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

Mass spectrometry (MS)-based immunopeptidomics is essential for characterizing MHC class I (MHC-I) immunopeptides and defining targets for tumor immunotherapies. Conventional workflows require large sample amounts, limiting their use in low-input scenarios, high-throughput screens, and kinetic studies. We developed Mild Acid Elution in a Tip (MAETi), an antibody-free, low-input method for MHC-I immunopeptidome profiling. An optimized {beta}-alanine MAE buffer reduces background, enhances peptide coverage, and improves reproducibility. Compared with bulk immunoprecipitation, {beta}-alanine MAE achieves similar or complementary peptide depth. Using two protocol formats we profiled 1E4-1E6 JY cells, detecting on average over 3,000 and 6,000 predicted binders, respectively. A personalized database with single amino acid variants (SAVs) enabled robust identification and quantitation of SAV epitopes from only 1E4 JY cells. Additionally, MAETi supported differential ligandome analysis of 1.5E4-5E4 FACS-sorted immune cells, revealing cell type-specific ligandome repertoires. MAETi provides a simple, fast, and scalable workflow for MS-based immunopeptidomics from minimal sample input.