Sensitive and high-throughput immunopeptidomics using the latest generation Orbitrap astral and ascend

Quantitative mass spectrometry is a central technology for characterizing human leukocyte antigen (HLA) landscape and represents a key data source for the development of therapeutic vaccines. However, a combination of faster throughput, higher sensitivity and accuracy is required for deeper coverage of peptides due to the recovery of low abundance HLA-peptides and complexity of samples. Here, we evaluate and extend DDA, DIA for the detection of HLA-I peptides using next generation of Thermo Scientific^TM Orbitrap^TM mass spectrometers (Orbitrap Ascend^TM and the Orbitrap Astral^TM) instruments. We optimized and validated DDA and DIA methods using synthetic peptide libraries, benchmarking their sensitivity through serial dilutions. After validation, these optimized methods were applied to the detection of HLA-I peptides from HCT116 cell lines and human plasma samples, aiming to explore the robustness of the techniques in complex biological matrices. Our results demonstrated that the latest generation Orbitrap mass spectrometers provide significantly enhanced sensitivity and throughput for immunopeptidomics applications. Moreover, the improved data acquisition methods led to a deeper coverage of the HLA-I peptide repertoire, even when working with limited sample inputs, thereby offering a powerful tool for immunopeptidomics.