Beyrle, J.; Distler, U.; Gomez-Zepeda, D.; Tenzer, S.

The identification of MHC class I presented ligands by mass spectrometry (MS)-based immunopeptidomics is an essential tool to characterize antigen processing pathways and to define targets for tumor immunotherapies. However, existing sample preparation workflows typically require large sample inputs, limiting the applicability in high throughput drug screenings, kinetic immunopeptidome studies and for scare samples in clinical contexts. To address this challenge, we developed Mild Acid Elution in a Tip (MAETi), a streamlined and antibody-free approach for high-sensitivity MHC-I immunopeptidome profiling from 1 Mio to as low as 25,000 cells. Using an optimized {beta}-alanine MAE-buffer for MAETi reduces background interferences, enhances peptide coverage, and boosts reproducibility. {beta}-alanine-based MAE achieves similar or complementary immunopeptidome depth compared to bulk immunoprecipitation (IP). This renders MAETi a low-cost and scalable method enabling robust MS-based immunopeptidomics for minimal sample inputs, enabling future applications such as kinetic studies, drug screening and the profiling of immunopeptidomies of rare cell populations.