Automated MHC-Associated Peptide Enrichment for Immunopeptidomics Analysis Using Agilent AssayMAP Bravo Large Capacity Cartridges

Significance of the Topic

Automated enrichment and analysis of MHC class I peptides is critical for understanding immune surveillance in viral infections and cancer immunotherapy
Characterization of the immunopeptidome enables rational design of vaccines and T cell based therapies
Traditional immunopeptidomics workflows are challenged by low peptide abundance and complexity requiring robust sample preparation and sensitive mass spectrometry

Aims and Study Overview

This study describes an automated workflow for MHC I peptide enrichment using the Agilent AssayMAP Bravo platform with large capacity 25 uL PAW cartridges and subsequent cleanup on 5 uL C18 cartridges
Objective was to demonstrate reproducibility and scalability of peptide capture from GRANTA 519 cell lysates and identification by nanoflow LC MS with data dependent acquisition and Byos data analysis

Methodology and Instrumentation

Antibody immobilization and immunoaffinity purification

• Anti human MHC I antibody crosslinked to protein A resin in 25 uL PAW cartridges using DMP crosslinker
• Cell lysates loaded at 3 mg per cartridge with controlled flow rates on AssayMAP Bravo liquid handler
• MHC I complexes eluted with 1 acetic acid and combined from six cartridges

Peptide cleanup and desalting

• Peptides separated from protein matrix on 5 uL C18 cartridges using acetonitrile trifluoroacetic acid gradients
• Eluates dried and stored at minus 80 across to LC MS

Nano LC MS analysis

• Agilent Infinity II LC converted to nanoflow with Infinity UHPLC Nanodapter
• Separation on 75 um x 25 cm C18 column at 60 and 90 minute gradient
• Detection on Agilent 6550 iFunnel Q TOF in data dependent acquisition with top 20 precursors

Data processing via Byos workflow

• Byonic search with nonspecific cleavage and variable modifications for oxidation and deamidation
• Byologic quantitation of MS1 extracted ion chromatograms with decoy filtering

Results and Discussion

Total ion chromatograms across three biological replicates showed highly consistent retention times and peak intensities indicating workflow reproducibility
Unique peptide identifications ranged from 2284 to 2426 per sample with a coefficient of variation below 3 percent and 3604 unique peptides across all runs
Quantitative reproducibility of total peptide abundance yielded a CV around 11 percent across days
Peptide length analysis confirmed the majority fall between 8 and 11 residues aligning with known MHC I ligands
Motif analysis of 9 mer peptides revealed strong preference for leucine or valine at anchor positions indicating high data quality

Benefits and Practical Applications

• Fully automated sample preparation reduces hands on time and variability
• High capacity cartridges enable processing of low abundance immunopeptidomes
• Sensitive nanoflow LC MS provides robust peptide identification and quantitation
• Reproducible results support vaccine target discovery and antigen profiling

Future Trends and Opportunities

• Scaling to multiple HLA alleles and patient derived samples for personalized immunopeptidomics
• Integration with data independent acquisition workflows for deeper coverage
• Advances in cartridge chemistry and microfluidics for single cell immunopeptidomics
• Automation of downstream bioinformatics for real time immune monitoring

Conclusion

The automated AssayMAP Bravo workflow with large capacity PAW cartridges delivers a robust and reproducible method for MHC I peptide enrichment
Combined with nanoflow LC MS and advanced data processing this approach accelerates immunopeptidomics studies
This scalable platform supports high throughput antigen discovery in research and clinical applications

Used Instrumentation

• Agilent AssayMAP Bravo with 25 uL PAW and 5 uL C18 cartridges
• Agilent 1290 Infinity II UHPLC with Infinity UHPLC Nanodapter
• Agilent 6550 iFunnel Q TOF LC MS
• Byos software incorporating Byonic and Byologic modules

References

1. Hunt DF et al Characterization of peptides bound to the class I MHC molecule HLA A2 1 by mass spectrometry Science 1992 255(5049) 1261 1263
2. Purcell AW Ramarathinam SH Ternette N Mass spectrometry based identification of MHC bound peptides for immunopeptidomics Nature Protocols 2019 14 1687 1707
3. Caron E et al Analysis of major histocompatibility complex immunopeptidomes using mass spectrometry Molecular and Cellular Proteomics 2015 14(12) 3105 3117
4. Wu S Wu LF Human breast cancer cell line phosphoproteome revealed by an automated and highly selective enrichment workflow Agilent Technologies Application Note 5994 0315EN 2018