{"database":"Pride","file_versions":[{"headers":{"Content-Type":["application/json"]},"body":{"files":{"Txt":["ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD060933/proteinGroupse_sEVs.txt","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD060933/peptides_Tissue.txt","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD060933/proteinGroups_Tissue.txt","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD060933/checksum.txt","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD060933/peptides_sEVs.txt"],"Xml":["ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD060933/mqpar_Tissue.xml","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD060933/mqpars_EVs.xml"],"Raw":["ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD060933/TMT_II_Tissue_F4.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD060933/TMT_III_sEVs_II_F6.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD060933/TMT_III_sEVs_II_F2.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD060933/TMT_II_Tissue_F1.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD060933/TMT_II_Tissue_F6.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD060933/TMT_III_sEVs_II_F4.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD060933/TMT_III_sEVs_II_F1.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD060933/TMT_II_Tissue_F2.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD060933/TMT_III_sEVs_II_F5.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD060933/TMT_III_sEVs_II_F3.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD060933/TMT_II_Tissue_F3.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD060933/TMT_II_Tissue_F5.raw"]},"type":"primary"},"statusCodeValue":200,"statusCode":"OK"}],"scores":null,"additional":{"labhead_mail":["r.barderasm@isciii.es"],"submitter":["Ana Montero Calle"],"technology_type":["Data-dependent acquisition","Mass Spectrometry","Bottom-up proteomics"],"disease":["Colorectal Cancer"],"software":[""],"submitter_keywords":[""],"full_dataset_link":["https://www.ebi.ac.uk/pride/archive/projects/PXD060933"],"tissue":["Blood Plasma","Colon"],"sample_protocol":["Paired PPFE tissue and plasma samples from CRC patients at stage II with or without CRC recurrence were used. Small extracellular vesicles (sEVs) were isolated from plasmas using the qEVoriginal 70 nm Columns (Izon).  Protein extracts from tissue samples and sEVs isolated from paired plasma samples were analyzed by TMT (Tandem Mass Tags) liquid chromatography tandem mass spectrometry (LC-MS/MS). Tissue and sEVs samples from stage II CRC patients with recurrent (n=5) and non-recurrent (n=5) disease were analyzed in separate, using a 10-plex TMT kit. For tissue samples, 10 µg of each protein extract in 100 µL of  RIPA buffer were used, and reduced with 10 mM tris(2-carboxyethyl)phosphine (TCEP, Sigma-Aldrich) and alkylated with 40 mM chloroacetamide (Sigma-Aldrich) prior digestion, as previously described. For sEVs samples, 10 µg of each sample were lysed with 250 µL of RIPA buffer and mechanically disaggregation using 25 gauge syringes. In addition, after lysis, sEVs samples were incubated 5 times at 95ºC for 5 min and in ice for 5 min to ensure complete lysis of sEVs. Then, samples were reduced and alkylated prior to digestion, as described above. Digestion was performed on SP3 magnetic beads mix (50% hydrophilic beads - 50% hydrophobic beads, GE Healthcare) after protein incubation with the beads and 50% acetonitrile (ACN) for 35 min at RT and in rotation. Digestion was performed with 0.4 µg of porcine trypsin (1:20, Thermo Fisher Scientific) in 100 μL 200 mM HEPES pH 8.0 overnight (O/N) and at 37ºC and 1000 rpm. After digestion, samples were separately labeled with 0.4 mg of each TMT reagent (Thermo Fisher Scientiﬁc). Briefly, the 0.8 mg of each TMT reagent that covalently labels primary amines of Lys and the N-termini of trypsin digested peptides were resuspended in 41 µL of 100% ACN. Samples were first incubated with 10 µL of the corresponding TMT reagent for 30 min at 600 rpm and RT, followed by a second incubation with 10 µL of the same reagent for 30 min at 600 rpm and RT. After labeling, the reaction was quenched with 10 µL of 1 M glycine incubated for 30 min at 600 rpm and RT. Then, the 10 different samples per TMT experiment were pooled together, dried under vacuum, and labeled peptides were separated according to their hydrophobicity using a High pH Reversed-Phase Peptide Fractionation Kit (Thermo Fisher Scientific). After fractionation, peptides were divided into 12 fractions of 300 µL each in a triethylamine-ACN gradient (2.5–100% ACN). Peptide fractions were mixed in six fractions, dried under vacuum, and stored at -80ºC until LC-MS/MS analysis using an Orbitrap Exploris 480 mass spectrometer (Thermo Fisher Scientific) coupled to the FAIMS Pro Due Interface (Thermo Fisher Scientific).  The 6 peptide fractions of the 2 TMT experiments were re-suspended in 10 μL of 0.1% formic acid (FA). For the tissue samples, 4 μL of each fraction were injected per run, whereas for the sEVs samples 6 μL of each fraction were injected  per run. Peptide separation was carried out on the Vanquish Neo UHPLC System (Thermo Fisher Scientific) as previously described (13). Briefly, peptide samples were loaded into a precolumn PepMap 100 C18 3 µm, 75 µm × 2 cm Nanoviper Trap 1200BA (Thermo Fisher Scientific) and eluted in an Easy-Spray PepMap RSLC C18 2 µm, 75 µm × 50 cm (Thermo Fisher Scientific) heated at 50°C. The mobile phase flow rate was 300 nL/min, with buffer A (0.1% FA in H2Omq) and buffer B (0.1% FA in 80% CAN) used for elution. A 2 h gradient was set as followed: 0–2% buffer B for 4 min, 2% buffer B for 2 min, 2–42% buffer B for 100 min, 42–72% buffer B for 14 min, 72–95% buffer B for 5 min, and 95% buffer B for 10 min. For ionization, 1900 V of liquid junction voltage and 280°C capillary temperature were used. The full scan method employed a m/z 350–1400 mass selection, an Orbitrap resolution of 60,000 (at m/z 200), an automatic gain control (AGC) value of 300%, and a maximum injection time (IT) of 25 ms. After the survey scan, the 12 most intense precursor ions were selected for MS/MS fragmentation. Fragmentation was performed with a normalized collision energy of 34, and MS/MS scans were acquired with a 100 m/z first mass, an AGC target of 100%, a resolution of 15,000 (at m/z 200), an intensity threshold of 2×104, an isolation window of 0.7 m/z units, a maximum IT of 22 ms, and the TurboTMT enabled. Charge state screening was enabled to reject unassigned, singly charged, and greater than or equal to seven protonated ions. A dynamic exclusion time of 30 s was used to discriminate against previously selected ions. For FAIMS, a gas flow of 4.7 L/min and CVs = −45 V and −60 V were used."],"repository":["Pride"],"quantification_method":[""],"modification":[""],"data_protocol":["MS data were analyzed with MaxQuant (version 1.6.6.0) using standardized workflows. Mass spectra *.raw files were searched against Uniprot UP000005640_9606.fasta Homo sapiens (human) 2019 database (20962 protein entries) using Reporter ion MS2 type. TMT10 was specified as variable modification on lysine and peptide N termini, and reporter ion intensities were bias corrected for the overlapping isotope contributions from the TMT tags according to manufacturer’s certificate. Precursor and reporter mass tolerance were set to 4.5 ppm and 0.003 Da, respectively, allowing 2 missed cleavages. Carbamidomethylation of cysteines was set as a fixed modification, and methionine oxidation, and acetylation N-terminal were set as variable modifications. Unique and Razor peptides were considered for quantification, with a minimal peptide length of 7 amino acids and a maximal peptide mass of 4600 Da. Identified peptides were filtered by their precursor intensity fraction (PIF) with a false discovery rate (FDR) threshold of 0.01. The protein sequence coverage was estimated for specific proteins by the percentage of matching amino acids from the identified peptides having confidence greater than or equal to 95% divided by the total number of amino acids in the sequence."],"omics_type":["Proteomics"],"labhead":["Rodrigo Barderas"],"instrument_platform":[""],"labhead_affiliation":["Chronic Disease Programme (UFIEC), Instituto de Salud Carlos III, Majadahonda, Madrid, Spain"],"submission_type":["PARTIAL"],"species":["Homo Sapiens (human)"],"submitter_mail":["ana.monteroc@isciii.es"],"publication":["10.1021/ACS.JPROTEOME.5C01148"],"submitter_affiliation":["Instituto de Salud Carlos III"],"submitter_country":["Spain"],"additional_accession":[]},"is_claimable":false,"name":"In-Depth Proteomic Analysis of Stage II Colorectal Cancer for the Identification of  Recurrence Biomarkers in FFPE Tissue and Extracellular Vesicles","description":"Colorectal cancer (CRC) is a leading cause of cancer-related death worldwide, with stage II CRC patients presenting unique challenges due to their heterogeneous outcomes. Despite curative surgical resection, 15-20% of stage II CRC patients experience recurrence within five years of diagnosis. Current prognostic models, based on clinicopathologic features such as vascular invasion, poor differentiation, and adverse molecular profiles, lack precision in predicting recurrence, highlighting the need for other complementary tools. Advances in proteomics have positioned formalin-fixed paraffin-embedded (FFPE) tissues and small extracellular vesicles (sEVs) as promising sources for biomarker identification. FFPE tissues offer a wealth of retrospective diagnostic material, while sEVs, particularly exosomes, encapsulate tumor-derived proteins and nucleic acids, providing minimally invasive insights into the tumor microenvironment. Proteomic profiling of these samples should allow the identification of molecular alterations linked to tumor aggressiveness and recurrence, paving the way for novel diagnostic and prognostic applications. In this study, we here combine proteomics analyses of paired FFPE tissues and sEVs to identify stage II CRC associated biomarkers and tumor biopsies, plasma of CRC patients and controls, and immunohistochemistry and in vitro and in vivo analyses to validate proteomics results. Among the identified candidates, CDCA2 emerged as a interesting driver of tumor formation, progression, and metastasis in CRC. Transient knockdown experiments demonstrated that its depletion impaired key tumorigenic processes, including proliferation, adhesion, migration, and invasion. Additionally, this study validated MANF as a plasma biomarker capable of distinguishing recurrent from non-recurrent CRC patients, offering potential for improving patient stratification and personalized treatment strategies in combination with other markers. Interestingly, our multifaceted approach allowed the identification of dysregulated proteins associated to CRC recurrence.","dates":{"publication":"2026-05-03","submission":"2025-02-18"},"accession":"PXD060933","cross_references":{"TAXONOMY":["NEWT:330879","NEWT:377960","NEWT:1131","NEWT:35795","NEWT:2042546","NEWT:259447","NEWT:295546","NCBITaxon:2719036","NEWT:1334565","NEWT:625351","NEWT:258594","NCBITaxon:1280","NEWT:112503","NEWT:1129","NEWT:309807","NEWT:59753","NEWT:89184","NEWT:309800","NEWT:281395","NEWT:247480","NEWT:1211601","NEWT:876138","NEWT:44271","NEWT:193516","NEWT:2315688","NEWT:592321","NEWT:1073882","NEWT:6819","NEWT:112509","NEWT:475174","NEWT:111205","NEWT:1599","NEWT:428386","NEWT:1117","NEWT:498257","NEWT:10036","NEWT:32264","NEWT:12215","NEWT:1590","NEWT:10034","NEWT:33114","NEWT:1596","NEWT:661410","NEWT:638632","NEWT:224326","NEWT:376619","NEWT:1748418","NCBITaxon:79857","NEWT:1096976","NEWT:3765","NEWT:3767","NEWT:1589","NEWT:1907535","NEWT:135622","NEWT:4615","NEWT:672127","NEWT:35786","NEWT:1580","NEWT:399784","NEWT:96731","NEWT:656064","NEWT:1582","NEWT:3760","NEWT:1584","NEWT:2086595","NEWT:36630","NEWT:1585","NEWT:383379","NEWT:381198","NEWT:418106","NCBITaxon:12721","NEWT:10029","NEWT:913645","NEWT:2492962","NEWT:641809","NEWT:1392488","NEWT:38815","NEWT:317447","NEWT:4688","NEWT:3357","NEWT:111225","NEWT:7719","NEWT:5530","NEWT:868565","NEWT:135674","NEWT:1562698","NEWT:79329","NEWT:30069","NEWT:12637","NEWT:59729","NEWT:2164133","NEWT:295105","NEWT:108061","NEWT:60711","NEWT:224308","NEWT:3347","NEWT:49928","NEWT:3349","NEWT:160621","NEWT:2502409","NEWT:212790","NEWT:110368","NEWT:55350","NEWT:1916073","NEWT:1310161","NEWT:77133","NEWT:145481","NEWT:1310165","NEWT:29058","NCBITaxon:79824","NEWT:1912919","NEWT:410658","NEWT:5516","NEWT:44688","NEWT:44689","NEWT:370038","NEWT:498211","NEWT:345076","NEWT:347256","NEWT:5518","NEWT:398007","NEWT:1527468","NEWT:498217","NEWT:498216","NEWT:11320","NEWT:246196","NEWT:246197","NEWT:145458","NEWT:1310173","NEWT:200795","NEWT:37919","NEWT:2579791","NEWT:44685","NEWT:161934","NEWT:156587","NEWT:1148","NEWT:5508","NEWT:3329","NEWT:419481","NEWT:5507","NEWT:260499","NEWT:410661","NEWT:329854","NEWT:1140","NCBITaxon:2157","NEWT:2066563","NEWT:2649329","NEWT:1143","NEWT:1287689","NEWT:1094343","NEWT:1462472","NEWT:1336795","NEWT:644042","NEWT:150808","NEWT:1182590","NEWT:3712","NEWT:3711","NEWT:3714","NCBITaxon:37162","NEWT:270643","NEWT:3715","NEWT:1538","NEWT:2065263","NEWT:177437","NEWT:717966","NEWT:10891","NEWT:10418","NEWT:118698","NEWT:1616117","NEWT:118696","NEWT:34865","NEWT:52283","NEWT:28174","NEWT:284812","NEWT:8175","NEWT:43330","NEWT:1603293","NEWT:44664","NEWT:43346","NEWT:3702","NEWT:1245466","NEWT:3704","NEWT:244366","NEWT:1246791","NEWT:176597","NEWT:118694","NEWT:2850","NEWT:118691","NEWT:34871","NEWT:9006","NEWT:33548","NEWT:52299","NEWT:8165","NEWT:96794","NEWT:3708","NEWT:332648","NEWT:44670","NEWT:926571","NEWT:536231","NEWT:376219","NEWT:219813","NEWT:1510","NEWT:460519","NEWT:1515","NEWT:572307","NEWT:1432138","NEWT:2714896","NEWT:1424507","NEWT:1194599","NEWT:272844","NEWT:1348799","NEWT:1608994","NEWT:33995","NEWT:55784","NEWT:1303443","NEWT:9483","NEWT:485","NEWT:56636","NEWT:2709072","NEWT:2499525","NEWT:2853422","NEWT:1122984","NEWT:487","NEWT:1538553","NEWT:1679718","NEWT:480","NEWT:67767","NEWT:45972","NEWT:46835","NEWT:109757","NEWT:582580","NEWT:1185650","NEWT:216778","NEWT:294607","NEWT:1502","NEWT:128017","NEWT:2836","NEWT:376686","NEWT:237610","NEWT:95486","EFO:0001352","NEWT:119562","NEWT:1883446","NEWT:1233435","NEWT:118223","NEWT:11757","NEWT:987059","NEWT:109760","NEWT:29031","NEWT:317047","NEWT:1509","NEWT:491","NEWT:460523","NEWT:45954","NEWT:235443","NEWT:1579","NEWT:1757312","NEWT:85694","NEWT:109786","NEWT:1922344","NEWT:108458","NEWT:5936","NEWT:85692","NEWT:321967","NEWT:320637","NEWT:1078283","NEWT:3750","NEWT:983964","NEWT:11706","NEWT:32644","NEWT:527796","NEWT:279010","NEWT:499175","NEWT:109779","NEWT:3745","NEWT:1715989","NCBITaxon:4751","NEWT:3747","NEWT:1116234","NEWT:415978","NEWT:1255228","NEWT:410289","NEWT:191701","NEWT:373153","NEWT:1640278","NEWT:9054","NEWT:472","NEWT:940825","NEWT:29459","NEWT:529507","NEWT:1071661","NEWT:28128","NEWT:470","NEWT:41122","NEWT:5911","NEWT:1328300","NEWT:748693","NCBITaxon:50557","NEWT:39251","NEWT:29491","NEWT:101841","NEWT:446","NEWT:153481","NEWT:2014887","NEWT:33952","NEWT:11723","NEWT:445","NEWT:153009","NEWT:261756","NEWT:166314","NEWT:166313","NEWT:63366","NEWT:63367","NEWT:29488","NEWT:215402","NEWT:1547","NEWT:1290454","NEWT:3726","NEWT:906914","NEWT:81231006","NEWT:30455","NEWT:28141","NEWT:9031","NEWT:27292","NEWT:1050722","NEWT:3728","NEWT:108931","NEWT:1293497","NEWT:1055524","NEWT:548","NEWT:150475","NEWT:267872","NEWT:381666","NEWT:172269","NEWT:9534","NEWT:2126348","NEWT:5180","NEWT:256737","NEWT:299467","NEWT:9541","NEWT:8694","NEWT:33936","NEWT:8692","NEWT:542","NEWT:2903","NEWT:185579","NEWT:872227","NEWT:13076","NEWT:2587410","NEWT:207340","NEWT:1006581","NEWT:8673","NEWT:33940","NEWT:2126337","NEWT:550","NEWT:10804","NEWT:33941","NEW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