Fig. 5: Comparison of Thunder-DDA-PASEF to previous MS immunopeptidomics methods analyzing JY HLA1p-enriched samples.

In this study, aliquots of a JY HLAIp sample were acquired on a timsTOF Pro-2 using Thunder-DDA-PASEF (Thunder; n = 3 inection replicates) or on an Orbitrap Exploris 480 using DDA without FAIMS (wo-FAIMS; n = 3) or with FAIMS either switching between three compensation voltages (CVs: −20, −50, −70) during the acquisition (FAIMS-3CVs; n = 3) or using single CVs per acquisition from −10 to −80 in steps of 10 (FAIMS-8CVs; n = 1 each). In addition, two previously reported JY HLAIp-profiling data sets were included: Demmers 2021⁴¹ using an Orbitrap Fusion Lumos in DDA with EThcD fragmentation (DDA EThcD; n = 3) and Pak 2021³³ using a Q Exactive (Q-ex.) HF-X in DIA and a big library for data analysis (DIA BigLib; n = 3). a Total HLAIps identified predicted to bind the JY HLA alleles (rank ≤ 2%). Colors indicate the instrument used. b Upset plot showing the number (barplot) and percentage (text) of HLAIps identified exclusively in each instrument or their combinations; the intersection matrix at the bottom indicates that the same peptides shown above (columns) were detected in the instruments (rows) highlighted with a blue dot. c Proportion of peptides (considering modifications) identified in function of their charge state exclusively as + 1, in multiple charge states including + 1 (+1, ≤ +2) or only as multiply charged (≤ +2). d Percentage of peptides identified exclusively by Thunder-DDA-PASEF (blue), exclusively by Orbitrap instruments (red) or in both type of instruments (pink) in function of the charge state.