Next-generation immunopeptidomics with hybrid-DIA: Comprehensive identification and enhanced quantitative performance

Significance of the topic

Immunopeptidomics characterizes peptides presented by MHC molecules and is central to understanding immune surveillance, vaccine design and neoantigen-driven immunotherapies. Improving both the depth of peptide discovery and the quantitative reliability of measurements is critical for translating immunopeptidome profiles into robust biological and clinical insights. The hybrid-DIA approach presented here aims to merge broad, library-free discovery with high-sensitivity targeted quantitation on modern high-speed mass analyzers, addressing common trade-offs between coverage and quantitation in current workflows.

Objectives and overview

The study evaluates a hybrid data-independent acquisition (hybrid-DIA) workflow implemented on the Orbitrap Astral Zoom mass spectrometer coupled to a Vanquish Neo UHPLC system. Goals were to demonstrate that hybrid-DIA preserves the breadth of conventional DIA (discovery) while embedding targeted MS2 events to achieve improved sensitivity and quantitative precision across a wide dynamic range, and to benchmark identification and quantitation performance for HLA class I peptides.

• Compare peptide identification yield between standard DIA and hybrid-DIA.
• Assess quantitative linearity, limit of quantitation (LOQ) and dynamic range for targeted HLA peptides.
• Demonstrate practical sample preparation and data analysis suitable for immunopeptidomics studies.

Methodology

The experimental workflow combined immunoaffinity enrichment of MHC class I peptides from cultured HCT116 cells with nanoLC–MS/MS analysis using hybrid acquisition. Key methodological points:

• Cell culture and enrichment: HCT116 cells were grown with/without IFN-γ. MHC class I complexes were immunoprecipitated using the W6/32 antibody coupled to magnetic agarose beads and peptides eluted with 1% TFA. Typical input corresponded to peptides from ~1e7 cells per capture and injections equivalent to 1e6 cells for LC–MS analysis.
• LC separation: Peptides were separated on IonOpticks Aurora Ultimate 25×75 XT C18 column using a Vanquish Neo UHPLC with a 72 min total gradient and analyzed via an EASY-Spray source.
• Acquisition strategy: Hybrid-DIA integrates wide-window DIA scans for unbiased discovery with embedded targeted (tMS2) scans directed at pre-specified peptide targets to boost sensitivity, taking advantage of the Orbitrap Astral Zoom analyzers high scan speed.
• Data processing: Library-free directDIA analysis was performed using Biognosys Spectronaut (version noted as Spectronaut 20). Targeted peak extraction and verification used Skyline. Database searches used UniProt human sequences with a no-enzyme search parameter appropriate for MHC peptides.

Used instrumentation

• Mass spectrometer: Thermo Scientific Orbitrap Astral Zoom MS (Astral analyzer with Zoom capability).
• UHPLC: Thermo Scientific Vanquish Neo UHPLC System.
• LC column: IonOpticks Aurora Ultimate 25 XT C18.
• Source: Thermo Scientific EASY-Spray.
• Sample prep automation: KingFisher Apex Purification System and Thermo Scientific Pierce MHC Class I Antibody Coupling Kit.
• Software: Spectronaut (directDIA), Skyline; UniProt human protein database used for searches.

Main results and discussion

The hybrid-DIA workflow showed parity with standard DIA in discovery coverage while substantially improving targeted sensitivity and quantitative performance:

• Identification depth: Average peptide counts for hybrid-DIA closely matched conventional DIA (both methods identified on the order of 20–25k peptide-spectrum matches/peptides in the reported experiments), indicating no loss of immunopeptidome coverage when embedding targeted scans.
• Quantitative agreement: Relative quantitation between hybrid-DIA and standard DIA was highly correlated, demonstrating that incorporating targeted scans does not bias comparative analyses across conditions.
• Sensitivity and linearity: Targeted peptides (20 HLA-I standards) exhibited high quantitative linearity (most targets R2 > 0.9). The hybrid approach achieved sensitivity into the attomole (amol) range for some peptides, with LOQs reported below 10 amol for the most responsive analytes and linear dynamic ranges spanning multiple orders of magnitude (up to ~5 orders reported for select peptides).
• Dynamic range and calibration: Calibration curves for example peptides showed strong linearity from amol to fmol, supporting precise quantitation across biologically relevant concentration ranges.
• Practical performance: Skyline-extracted peaks for low-level calibration points (e.g., ~1.5 amol) were reliably detected and quantified when targeted MS2 events were included, highlighting the added sensitivity of the Astral Zoom analyzer within the hybrid format.

Discussion points emphasize that the hybrid-DIA approach leverages the high scan speed and sensitivity of modern Orbitrap analyzers to deliver targeted-level detection without sacrificing DIA's comprehensive sampling. Library-free directDIA processing preserves flexibility for discovery while targeted extraction improves confidence at low abundance.

Benefits and practical applications

• Combined discovery and quantitation: Allows researchers to survey broad peptide repertoires while simultaneously obtaining high-quality quantitative data for predefined targets (e.g., putative neoantigens).
• High sensitivity: Attomole-level LOQs enable detection of low-abundance HLA peptides that are often missed with discovery-only methods.
• Robust relative quantitation: High correlation with conventional DIA supports adoption in comparative studies (e.g., treated vs. untreated cells) without introducing quantitation bias.
• Compatibility with library-free workflows: Reduces dependence on spectral libraries, facilitating experiments across diverse sample sets and conditions.

Practical use cases include neoantigen discovery pipelines, biomarker validation for immunotherapies, and deeper characterization of antigen presentation dynamics in cell models or clinical samples.

Future trends and applications

• Integration with AI and advanced spectral prediction to further increase identification confidence and reduce the need for empirical libraries.
• Scaling to clinical throughput: Shorter gradients, optimized sample prep and automated immunoenrichment could enable larger cohorts and more routine clinical applications.
• Enhanced multiplexing and parallelized targeted assays to monitor large panels of candidate epitopes in the same run without compromising discovery capacity.
• Improvements in front-end sample handling and single-cell-compatible immunopeptidomics to expand the method to scarce clinical materials.
• Standardization of hybrid-DIA acquisition and data-processing pipelines to improve reproducibility across labs and instruments.

Conclusion

The hybrid-DIA strategy implemented on the Orbitrap Astral Zoom MS effectively combines the comprehensive sampling of DIA with the sensitivity and quantitative precision of targeted MS2, delivering high peptide coverage and attomole-level quantitation for HLA class I peptides. Library-free data processing (directDIA) preserves discovery flexibility while embedded targeted scans enhance sensitivity for predefined targets, making hybrid-DIA a valuable approach for translational immunopeptidomics and neoantigen research.

References

The summary is based on the presented experimental report describing hybrid-DIA implementation on the Thermo Scientific Orbitrap Astral Zoom MS and Vanquish Neo UHPLC system; data processing with Biognosys Spectronaut (directDIA) and Skyline; and sample preparation using Thermo Scientific Pierce kits and KingFisher Apex automation. No separate literature list was provided in the source document.