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A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes
Published on: March 25, 2014
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Targeted Analyses of MHC-I Peptides Using TOMAHAQ
Marlene L Heberling1, Sam Pollock2, Qui T Phung1
1Proteomic and Genomic Technologies, Genentech, South San Francisco, CA, USA.
Methods in Molecular Biology (Clifton, N.J.)
|October 14, 2025
Summary
We developed a semiautomated method for enriching major histocompatibility complex class I (MHC-I) peptides, improving throughput and sensitivity for immune-therapeutics research. This technique enables rapid, reproducible analysis of MHC-I peptidomes for better understanding of immune regulation.
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.
