Enhancing Immunopeptide Profiling with Orbitrap Astral Mass Spectrometer for Unbiased Discovery of Neoantigens

Significance of the Topic

Immunopeptidomics reveals peptides presented by MHC molecules on the surface of cells, providing a direct window into antigen presentation and enabling the discovery of tumor-specific neoantigens for immunotherapy and personalized medicine.

Objectives and Study Overview

This work demonstrates the performance of the Thermo Scientific Orbitrap Astral mass spectrometer coupled to the Vanquish Neo UHPLC system in label-free data-dependent acquisition (DDA) analysis of MHC class I peptides from HCT-116 cells. The study focuses on sensitivity across sample loads, optimization of FAIMS compensation voltages (CVs), and isolation width settings to maximize peptide identification.

Methodology and Instrumentation

• Sample Preparation: Immunocapture of class I MHC peptides from 1×10⁸ HCT-116 cells using W6/32 antibody resin, cleanup on StageTips, and dilution to represent 1×10⁴–1×10⁶ cell equivalents.
• Chromatography: Vanquish Neo UHPLC in trap-and-elute mode with PepMap Neo Trap cartridge and Aurora Ultimate 25 cm×75 µm column, 72 min gradient.
• Mass Spectrometry: Orbitrap Astral MS with EASY-Spray Ion Source and FAIMS Pro Duo interface. Full MS resolution at 120 000, MS2 cycle time 0.6 s, optimized FAIMS CVs at −50 V and −60 V, isolation window 1 m/z.
• Data Analysis: PEAKS Studio 11 with DeepNovo workflow, no-enzyme database search against UniProt human proteome for combined database and de novo peptide identification.

Main Results and Discussion

The platform achieved high sensitivity across sample loads from 1×10⁵ to 1×10⁷ cell equivalents, identifying up to ~17 700 peptides per run. Optimal FAIMS CV settings increased coverage by 15–25%, and a narrow isolation window improved the PSM-to-MS2 ratio. Peptide length and charge state distributions matched expected HLA-I patterns (predominantly +2 charge, 8–11 residues).

Benefits and Practical Applications

• Enhanced sensitivity enables reliable analysis of low-input samples down to 1×10⁵ cell equivalents.
• High dynamic range and throughput support deep immunopeptidome profiling for neoantigen discovery.
• Compatibility with clinical specimens, including small biopsies, improves translational research potential.
• FAIMS and optimized acquisition parameters refine selectivity and maximize identification depth.

Future Trends and Opportunities

Emerging developments may include integration with data-independent acquisition (DIA) methods, single-cell immunopeptidomics, and machine learning–driven peptide annotation to further enhance sensitivity, throughput, and biological insight.

Conclusion

The combination of the Orbitrap Astral mass spectrometer and Vanquish Neo UHPLC system offers a robust, high-sensitivity platform for comprehensive immunopeptidome profiling. Optimized FAIMS CVs and acquisition settings enable deep coverage across a broad range of sample loads, facilitating unbiased neoantigen discovery and supporting both basic research and clinical applications.