MHC-associated peptides powerfully modulate T cell immunity and play a critical role in generating effective anti-tumor immune responses. Characterization of these peptides helps to generate therapeutic treatments and gain information on T cell mediated biomarkers. These peptides are challenging to characterize due to similar length, sequence conservation and lacking defined termini when compared to peptides generated upon enzymatic digestion. To overcome these challenges, use of PASEF (Parallel Accumulation and Serial Fragmentation) enables to generate high quality peptide spectra and resolve coeluting and isobaric peptides. Moreover, the capability to easily tailor the mobility space enables preferential detection of groups and sub-groups of relevant peptides.