Peptides presented on the cell surface by the major histocompatibility complex (MHC) play a vital role in immune response, making identification of neoantigens critical for immunotherapy. Mass spectrometry (MS) serves as a valuable tool for immunopeptidomics. These measurements can be challenging due to low starting material and low sample preparation recovery. The Orbitrap Astral™ mass spectrometer enables researchers to delve deeper, with the new levels of sensitivity and selectivity, providing comprehensive insights into the immunopeptidome and neoantigen discoveries.

Class I MHC peptides were obtained by immunocapture with W6/32-conjugated resin on 100 million HCT-116 cells. For proof-of-concept studies, performance on low-loads was performed by diluting the starting material down to lower amounts. The MHC-bound peptides were eluted and subjected Liquid Chromatography (LC) coupled to MS analysis on an the Orbitrap Astral mass spectrometer interfaced with a Thermo Scientific™ Vanquish™ Neo LC and FAIMS Pro™ Interface.

Preliminary findings suggest that the Orbitrap Astral MS enables deep immunopeptidome coverage from low starting inputs without compromising data quality. Over 3,500 peptides from 1e5 cell equivalents, >9,000 peptides from 1e6 cell equivalents were identified using our platform. Our initial data also suggests that the peptide identifications are of high quality. Results are validated on two different search engines and show similarly high coverage. We successfully achieved high reproducibility between runs with over 75% of peptides detected across all three technical injections. Overall, our preliminary results suggest that the Orbitrap Astral produces deep coverage with exceptional data quality, enabling vital biological discovery in the field of immunopeptidomics.