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BaileyNot availableLungSnap frozen tissue samples were briefly thawed and weighed prior to 30 S of mechanical homogenization (Fisher 150 mechanical homogenizer, using disposable generator probes) in 6 mL lysis buffer (0.02M Tris, 0.5% (w/v) IGEPAL, 0.25% (w/v) sodium deoxycholate, 0.15mM NaCl, 1mM EDTA, 0.2mM iodoacetamide supplemented with EDTA-free protease inhibitor mix). Homogenates were clarified for 10 min at 2,000g, 4°C and then for a further 60 min at 13,500g, 4°C. 2 mg of anti-MHC-I mouse monoclonal antibodies (W6/32) covalently conjugated to 1 mL of protein A sepharose (Repligen) using DMP as previously described (Purcell et al., 2019) were added to the clarified supernatants and incubated with constant agitation for 2 h at 4°C. The captured MHC-I/𝛽2m/immunopeptide complex on the beads was washed sequentially with 10 column volumes of low (isotonic, 0.15M NaCl) and high (hypertonic, 0.4M NaCl) TBS washes prior to elution in 10% acetic acid and dried under vacuum. The MHC-I-depleted lysate was then incubated with anti-MHC-II mouse monoclonal antibodies (HB-145) and MHC-II bound peptides were captured and eluted in the same conditions. Immunopeptides were separated from MHC-I/𝛽2m complex or MHC-II and simultaneously desalted by C18 capture using HLB prime columns (Waters, UK). Briefly, dried immunoprecipitates were reconstituted in buffer (1% acetonitrile,0.1% TFA) and applied to a HLB-prime columns and eluted sequentially under vacuum with increasing concentrations of acetonitrile up to 30% (v/v). Alternate fractions were combined and dried under vacuum. LC-MS/MS analysis of HLA-I and -II peptides HLA peptides were separated by an Ultimate 3000 RSLC nano system (Thermo Scientific) using a PepMap C18 EASY-Spray LC column, 2 µm particle size, 75 µm x 75 cm column (Thermo Scientific) in buffer A (0.1% Formic acid) and coupled on-line to an Orbitrap Fusion Tribrid Mass Spectrometer (Thermo Fisher Scientific,UK) with a nano-electrospray ion source. Peptides were eluted with a linear gradient of 3%-30% buffer B (Acetonitrile and 0.1% Formic acid) at a flow rate of 300 nL/min over 110 minutes. Full scans were acquired in the Orbitrap analyser using the Top Speed data dependent mode, preforming a MS scan every 3 second cycle, followed by higher energy collision-induced dissociation (HCD) MS/MS scans. MS spectra were acquired at resolution of 120,000 at 300 m/z, RF lens 60% and an automatic gain control (AGC) ion target value of 4.0e5 for a maximum of 100 ms. MS/MS resolution was 30,000 at 100 m/z. Higher-energy collisional dissociation (HCD) fragmentation was induced at an energy setting of 28 for peptides with a charge state of 2-4, while singly charged peptides were fragmented at an energy setting of 32 at lower priority. Fragments were analysed in the Orbitrap at 30,000 resolution. Fragmented m/z values were dynamically excluded for 30 seconds.PridePaul J SkippCentre for Proteomic Research B85, Life Sciences Building University of Southampton University Road Highfield Southampton, Hants. SO17 1BJ United KingdomCOMPLETE42270771 Nicholas B, Bailey A, McCann KJ, Wood O, Currall E, Johnson P, Elliott T, Ottensmeier C, Skipp P. Immunopeptidomics-guided identification of functional neoantigens in non-small cell lung cancer. NPJ Precis Oncol. 2026 10.1038/s41698-026-01539-2University of Southamptonpjss@soton.ac.ukAffinity purification coupled with mass spectrometry proteomicsMass SpectrometryLung CancerLungHlaNeoantigenCancerPeptidomehttps://www.ebi.ac.uk/pride/archive/projects/PXD028990Not availableRaw spectrum files were analyzed using Peaks Studio 10.6 build 20201015 (Zhang et al., 2012; Tran et al., 2017) and the data processed to generate reduced charge state and deisotoped precursorand associated product ion peak lists which were searched against the Uniprot database (20,350 entries, 2020-04-07) plus the corresponding mutanome for each sample (~1,000-5,000 sequences) and contaminants list in unspecific digest mode. Parent mass error tolerance was set a 5ppm and fragment mass error tolerance at 0.03 Da. Variable modifications were set for N-term acetylation (42.01 Da), methionine oxidation (15.99 Da), carboxyamidomethylation (57.02 Da) of cysteine. A maximum of three variable modifications per peptide was set. The false discovery rate (FDR) was estimated with decoy-fusion database searches (Zhang et al., 2012) and were filtered to 1% FDR. Downstream analysis and data visualizations of the Peaks Studio identifications was performed in R using associated packages (R Core Team, 2018; Wickham et al., 2019).ProteomicsHomo Sapiens (human)ab604@soton.ac.ukUnited Kingdom10.6019/PXD028990Non-small cell lung cancer (NSCLC) has poor survival even with modern checkpoint inhibitor therapies. Personalised vaccines based on short peptide neoantigens containing tumour mutations are an attractive precision medicine strategy, but identifying therapeutically relevant neoantigens remains challenging, with existing methods yielding positive responses in only 6% of candidates tested. We developed an immunopeptidomics approach to improve neoantigen identification in 24 NSCLC patients (15 adenocarcinoma, 9 squamous cell carcinoma). We directly identified one neoantigen and using whole exome sequencing, transcriptomics and mass spectrometry-based immunopeptidomics, we filtered predicted neoantigens based on observed cohort HLA peptide presentation. This approach achieved positive functional responses in 5 of 6 patients tested (83% success rate) with 13% of putative neoantigens (9 out of 70) eliciting strong responses. Bayesian modelling of our initial rules-based neoantigen selection further revealed patient specific peptide presentation patterns and propensities. Our findings demonstrate that incorporating donor-specific HLA peptide presentation data substantially improves neoantigen identification success rates and immune response specificity, advancing personalised cancer vaccine development.Immunopeptidomics-guided identification of functional neoantigens in non-small cell lung cancer.Nicholas Ben B, Bailey Alistair A, McCann Katy J KJ, Wood Oliver O, Currall Eve E, Johnson Peter P, Elliott Tim T, Ottensmeier Christian C, Skipp Paul PfalseImmunopeptidomics guided identification of neoantigens in non-small cell lung cancerDiscovery of tumour specific peptides (neoantigens) generating target specific personalised T cell therapies is difficult because of the complex relationship between genetic mutations and neoantigen presentation. Studies predicting neoantigens have a 5% success rate, and direct observations of neoantigens are even rarer. We have developed a mass spectrometry proteomics guided approach to identify neoantigens in non-small cell lung cancer (NSCLC). We hypothesised that by characterising the observable peptides presented by HLA molecules (the immunopeptidome) we might better predict which mutations lead to neoantigens. From a cohort of 24 NSCLC patients, we performed a detailed analysis of 3 adenocarcinoma and 3 squamous cell carcinoma samples. Our workflow identified neoantigens generating functional T cell responses in 5 out of 6 patients, with one directly observed neoantigen, and an overall response rate of 20% of predicted neoantigens.2026-07-032021-10-07PXD028990NEWT:6945NEWT:3555NEWT:2NEWT:157546NEWT:35554NEWT:38942NEWT:307972NEWT:32046NEWT:544496NEWT:2042546NEWT:45351NEWT:43179NEWT:4513NEWT:5722NEWT:376741NEWT:55153NCBITaxon:10407NEWT:1736309NEWT:1211601NEWT:876138NEWT:237561NEWT:5833NEWT:6928NEWT:10036NEWT:36745NEWT:1351NEWT:1438992NEWT:2649997NEWT:272563NEWT:224326NCBITaxon:79857NEWT:1096976NEWT:95648NEWT:3885NEWT:3888NEWT:1589NEWT:135622NEWT:6915NEWT:3649NEWT:101510NEWT:3880NEWT:272559NEWT:3641NEWT:383379NEWT:10029NEWT:1000589NEWT:85963NEWT:85962NEWT:317447NEWT:7955NEWT:7959NEWT:2261NEWT:31156NEWT:4565NEWT:1264690NEWT:515619NEWT:192875NEWT:34305NEWT:59729NCBITaxon:183674NEWT:224308NEWT:84645NEWT:626528NEWT:139927NEWT:4558NEWT:209285NEWT:1283NEWT:931281NEWT:4550NEWT:1000561NEWT:197NCBITaxon:79824NEWT:4787NCBITaxon:4563NEWT:5755NEWT:44689NEWT:3218NEWT:5759NEWT:1736231NEWT:1270NEWT:2242NEWT:4784NEWT:11320NEWT:360106NEWT:287NEWT:10117NEWT:10239NEWT:10116NEWT:1280NEWT:1735272NEWT:83334NEWT:83332NEWT:44685NEWT:317513NEWT:1148NEWT:580240NEWT:294128NEWT:11676NEWT:55571NEWT:100226NEWT:4530NEWT:4896NEWT:75058NEWT:13616NEWT:1094343NEWT:296543NEWT:1773NEWT:1895NEWT:1182590NEWT:935293NEWT:64152NEWT:4924NEWT:749200NEWT:990346NEWT:145953NEWT:257309NEWT:100816NEWT:263NEWT:230741NEWT:52283NEWT:284812NCBITaxon:1313NEWT:43330NEWT:408169NEWT:44544NEWT:4911NEWT:645463NEWT:3702NEWT:129249NEWT:990119NEWT:408172NEWT:408170NEWT:493760NEWT:260710NEWT:257313NEWT:400772NEWT:3708NEWT:128161NEWT:332648NEWT:106592NEWT:536231NEWT:460519NEWT:1187947NEWT:1432138NEWT:10312NEWT:1424507NCBITaxon:1773NEWT:9598NEWT:8030NEWT:1639NEWT:188229NEWT:3818NEWT:480NEWT:4909NEWT:67767NEWT:432359NEWT:46835NEWT:2711NEWT:376686NEWT:95486NEWT:9103NEWT:29159NEWT:10306NCBITaxon:2759NEWT:1233435NEWT:8022NEWT:145943NCBITaxon:4932NEWT:595536NEWT:240906NEWT:593117NEWT:3635NEWT:5811NEWT:235443NEWT:272624NEWT:411483NEWT:884019NEWT:198215NEWT:411490NEWT:983964NEWT:169963NEWT:32644NEWT:499175NEWT:476272NEWT:3747NEWT:367830NEWT:178616NEWT:410289NEWT:373153NEWT:352472NEWT:360094NEWT:470NEWT:1313NEWT:411469NEWT:84023NEWT:559292NCBITaxon:5811NEWT:411464NEWT:411460NEWT:2762NEWT:1174673NEWT:562NEWT:411470NEWT:33952NEWT:2094720NCBITaxon:2697049NEWT:28038NEWT:1423NEWT:4932NEWT:3603NEWT:2759NEWT:3847NEWT:327159NEWT:178876NEWT:327160NEWT:573NEWT:9031NEWT:7091NEWT:241368NEWT:42528NEWT:190802NEWT:9778NEWT:150475NEWT:9417NEWT:7111NEWT:347515NEWT:1216979NEWT:5180NEWT:256737NEWT:115104NEWT:1121114NEWT:1081927NEWT:1238993NEWT:67825NEWT:185579NEWT:13076NEWT:1249668NEWT:317NEWT:7227NEWT:7469NEWT:885318NEWT:9402NEWT:415540NEWT:550NEWT:675060NEWT:4081NEWT:554NEWT:98334NEWT:426428NEWT:7574NEWT:7215NEWT:575412NEWT:29204NEWT:2172103NEWT:507601NCBITaxon:6157NEWT:746360NEWT:6239NEWT:470150NEWT:216257NEWT:102169NEWT:9986NEWT:4054NEWT:73239NEWT:226186NEWT:1268063NEWT:8782NEWT:1263854NEWT:435590NEWT:1902NEWT:160488NEWT:28104NEWT:1908NCBITaxon:2NEWT:985076NEWT:1215323NEWT:52641NEWT:7038NEWT:6192NEWT:28532NCBITaxon:38727NEWT:353152NEWT:2829NEWT:366581NEWT:216599NEWT:216595NEWT:243230NEWT:8355NEWT:9685NEWT:7029NEWT:1080772NEWT:1283300NEWT:6183NEWT:6063NEWT:334747NEWT:61235NEWT:6289NEWT:436486NEWT:6287NEWT:300641NEWT:727NEWT:9796NEWT:725NEWT:170187NEWT:260707NCBITaxon:6191NEWT:1836NEWT:185431NEWT:29760NEWT:260704NEWT:703612NEWT:260705NEWT:80863NEWT:2697049NEWT:105231NEWT:1216981NCBITaxon:6073NEWT:884204NEWT:6279NEWT:1123869NEWT:9544NEWT:7370NEWT:83906NEWT:607699NEWT:6282NEWT:208964NEWT:1134506NEWT:575584NEWT:38783NEWT:8727NEWT:4006NEWT:8726NEWT:6426NEWT:6669NEWT:10090NEWT:4120NEWT:51515NEWT:5693NEWT:8724NEWT:51511NEWT:92867NEWT:8723NEWT:5334NEWT:37334NEWT:382352NCBITaxon:10359NEWT:242619NEWT:632957NEWT:373995NEWT:5689NEWT:544404NEWT:9925NEWT:8839NEWT:4232NEWT:2758385NEWT:4113NEWT:837NEWT:11298NEWT:171101NEWT:714NEWT:421932NEWT:196627NEWT:5691NEWT:48479NEWT:627025NEWT:1097677NEWT:61674NEWT:1117957NEWT:9913NEWT:4100NEWT:1076NEWT:6763NEWT:803NEWT:29722NEWT:380394NEWT:1692259NEWT:180066NEWT:135588NEWT:1843183NEWT:58002NEWT:326423NEWT:4577NEWT:1416333NEWT:5664NEWT:2157NEWT:1678078NEWT:749906NEWT:418985NEWT:146479NEWT:1911079NEWT:264203NEWT:1480154NEWT:1274414NEWT:27606NEWT:59202NEWT:9975NEWT:9612NEWT:38865NEWT:51953NEWT:3197NEWT:9615NEWT:10299NEWT:860688NEWT:36329NEWT:1147787NCBITaxon:3044782NEWT:72407NEWT:349741NEWT:9605NEWT:9606NEWT:157295NEWT:641501NEWT:7668NEWT:915099NEWT:74940NEWT:9721NEWT:137221NEWT:1450511NEWT:1143193NEWT:411901NEWT:9838NEWT:105884NCBITaxon:9615NEWT:58334NEWT:1193501NEWT:3055NEWT:58331NEWT:6326NEWT:6689NEWT:5476NEWT:1274432NEWT:1274426NEWT:70448NEWT:9825NEWT:1274423NEWT:393765NEWT:698936NEWT:39946NEWT:9823NEWT:9940NEWT:521001NEWT:39947NEWT:1274420NEWT:578458422707710000-0003-0023-8679