Validation and quantification of peptide antigens presented on MHCs using SureQuant.

Area of Science:

• Immunology
• Mass Spectrometry
• Proteomics

Background:

• Peptide-major histocompatibility complexes (peptide-MHCs) are crucial targets for vaccines and immunotherapies in cancer and infectious diseases.
• Accurate identification and quantification of target peptides are essential for designing effective therapeutic strategies.
• Existing methods may require enhanced validation and quantification capabilities for MHC peptides.

Purpose of the Study:

• To introduce and validate SureQuant MHC as a robust method for peptide-MHC analysis.
• To demonstrate the utility of SureQuant MHC for confirming peptide identity, performing relative quantification, and enabling absolute quantification.
• To provide immunologists with a reliable tool for validating antigenic targets and understanding MHC repertoire dynamics.

Main Methods:

• Utilizing internal standard parallel reaction monitoring (SureQuant) with synthetic stable isotope labeled (SIL) peptide standards.
• Employing pre-formed heavy isotope-labeled peptide-MHC complexes (hipMHCs) with SIL peptides for relative quantification.
• Developing an internal standard curve using varying amounts of hipMHCs loaded with SIL peptides for absolute quantification.

Main Results:

• Conclusive confirmation of putative MHC peptide identities through comparison with SIL peptide standards.
• Accurate relative quantification of MHC peptides using hipMHCs as internal controls.
• Precise absolute quantification of target peptides, crucial for assessing therapeutic modality suitability.

Conclusions:

• SureQuant MHC offers a powerful solution for validating antigenic targets and quantifying peptide-MHCs.
• The method supports diverse applications, from confirming peptide presence to determining abundance for therapeutic development.
• This approach enhances the ability to design and optimize peptide-MHC-based vaccines and immunotherapies.