Prideapplication/xmlftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD069883/checksum.txtftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD069883/peptides_totales.csvftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD069883/Total_Proteins.csvftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD069883/20210512_127_UDMSE_MDA_MB_231_p21_R001.raw.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD069883/20210512_123_UDMSE_T4TD_p28_R001.raw.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD069883/20210512_126_UDMSE_MDA_MB_231_p18_R001.raw.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD069883/20210512_126_UDMSE_MDA_MB_231_p18_R002.raw.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD069883/20210512_124_UDMSE_T4TD_p31_R002.raw.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD069883/20210512_123_UDMSE_T4TD_p28_R002.raw.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD069883/20210512_124_UDMSE_T4TD_p31_R001.raw.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD069883/20210512_117_UDMSE_MCF_12F_p30_R001.raw.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD069883/20210512_127_UDMSE_MDA_MB_231_p21_R002.raw.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD069883/20210512_125_UDMSE_MDA_MB_231_p10_R001.raw.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD069883/20210512_121_UDMSE_T47D_p20_R001.raw.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD069883/20210512_125_UDMSE_MDA_MB_231_p10_R002.raw.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD069883/20210512_118_UDMSE_MCF_12F_p32_R001.raw.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD069883/20210512_122_UDMSE_T4TD_p20_pgel_nativo_R002.raw.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD069883/20210512_117_UDMSE_MCF_12F_p30_R002.raw.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD069883/20210512_121_UDMSE_T47D_p20_R002.raw.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD069883/20210512_118_UDMSE_MCF_12F_p32_R002.raw.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD069883/20210512_122_UDMSE_T4TD_p20_pgel_nativo_R001.raw.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD069883/20210512_119_UDMSE_T47D_p14_R001.raw.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD069883/20210512_120_UDMSE_T47D_p17_R002.raw.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD069883/20210512_116_UDMSE_MCF_12F_p28_R002.raw.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD069883/20210512_119_UDMSE_T47D_p14_R002.raw.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD069883/20210512_116_UDMSE_MCF_12F_p28_R001.raw.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD069883/20210512_120_UDMSE_T47D_p17_R001.raw.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD069883/20210512_128_UDMSE_MDA_MB_231_p24_R001.raw.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD069883/20210512_128_UDMSE_MDA_MB_231_p24_R002.raw.zipprimaryOK200jpluna@cinvestav.mxRodolfo Moreno CastilloMass SpectrometryData-independent acquisitionBreast Cancerlabel-free proteomics analysisT47d and mda-mb-231 breast cancer cell lines; healthy breast mcf-12f cell line; mitochondria; differentially expressed proteins; label-free proteomics analysismitochondriadifferentially expressed proteinshealthy breast mcf-12f cell lineT47d and mda-mb-231 breast cancer cell lineshttps://www.ebi.ac.uk/pride/archive/projects/PXD069883Epithelial CellBreast Cancer Cell LinePreOmics iST sample preparation kit was used to prepare samples from mitochondria enriched fractions for tandem mass spectrometry analysis, following the manufacturer´s manual.For mass spectrometry analysis, we followed the Rios-Castro and coworkers’ methodology on purified peptides of 3, 4, and 6 samples from independent biological experiments from MCF-12F, MDA-MB-23, and T47D, respectively. Each cell line counted with two technical replications per sample. The samples were normalized before to be injected to the SynapG2-Si (Waters®, Milford, Ma) mass spectrometer, employing the ion mobility cell to use Ultra Definition MultiplexedMS/MS (UDMSE) in Data-independent acquisition (DIA) mode along with ion mobility (IMS) [31-33]. The peptides separation was carried out using a C18 reverse phase chromatography column using a Waters nano Ultra Performance Liquid Chromatography (UPCL) Acquity equipment. The same amount of peptides in each condition was charged on a C18 pre-column Trap V/M 180 µm × 20 mm, 20 Å pore size, 5 µm particle size, and desalted using as mobile phase A of 0.1% formic acid (FA) in water, and as mobile phase B 0.1% FA in acetonitrile (ACN) under the following isocratic gradient: 99.9% mobile phase A and 0.1% mobile phase B with a flow rate of 5 µL/min for 3 minutes. Next, the peptides were charged and separated in a HSS T3 C18 column, 75 µm ×150 mm, 100 Å pore size, 1.8 µm particle size using a Waters Acquity UPLC System M class with the same mobile phases under the following gradient: 0 minutes 7% B, 0 minutes, 40% B for 121.49 minutes, 85% B for 123.15 to 126.46 minutes,7% B for 129 to 130, using a flow rate of 400 nL/min and 45°C. For ionization source, the parameters were set up with the following values: 2.75 kV in the capillary sampler, 30 V in the sampling cone, 30 V in the source of compensation, 70°C for the temperature source, gas flow at 0.5 bar, and 150 L/h for the purge of gas flow. Two chromatograms (down and high energy) were obtained in positive mode in a m/z range between 50 and 2,000 with a scan time of 500 milliseconds. The collision energy applied was 4 eV to obtain the chromatogram of down energy, whereas for chronograms of high energy,the ionic precursors were fragmented in the chamber using a collision energy ramp of 19 to 55 eV. The generated raw files were analyzed with the driftScope v2.8 (Waters, Mildford, MA). The goal was to obtain energy of collisions quasi specifics based in the drift time for each m/z with more of [M +2H]2+. A rules file (*.rul) was generated, which was used applying a specific collision energy for each detected peptide in UDMSE mode.PrideBased on Rios-Castro et al., the measurements of MS and MS/MS contained in the generated *raw files was normalized, aligned, identified, compared, and relatively quantified using Progenesis QI v3.03 (Waters, Milford, Ma) compared against the Homo sapiens database downloaded from Uniprot (UP000005640); one of many virtues of this software is that use the absolute deviation of the mean, based on all detected abundances to compute a factor that adjusts the differences between the conditions by minimizing any bias in the calculation, as well as noise in the data, for it; the mathematical model used by software is based on the formula 𝑦′ 𝑖 = 𝛼𝑘𝑦𝑖 , where, 𝑦′ 𝑖 is the normalized abundance, 𝑦𝑖 are the abundances of ion compounds, and 𝛼𝑘 is the computed scalar factor. False Discovery Rate (FDR) less than 4% (given by default) was used. Estimating the proportion of false negatives was carried out using the target decoy strategy, which is the ratio between the number of incorrectly assigned identifications (decoy) and the correct identifications (target). The formula used was 𝐹𝐷𝑅 = # 𝑑𝑒𝑐𝑜𝑦 # 𝑡𝑎𝑟𝑔𝑒𝑡 The generated results by Progenesis were exported as *.xlsx files. The fold changes of each peptide were computed based to the average of the MS signal answer of the three peptides more intensity, Hi3 or Top3 described for. This method is used because it gives a universally linear correlation when comparing the three peptides with the highest intensity(y axis) versus the total protein concentration (x axis). Therefore, the fold change computing is the Hi3 ratio of the breast cancer cell lines between Hi3 ratio of the control breast cell line. To normalize the raw values of the intensity of peptides, these were transformed using logarithm base 2. For the replicates whose value was 0 in at least 3 of them, imputations were performed, being adjusted to the lower limit according to the Gaussian bell curve. All these adjustments were made using the Perseus software (MaxQuant). To identify the mitochondrial proteins obtained in the proteomics analysis, Human MitoCarta 3.0 (BROAD Institute), mitoXplorer 2.0 (Aix-Marseille University) y Integrated Mitochondrial Protein Index (IMPI, University of Cambridge) databases were downloaded. We detected proteins with mitochondrial localization comparing, filtering, and using logical formulas from Excel between the download databases with our database.ProteomicsJuan Pedro Luna-AriasPARTIALDepartamento de Biología Celular. Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional.Avenida Instituto Politécnico Nacional 2508, Col. San Pedro Zacatenco, Alcaldía Gustavo A. Madero, Ciudad de México, Código Postal 07360.Homo Sapiens (human)42025918 Vázquez-Narváez HD, Labra-Barrios ML, Ríos-Castro E, Castañón-Sánchez CA, Uribe-Carvajal S, Hernández-Martínez E, Hernández-Hernández JM, León-Ávila G, Pérez-Rangel A, Ángeles-Morales EB, Luna-Arias JP. The proteomics analysis of mitochondria-enriched fractions reveals alteration of key mitochondrial pathways in breast cancer cell lines. J Proteomics. 2026 328:105661 10.1016/j.jprot.2026.105661rmorenoc1700@alumno.ipn.mxInstituto Politécnico NacionalMexicoBreast cancer, the leading cause of death in women worldwide, shows significant heterogeneity that makes this disease extremely difficult to treat. Many reports point to metabolic shifts, mainly those carried out into mitochondria, as key processes governing the behavior and heterogeneity of several types of breast cancer. In this study, we performed label-free proteomics analysis on mitochondria-enriched fractions from T47D and MDA-MB-231 breast cancer cell lines, which have distinct molecular classifications, using bioinformatics analyses to identify differentially expressed proteins compared to MCF-12F healthy breast cells. Cancer cells exhibited down-regulated protein levels of subunits from the respiratory chain's Complex I. However, both showed differentially abundant proteins involved in ligase and oxidoreductase activities, including enzymes of glycolysis, pyruvate metabolism, the Krebs cycle, and gluconeogenesis. Many of these enzymes also participate in other metabolic processes, such as mitochondrial localization, mitochondrial gene expression, and the metabolism of amino acids, fatty acids, purines, and pyrimidines. Gene Set Enrichment Analysis revealed that OXPHOS subunits are integrated as signatures of neurodegenerative disease pathways. A protein set with little or no evidence in breast cancer was identified, which could lead to future research in breast cancer mitochondrial metabolism. Data are available via ProteomeXchange with identifier PXD069883. SIGNIFICANCE: This manuscript determined the protein expression profiles of mitochondria-enriched fractions from T47D (Luminal A, stage IV) and MDA-MB-231 (triple negative, Stage IV) breast cancer cell lines compared to the MCF-12F healthy breast cell line. We found that breast cancer cell lines exhibited low expression levels of Complex I subunits from the respiratory chain. However, both breast cancer cell lines presented high expression levels of some proteins related to ligase and oxidoreductase activities, the latter on CH-OH groups in cellular respiration processes, such as some enzymes from glycolysis, pyruvate metabolism, Krebs cycle and gluconeogenesis. Moreover, many of these enzymes also participate in other metabolic processes, such as localization to the mitochondrion, mitochondrial gene expression, amino acid, fatty acid, purine, and pyrimidine metabolism. We also observed through Gene Set Enrichment Analysis that OXPHOS enzymes have a key role in many neurodegenerative disease pathways as well. Finally, we found a protein set with little or no evidence in breast cancer that could lead to future pivotal research in the mitochondrial metabolism of breast cancer.The proteomics analysis of mitochondria-enriched fractions reveals alteration of key mitochondrial pathways in breast cancer cell lines.Vázquez-Narváez Hersson David HD, Labra-Barrios María Luisa ML, Ríos-Castro Emmanuel E, Castañón-Sánchez Carlos Alberto CA, Uribe-Carvajal Salvador S, Hernández-Martínez Edgar E, Hernández-Hernández José Manuel JM, León-Ávila Gloria G, Pérez-Rangel Armando A, Ángeles-Morales Erika Beatriz EB, Luna-Arias Juan Pedro JPfalseThe proteomics analysis of mitochondria-enriched fractions reveals alteration of key mitochondrial pathways in breast cancer cell linesBreast cancer is the leading cause of death in women worldwide. Plasticity and heterogeneity of breast cancer cells make this disease extremely difficult to treat. Many reports point to metabolic shifts, mainly those carried out in o mitochondria, as key processes governing the behavior and heterogeneity of several types of breast cancer. In this study, we performed label-free proteomics analysis on mitochondria-enriched fractions from T47D and MDAMB-231 breast cancer cell lines, which have distinct molecular classifications, using bioinformatics analyses to identify differentially expressed proteins compared to MCF-12F healthy breast cells. Cancer cells exhibited down-regulated protein levels of subunits from the respiratory chain's Complex I. However, both presented differentially abundant proteins related to ligase and oxidoreductase activities, such as enzymes involved in glycolysis, pyruvate metabolism, the Krebs cycle, and gluconeogenesis. Many of these enzymes also participate in other metabolic processes, such as localization to the mitochondrion, mitochondrial gene expression, and the metabolism of amino acids, fatty acids, purines, and pyrimidines. Gene Set Enrichment Analysis revealed that OXPHOS enzymes play key roles in many neurodegenerative disease pathways. A protein set with little or no evidence in breast cancer was identified, which could lead to future research in breast cancer mitochondrial metabolism.2026-04-272025-10-24PXD069883NEWT:330879NEWT:377960NEWT:2042546NEWT:259447NEWT:295546NCBITaxon:1280NEWT:112503NEWT:1129NEWT:309807NEWT:59753NEWT:89184NEWT:309800NEWT:281395NEWT:1211601NEWT:876138NEWT:44271NEWT:193516NEWT:1073882NEWT:475174NEWT:1117NEWT:498257NEWT:10036NEWT:1590NEWT:10034NEWT:661410NEWT:638632NEWT:224326NEWT:376619NCBITaxon:79857NEWT:1096976NEWT:1589NEWT:135622NEWT:35786NEWT:1580NEWT:399784NEWT:96731NEWT:383379NEWT:418106NEWT:10029NEWT:913645NEWT:641809NEWT:38815NEWT:317447NEWT:4688NEWT:7719NEWT:868565NEWT:135674NEWT:79329NEWT:30069NEWT:12637NEWT:59729NEWT:2164133NEWT:295105NEWT:108061NEWT:60711NEWT:224308NEWT:3347NEWT:160621NEWT:212790NEWT:110368NEWT:1310161NEWT:77133NEWT:145481NEWT:1310165NEWT:29058NCBITaxon:79824NEWT:44688NEWT:44689NEWT:498211NEWT:347256NEWT:5518NEWT:398007NEWT:1527468NEWT:498217NEWT:498216NEWT:11320NEWT:246196NEWT:246197NEWT:145458NEWT:44685NEWT:161934NEWT:1148NEWT:5508NEWT:3329NEWT:419481NEWT:5507NEWT:410661NEWT:1140NCBITaxon:2157NEWT:1143NEWT:1287689NEWT:1094343NEWT:1462472NEWT:1336795NEWT:644042NEWT:1182590NEWT:3712NEWT:3711NEWT:2065263NEWT:177437NEWT:10418NEWT:118698NEWT:1616117NEWT:118696NEWT:34865NEWT:52283NEWT:284812NEWT:8175NEWT:43330NEWT:1603293NEWT:44664NEWT:43346NEWT:3702NEWT:1245466NEWT:244366NEWT:1246791NEWT:118694NEWT:2850NEWT:118691NEWT:34871NEWT:33548NEWT:52299NEWT:96794NEWT:3708NEWT:332648NEWT:44670NEWT:536231NEWT:376219NEWT:219813NEWT:1510NEWT:460519NEWT:1515NEWT:572307NEWT:1432138NEWT:1424507NEWT:1194599NEWT:272844NEWT:1303443NEWT:9483NEWT:485NEWT:56636NEWT:2853422NEWT:1679718NEWT:480NEWT:67767NEWT:46835NEWT:109757NEWT:582580NEWT:1185650NEWT:216778NEWT:1502NEWT:128017NEWT:376686NEWT:95486NEWT:1883446NEWT:1233435NEWT:109760NEWT:29031NEWT:317047NEWT:460523NEWT:235443NEWT:1757312NEWT:85694NEWT:108458NEWT:5936NEWT:320637NEWT:1078283NEWT:3750NEWT:983964NEWT:11706NEWT:32644NEWT:527796NEWT:499175NEWT:109779NEWT:3745NEWT:1715989NCBITaxon:4751NEWT:3747NEWT:1116234NEWT:1255228NEWT:410289NEWT:373153NEWT:472NEWT:940825NEWT:529507NEWT:1071661NEWT:470NEWT:5911NCBITaxon:50557NEWT:39251NEWT:29491NEWT:101841NEWT:446NEWT:153481NEWT:2014887NEWT:33952NEWT:445NEWT:153009NEWT:261756NEWT:166313NEWT:63366NEWT:63367NEWT:215402NEWT:1547NEWT:906914NEWT:9031NEWT:27292NEWT:1050722NEWT:108931NEWT:1293497NEWT:1055524NEWT:150475NEWT:267872NEWT:172269NEWT:9534NEWT:5180NEWT:256737NEWT:9541NEWT:8694NEWT:33936NEWT:8692NEWT:2903NEWT:185579NEWT:13076NEWT:1006581NEWT:8673NEWT:33940NEWT:550NEWT:554NEWT:451516NEWT:552NEWT:1325291NEWT:36185NEWT:1054211NEWT:1225786NEWT:51338NEWT:575412NEWT:28112NEWT:6493NEWT:6494NEWT:6491NEWT:507601NEWT:520NEWT:186441NEWT:643680NEWT:214092NCBITaxon:6157NEWT:13095NEWT:162425NEWT:104105NEWT:216257NEWT:9986NEWT:8654NEWT:8658NEWT:1268063NEWT:8657NEWT:8655NEWT:5147NEWT:28108NEWT:28104NEWT:407821NEWT:568703NCBITaxon:2NEWT:568708NEWT:986NEWT:52641NEWT:28532NEWT:353152NEWT:298176NEWT:40674NEWT:1194669NEWT:443906NEWT:519NEWT:2510939NEWT:7394NEWT:6063NEWT:1328388NEWT:1548728NEWT:667127NEWT:9557NEWT:377586NEWT:300641NEWT:39655NEWT:9554NEWT:38323NEWT:256318NEWT:206411NCBITaxon:6191NEWT:229533NEWT:214053NEWT:80863NEWT:90675NEWT:52638NEWT:57075NEWT:1666905NEWT:8697NEWT:8695NEWT:884204NEWT:1123869NEWT:9544NEWT:9545NEWT:979NEWT:7370NEWT:83906NEWT:99488NEWT:1134506NEWT:255470NEWT:38783NEWT:6426NEWT:33090NEWT:9935NEWT:287889NEWT:305959NEWT:13489NEWT:29829NEWT:92867NEWT:92866NCBITaxon:3055NEWT:51750NEWT:202950NEWT:295027NCBITaxon:11320NEWT:632957NEWT:9925NCBITaxon:9606NEWT:90690NEWT:1436183NEWT:300273NEWT:4232NEWT:416348NEWT:11298NEWT:4236NEWT:7742NEWT:196627NEWT:200308NEWT:200302NEWT:870435NEWT:9913NEWT:9915NEWT:105841NEWT:2666255NEWT:999810NCBITaxon:5693NEWT:28995NEWT:1392998NEWT:380394NEWT:226900NEWT:1266738NEWT:231490NEWT:244704NEWT:7725NEWT:9909NEWT:9902NEWT:430615NEWT:72664NEWT:563041NEWT:36599NEWT:41949NEWT:326423NEWT:452467NEWT:198822NEWT:36111NEWT:326424NEWT:1678078NEWT:749906NEWT:418985NEWT:749907NEWT:150847NEWT:29835NEWT:431947NEWT:69014NEWT:142809NEWT:302409NEWT:130821NEWT:27606NEWT:1519788NEWT:59202NEWT:9975NEWT:8643NEWT:1159899NEWT:502780NEWT:860688NEWT:13443NEWT:35231NEWT:8644NEWT:5141NEWT:46360NCBITaxon:3044782NEWT:202914NEWT:160235NEWT:502779NEWT:297246NEWT:8639NEWT:1928434NEWT:8637NEWT:1114970NEWT:2096NEWT:192222NEWT:9963NEWT:1617910NEWT:637379NEWT:2785785NEWT:411901NEWT:1298722NEWT:8626NEWT:186801NEWT:105884NCBITaxon:9615NEWT:8621NEWT:58334NEWT:58331NEWT:8624NEWT:5116NEWT:8623NEWT:5597NEWT:194439NCBITaxon:12637NEWT:38727NEWT:218496NEWT:8618NEWT:70448NEWT:300251NEWT:9940NEWT:1247190NEWT:118702NEWT:118700NEWT:2126719NEWT:118705NEWT:6945NEWT:2104NEWT:2627626NEWT:6943NEWT:157546NEWT:870726NEWT:91374NEWT:91373NEWT:474186NEWT:54195NEWT:10136NEWT:38942NEWT:4762NEWT:6941NEWT:307972NEWT:2102NEWT:34NEWT:38949NEWT:1299114NEWT:1590841NEWT:1276473NEWT:2282334NEWT:1247NEWT:70667NEWT:869728NEWT:6938NCBITaxon:39054NEWT:1736309NEWT:4751NEWT:194963NEWT:15819NEWT:109461NEWT:1085956NEWT:6928NEWT:1239NEWT:36745NEWT:400727NEWT:272563NEWT:11009NEWT:913998NEWT:68335NEWT:388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