Mild acid elution and MHC immunoaffinity chromatography reveal similar albeit not identical profiles of the HLA immunopeptidome

ABSTRACT: To understand and treat immunology-related diseases, a comprehensive, unbiased characterization of major histocompatibility complex (MHC) peptide ligands is of key importance. Preceding the analysis by mass spectrometry, MHC peptide ligands are typically isolated by MHC immunoaffinity chromatography (MHC-IAC). Less often, mild acid elution (MAE) is used to extract MHC class I peptide ligands. MAE may provide a cheap alternative to MHC-IAC for suspension cells, but it is thought to be hampered by the high number of contaminating peptides not derived from the MHC. Here, we optimized the MAE protocol yielding MHC peptide ligand purities of more than 80%. Subsequently, the qualitative and quantitative performance of MAE was benchmarked in direct comparisons to MHC-IAC. Peptides obtained by MAE and MHC-IAC were similar in numbers, identities and to a large extent intensities. A striking difference was the percentage of peptides containing cysteinylated cysteine residues, which was more than five times higher in MAE as compared to MHC-IAC. This benefitted the discovery of MHC allotype specific, distinct cysteinylation frequencies at individual positions of MHC peptide ligands. MAE revealed many MHC ligands with unmodified, N-terminal cysteine residues which get lost in MHC-IAC workflows. The results support the idea that MAE is particularly valuable for the high-confidence analysis of post-translational modifications by avoiding the exposure of the investigated peptides to enzymes and reactive molecules in the cell lysate. Our improved and carefully documented MAE workflow represents a high-quality, cost-effective alternative to MHC-IAC for suspension cells and should be applicable also in laboratories not specialized in MHC peptide ligand analyses.

INSTRUMENT(S): LTQ Orbitrap XL

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): B Cell, Monocyte, Leukocyte

SUBMITTER: Theo Sturm  

LAB HEAD: Hans-Georg Rammensee

PROVIDER: PXD012771 | Pride | 2020-11-11

REPOSITORIES: Pride