Optimized enrichment and analysis of MHC-I peptides for comprehensive immunopeptidome profiling

Significance of the topic

The immunopeptidome consists of peptides bound to MHC-I molecules and defines the landscape of antigen presentation. Detailed profiling of this repertoire is crucial for understanding immune surveillance, guiding cancer vaccine development, and advancing personalized immunotherapies.

Objectives and study overview

This study aimed to develop a rapid, reproducible, and automation-compatible workflow for isolating and analyzing MHC-I-bound peptides from cell cultures. By optimizing immunoprecipitation supports, clean-up methods, and mass spectrometric detection, the authors sought to achieve high-depth coverage and precise quantitation of the immunopeptidome within a single working day.

Methodology

The protocol includes the following key steps:

• Cell culture and lysis: HCT116 cells were grown with or without IFN-γ stimulation and lysed using four different buffers to assess peptide yield.
• Antibody coupling: W6/32 anti-HLA antibody was covalently linked to Protein A/G magnetic agarose beads or CNBr-activated Sepharose.
• Immunoprecipitation: MHC-I complexes were captured from cell lysates on magnetic beads at 4 °C, followed by stringent washes.
• Peptide elution and clean-up: Elution at pH 1% TFA was spiked with a PRTC internal standard; peptides were cleaned using C18 spin tips or alternative formats.
• LC-MS/MS analysis: Peptides were separated by nanoLC and analyzed on Orbitrap Exploris 480 and Orbitrap Astral mass spectrometers with data processed by PEAKS Studio and DeepNovo.

Instrumentation

The following equipment and consumables were essential:

• Pierce Protein A/G Magnetic Agarose beads
• CNBr-activated Sepharose
• IonOpticks Aurora Ultimate XT nanoUPLC system
• Thermo Scientific Orbitrap Exploris 480 MS and Orbitrap Astral MS
• Pierce Peptide Retention Time Calibration Mixture (PRTC)

Main results and discussion

Optimization of lysis buffers showed that Mem-PER solubilization achieved the highest peptide identifications with CVs below 10%. Magnetic agarose beads outperformed CNBr‐Sepharose by yielding 15% more peptides. Comparison of clean-up formats revealed C18 spin tips delivered up to 7.5% greater peptide recovery versus SDB tips. Use of the Orbitrap Astral MS improved detection of low-abundance peptides, resulting in over 6 800 unique identifications and deeper coverage of 9-mer epitopes. Reproducibility was confirmed over five days with CVs under 5% for internal standard recovery and consistent chromatographic profiles.

Benefits and practical applications

The optimized workflow offers:

• High-throughput and automation capability with magnetic bead formats
• Robust recovery and quantitative reproducibility using internal standards
• Enhanced depth of immunopeptidome coverage on advanced MS platforms
• Compatibility with standard laboratory equipment and commercially available reagents

Future trends and potential applications

Advances may include integration with fully automated platforms for large cohort studies, coupling to next-generation MS instruments for greater sensitivity, and application in clinical biomarker discovery. Machine learning algorithms can further refine de novo sequencing and epitope prediction, enhancing immunotherapeutic target identification.

Conclusion

This work establishes a reproducible, scalable, and sensitive immunopeptidomics workflow. By combining magnetic bead immunoprecipitation, optimized clean-up, and high-resolution mass spectrometry, it enables comprehensive profiling of MHC-I peptides within a standard workday.

References

Hesnard L, Thériault C, Cahuzac M, Durette C, Vincent K, Hardy M-P, Lanoix J, Lavallée GO, Humeau J, Thibault P, Perreault C. Immunogenicity of Non-Mutated Ovarian Cancer-Specific Antigens. Current Oncology. 2024;31(6):3099-3121. DOI:10.3390/curroncol31060236