Personalized immunopeptidomics using differential ion mobility technology enables direct profiling of neoantigens on colorectal cancer tissues

Knowing the nature of human leukocyte antigen (HLA)-presented peptides (HLAp) is indispensable to realize the precision medicine 12 while direct identification of HLAp from scarce tissue specimens still confronts technical challenges 3. Here we optimized the isolation procedures for HLAp and employed the technology of multiple gas phase fractionation differential ion mobility mass spectrometry (DIM-MS) to overcome that limitation. From 1e8 cells of HCT116, our method detected 9,249 unique HLAp including 11 peptides with missense mutations (neoantigens). Next, from individual differential immunopeptidome analysis using 40 mg each of colon cancer and adjacent normal tissues (n = 17), 44,785 HLAp including 2 neoantigens, KRAS7-16(G12V) and CPPED1226-234(R228Q), were identified. Furthermore, colon cancer-specific amino acid usage at C-terminus of HLAp was also found. Thus, this technology drastically improves the depth of immunopeptidomic analysis and allows direct determination of clinically important cancer antigens.