Immunopeptidomics: Harnessing cutting-edge technology for unprecedented depth and accessibility in analysis to leverage the immune response for precision therapy

Significance of the Topic

Immunopeptidomics maps peptides bound to MHC molecules to guide precision immunotherapies, enabling identification of tumor specific and pathogen derived antigens and informing personalized treatment strategies.

Objectives and Study Overview

This study evaluates the performance of a new LC FAIMS Orbitrap Astral mass spectrometer for deep immunopeptidome profiling of IM-9 human multiple myeloma cells, aiming to detect and annotate potential neoantigens from low material inputs with high throughput.

Methodology and Instrumentation

Cell lysis with NP-40 and automated immunopeptide enrichment using AssayMAP Bravo with pan-HLA class I and II antibodies
Acid elution, C18 desalting and nano-UHPLC separation on Vanquish Neo system
Data-dependent acquisition with FAIMS Pro Duo interface, Orbitrap MS1 and Astral MS/MS analyzers synchronized to process multiple ion packets
FAIMS compensation voltages: class I at -25, -50, -70 V; class II at -40, -60 V

Main Results and Discussion

Class I peptides: up to 40 551 unique 8–12 mer peptides (≈65 % 9-mers) from 1e7 cell equivalent input, charge states predominantly +2 and +3; sequence motifs matched HLA alleles including A*02:01
Class II peptides: 8 927 to 23 435 peptides (13–17 AA) from 2e5 to 5e6 cells
High-dynamic range MS1 survey and high-accuracy Astral MS/MS enabled confident sequence assignment and low-abundance precursor detection from limited samples

Benefits and Practical Applications

• Four-fold faster throughput and double proteome depth compared to previous platforms
• Antigen discovery from biopsy-scale tissue down to 25 mg
• Quantitative and qualitative profiling for neoantigen identification in oncology and infectious disease

Future Trends and Potential Applications

1. Routine immunopeptidome analysis from small clinical biopsies to guide personalized therapy
2. Development of off-the-shelf TCR, CAR-T and vaccine modalities based on rapid antigen mapping
3. Real-time monitoring of treatment response via sequential immunopeptidome profiling

Conclusion

The integration of FAIMS and the Orbitrap Astral mass spectrometer with nano-UHPLC provides unprecedented sensitivity and throughput for immunopeptidomics, enabling deep coverage from minimal input material and accelerating antigen discovery for precision immunotherapy.

Reference

• Salzler RR, Cheung N, Salvato F et al. Immunopeptidomics using Vanquish Neo UHPLC and Orbitrap Astral MS. Thermo Fisher Scientific Case Study CS003393-EN, 2024.