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Jourdain"],"technology_type":["Mass Spectrometry","Data-independent acquisition"],"software":[""],"submitter_keywords":["Immunometabolism","Mitochondria","Mtdna","Integrated stress response","Proteomics","Il-4","Il-13","Membrane potential","Oxphos","Macrophage"],"full_dataset_link":["https://www.ebi.ac.uk/pride/archive/projects/PXD069191"],"tissue":["Bone Marrow","Macrophage"],"sample_protocol":["Lysates were digested following a modified version of the iST method (Kulak, et al., 2014) (named miST method).  Washed cell pellets were lysed in 100 ul of miST lysis buffer (1% Sodium deoxycholate, 100mM Tris pH 8.6, 10 mM DTT) and heated for 10min at 75C. After 1:1 (v:v) dilution with water containing 4mM MgCl2 and benzonase (Merck #70746, 100x dil of stock = 250 Units/ul), samples were incubated for 15 minutes at RT to digest nucleic acids. Based on tryptophane fluorescence quantification (Wisniewski, et al., 2015), 100 ug of proteins at 1-2ug/ul were transferred to new tubes. Reduced disulfides were alkylated by adding ¼ vol. of 160 mM chloroacetamide (32 mM final) and incubating for 45min at RT in the dark. Samples were adjusted to 3 mM EDTA and digested with 1 ug Trypsin/LysC mix (Promega #V5073) for 2h at 37°C, followed by a second 1h digestion with an additional 1 ug of proteases. To remove sodium deoxycholate, two sample volumes of isopropanol containing 1% TFA were added to the digests, and the samples were desalted on a strong cation exchange (SCX) plate (Oasis MCX; Waters Corp., Milford, MA) by centrifugation. After washing with isopropanol/1%TFA, peptides were eluted in 200ul of 80% MeCN, 19% water, 1% (v/v) ammonia, and dried by centrifugal evaporation.  Samples derived from mitochondrial enrichment (washed pellets) and MACS-beads immunoprecipitation were processed similarly to cell pellets, but without benzonase digestion step. For immunoprecipitated samples, digestion was performed on-beads for 2h with 1 ug Trypsin under gentle agitation. The supernatant was then collected after centrifugation and desalted by SCX solid phase extraction as described above.  A dedicated library for data-independent analysis (DIA) was prepared for each type of sample. Aliquots (1/12) of samples after digestion were pooled (final amount 12-30 ug) and separated into 6 fractions by off-line basic reversed-phase (bRP) using the Pierce High pH Reversed-Phase Peptide Fractionation Kit (Thermo Fisher Scientific). The fractions were collected in 7.5, 10, 12.5, 15, 20 and 50% acetonitrile in 0.1 % triethylamine (~pH 10). Dried bRP fractions were redissolved in 50-100 ul 2% acetonitrile with 0.5% TFA, and 5-10 ul were injected for LC-MS/MS analyses in data-dependent acquisition (DDA) mode. LC-MS/MS analyses were carried out on a TIMS-TOF Pro (Bruker, Bremen, Germany) mass spectrometer interfaced through a nanospray ion source (“captive spray”) to an Ultimate 3000 RSLCnano HPLC system (Dionex). Peptides were separated on a reversed-phase custom packed 45 cm C18 column (75 μm ID, 100Å, Reprosil Pur 1.9 um particles, Dr. Maisch, Germany) at a flow rate of 250 nl/min with a 2-27% acetonitrile gradient in 93 min followed by a ramp to 45% in 15 min and to 90% in 5 min (total method time: 140 min, all solvents contained 0.1% formic acid). Identical LC gradients were used for DDA and DIA measurements.   For creation of the spectral library, data-dependent acquisitions (DDA) were carried out on the 6 bRP fractions sample pool using a standard TIMS PASEF method (Meier, et al. 2018) with ion accumulation for 100 ms for each survey MS1 scan and the TIMS-coupled MS2 scans. Duty cycle was kept at 100%. Up to 10 precursors were targeted per TIMS scan. Precursor isolation was done with a 2 Th or 3 Th windows below or above m/z 800, respectively. The minimum threshold intensity for precursor selection was 2500.  If the inclusion list allowed it, precursors were targeted more than one time to reach a minimum target total intensity of 20’000. Collision energy was ramped linearly based uniquely on the 1/k0 values from 20 (at 1/k0=0.6) to 59 eV (at 1/k0=1.6). Total duration of a scan cycle including one survey and 10 MS2 TIMS scans was 1.16 s. Precursors could be targeted again in subsequent cycles if their signal increased by a factor 4.0 or more.  After selection in one cycle, precursors were excluded from further selection for 60s. Mass resolution in all MS measurements was approximately 35’000.  The data-independent acquisition (DIA) used mostly the same instrument parameters as the DDA method and was as reported previously (Meier, et al. 2020). Per cycle, the mass range 400-1200 m/z was covered by a total of 32 windows, each 25 Th wide and a 1/k0 range of 0.3. Collision energy and resolution settings were the same as in the DDA method. Two windows were acquired per TIMS scan (100ms) so that the total cycle time was 1.7 s."],"repository":["Pride"],"quantification_method":[""],"modification":[""],"data_protocol":["Raw Bruker MS data were processed directly with Spectronaut 16.0 (Biognosys, Schlieren, Switzerland). A library was constructed from the DDA bRP fraction data by searching the reference mouse proteome (www.uniprot.org) database of June 4th, 2021 (55’364 sequences), and a contaminant database containing  as defined in the MaxQuant 16.1.4 software (Cox et al.2008, Cox et al 2011). For identification, peptides of 7-52 AA length were considered, cleaved with trypsin/P specificity and a maximum of 2 missed cleavages. Carbamidomethylation of cysteine (fixed), methionine oxidation and N-terminal protein acetylation (variable) were the modifications applied. Mass calibration was dynamic and based on a first database search. The Pulsar engine was used for peptide identification. Protein inference was performed with the IDPicker algorithm. Spectra, peptide and protein identifications were all filtered at 1% FDR against a decoy database.  Specific filtering for library construction removed fragments corresponding to less than 3 AA and fragments outside the 300-1800 m/z range. Also, only fragments with a minimum base peak intensity of 5% were kept.  Precursors with less than 3 fragments were also eliminated and only the best 6 fragments were kept per precursor. No filtering was done on the basis of charge state and a maximum of 2 missed cleavages was allowed. Shared (non proteotypic) peptides were kept.   Three libraries were created separately for total cell extracts, enriched and immunoprecipitated mitochondria but were later combined in a single quantification run with Spectronaut, including all datafiles. The combined library created contained 148’201 unique precursors mapping to 103’792 peptides. Peptide-centric analysis of DIA data was done with Spectronaut 16.0 using the library described above. Single hits proteins (defined as matched by one stripped sequence only) were kept in the Spectronaut analysis. Peptide quantitation was based on XIC area, for which a minimum of 1 and a maximum of 3 (the 3 best) precursors were considered for each peptide, from which the median value was selected.  Quantities for protein groups were derived from inter-run peptide ratios based on MaxLFQ algorithm (Cox et al 2014). Global normalization of runs/samples was done based on the median of peptides.  Overall 147’177 precursors were quantified in the dataset, mapped to 8’354 protein groups. Between 6728 and 8167 protein groups were quantified in individual samples (between 67’591 and 92720 peptides)."],"omics_type":["Proteomics"],"labhead":["Alexis Jourdain"],"instrument_platform":[""],"labhead_affiliation":["Dpt. of Immunobiology, University of Lausanne (UNIL). Ch.Boveresses 155 CH-1066 Epalinges"],"submission_type":["PARTIAL"],"species":["Mus Musculus (mouse)"],"publication":["Not available"],"submitter_mail":["alexis.jourdain@unil.ch"],"submitter_affiliation":["Department of Immunobiology, University of Lausanne"],"submitter_country":["Switzerland"],"additional_accession":[]},"is_claimable":false,"name":"Integrated mitochondrial profiling identifies mitochondrial gene expression and membrane potential in IL-4/IL-13 macrophages","description":"Macrophage activation triggers intense genetic and immunometabolic reprograming that support distinct macrophage functions, and mitochondria are central to this process. To date, the extent to which they are remodeled and how this impacts macrophage function remains unclear. Here, we integrate transcriptomics with whole-cell and highly purified mitochondrial proteomics to profile LPS/IFNγ- andIL-4/IL-13-stimulated bone-marrow–derived macrophages (BMDM). We uncover a striking disconnect between mitochondrial transcripts and proteins and identify a STAT6-dependent increase in mitochondrial gene expression and intramitochondrial translation as hallmarks of IL-4/IL-13 stimulation. Pharmacological blockade of mitochondrial translation or individual respiratory chain complexes impairs activation, while ATP synthase inhibition uniquely triggers mitochondrial hyperpolarization and the integrated stress response, halting IL-4/IL-13 activation. Restoring mitochondrial membrane potential or inhibiting the stress response rescues effector synthesis. Together, we establish mitochondrial gene expression and membrane potential as regulators of macrophage immunometabolism and identify mitochondrial translation as a critical, drug-sensitive determinant of IL-4/IL-13 macrophage function.","dates":{"publication":"2026-06-29","submission":"2025-10-07"},"accession":"PXD069191","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: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:294671","NEWT:376619","NEWT:1748418","NCBITaxon:79857","NEWT:1096976","NEWT:3765","NEWT:3767","NEWT:1589","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:29073","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:2020713","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: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:199225","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:1122981","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:31345","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:1380096","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:51350","NEWT:8694","NEWT:33936","NEWT:8692","NEWT:542","NEWT:2903","NEWT:185579","NEWT:872227","NEWT:13076","NEWT:2587410","NEWT:207340","NEWT:1006581","NEWT:8673","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