MAETi enables high resolution MHC-I ligandome profiling
The identification of MHC class I (MHC-1) presented peptides (MHC1ps) by mass spectrometry (MS)-based immunopeptidomics is an essential approach to characterize antigen processing pathways and to define targets for tumor immunotherapies. However, existing sample preparation workflows typically require large sample amounts of sample material, limiting the applicability in high-throughput drug screenings, kinetic immunopeptidome studies and in clinical contexts where samples are scarce. 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 ß-alanine MAE-buffer for MAETi reduces background interferences, enhances peptide coverage, and boosts reproducibility. Comparing bulk ß-alanine-based MAE with bulk immunoprecipitation (IP), achieves similar or complementary immunopeptidome depth. Using two protocol layouts and HLA-tailored DDA- and DIA-PASEF schemes, we profiled immunopeptidomes from 1e4 to 1e6 JY cells yielding on average over 3,000 and 6,000 predicted binders in diaPASEF, respectively. Using a personalized database and single amino acid variants (SAVs) as proxy for neoantigens reveals robust detection and quantitation of SAVs down to 1e4 cells. Finally, MAETi enabled differential ligandome analysis of 1.5e4-5e4 immune cells isolated via fluorescence-based cell sorting providing biological insights into cell type-associated immunopeptidomes.  This renders MAETi a simple, fast and scalable method enabling robust MS-based immunopeptidomics for minimal sample inputs.
The identification of MHC class I (MHC-1) presented peptides (MHC1ps) by mass spectrometry (MS)-based immunopeptidomics is an essential approach to characterize antigen processing pathways and to define targets for tumor immunotherapies. However, existing sample preparation workflows typically require large sample amounts of sample material, limiting the applicability in high-throughput drug screenings, kinetic immunopeptidome studies and in clinical contexts where samples are scarce. 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 ß-alanine MAE-buffer for MAETi reduces background interferences, enhances peptide coverage, and boosts reproducibility. Comparing bulk ß-alanine-based MAE with bulk immunoprecipitation (IP), achieves similar or complementary immunopeptidome depth. Using two protocol layouts and HLA-tailored DDA- and DIA-PASEF schemes, we profiled immunopeptidomes from 1e4 to 1e6 JY cells yielding on average over 3,000 and 6,000 predicted binders in diaPASEF, respectively. Using a personalized database and single amino acid variants (SAVs) as proxy for neoantigens reveals robust detection and quantitation of SAVs down to 1e4 cells. Finally, MAETi enabled differential ligandome analysis of 1.5e4-5e4 immune cells isolated via fluorescence-based cell sorting providing biological insights into cell type-associated immunopeptidomes.  This renders MAETi a simple, fast and scalable method enabling robust MS-based immunopeptidomics for minimal sample inputs.
German cancer research center