MAETi: Mild acid elution in a tip enables immunopeptidome profiling from 25,000 cells

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), an antibody-free approach for low input MHC-1 immunopeptidome profiling. Using an optimized β-alanine MAE-buffer for MAETi reduces background interferences, enhances peptide coverage, and boosts reproducibility. Comparing bulk β-alanine-based MAE with bulk immunoprecipitation (IP), achieves similar or complementary immunopeptidome depth. Using two protocol layouts we further profiled initial inputs from 25,000 to 1 Million cells with HLA-tailored DDA- and DIA-PASEF schemes yielding on average over 1,000 and 3,000 predicted binders, respectively (DIA). This renders MAETi a facile, fast and scalable method enabling robust MS-based immunopeptidomics for minimal sample inputs.