Comparison of the MHC I immunopeptidome repertoire of B-cell lymphoblasts using two isolation methods

Significant technological advances in both affinity chromatography and mass spectrometry have facilitated the identification of peptides associated with the major histocompatibility complex class I (MHC I) molecules, and enabled a greater understanding of the dynamic nature of the immunopeptidome of normal and neoplastic cells. While the isolation of MHC I-associated peptides (MIPs) typically used mild acid elution (MAE) or immunoprecipitation (IP), limited information currently exists regarding their respective analytical merits. Here, we present a comparison of these approaches for the isolation of two different B-cell lymphoblasts cell models, and report on the recovery, reproducibility, scalability and complementarity of identification from each method. Both approaches yielded reproducible datasets for peptide extracts obtained from 2 to 100 million cells, with 2016 to 5093 MIPs, respectively. The IP typically provides up to 6.4 fold increase in MIPs compared to the MAE. We extended the comprehensiveness of these immunopeptidome analyses using personalized genomic database of B-cell lymphoblasts, and discovered that 0.4 % of their respective MIP repertoire harbored non-synonymous single nucleotide variations (also known as minor histocompatibility antigens, MiHAs).