75% of the expected sequences per single injection even at >10,000 peptide Pepyrus library. Pepyrus-assisted identification of rare neoantigens from 0.1 fmol in a complex background was feasible. Personalized Pepyrus libraries facilitated the identification of clinically relevant peptides from melanoma patients, several of which were previously undetected using MS. The customization of Pepyrus allows rapid generation of patient- or disease-specific peptide libraries that enable the confident identification of rare HLA peptides from immunopeptidomics data as well as the generation of large training datasets to improve spectrum, retention time, and ion mobility prediction tools.]]>