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Mathieu"],"software":["Not available"],"tissue":["Colon"],"sample_protocol":["Primary tissues Primary human colon adenocarcinoma tumor samples were purchased from BioIVT (n=15) and Reprocell (n=11). Tissue samples were taken from patients receiving surgery as the first line of treatment and were flash-frozen in liquid nitrogen.  Isolation of MAPs  The W6/32 antibodies (BioXcell) were coupled to CNBR-activated sepharose 4B beads (Cytivia) as described in Sirois et al, and the beads were stored at 4°C in PBS pH 7.2 and 0.02% NaN3 until use. CRC adenocarcinoma primary samples (573-1346 mg) were cut into small pieces (cubes, ~3 mm in size) and 6 ml of ice-cold PBS containing protein inhibitor cocktail (Sigma) was added. Tissues were first homogenized twice for 20 seconds using an Ultra Turrax T25 homogenizer (IKA-Labortechnik) set at a speed of 20 000 rpm. Then, 700 μL of ice-cold 10X lysis buffer (5% w/v CHAPS) was added to each sample. After 60-minute incubation with tumbling at 4°C, tissue samples were spun at 16,600g for 20 minutes at 4°C. Supernatants were transferred into new tubes containing 1 mg of W6/32 antibody covalently-cross-linked to Sepharose beads and incubated with tumbling for 3 hours at 4°C. The samples were transferred into poly prep chromatography columns (Biorad) and the liquid mixture was eluted by gravity. Sepharose beads were first washed with 11 mL PBS, then with 11 mL of 0.1X PBS and finally with 11 mL of water. MHC I complexes were eluted from sepharose beads by acidic treatment using 1% trifluoroacetic acid (TFA). Filtrates containing peptides were separated from MHC I subunits (HLA molecules and β-2 macroglobulin) using home-made stage tips packed with two 1 mm diameter octadecyl (C-18) solid-phase extraction disks (EMPORE). Stage tips were pre-washed first with methanol then with 80% acetonitrile (ACN) in 0.1% TFA, followed by 0.1 % TFA and finally with 1% TFA. Samples were loaded onto the stage tips and the peptides were retained on the stage tips while the HLA molecules and β-2 macroglobulin were found in the flow through. Stage tips were washed with 1% TFA and then with 0.1% TFA and peptides were eluted with 30% ACN in 0.1%TFA. The peptides were dried using vacuum centrifugation and then stored at -20°C until MS analysis.  Mass spectrometry analyses  Dried peptide extracts were resuspended in 4% FA and loaded on an IonOpticks Aurora 25-cm C18 column. Peptides were eluted using a 106 min linear gradient from 0% to 38% ACN (0.2% FA) and a 300 nL/min flow rate on a Neo Vanquish nano LC system. Samples were analyzed with an Orbitrap Tribrid Ascend mass spectrometer (Thermo Fisher Scientific) in positive ion mode with Nanoflex source at 3kV. Each full MS spectrum, acquired with a 120 000 resolution was followed by 40 MS/MS spectra, where the most abundant multiply charged ions were selected for MS/MS sequencing with a resolution of 30 000, a normalized automatic gain control target of 100%, an injection time of 59 ms, and collisional energy of 27%. LC-MS instrument was controlled using Xcalibur version 4.6 (Thermo Fisher Scientific)."],"repository":["Pride"],"labhead":["Pierre Thibault"],"instrument_platform":[""],"labhead_affiliation":["Institute for Research in Immunology and Cancer, Department of Biochemistry, Department of Chemistry, Université de Montréal, Québec, Canada H3T 1J4"],"submission_type":["COMPLETE"],"publication":["42106149 Courcelles M, Hardy MP, Durette C, Lanoix J, Minati R, Laverdure JP, Vincent K, Perreault C, Thibault P. Non-canonical transcription and splicing shape the colorectal cancer immunopeptidome in MSI and MSS tumors. Mol Cell Proteomics. 2026:101581 10.1016/j.mcpro.2026.101581"],"submitter_affiliation":["IRIC"],"labhead_mail":["pierre.thibault@umontreal.ca"],"technology_type":["Immunopeptidomics","Data-dependent acquisition","Mass Spectrometry"],"disease":["Colon Cancer"],"submitter_keywords":["Immunopeptidomics","Colorectal cancer","Cancer immunotherapy","Tumor-specific antigen","Mass spectrometry"],"full_dataset_link":["https://www.ebi.ac.uk/pride/archive/projects/PXD071022"],"quantification_method":["Not available"],"modification":[""],"data_protocol":["Database generation  Global cancer databases were constructed as previously described. In brief, each database is composed of two parts:  the personalized canonical proteome and the cancer-specific proteome. For the canonical proteome, in each sample the single-base mutations with a minimum alternate count setting of 5 were identified using freeBayes v1.0.2. Transcript expression was quantified in transcripts per million (tpm) with kallisto v0.43.0 in stranded mode with “-b 100” and other default parameters. We then used pyGeno v2.0.0 to insert high-quality sample-specific single-base mutations (freeBayes quality > 20) in the reference exome and export sample-specific sequences of known proteins generated by expressed transcripts (tpm > 0) to generate FASTA files of personalized canonical proteomes. The cancer-specific proteome was generated as described previously, with the following exceptions: 8 mTEC samples (GEO accessions GSE127825, GSE127826) were used instead of 6 mTECs, the k-mer occurrence allowed in mTECs was 1 instead of 0, and polypeptides were linked with “JJ” linkers before concatenation to the personalized canonical proteome. Briefly, the R1 and R2 fastq files of each sample were trimmed as reported above, and the reverse mapping reads were reverse complemented using the fastx_reverse_complement function of the FASTX-Toolkit v0.0.14. K-mer databases (24 or 33-long) were generated using Jellyfish v2.2.377. A single k-mer database was generated for each tumor sample, while the eight mTEC samples were combined in a unique database by concatenating their fastq files. Because the duration of k-mer assembly increases exponentially above 30 million k-mers, each cancer 33-nucleotide-long k-mer database was filtered based on a sample-specific threshold on occurrence (the number of times that a given k-mer is present in the database) to reach a maximum of 30 million k-mers for the assembly step. After this filtering, k-mers present more than once in the mTECs k-mer database were removed from each sample database. The remaining k-mers were assembled into contigs with NEKTAR, an in-house developed software. Briefly, one of the submitted 33-nucleotide-long k-mer is randomly selected as a seed that is extended from both ends with consecutive k-mers overlapping by 32 nucleotides on the same strand (-r option disabled, as we were working with stranded sets of k-mers). The assembly process stops when either no k-mers can be assembled or when more than one k-mer fits (–a 1 option for linear assembly). Then a new seed is selected, and the assembly process resumes until all k-mers from the submitted list have been used once. Finally, we 3-frame translated the contigs using an in-house python script and split amino acid sequences at internal stop codons. The resulting polypeptides were linked with “JJ” linkers and concatenated with the respective personalized canonical proteome for each sample. is generated using an alignment-free RNA-seq workflow called k-mer profiling because current mappers and variant callers poorly identify structural variants. This second dataset enabled the detection of peptides encoded by any reading frame of any genomic origin (including structural variants), as long as they were cancer specific (that is, absent from normal cells). We used MHC IIhi medullary thymic epithelial cells (mTEChi) cells as a “normal control”. To identify RNA sequences that were cancer specific, we chopped cancer RNA-seq reads into 33-nucleotide long sequences, called k-mers with Jellyfish 2.2.3, from which we removed k-mers present in syngeneic mTEChi cells. Redundancy inherent to the k-mer space was removed by assembling overlapping cancer-specific k-mers into longer sequences, called contigs, which were 3-frame translated in silico. We then concatenated the canonical and cancer-specific proteomes to create a global cancer database, one for each analyzed sample.   MAP Identification  Database searches were conducted using the PeaksXPro software, version 10.6 (Bioinformatics Solutions Inc.). Error tolerances for precursor mass and fragment ions were set to 10.0 ppm and 0.01 Da, respectively. Variable modifications included Oxidation (M), and Deamidation (NQ). Peaks searches were then loaded into MAPDP, which was used to apply the following filters: selecting peptides of 8-11 amino acids in length, with rank eluted ligand threshold ≤ 2% based on NetMHCpan-4.1b predictions, using a 5% false discovery rate (FDR). FDR was calculated using the decoy hits imported from Peaks, which employ the decoy-fusion strategy."],"omics_type":["Proteomics"],"species":["Homo Sapiens (human)"],"submitter_mail":["mathieu.courcelles@umontreal.ca"],"submitter_country":["Canada"],"doi":["10.6019/PXD071022"],"pubmed_abstract":["Treatment with immune checkpoint inhibitors in colorectal cancer (CRC) has largely benefited patients with microsatellite instability-high (MSI-H) and not the larger proportion of patient with microsatellite-stable (MSS) tumors. This clinical dichotomy has fueled the view that high mutational burden is the dominant driver of tumor immunogenicity and that MSS CRC fails to respond because it is \"antigen poor\". To directly test this premise and define the origins of presented tumor antigens, we integrated HLA class I immunopeptidomics and matched RNA-seq from 26 primary CRC tumors spanning MSI-H and MSS subtypes. Using patient-specific canonical and cancer-specific proteogenomic databases, we identified 115,292 unique MHC-associated peptides (MAPs) across 61 HLA alleles, with a mean of 9,292 MAPs per tumor and no significant difference in MAP counts between MSI-H and MSS tumors. In toto, we identified 266 tumor antigens, all coded by unmutated genomic sequences, comprising 70 aberrantly expressed tumor-specific antigens (aeTSAs) and 196 tumor-associated antigens (TAAs). In our cohort, MSS tumors presented more TAAs and a comparable number of aeTSAs per tumor relative to MSI-H tumors. In TCGA-COAD stratified analyses (483 tumors), MSS tumors yielded more presentable aeTSAs and TAAs per patient than MSI-H tumors. Across both subtypes, aeTSAs arose predominantly from intronic translation, UTR usage, retroelement activation, and germline-like transcription, including recurrent aeTSAs from PIWIL1, L1TD1, and endogenous retroviral loci. Together, these data demonstrate that MSS CRC is not antigen poor and highlight non-canonical translation as a major, previously underappreciated contributor to the CRC immunopeptidome."],"pubmed_title":["Non-canonical transcription and splicing shape the colorectal cancer immunopeptidome in MSI and MSS tumors."],"pubmed_authors":["Courcelles Mathieu M, Hardy Marie-Pierre MP, Durette Chantal C, Lanoix Joel J, Minati Robin R, Laverdure Jean-Philippe JP, Vincent Krystel K, Perreault Claude C, Thibault Pierre P"],"additional_accession":[]},"is_claimable":false,"name":"Comprehensive proteogenomic analysis identifies immune targets in microsatellite stable and unstable colorectal cancers","description":"Immunotherapy in colorectal cancer (CRC) has largely focused on microsatellite instability (MSI) tumors, based on the assumption that high mutational burden drives immunogenicity. However, most CRCs are microsatellite-stable (MSS) and remain unresponsive to checkpoint blockade. Using proteogenomic profiling of 26 primary CRC tumors, we identified 115,292 MHC-I–associated peptides across 61 HLA alleles, yielding 196 tumor-associated antigens (TAAs) and 70 aberrantly expressed tumor-specific antigens (aeTSAs).","dates":{"publication":"2026-05-26","submission":"2025-11-21"},"accession":"PXD071022","cross_references":{"TAXONOMY":["NEWT:108018","NEWT:377960","NEWT:1131","NEWT:190486","NEWT:2737160","NEWT:2042546","NEWT:148942","NEWT:193528","NEWT:295546","NEWT:258594","NEWT:1126","NEWT:1129","NEWT:89187","NEWT:309807","NEWT:89184","NEWT:309800","NEWT:57577","NEWT:156563","NEWT:1323429","NEWT:876138","NEWT:44271","NEWT:193516","NEWT:2315688","NEWT:44270","NEWT:592321","NEWT:1073882","NEWT:6819","NEWT:93217","NEWT:428386","NEWT:1117","NEWT:498257","NEWT:10036","NEWT:32264","NEWT:10034","NEWT:10031","NEWT:82569","NEWT:661410","NEWT:488003","NEWT:224326","NEWT:109376","NEWT:376619","NEWT:3765","NEWT:2433","NEWT:1190602","NEWT:3767","NEWT:433727","NEWT:135622","NEWT:672127","NEWT:656061","NEWT:37965","NEWT:656064","NEWT:3760","NEWT:36630","NEWT:381198","NEWT:1176302","NEWT:418106","NEWT:10029","NEWT:913645","NEWT:641809","NEWT:44250","NEWT:77151","NEWT:317447","NEWT:698761","NEWT:5530","NEWT:868565","NEWT:135674","NEWT:1562698","NEWT:11308","NEWT:12637","NEWT:2164133","NEWT:1180","NEWT:2767846","NEWT:1343074","NEWT:314344","NEWT:108061","NEWT:143292","NEWT:2767842","NEWT:314345","NEWT:224308","NEWT:3134131","NEWT:49928","NEWT:160621","NEWT:110368","NCBITaxon:5204","NEWT:1916073","NEWT:1303873","NEWT:305400","NEWT:1310161","NEWT:1230383","NEWT:77133","NEWT:160620","NCBITaxon:77643","NEWT:1310165","NEWT:114727","NEWT:5516","NEWT:498211","NEWT:347256","NEWT:5518","NEWT:398007","NEWT:1527468","NEWT:498217","NEWT:498216","NEWT:32224","NEWT:11320","NEWT:1410384","NEWT:1310173","NEWT:1072569","NEWT:1072568","NEWT:161934","NEWT:1148","NEWT:5508","NEWT:135666","NEWT:419481","NEWT:5507","NEWT:1140","NCBITaxon:2157","NEWT:2066563","NEWT:1143","NEWT:1336794","NEWT:1462472","NEWT:1336795","NEWT:644042","NEWT:709964","NEWT:3710","NEWT:65125","NEWT:3712","NEWT:3711","NEWT:3714","NEWT:65121","NEWT:3716","NEWT:3715","NEWT:2065263","NEWT:1549675","NEWT:177437","NEWT:9014","NEWT:10891","NEWT:118221","NEWT:32201","NEWT:34869","NEWT:226126","NEWT:1616117","NEWT:34865","NEWT:28174","NEWT:550537","NEWT:284812","NEWT:284811","NEWT:3701","NEWT:3702","NEWT:1245466","NEWT:3704","NEWT:1246791","NEWT:34875","NEWT:10882","NEWT:1431297","NEWT:9005","NEWT:34871","NEWT:9006","NEWT:33548","NEWT:1883426","NEWT:657321","NEWT:1423687","NEWT:657322","NEWT:10886","NEWT:3707","NEWT:3708","NEWT:536231","NEWT:219813","NEWT:1301262","NEWT:319224","NEWT:2305479","NEWT:215451","NEWT:460519","NEWT:272843","NEWT:272844","NEWT:1348799","NEWT:55784","NEWT:485","NEWT:223074","NEWT:2499525","NEWT:467144","NEWT:142786","NEWT:28196","NEWT:2853422","NEWT:487","NEWT:1679718","NEWT:387344","NEWT:657318","NEWT:480","NEWT:67767","NEWT:46835","NEWT:216778","NEWT:376686","NEWT:237610","EFO:0001352","NEWT:219809","NEWT:219808","NEWT:119562","NEWT:1883446","NEWT:1233435","NEWT:118223","NEWT:141469","NEWT:272831","NEWT:987059","NEWT:491","NEWT:422203","NEWT:460523","NEWT:2422","NEWT:2423","NEWT:3755","NEWT:85694","NEWT:85692","NEWT:911045","NEWT:3750","NEWT:1195466","NEWT:2483192","NEWT:85698","NEWT:291146","NEWT:233271","NEWT:3745","NEWT:1715989","NCBITaxon:4751","NEWT:3747","NEWT:278161","NEWT:415978","NEWT:410289","NEWT:191701","NEWT:9054","NEWT:472","NEWT:29459","NEWT:34839","NEWT:476","NEWT:28128","NEWT:470","NEWT:41122","NEWT:1328300","NEWT:748693","NEWT:106299","NCBITaxon:50557","NEWT:63363","NEWT:3735","NEWT:29491","NEWT:911022","NEWT:446","NEWT:153481","NEWT:714485","NEWT:445","NEWT:427924","NEWT:2003758","NEWT:29486","NEWT:63366","NEWT:63367","NEWT:29488","NEWT:215402","NEWT:3726","NEWT:1120755","NEWT:1862567","NEWT:591090","NEWT:30455","NEWT:28141","NEWT:9030","NEWT:9031","NEWT:3728","NEWT:29471","NEWT:108931","NEWT:219799","NEWT:1325292","NEWT:219798","NEWT:548","NEWT:150475","NEWT:546","NEWT:1431340","NEWT:256737","NEWT:51350","NEWT:8694","NEWT:8692","NEWT:542","NEWT:394503","NEWT:13076","NEWT:219785","NEWT:451515","NEWT:8673","NEWT:550","NEWT:10804","NEWT:52694","NEWT:554","NEWT:451516","NEWT:552","NEWT:1325291","NEWT:42410","NEWT:40686","NEWT:1225786","NEWT:366497","NEWT:771870","NEWT:8665","NEWT:51338","NEWT:771875","NEWT:377142","NEWT:9995","NEWT:28112","NEWT:49012","NEWT:28110","NEWT:10832","NEWT:311339","NEWT:507601","NEWT:520","NEWT:64657","NEWT:186441","NEWT:2585209","NEWT:643680","NEWT:214092","NEWT:40697","NEWT:13095","NEWT:162425","NEWT:242124","NEWT:9986","NEWT:8654","NEWT:536","NEWT:8658","NEWT:1268063","NEWT:8657","NEWT:9983","NEWT:8655","NEWT:80833","NEWT:479096","NEWT:28108","NEWT:28104","NEWT:1114957","NEWT:568703","NEWT:568708","NEWT:480020","NEWT:51315","NEWT:52641","NEWT:353152","NEWT:116597","NEWT:353153","NEWT:116598","NEWT:227377","NEWT:1774284","NEWT:27689","NEWT:207769","NEWT:40674","NEWT:51329","NCBITaxon:1763","NEWT:443906","NEWT:6055","NEWT:13037","NEWT:518","NEWT:519","NEWT:941638","NEWT:511","NEWT:7394","NEWT:6063","NEWT:404330","NEWT:1328388","NEWT:13064","NEWT:529120","NEWT:7375","NEWT:300641","NEWT:39655","NEWT:38323","NEWT:206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