Development of an automated MHC-associated peptide enrichment method for immunopeptidomics analysis using AssayMAP large capacity cartridges

Significance of the Topic

The identification of MHC-bound peptides is essential for understanding immune recognition and developing immunotherapies, yet these peptides are present at extremely low abundance and require effective enrichment strategies.

Objectives and Overview of the Study

This work aimed to establish a fully automated immunopeptidomics workflow using large-capacity AssayMAP cartridges for MHC peptide enrichment and to assess its performance on a nanoflow Q-TOF mass spectrometer in terms of throughput and reproducibility.

Methodology and Instrumentation

The protocol integrates immunoaffinity capture on an Agilent AssayMAP Bravo platform with high-resolution mass spectrometry.

• Antibody immobilization: Anti-human MHC-I w6/32 antibody was cross-linked to 25 μL Protein A cartridges using dimethyl pimelimidate.
• Sample preparation: GRANTA-519 cell lysates were loaded (≈3 mg per cartridge) under non-denaturing conditions and processed in triplicate to enrich MHC complexes.
• Peptide cleanup: Eluted peptides were desalted on 5 μL C18 cartridges.
• LC-MS/MS analysis: An Agilent 1290 Infinity II LC system converted to nanoflow via UHPLC Nanodapter, coupled to a nanoESI source and 6550 iFunnel Q-TOF; samples were run using a 90-minute gradient and data-dependent acquisition.

Main Results and Discussion

The method demonstrated consistent retention times and signal intensities across three replicates. Each sample yielded 2282–2424 unique MHC class I peptides (3604 total unique), with a CV of ~3 % for peptide identification and ~11 % for peptide abundance quantitation. Analysis of peptide lengths revealed that 94 % were 8–11 residues long, predominantly 9-mers (~75 %). Motif analysis of nonamer peptides confirmed expected HLA-binding preferences, notably leucine or valine at the C terminus and conserved anchor residues at position 2.

Benefits and Practical Applications

The automated platform reduces hands-on time and variability, enabling high-throughput, reproducible immunopeptidomic profiling. It is adaptable to low-abundance samples and supports comparative studies and target discovery in immunology and clinical research.

Future Trends and Opportunities

Advances may include integration with bioinformatics pipelines for deeper epitope mapping, adaptation to multiplexed or single-cell workflows, implementation of data-independent acquisition to enhance coverage, and applications in personalized vaccine design and immune monitoring.

Conclusion

The automated MHC-associated peptide enrichment workflow using AssayMAP cartridges combined with nanoflow Q-TOF MS provides a robust, high-throughput solution for immunopeptidomic studies, delivering reliable peptide identification and quantitation with strong reproducibility.

References

No explicit literature references were provided in the source document.