Targeted Analyses of MHC-I Peptides Using TOMAHAQ.

Area of Science:

• Immunology
• Proteomics
• Mass Spectrometry

Background:

• Major histocompatibility complex (MHC) peptidomics is crucial for understanding immune regulation and developing immune-therapeutics.
• Current methods for isolating and quantifying MHC-bound peptides are challenging, limiting throughput, sensitivity, and reproducibility.
• Improvements are needed for rapid characterization of MHC peptidomes under various cellular conditions.

Purpose of the Study:

• To describe a robust, sensitive, and quantitative method for enriching MHC-I bound peptides.
• To enable high-throughput, semiautomated isolation of MHC-I peptides from diverse cell lines.
• To introduce TOMAHAQ (Triggered by Offset, Multiplexed, Accurate-mass, High-resolution, and Absolute Quantification) for targeted mass spectrometry of MHC-I peptides.

Main Methods:

• Semiautomated enrichment of MHC-I peptides using reusable antibody cartridges.
• Application to challenging adherent cell lines like MC38.
• Utilizing TOMAHAQ, a targeted mass spectrometry technique for multiplexed and absolute quantification.

Main Results:

• Achieved reproducible enrichment of MHC-I peptides with minimal hands-on time, enabling up to 96 simultaneous enrichments per day.
• Demonstrated high-throughput analysis of over 4000 unique MHC-I peptides from 250 million cells using nontargeted methods.
• TOMAHAQ provided quantitative sensitivity down to the low amol/μL level for neoepitope characterization.

Conclusions:

• The developed semiautomated MHC-I peptide enrichment method significantly enhances throughput, sensitivity, and reproducibility.
• This approach facilitates detailed characterization of MHC peptidomes and T-cell receptor targets for immune-therapeutics.
• TOMAHAQ coupled with robust peptide isolation offers a powerful tool for quantitative neoantigen analysis in cancer research.