice, and method for antibody crosslinking to the purification support.
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
| MHC Class I | MHC Class II | |
| Human HLA Class I antigens are HLA-A represented by HLA-A, HLA-B and HLA-C molecules | Human HLA Class II antigens are HLA-D including DP, DQ, DR2, DR3, and DR4 molecules | |
| Anti-pan MHC or class specific antibodies | HLA-ABC Monoclonal Antibody (W6/32) commonly used in published immunopeptidomics experiments to react with human MHC Class I including HLA-A, HLA-B, and HLA-C | MHC Class II Monoclonal Antibody (M5/114) reacts with mouse MHC Class II molecules |
| Allele-specific antibodies | HLA-A2 (MHC Class I) Monoclonal Antibody (BB7.2) | HLA-DR (MHC Class II) Monoclonal Antibody (L243) |
| H-2Kb (MHC Class I) Monoclonal Antibody (Y-3) | HLA-DQ (MHC Class II) Monoclonal Antibody (NKI(SPV)L3) | |
| H-2Db (MHC Class I) Monoclonal Antibody (B22-249.R1) | I-A/I-E (MHC Class II) Monoclonal Antibody (M5/114.15.2) |
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*
| Format | Dynabeads and Pierce magnetic beads | Agarose/Sepharose beads |
| Features and benefits |
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| Protein A products | ||
| Protein G products | ||
| Protein A/G products | ||
| Streptavidin products | ||
| Reactive products |
*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
| Stand-alone crosslinkers | Crosslinking immunoprecipitation kits | Activated protein immobilization supports | |
| DSS (disuccinimidyl suberate) | DMP (dimethyl pimelimidate) | ||
| Contains an amine-reactive N-hydroxysuccinimide (NHS) ester at each end of an 8-carbon spacer arm | Contains an amine-reactive imidoester group at each end of a 7-atom spacer arm | Contains Protein A/G magnetic or agarose beads to bind antibody. The antibody is then crosslinked to the beads using DSS (disuccinmidyl suberate). |
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Step 2: Lyse the cell or tissue sample for MAP extraction
Cells or tissue are lysed to release the MHC molecule-peptide complexes into solution for further purification. Various cell lysis and protein extraction reagents are available, tailored to different sample types and downstream applications. To help ensure the integrity of the peptides, protease and phosphatase inhibitors are added during the lysis process to help prevent peptide degradation. After lysis, the cell lysate is cleared by centrifugation, and protein quantitation is performed to determine the amount of protein present before proceeding to immunoprecipitation of the MHC complex.
| Cell and tissue lysis reagents | Protease and phosphatase inhibitors | |
| Optimized reagents | Stand-alone detergents used in immunopeptidomics publications | |
| Pierce IP Lysis Buffer Cell lysis buffer that contains NP-40 and is optimized for immunoprecipitation assays |
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Halt Protease and Phosphatase Inhibitor Cocktail, EDTA-free (100X) Compatible with mass spectrometry since it does not contain interfering components |
| M-PER Mammalian Protein Extraction Reagent Cell lysis buffer containing a mild, nondenaturing detergent for total protein extraction |
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| Mem-PER Plus Membrane Protein Extraction Kit Cell and tissue lysis reagent for membrane protein extraction |
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Lysate clearing and quantitation:
- Clear the lysate by centrifugation
- Perform total protein quantitation of the lysate using the BCA assay
- Preclear the lysate by incubating with the immunoaffinity purification support without the antibodies to remove abundant proteins and decrease nonspecific binding
Step 3: Purify the MAPs using immunoprecipitation
MHC Class I or II molecules and their associated peptides are isolated from cell and tissue lysates using MHC immunoprecipitation with immunoaffinity columns. MHC-peptide complexes are then eluted from the beads using acid elution, releasing the isolated complexes and dissociating the peptides from the MHC molecules.
Perform IP to isolate the MHC-peptide complexes
Immunoprecipitation and immunoaffinity purification involve the following steps:
- Bind—incubate the antibody-beads with the lysate so that the MHC-peptide complexes bind to the antibody-bead support
- Wash—collect the beads through centrifugation or magnification and wash to remove unbound sample components
- Elute—elute the MHC molecules and peptides from the bead and dissociate the peptides from the MHC molecules using 1% TFA
Follow guidelines from the manufacturer of the antibody and immunoaffinity purification support along with published protocols when performing the immunoprecipitation procedure.
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).
| Size exclusion chromatography products | Solid phase extraction products | |
| Protein Concentrators | C18 Columns and Peptide Desalting for Mass Spectrometry | RP-HPLC instruments and columns |
| Pierce Protein Concentrator, 3K MWCO, 100–500 µL | Reversed-phase high performance liquid chromatography (RP-HPLC) systems | |
| Reversed Phase HPLC Columns | ||
| C18 Reversed Phase HPLC Columns | ||
Step 4: Discover and characterize the MAPs with MS
Once the MAPs are isolated and separated from the MHC molecules, they are analyzed with high-performance liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Both targeted and discovery mass spectrometry workflows are employed in immunopeptidomics to study the repertoire of peptides presented by MHC molecules.
- Discovery mass spectrometry—enables the comprehensive identification and characterization of the peptides presented by MHC molecules. This unbiased approach helps in discovering novel peptides and understanding the breadth of the MHC peptide repertoire.
- Targeted mass spectrometry—used for the precise quantitation of specific peptide antigens of interest. This approach is often applied to validate and quantify known peptides presented by MHC molecules.
MS quantitation methods used for immunopeptide analysis
| Quantitation method | Technique | Features | Usage |
| Relative | Label-free quantitation (LFQ) | Compared samples are tested and analyzed individually | Used in discovery (untargeted) proteomics applications |
| Isobaric labeling with TMT (Tandem Mass Tags) | Multiplexed quantitation of up to 18 samples within a single analysis using isobaric mass tags | ||
| Metabolic labeling using SILAC (stable isotope labeling using amino acids in cell culture) | Multiplexed quantitation with cells cultured in the presence of media containing heavy or light isotope-labeled amino acids and compared for the light to heavy ratio | ||
| Absolute | Heavy-labeled spike-in standard | A synthetic, stable-isotope-labeled (SIL) peptide standard is added to the sample and quantitation is performed by comparing the signal intensity of the endogenous (light) peptide with a synthetic peptide standard (heavy) such as HeavyPeptide AQUA custom peptides | Used in discovery (untargeted) proteomics applications for quality control and in targeted quantitation proteomics applications |
Step 5: Validate the MAPs
Once the MAPs are identified, targeted quantitation using mass spectrometry can be used for validation of the peptide antigens. Targeted quantitation involves spiking an MHC peptide sample with known amounts of synthetic peptides containing heavy stable isotopes, which act as internal standards for quantitation of the MHC peptide antigens. SureQuant targeted quantitation using SureQuant targeted mass spec acquisition kits and reagents enables quantitation of target peptides with the absolute or relative quantitation module, which includes a system suitability standard and AQUA Ultimate Heavy and/or Light Peptide mixtures.
For Research Use Only. Not for use in diagnostic procedures.