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KlattData-dependent acquisitionMass SpectrometryNot availableMachine learningImmunopeptidomeHla assignmenthttps://www.ebi.ac.uk/pride/archive/projects/PXD061101Cell Suspension CultureImmunopurification of MHC class I ligands For immunopurification suspension cells were harvested through direct resuspension, adherent cell lines after incubating for 15 min with CellStripper solution (Corning™, Cat# 25056CI). Harvested cells were pelleted and washed three times in ice-cold sterile PBS (Gibco). For experiments with BV173, AML14, and JMN cell lines 50*106 cells were used, for experiments with LP-1, JJN3, and Nalm-6 cell lines 150*106 cells were used. Cells were lysed in 7.5 ml of 1% CHAPS (Sigma-Aldrich, Cat# C3023) dissolved in PBS and supplemented with protease inhibitors (cOmplete, Cat# 11836145001). Cell lysis was performed for 1 hour at 4°C, lysates were spun down for 1 hour with 20,000 g at 4°C, and supernatant fluids were isolated. Affinity columns were prepared as follows: 40 mg of Cyanogen bromide-activated-Sepharose 4B (Sigma-Aldrich, Cat# C9142) were activated with 1mM hydrochloric acid (Sigma-Aldrich, Cat# 320331) for 30 min. Subsequently, 1 mg of W6/32 antibody (BioXCell, Cat #BE0079) was coupled to sepharose in the presence of binding buffer (150 mM sodium chloride, 50 mM sodium bicarbonate, pH 8.3; sodium chloride: Sigma-Aldrich, Cat# S9888, sodium bicarbonate: Sigma-Aldrich, Cat#S6014) for at least 2 hours at room temperature. Sepharose was blocked for 1 h with glycine (Sigma-Aldrich, Cat# 410225) and washed 3 times with PBS. Supernatants of cell lysates were run over the columns through peristaltic pumps with a 1 ml/min flow rate overnight in a cold room. Affinity columns were washed with PBS for 30 min, water for 30 min, then run dry, and MHC complexes were subsequently eluted five times with 200 μl 1% trifluoracetic acid (TFA, Sigma/Aldrich, Cat# 02031). The TFA eluates were pooled and then split in as many portions as settings were investigated (either 3 portions with 30%, 40%, and 50% ACN settings and 5 portions with 5%, 20%, 35%, 50%, and “mix” settings). For separation of MHC ligands and their MHC complexes C18 columns (Sep-Pak C18 1 cc Vac Cartridge, 50 mg Sorbent per Cartridge, 37-55 μm Particle Size, Waters, Cat# WAT054955) were prewashed with 80% acetonitrile (ACN, Sigma-Aldrich, Cat# 34998) in 0.1% TFA and equilibrated with two washes of 0.1% TFA. Samples were loaded, washed again with 0.1% TFA and eluted in 400 μl of either 30%, 40%, or 50% ACN in 0.1%TFA for BV173, AML14, and JMN cells or for LP-1, JJN3, and Nalm-6 cells with 4x200 ul of either 5%, 20%, 35% and 50% ACN in 0.1%TFA or 200ul each of 5%, 20%, 35% and 50% ACN in 0.1%TFA for the “mix” setting. The sample volume was reduced by vacuum centrifugation for mass spectrometry analysis. Solid Phase Extractions (SPE) In-house C18 mini columns were prepared as follows: for SPE of one sample two small disks of C18 material (1mm in diameter) were punched out from CDS Empore™ C18 disks (Fisher Scientific, Cat# 13-110-018) and transferred to the bottom of a 200 µl Axygen pipette tip (Fisher Scientific, Cat# 12639535). Columns were washed once with 100 µl 80%ACN/0.1%TFA and equilibrated with 3 times 100 µl 1%TFA. All fluids were run through the column by centrifugation in mini tabletop centrifuges and eluates were collected in Eppendorf tubes. Then, dried samples were resuspended in 100 µl 1%TFA and loaded onto the columns, washed twice with 100 µl 1%TFA, ran dry, and eluted with 50 µl 80%ACN/0.1% TFA. Again, the sample volume was reduced by vacuum centrifugation. LC-MS/MS analysis of MHC ligands Samples were analyzed by LC-MS/MS (Orbitrap Exploris, Thermo Fisher). 480 mass spectrometer (Thermo Fisher Scientific). A 44 min gradient was applied using an EASY-nLC 1200 system (Thermo Fisher Scientific) with an in-house packed column (C18-AQ 1.9 µm beads; Dr. Maisch Reprosil-Pur 120). MS1 resolution was set to 120’000, an MIPS peptide filter with relaxed restrictions was applied, the minimum intensity threshold was specified to 50’000, dynamic exclusion occurred for 20 s and charge states 1-5+ were allowed as precursors.PrideNot availableMass spectrometry data were processed using Byonic software (version 4.5.2, Protein Metrics, Palo Alto, CA) through a custom-built computer server. Mass accuracy for MS1 was set to 10 ppm and 20 ppm for MS2, respectively. Digestion specificity was defined as unspecific and only precursors with charges 1,2, 3, and up to 2 kDa were allowed. Protein FDR was disabled to allow a complete assessment of potential peptide identifications. Oxidation of methionine, phosphorylation of serine, threonine, and tyrosine as well as cysteinylation of cysteine were set as variable modifications for all samples. Samples were searched against the UniProt Human Reviewed Database with common contaminants added. Peptides were selected with a minimal log prob value of 1.3 indicating p-values for PSMs of <0.05 and duplicates removed.ProteomicsMartin KlattCOMPLETECharité-University Medicine BerlinHomo Sapiens (human)41903651 Mecklenbräuker S, Skoczylas P, Biernat P, Zaghla BKQ, Atay B, Hossam M, Król-Józaga B, Jasiński M, Pienkowski VM, Sanecka-Duin A, Popp O, Haji M, Szatanek R, Mertins P, Kaczmarczyk J, Keller U, Blum A, Klatt MG. Identification of MHC Ligands Through Allele-Guided Isolation Combined With Machine Learning for Improved MHC Assignment Using ARDisplay-I. Mol Cell Proteomics. 2026 25(5):101560 10.1016/j.mcpro.2026.101560martin.g.klatt@gmail.comCharite - University Medicine BerlinGermany10.6019/PXD061101The isolation of major histocompatibility complex (MHC) ligands and subsequent analysis by mass spectrometry is considered the gold standard for defining targets for T cell-based immunotherapies. However, as many targets of high tumor specificity are only presented at low abundance on the cell surface of tumor cells, the efficient isolation of these peptides is crucial for their successful detection. Here, we demonstrate how optimizing the MHC ligand isolation strategy, based on both the presenting MHC alleles and the individual peptide level, enhances the identification of specific MHC ligands. This ideally acknowledges not only the hydrophobicity but also the post-translational modifications of the respective MHC ligands. To further improve the identification and characterization of MHC ligands, we developed an MHC class I ligand prediction algorithm (ARDisplay-I) that outperforms current state-of-the-art tools when benchmarked against competitors such as netMHCpan 4.1, MixMHCpred, or MHCflurry. Implementing these strategies can augment the development of T cell receptor-based therapies by improving the identification of novel immunotherapy targets and enriching the resources available in the computational immunology field through a superior MHC presentation prediction algorithm.Identification of MHC Ligands Through Allele-Guided Isolation Combined With Machine Learning for Improved MHC Assignment Using ARDisplay-I.Mecklenbräuker Shima S, Skoczylas Piotr P, Biernat Paweł P, Zaghla Badeel Kh Q BKQ, Atay Bilge B, Hossam Mai M, Król-Józaga Bartłomiej B, Jasiński Maciej M, Pienkowski Victor Murcia VM, Sanecka-Duin Anna A, Popp Oliver O, Haji Mohamed M, Szatanek Rafał R, Mertins Philipp P, Kaczmarczyk Jan J, Keller Ulrich U, Blum Agnieszka A, Klatt Martin G MGfalseIdentification of MHC ligands through allele guided isolation combined with machine learning for specific MHC assignmentIsolation of MHC ligands and subsequent analysis by mass spectrometry is considered the gold standard for defining targets for T cell-based immunotherapies. However, as many targets of high tumor-specificity are only presented at low abundance on the cell surface of tumor cells, the efficient isolation of these peptides is crucial for their successful detection. Furthermore, since multiple MHC alleles can present the same MHC ligand, improving the prediction and assignment to a specific MHC allele is highly desirable. Here, we demonstrate how optimizations of the MHC ligand isolation strategy support the identification of specific MHC ligands and how the hydrophobicity of presented peptides as well as their post-translational modifications need to be considered as exemplified by, the detection of cysteinylated MHC ligands from cancer germline antigens or point-mutated neoepitopes. To further improve the identification and characterization of MHC ligands, we developed a novel MHC class I ligand prediction algorithm (ARDisplay-I) that outperforms the current state-of-the-art. Moreover, ARDisplay-I facilitates the assignment of peptides to the appropriate MHC allele, especially if multiple alleles have to be considered. Implementing these strategies can augment the development of T cell receptor-based therapies by facilitating the identification of novel immunotherapy targets and enriching the resources available in the computational immunology field.2026-06-082025-02-23PXD061101NEWT:3555NEWT:71647NEWT:35554NEWT:32046NEWT:544496NEWT:2042546NEWT:32049NEWT:1392696NEWT:259447NEWT:45351NEWT:445974NEWT:43179NEWT:180454NEWT:5722NCBITaxon:1280NEWT:1129NEWT:309807NEWT:55153NEWT:309800NEWT:281395NEWT:1211601NEWT:876138NEWT:44271NEWT:10036NEWT:1590NEWT:498019NEWT:1351NEWT:1438992NEWT:1352NEWT:2649997NEWT:1147161NEWT:224326NEWT:1333499NCBITaxon:79857NEWT:1096976NEWT:95648NEWT:1589NEWT:135622NEWT:1349NEWT:96731NEWT:383379NEWT:10029NEWT:913645NEWT:44491NEWT:556484NEWT:317447NEWT:4688NEWT:7955NEWT:7959NEWT:2261NEWT:31156NEWT:3112NEWT:4442NEWT:192875NEWT:1214915NEWT:12637NEWT:59729NEWT:2164133NEWT:108061NEWT:1316931NEWT:224308NEWT:3347NEWT:511145NEWT:931281NEWT:658457NEWT:1310161NEWT:77133NEWT:295358NEWT:145481NCBITaxon:79824NCBITaxon:4563NEWT:5755NEWT:44689NEWT:44447NEWT:5759NEWT:1736231NEWT:1392NEWT:498217NEWT:2242NEWT:11320NEWT:286NEWT:391619NEWT:246196NEWT:287NEWT:246197NEWT:633149NEWT:10239NEWT:44685NEWT:161934NEWT:1148NEWT:5508NEWT:5507NEWT:410661NEWT:55571NEWT:35500NEWT:1140NCBITaxon:2157NEWT:1143NEWT:4896NEWT:1390NEWT:11557NEWT:1094343NEWT:1336795NEWT:644042NEWT:294NEWT:1773NEWT:1182590NEWT:3712NEWT:82380NEWT:935293NEWT:375146NEWT:118698NEWT:263NEWT:118696NEWT:52283NEWT:284812NEWT:8175NEWT:43330NEWT:1603293NEWT:408169NEWT:44664NEWT:47946NEWT:3702NEWT:243277NEWT:7067NEWT:118694NEWT:2850NEWT:118691NEWT:33548NEWT:274NEWT:408172NEWT:408170NEWT:3708NEWT:332648NEWT:44670NEWT:536231NEWT:376219NEWT:1436733NEWT:460519NEWT:572307NEWT:1432138NEWT:1424507NEWT:1194599NEWT:272844NEWT:9483NEWT:1513458NCBITaxon:40559NEWT:84588NEWT:1679718NEWT:480NEWT:67767NEWT:46835NEWT:109757NEWT:582580NEWT:300852NEWT:1502NEWT:376686NEWT:95486NEWT:508771NEWT:1883446NEWT:253NCBITaxon:2759NEWT:1233435NEWT:93061NEWT:93062NCBITaxon:4932NEWT:644223NEWT:235443NEWT:108458NEWT:272623NEWT:272624NEWT:320637NEWT:983964NEWT:118499NEWT:32644NEWT:527796NEWT:499175NEWT:109779NEWT:3745NEWT:1715989NCBITaxon:4751NEWT:476272NEWT:3747NEWT:195051NEWT:3988NEWT:1255228NEWT:649908NEWT:410289NEWT:373153NEWT:352472NEWT:1071661NEWT:360094NEWT:470NEWT:41364NEWT:1313NEWT:39491NCBITaxon:5811NEWT:29491NEWT:2014887NEWT:33952NEWT:261756NEWT:571256NEWT:40483NEWT:272634NEWT:9031NEWT:1872122NEWT:7091NEWT:141262NEWT:108931NEWT:1055524NEWT:4087NEWT:9778NEWT:306NEWT:150475NEWT:303NEWT:267872NEWT:7111NEWT:347515NEWT:9534NEWT:5180NEWT:256737NEWT:4090NEWT:4092NEWT:9541NEWT:4093NEWT:4096NEWT:185579NEWT:941442NEWT:13076NEWT:7108NEWT:40479NEWT:4076NEWT:317NEWT:1006581NEWT:885318NEWT:39488NEWT:550NEWT:4081NEWT:273068NEWT:554NEWT:98334NEWT:451516NEWT:36185NEWT:1225786NEWT:7574NEWT:1715256NEWT:30640NEWT:575412NEWT:929793NEWT:29204NEWT:28112NEWT:114155NEWT:6494NEWT:6491NEWT:507601NEWT:643680NEWT:214092NCBITaxon:6157NEWT:6239NEWT:162425NEWT:216257NEWT:102169NEWT:9986NEWT:4054NEWT:1268063NEWT:1263854NEWT:118503NEWT:2059687NEWT:160488NEWT:28104NEWT:407821NCBITaxon:2NEWT:985076NEWT:1215323NEWT:986NEWT:52641NEWT:7159NEWT:28532NEWT:353152NEWT:8496NEWT:27448NEWT:1194669NEWT:51329NEWT:243230NEWT:1080772NEWT:519NEWT:8479NEWT:8485NEWT:6063NEWT:49240NEWT:104395NEWT:6289NEWT:6287NEWT:300641NEWT:727NEWT:9796NEWT:725NEWT:469008NEWT:256318NEWT:9321NEWT:206411NCBITaxon:6191NEWT:596153NEWT:229533NEWT:1836NEWT:80863NEWT:105231NEWT:52638NEWT:57075NEWT:4097NEWT:884204NEWT:9785NEWT:6279NEWT:1123869NEWT:9544NEWT:7370NEWT:83906NCBITaxon:780NCBITaxon:1502NEWT:6282NEWT:1134506NEWT:38783NEWT:4006NEWT:6426NCBITaxon:5476NEWT:6669NEWT:9935NEWT:89453NEWT:287889NEWT:51515NEWT:588596NEWT:51511NEWT:8845NEWT:92867NEWT:92866NEWT:5334NEWT:51750NEWT:202950NEWT:382352NEWT:413071NEWT:632957NEWT:9925NEWT:89462NCBITaxon:9606NEWT:8839NEWT:90690NEWT:4232NEWT:2758385NEWT:11298NEWT:171101NEWT:714NEWT:421932NEWT:312017NEWT:196627NEWT:200308NEWT:200302NEWT:870435NEWT:284593NEWT:100392NEWT:5315NEWT:9913NEWT:265872NEWT:105841NEWT:337330NEWT:2666255NEWT:2285NEWT:999810NCBITaxon:5693NEWT:1392998NEWT:380394NEWT:981087NEWT:180066NEWT:226900NEWT:231490NEWT:430615NEWT:563041NEWT:326423NEWT:452467NEWT:198822NEWT:36111NEWT:326424NEWT:1416333NEWT:1678078NEWT:749906NEWT:418985NEWT:1817808NEWT:749907NEWT:145263NEWT:694569NEWT:27606NEWT:9739NEWT:59202NEWT:9975NEWT:51953NEWT:3197NEWT:860688NEWT:13443NEWT:36329NEWT:62615NCBITaxon:3044782NEWT:160235NEWT:72407NEWT:349741NEWT:157295NEWT:2096NEWT:192222NEWT:9721NEWT:137221NEWT:9963NEWT:1617910NEWT:565050NEWT:2785785NEWT:411901NEWT:105884NCBITaxon:9615NEWT:58334NEWT:1193501NEWT:58331NEWT:6689NEWT:298315NEWT:70448NEWT:698936NEWT:9940NEWT:3168NEWT:521001NEWT:192011NEWT:1813887NEWT:118702NEWT:118700NEWT:224911NEWT:118705NCBITaxon:11552NEWT:6945NEWT:184922NEWT:6703NEWT:2104NEWT:2NEWT:157546NEWT:474186NEWT:38942NEWT:307972NEWT:1496NEWT:2102NEWT:45474NEWT:1590841NEWT:4513NEWT:1247NEWT:70667NEWT:6938NCBITaxon:10407NEWT:1736309NEWT:4751NEWT:10360NEWT:47664NEWT:317548NEWT:15819NEWT:3654NEWT:237561NEWT:5833NEWT:6928NEWT:1239NEWT:36745NEWT:272563NEWT:82688NEWT:3885NEWT:3888NEWT:5821NEWT:44139NCBITaxon:4896NEWT:6915NEWT:3649NEWT:101510NEWT:28903NEWT:3880NEWT:272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