Unprecedented depth and data quality in immunopeptidome profiling with the Orbitrap Astral mass spectrometer

Unprecedented depth and data quality in immunopeptidome profiling with the Orbitrap Astral MS — Technical Note 003570

Importance of the topic

Immunopeptidomics identifies peptides presented by MHC class I molecules and is central to understanding immune recognition, neoantigen discovery, and personalized immunotherapies. The analytical challenge stems from extreme dynamic range, low-abundance peptides, lack of predictable proteolytic termini, and limited clinical sample amounts. Advances in mass spectrometry and gas-phase ion separation can dramatically increase identification depth and confidence, enabling discovery of tumor-specific antigens from scarce material.

Objectives and study overview

This technical note evaluates data-dependent acquisition (DDA) performance for MHC class I peptide profiling using the Thermo Scientific Orbitrap Astral mass spectrometer coupled to a Vanquish Neo UHPLC and FAIMS Pro Duo interface. The study aims to: (1) assess peptide identification depth across different sample loads (cell-equivalent E+5, E+6, E+7), (2) optimize FAIMS compensation voltages (CVs) and quadrupole isolation windows, and (3) demonstrate spectral quality and dynamic range for confident peptide identification and quantitation.

Methodology

Sample preparation and types:

• MHC class I peptides were immunoaffinity-captured (W6/32 antibody) from HCT-116 cells; HeLa proteins were elastase-digested to produce non-tryptic peptides for method tests.
• Extracted peptide material was diluted to represent injections equivalent to 1E5, 1E6, and 1E7 cells.

LC and separation:

• Trap-and-elute UHPLC workflow on a Vanquish Neo system using an IonOpticks Aurora Ultimate XT 25 cm × 75 μm C18 column at 50 °C; flow ~0.2 μL/min; mobile phases: 0.1% FA in water (A) and 0.1% FA in 80% ACN (B).
• Backward-flush trap-elute operation and rigorous wash procedures to minimize carryover for low-input samples.

MS acquisition and gas-phase separation:

• Orbitrap Astral MS operated in DDA with FAIMS Pro Duo. Single-CV scans (-80 to -10) and optimized double-CV combinations (notably -60/-50) were compared.
• Full MS: 120,000 resolution, m/z 350–800, AGC target high, max IT 100 ms. MS2: narrow isolation windows (1.0–1.2 m/z depending on load), HCD NCE ~29%, fragment scan range 110–1500 m/z, max IT 100 ms.
• FAIMS enhanced S/N and selectivity, shifting charge state distributions across CVs and enriching multiply charged species at lower CVs.

Data analysis:

• PEAKS Studio 12 with the DeepNovo Peptidome workflow was used for combined database (no-enzyme) and de novo identification against UniProt human (20,607 entries), with variable Ox(M) and carbamidomethyl(C) set, precursor tolerance 10 ppm, fragment 0.02 Da, peptide-level FDR 1%, DeepNovo score ≥70.

Instrumentation used

• Thermo Scientific Orbitrap Astral mass spectrometer.
• Thermo Scientific FAIMS Pro Duo interface.
• Thermo Scientific Vanquish Neo UHPLC system with PepMap Neo trap cartridge and IonOpticks Aurora Ultimate XT 25 cm × 75 μm C18 analytical column.
• Thermo Scientific EASY-Spray ion source.

Main results and discussion

FAIMS optimization and CV selection:

• FAIMS CVs strongly affected peptide identifications and charge distributions. For HCT-116 MHC-I material, the double CV combination -60 and -50 delivered the deepest coverage, accounting for ~67% of peptides across injections; overlap between the two CVs was ~22%, indicating complementary ion populations.
• For HeLa elastase-derived peptides, -50 and -40 CVs identified the most unique peptides, illustrating sample-dependent optimal CV choice. Practical recommendation: test several CVs on representative samples, or use -40/-60 as broadly effective defaults.

Isolation window optimization and load dependence:

• Narrower quadrupole isolation windows reduce spectral complexity and improve PSM rates but can limit ion transmission at very low sample loads. The study found windows of 1.0 m/z for higher loads (E+6, E+7) and 1.2 m/z for the lowest load (E+5) as effective compromises.
• PSM:MS2 ratios declined at very high loads when isolation windows were too narrow, highlighting the need to tune isolation width based on sample amount to maximize identification efficiency.

Depth, dynamic range, and spectral quality:

• Average peptide identifications (n=3) scaled with input: ~5,299 (E+5), 9,867 (E+6), and 15,501 (E+7) unique peptides using the optimized -60/-50 FAIMS method.
• Peptide length distribution centered on 8–9 amino acids, matching expectations for Class I peptides and validating specificity.
• Measured dynamic range extended up to seven orders of magnitude across injection conditions, demonstrating sensitivity to both highly abundant and scarce peptides.
• Spectral quality was maintained across loads due to AGC-controlled injection times: example peptide SADPGNLKY showed consistent annotated MS2 spectra and linear MS1 peak area across E+5 to E+7 loads, supporting robust identification and quantitation.
• Orbitrap Astral provided MS2 information below 200 m/z, capturing small fragments and immonium ions important for confirming non-tryptic immunopeptides.

Search engine metrics:

• PEAKS score distributions (–10LogP and CAA) indicated high-confidence peptide-spectrum matches and good agreement between de novo tags and database hits, supporting annotation reliability.

Benefits and practical applications

• The combined Orbitrap Astral + FAIMS workflow substantially increases immunopeptidome coverage from limited sample inputs, enabling deeper discovery of MHC-bound peptides including potential tumor-specific antigens from biopsy-scale material.
• Flexible FAIMS CV combinations and tunable isolation windows allow tailoring to sample composition and load, improving identification rates and selectivity.
• AGC-driven spectral consistency facilitates quantitative comparisons across samples and conditions without manual per-peptide tuning.
• Improved low-mass fragment detection supports confident assignment of non-tryptic peptides common in immunopeptidomics.

Future trends and potential uses

• Integration of optimized FAIMS gas-phase fractionation with advanced MS platforms will continue to push sensitivity, enabling routine immunopeptidome profiling from clinical biopsies and small tumor samples.
• Coupling DDA improvements with complementary acquisition strategies (e.g., DIA) and enhanced de novo algorithms will increase identification of novel TSAs and post-translationally modified epitopes.
• Automated CV-selection schemes and adaptive acquisition strategies that respond to sample-specific ion populations could simplify method transfer between sample types.
• Higher-throughput sample processing and standardized immunoaffinity workflows will support translational research, vaccine target discovery, and personalized cancer immunotherapy pipelines.

Conclusions

The Orbitrap Astral MS combined with FAIMS Pro Duo and a high-performance nanoLC system provides a sensitive, high-dynamic-range platform for immunopeptidome profiling. Careful optimization of FAIMS CVs and quadrupole isolation width yields substantial gains in peptide identifications from limited inputs, while AGC-mediated acquisition preserves spectral quality across loads. The workflow facilitates confident detection and quantitation of MHC class I peptides, supporting discovery applications in immunology and oncology.

Reference

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4. Hassan C, et al. Accurate quantitation of MHC‑bound peptides by application of isotopically labeled peptide‑MHC complexes. Journal of Proteomics. 2014;109:240–244.