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PitteriData-dependent acquisitionMass SpectrometryBottom-up proteomicsBreast CancerNot availablePlasmaProteomicsBreast cancerRadiotherapyhttps://www.ebi.ac.uk/pride/archive/projects/PXD077995Blood PlasmaPlasma was collected from six breast cancer patients at four timepoints: prior to radiotherapy (RT), one week after the start of RT, one month after the start of RT, and at least one month after the end of RT. Plasma samples were depleted of the Top 14 most abundant proteins and concentrated. Protein was quantified with a Bradford assay, and samples were reduced with DTT. Heavy (13C) or light (12C) acrylamide was added to the reduced sample. All samples during or after RT were labeled with heavy acrylamide, and all samples prior to RT (control or baseline samples) were labeled with light acrylamide. For each patient, the four timepoints formed three pairs based on heavy/light acrylamide labeling in a 1:1 ratio. Labeled protein was fractionated by reversed-phase liquid chromatography and then digested with trypsin. Fractions were analyzed on an LTQ-Orbitrap Elite mass spectrometer. Data-dependent acquisition (DDA) was performed by fragmenting the Top 10 most abundant precursor ions with CID fragmentation. Fragment ions were detected in the linear trap with a normal scan rate.PrideNot availableRaw files were converted to mzXML using MSconvert from ProteoWizard. The mzXML files were searched against the human UniProtKB database (43,390 entries, 2015) using X!Tandem with a default fragment ion mass accuracy of ±0.5 Da. Search results were analyzed by PeptideProphet and ProteinProphet. Peptides with an X!Tandem expectation value ≤0.05 and PeptideProphet score ≥0.9 were retained for protein identification and quantitation. Only peptides with precursor fractional delta mass <20 ppm were used for quantitation. Heavy-to-light ratios were computed for cysteine-containing peptides labeled with acrylamide. Peptide-spectrum match information, peptide sequences, protein accessions, search scores, and q3 quantitative values were extracted from pepXML files and combined into a single dataset. Quantitative values were summarized at the gene level by summing light and heavy peptide intensities separately after combining redundant Swiss-Prot/TrEMBL entries. Log₂ heavy/light ratios were calculated, normalized, and standardized across samples. Temporal protein changes were analyzed using permutation-based clustering, Student’s t-tests, and functional enrichment with WebGESTALT.ProteomicsSharon PitteriDepartment of Radiology Canary Center at Stanford for Cancer Early Detection Stanford School of Medicine Stanford UniversityPARTIALHomo Sapiens (human)spitteri@stanford.edu42503768 Kapp KL, Going CC, Garcia-Marques FJ, Leow K, Maltos A, Bermudez A, Vilalta M, Rafat M, Jenkins M, Chen JJ, Mallick P, Horst K, Graves E, Pitteri SJ. Quantifying Protein Temporal Changes with Mass Spectrometry in Plasma Samples from Breast Cancer Patients Undergoing Radiotherapy. J Am Soc Mass Spectrom. 2026 37(9):2141-2154 10.1021/jasms.6c00168Stanford UniversityUnited StatesRadiation therapy (RT) is a central component of cancer treatment, including breast cancer, and primarily damages DNA in cancer cells, causing cell death. However, secondary effects of RT that may influence tumor and normal tissue both within and beyond the site of irradiation are not well characterized, particularly at the level of the circulating blood proteome. In this study, isotopic acrylamide labeling of proteins and high-resolution LC-MS/MS were used to analyze plasma proteins from breast cancer patients before, during, and after RT. Clustering analysis was used to group proteins according to how their levels changed over time. A total of 31 proteins were identified whose levels were altered during RT but returned to baseline following the conclusion of treatment, representing key parts of the immune response to RT. Another set of 31 proteins related to the extracellular matrix and other functions was altered during RT and remained altered after RT. Specific time points were also examined, and acute changes were identified at the start of RT in the levels of 63 proteins involved in coagulation, cell-cell adhesion, and carbohydrate metabolism. After the end of RT, the circulating levels of 23 extracellular matrix proteins remained altered. These LC-MS/MS findings were validated orthogonally by ELISAs of three proteins in a larger group of breast cancer patients undergoing RT. Overall, in this study, temporal changes in the circulating blood proteome of breast cancer patients undergoing RT were characterized, and these altered protein levels were linked to potential alterations in biological pathways.Quantifying Protein Temporal Changes with Mass Spectrometry in Plasma Samples from Breast Cancer Patients Undergoing Radiotherapy.Kapp Kathryn L KL, Going Catherine C CC, Garcia-Marques Fernando J FJ, Leow Ke K, Maltos Alisha A, Bermudez Abel A, Vilalta Marta M, Rafat Marjan M, Jenkins Melissa M, Chen Jie Jane JJ, Mallick Parag P, Horst Kathleen K, Graves Edward E, Pitteri Sharon J SJfalseQuantifying Protein Temporal Changes in Plasma Samples from Breast Cancer Patients Undergoing RadiotherapyShotgun proteomics analysis was performed on plasma samples from breast cancer patients undergoing radiotherapy (RT) treatment. Samples were obtained at 4 timepoints before, during, and after RT.2026-09-292026-05-04PXD077995NEWT:6945NEWT:3555NEWT:2NEWT:157546NEWT:35554NEWT:38942NEWT:307972NEWT:32046NEWT:544496NEWT:2042546NEWT:45351NEWT:43179NEWT:4513NEWT:5722NEWT:376741NEWT:55153NCBITaxon:10407NEWT:1736309NEWT:309800NEWT:1211601NEWT:876138NEWT:557436NEWT:237561NEWT:5833NEWT:6928NEWT:10036NEWT:36745NEWT:1351NEWT:1438992NEWT:2649997NEWT:272563NEWT:79220NEWT:224326NCBITaxon:79857NEWT:1096976NEWT:95648NEWT:3885NEWT:3888NEWT:1589NEWT:135622NCBITaxon:4896NEWT:6915NEWT:3649NEWT:101510NEWT:3880NEWT:272559NEWT:3641NEWT:383379NEWT:466585NEWT:10029NEWT:1000589NEWT:85963NEWT:85962NEWT:317447NEWT:7955NEWT:7959NEWT:2261NEWT:31156NEWT:398580NEWT:4565NEWT:1264690NEWT:515619NEWT:192875NEWT:34305NEWT:59729NCBITaxon:183674NEWT:224308NEWT:84645NEWT:626528NEWT:139927NEWT:4558NEWT:209285NEWT:211586NEWT:1283NEWT:931281NEWT:4550NEWT:1000561NEWT:197NCBITaxon:79824NEWT:4787NCBITaxon:4563NEWT:5755NEWT:44689NEWT:3218NEWT:5759NEWT:1736231NEWT:1270NEWT:2242NEWT:4784NEWT:11320NEWT:360106NEWT:286NEWT: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:3712NEWT:935293NEWT:64152NEWT:4924NEWT:749200NEWT:990346NEWT:145953NEWT:257309NEWT:100816NEWT:263NEWT:230741NEWT:52283NEWT:284812NCBITaxon:1313NEWT:43330NEWT:980415NEWT:1603293NEWT:408169NEWT:44544NEWT:4911NEWT:645463NEWT:3702NEWT:129249NEWT:243277NEWT: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:1182263NEWT:2711NEWT:376686NEWT:95486NEWT:9103NEWT:29159NEWT:253NEWT:10306NCBITaxon:2759NEWT:1233435NEWT:8022NEWT:145943NCBITaxon:4932NEWT:595536NEWT:240906NEWT:593117NEWT:3635NEWT:5811NEWT:235443NEWT:272624NEWT:2719099NEWT:411483NEWT:884019NEWT:198215NEWT:411490NEWT:983964NEWT:169963NEWT:32644NEWT:499175NEWT:109779NEWT:476272NEWT:3747NEWT:195051NEWT:367830NEWT:1255228NEWT:178616NEWT:410289NEWT:373153NEWT:352472NEWT:357NEWT:360094NEWT:470NEWT:1313NEWT:411469NEWT:84023NEWT:559292NEWT:39491NCBITaxon:5811NEWT:411464NEWT:411460NEWT:2014887NEWT:2762NEWT:1174673NEWT:562NEWT:411470NEWT:33952NEWT:2094720NCBITaxon:2697049NEWT:571256NEWT:28038NEWT:1663NEWT:1423NEWT:4932NEWT:3603NEWT:2759NEWT:3847NEWT:327159NEWT:178876NEWT:327160NEWT:573NEWT:9031NEWT:7091NEWT:241368NEWT:42528NEWT:190802NEWT:9778NEWT:150475NEWT:303NEWT:9417NEWT:7111NEWT:347515NEWT:1216979NEWT:5180NEWT:256737NEWT:115104NEWT:1121114NEWT:663NEWT:1081927NEWT:1238993NEWT:67825NEWT:185579NEWT:941442NEWT:220668NEWT:13076NEWT:1249668NEWT:7108NEWT:317NEWT:7227NEWT:7469NEWT:885318NEWT:9402NEWT:415540NEWT:550NEWT:675060NEWT:4081NEWT:334542NEWT:554NEWT:98334NEWT:426428NEWT:7574NEWT:1715256NEWT:7215NEWT:575412NEWT:29204NEWT:2172103NEWT:507601NEWT:643680NCBITaxon:6157NEWT:746360NEWT:6239NEWT:470150NEWT:216257NEWT:102169NEWT:9986NEWT:4054NEWT:73239NEWT:226186NEWT:1268063NEWT:8782NEWT:1263854NEWT:435590NEWT:1902NEWT:160488NEWT:28104NEWT:1908NCBITaxon:2NEWT:985076NEWT:1215323NEWT:623NEWT:52641NEWT:7038NEWT:6192NEWT:28532NCBITaxon:38727NEWT:353152NEWT:2829NEWT:366581NEWT:216599NEWT:216595NEWT:51329NEWT: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:426688NEWT:208964NEWT:1134506NEWT:575584NEWT:38783NEWT:8727NEWT:4006NEWT:8726NEWT:6426NEWT:6669NEWT:10090NEWT:4120NEWT:51515NEWT:5693NEWT:8724NEWT:51511NEWT:92867NEWT:8723NEWT:5334NEWT:37334NEWT:382352NCBITaxon:10359NEWT:242619NEWT:632957NEWT:34073NEWT:373995NEWT:5689NEWT:544404NEWT:9925NEWT:8839NEWT:4232NEWT:2758385NEWT:4113NEWT:837NEWT:11298NEWT:171101NEWT:714NEWT:36015NEWT:421932NEWT:196627NEWT:5691NEWT:48479NEWT:627025NEWT:1097677NEWT:242507NEWT:61674NEWT:1117957NEWT:9913NEWT:4100NEWT:1076NEWT:6763NEWT:3498NEWT:803NEWT:29722NEWT:380394NEWT:1692259NEWT:180066NEWT:135588NEWT:1843183NEWT:58002NEWT:326423NEWT:36111NEWT:4577NEWT:1416333NEWT:5664NEWT:2157NEWT:1678078NEWT:749906NEWT:418985NEWT:146479NEWT:1911079NEWT:69373NCBITaxon:620NEWT: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:9839NEWT:58334NEWT:1193501NEWT:3055NEWT:58331NEWT:6326NEWT:6689NEWT:5476NEWT:299767NEWT:1274432NEWT:1274426NEWT:70448NEWT:9825NEWT:1274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