Leveraging advanced mass spectrometry technology with Orbitrap Astral Zoom MS for in depth immunopeptidome profiling

Significance of the Topic

Immunopeptidomics elucidates peptides bound to MHC class I molecules on the cell surface, which are pivotal for immune recognition, vaccine design, and personalized cancer therapies.

Objectives and Study Overview

This study aims to evaluate the performance of the Thermo Scientific Orbitrap Astral Zoom mass spectrometer coupled with the Vanquish Neo UHPLC system for deep profiling of the immunopeptidome using a label-free data-dependent acquisition method that discriminates singly and multiply charged MHC class I peptides across a wide range of sample inputs.

Methodology

The methodology involved enriching MHC class I peptides from IM-9 human B-lymphocytes using a pan-specific W6/32 antibody on an automated platform. Peptides were extracted, dried, and injected to represent loads equivalent to 1e5–1e8 cells. Separation was performed on an Aurora Ultimate 25 cm×75 µm column over a 72-minute gradient. Data were acquired using data-dependent acquisition with differentiated FAIMS compensation voltages for singly (–20 V) and multiply (–45 V, –60 V) charged ions.

Instrument Used

• Orbitrap Astral Zoom mass spectrometer
• Vanquish Neo UHPLC system
• FAIMS Pro Duo interface
• Aurora Ultimate UHPLC column
• EASY-Spray ion source
• PEAKS Studio 12 (DeepNovo workflow)

Main Results and Discussion

The platform demonstrated high sensitivity, selectivity, and a dynamic range spanning seven orders of magnitude. Over 18 000 unique peptides were identified, revealing classical MHC-I binding motifs for 9-mer peptides. Quantitative linearity (R² = 0.99) was maintained across all loads (1e5–1e8 cells). Spectral quality was consistent from low to high inputs, ensuring reliable b and y ion series for confident identification.

Benefits and Practical Applications

• Enables deep immunopeptidome coverage with minimal sample input, suitable for tissue biopsies.
• FAIMS technology enhances detection of both singly and multiply charged peptides.
• High dynamic range and quantitative robustness support biomarker discovery and neoantigen profiling.
• Adaptable loading capacity accommodates diverse experimental designs from ultra-low to high sample amounts.

Future Trends and Opportunities

Advancements may include integration with real-time data analysis, higher-throughput FAIMS cycles, and refined workflows for neoantigen validation. Emerging applications encompass personalized cancer vaccines, autoimmune disease monitoring, and in-depth quality control for therapeutic protein characterization. Combining immunopeptidomics with single-cell analysis and artificial intelligence–driven motif prediction will further drive precision immunology.

Conclusion

The Thermo Scientific Orbitrap Astral Zoom MS with Vanquish Neo UHPLC and FAIMS offers unprecedented sensitivity, selectivity, and dynamic range for immunopeptidomics. Its ability to profile MHC class I peptides across a wide input range makes it a powerful tool for immunology research, biomarker discovery, and clinical applications.