Prideapplication/xmlftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/03/PXD063788/6349_20231009_maxquant_proteinGroups.xlsxftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/03/PXD063788/pEX1_pp_6349_20240125_TMT4plex_BRP_PD_Peptides.xlsxftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/03/PXD063788/pEX1_pp_6349_20240125_TMT4plex_BRP_PD_Proteins.xlsxftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/03/PXD063788/6349_20231009_maxquant_peptides.xlsxftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/03/PXD063788/pEX1_pp_6349_20240125_TMT4plex_BRP_F5_0pt3uL_5.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/03/PXD063788/pEX1_pp_6349_20240125_TMT4plex_BRP_F8_1uL_8.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/03/PXD063788/pEX1_pp_6349_20240125_TMT4plex_BRP_F2_0pt4uL_2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/03/PXD063788/pEX1_pp_6349_20231002_S3_0Gy_0906_1uL_3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/03/PXD063788/pEX1_pp_6349_20231002_S1_0Gy_0801_1uL_1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/03/PXD063788/pEX1_pp_6349_20231002_S5_10Gy_0823_1uL_5.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/03/PXD063788/pEX1_pp_6349_20231002_S3_0Gy_0906_1pt2uL_3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/03/PXD063788/pEX1_pp_6349_20231002_S2_0Gy_0823_1uL_2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/03/PXD063788/pEX1_pp_6349_20240125_TMT4plex_BRP_F1_0pt3uL_1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/03/PXD063788/pEX1_pp_6349_20231002_S1_0Gy_0801_0pt5uL_1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/03/PXD063788/pEX1_pp_6349_20231002_S2_0Gy_0823_0pt8uL_2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/03/PXD063788/pEX1_pp_6349_20240125_TMT4plex_BRP_F4_0pt3uL_4.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/03/PXD063788/pEX1_pp_6349_20240125_TMT4plex_BRP_F7_0pt2uL_7.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/03/PXD063788/pEX1_pp_6349_20240125_TMT4plex_BRP_F3_0pt3uL_3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/03/PXD063788/pEX1_pp_6349_20231002_S6_10Gy_0906_1uL_6.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/03/PXD063788/pEX1_pp_6349_20240125_TMT4plex_BRP_F6_0pt3uL_6.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/03/PXD063788/pEX1_pp_6349_20231002_S4_10Gy_0801_1uL_4.rawprimaryOK200kristie.rose@vanderbilt.eduKristie Lindsey RoseData-dependent acquisitionMass SpectrometryBottom-up proteomicsIonizing radiationTmt labelingExtracellular vesiclesComparative proteomicsLabel-free quantificationhttps://www.ebi.ac.uk/pride/archive/projects/PXD063788Fibroblast Of BreastBreastEV protein extracts from control and irradiated fibroblasts were digested with trypsin using S-Trap micro spin columns (ProtiFi) according to manufacturer’s instructions. Peptides were eluted, dried in a speed vac concentrator, and reconstituted in aqueous 0.2% formic acid. Aliquots were taken for LC-MS/MS and label-free quantitative analysis, and remaining volumes were dried. Dried aliquots were next analyzed using Tandem Mass Tags (TMTsixplex™ reagents, Thermo Scientific). Peptides were reconstituted in 50 mM TEAB and labeled with TMT tags following manufacturer’s instructions. Labeled peptides were combined and fractionated by high pH reversed-phase fractionation. Peptide fractions were reconstituted in 0.2% formic acid for LC-MS/MS analysis.PrideLabel-free samples and TMT-labeled peptide fractions were analyzed by nanoflow LC-MS/MS using a Dionex Ultimate 3000 nanoLC and an Exploris 480 Orbitrap mass spectrometer using a data-dependent (top 20) method. For label-free samples, mass spectra were searched with MaxQuant version 2.0.3.0 against a human database created from the UniprotKB protein database (www.uniprot.org). The false discovery rate was set to 0.01 for peptide and protein identifications. For TMT-labeled peptide fractions, mass spectra were searched using SequestHT in Proteome Discoverer (Thermo Scientific). Percolator validation was performed with a 1% false discovery rate.ProteomicsKristie Lindsey RoseVanderbilt University Proteomics CorePARTIALHomo Sapiens (human)41472394 Berumen Sánchez G, Patel P, Rose KL, Rafat M. Proteomics in Practice: A Case Study Highlighting Tandem Mass Tag-Based MS for Quantitative Profiling of Extracellular Vesicles and Application to Irradiated Fibroblasts. Proteomics. 2025:e70103 10.1002/pmic.70103kristie.rose@vanderbilt.eduVanderbilt UniversityUnited StatesExtracellular vesicles (EVs) are critical mediators of intercellular communication, and valuable sources of biomarkers for diagnostic and therapeutic applications. However, the complexity and heterogeneity of EVs present significant challenges for their proteomic characterization. Major challenges of EV samples include low yield, technical variability, and the need for sensitive and high throughput quantification approaches. In this study, we implement a tandem mass tag (TMT)-based MS workflow for comprehensive, quantitative proteomic profiling of isolated EVs. Through comparison with label-free quantitation (LFQ), we highlight the potential pitfalls and limitations of methodological choices in EV proteomic analyses. Our study integrates standard EV isolation with robust TMT labeling and high-resolution MS, providing insights into practical EV analysis. Utilizing this approach, we profiled EVs isolated from human fibroblasts treated with ionizing radiation. The TMT workflow uncovered an EV proteomic signature reflective of the cellular origins and potential functional roles of irradiated fibroblasts, compared to the LFQ workflow. Our case study underscores the potential of TMT-based MS to overcome common barriers in EV proteomics, facilitating new discoveries in EV biology and advancing their application in biomarker development and translational research.Proteomics in Practice: A Case Study Highlighting Tandem Mass Tag-Based MS for Quantitative Profiling of Extracellular Vesicles and Application to Irradiated Fibroblasts.Berumen Sánchez Greg G, Patel Purvi P, Rose Kristie Lindsey KL, Rafat Marjan MfalseQuantitative Proteomic Analysis of Extracellular Vesicles Derived from γ-Irradiated Normal Human Breast FibroblastsSmall extracellular vesicles (EVs) were isolated from a model of normal breast fibroblasts— immortalized cells derived from healthy reduction mammoplasty tissue—treated with γ-radiation to study the effects of radiation therapy on intercellular communication within the breast stroma. EVs were isolated using the gold-standard differential speed centrifugation protocol. Proteins were subsequently extracted, and a quantitative examination of the alterations in the EV proteome was pursued. Both label-free and isobaric label-based methodologies were conducted to comprehensively characterize proteomic changes.2026-03-022025-05-09PXD063788NEWT:3555NEWT:71647NEWT:35554NEWT:32046NEWT:544496NEWT:2042546NEWT:32049NEWT:1392696NEWT:259447NEWT:45351NEWT:445974NEWT:43179NEWT:180454NEWT:5722NCBITaxon:1280NEWT:1129NEWT: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:12637NEWT:59729NEWT:2164133NEWT:108061NEWT:1316931NEWT:224308NEWT:3347NEWT:511145NEWT:931281NEWT:658457NEWT: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: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: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:243230NEWT:1080772NEWT:519NEWT:8479NEWT:8485NEWT:6063NEWT:49240NEWT: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:89462NEWT: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: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: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:272559NEWT:28909NEWT:515849NEWT:3641NEWT:466585NEWT:1000589NEWT:85963NEWT:85962NEWT:143361NEWT:398580NEWT:4565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