The Choice of HLA‐Associated Peptide Enrichment and Purification Strategy Affects Peptide Yields and Creates a Bias in Detected Sequence Repertoire

: Understanding the most appropriate workflow for biochemical HLA-associated peptide enrichment prior to ligand sequencing is essential to achieve optimal sensitivity in immunopeptidomics workflows. We describe the use of different detergents for HLA solubilisation as well as complementary workflows to separate HLA-bound peptides from HLA protein complex components after their immunoprecipitation including HPLC, C18 cartridge and 5 kDa Filter. We observed that all solubilisation approaches tested led to similar peptide ligand identification rates, however, a higher number of peptides were identified in samples lysed with CHAPS compared with other methods. The HPLC method was superior in terms of HLA-I peptide recovery compared with 5 kDa filter and C18 cartridge peptide purification methods. Most importantly, we observed that both the choice of detergent and peptide purification strategy created a significant bias for the identified peptide sequence, and that therefore peptide repertoires were affected depending on the workflow of choice. Our results highlight the importance of employing a suitable strategy for HLA peptide enrichment and that obtained peptide repertoires do not necessarily reflect the true distributions of peptide sequences in the sample. This article is protected by copyright. All rights reserved.