Optimized Liquid and Gas Phase Fractionation Increases HLA-Peptidome Coverage for Primary Cell and Tissue Samples

MS is the most effective method to directly identify peptides presented on human leukocyte antigen (HLA) molecules. However, current standard approaches often use 500 million or more cells as input to achieve high coverage of the immunopeptidome, and therefore, these methods are not compatible with the often limited amounts of tissue available from clinical tumor samples. Here, we evaluated microscaled basic reversed-phase fractionation to separate HLA peptide samples offline followed by ion mobility coupled to LC–MS/MS for analysis. The combination of these two separation methods enabled identification of 20% to 50% more peptides compared with samples analyzed without either prior fractionation or use of ion mobility alone. We demonstrate coverage of HLA immunopeptidomes with up to 8107 distinct peptides starting with as few as 100 million cells. The increased sensitivity obtained using our methods can provide data useful to improve HLA-binding prediction algorithms as well as to enable detection of clinically relevant epitopes such as neoantigens. [Display omitted] •Deep immunopeptidome coverage using liquid and gas phase separation.•Up to 50% more HLA-I peptides using microscaled basic reversed-phase fractionation.•Ion mobility separation (FAIMS) increases HLA-I peptide identifications by up to 58%.•Increased sensitivity provided by these methods enables detection of neoantigens. Here, we evaluated off-line microscaled basic reversed-phase fractionation as well as the use of ion mobility coupled to LC–MS/MS for analysis of peptides presented on HLA-I. The two separation methods enabled identification of 20% to 50% more peptides compared with samples analyzed without either prior fractionation or use of ion mobility alone starting with as few as 100 million cells. The increased sensitivity obtained using our methods can enable detection of low abundant but clinically relevant epitopes such as neoantigens.