Immunopeptidomics is a rapidly evolving field that focuses on the identification and characterization of peptides presented by the major histocompatibility complex (MHC) on the cell surface. These peptides play a crucial role in immune surveillance and response, making their comprehensive analysis vital for understanding immune mechanisms and developing targeted immunotherapies. The advent of advanced mass spectrometry techniques, particularly those incorporating high sensitivity and accuracy, has significantly enhanced our ability to profile the immunopeptidome. Here we explores the latest advancements in immunopeptidomics, with a focus on the integration of data-independent acquisition (DIA) methods and the utilization of cutting-edge instruments like the modified Orbitrap Astral mass spectrometer. These innovations are paving the way for deeper insights into antigen presentation and neoantigen discovery, ultimately contributing to improved therapeutic strategies.
Class I MHC peptides were analyzed using liquid chromatography coupled with a modified Orbitrap Astral mass spectrometer. The system was interfaced with a Thermo Scientific™ Vanquish™ Neo UHPLC and a FAIMS Pro™ Duo interface. Various peptide loads were tested, ranging from ultra-low to higher sample inputs.
Preliminary results indicate that our workflow provides comprehensive immunopeptidome coverage across a range of sample loads, from ultra-low to high, without compromising data quality. Over 4,000 peptides were detected from ultra-low loads, with spectral quality remaining consistent across all loads. Additionally, more than 80% of peptides achieved MS2 correlation scores above 70%. We also intend to demonstrate a hybrid-DIA method, combining DIA and PRM immunopeptidomics workflows. This complementary workflow will allow for both comprehensive immunopeptidome profiling and sensitive quantification of specific markers.
Our preliminary results reveal that the modified Orbitrap Astral mass spectrometer delivers unmatched coverage and outstanding data quality, poised to revolutionize biological discoveries in immunopeptidomics.