Prideapplication/xmlftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD079623/checksum.txtftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD079623/20260223_134747_DF_DC_ex_vivo_spectronaut19_Report.setup.txtftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD079623/20260223_134747_DF_DC_ex_vivo_spectronaut19_Report.csvftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD079623/012_2025_DUN-TCD-DF-DAC-8-cDC1-2-6K.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD079623/012_2025_DUN-TCD-DF-DAC-7-cDC1-2-6K.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD079623/012_2025_DUN-TCD-DF-DAC-4-pDC-10K.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD079623/012_2025_DUN-TCD-DF-DAC-10-cDC2-10K.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD079623/012_2025_DUN-TCD-DF-DAC-11-cDC2-10K.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD079623/012_2025_DUN-TCD-DF-DAC-3-pDC-10K.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD079623/012_2025_DUN-TCD-DF-DAC-1-pDC-10K.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD079623/012_2025_DUN-TCD-DF-DAC-13-cDC2-10K.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD079623/012_2025_DUN-TCD-DF-DAC-2-pDC-10K.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD079623/012_2025_DUN-TCD-DF-DAC-6-cDC1-2-6K.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD079623/012_2025_DUN-TCD-DF-DAC-12-cDC2-10K.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD079623/012_2025_DUN-TCD-DF-DAC-5-pDC-2-6K.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD079623/012_2025_DUN-TCD-DF-DAC-9-cDC1-2-6K.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD079623/20260223_134747_DF_DC_ex_vivo_spectronaut19.sneprimaryOK200FinlayD@tcd.ieLinda SinclairMass SpectrometryData-independent acquisitionTransferrin receptor; dendritic cell; ironironTransferrin receptordendritic cellhttps://www.ebi.ac.uk/pride/archive/projects/PXD079623Dendritic CellPreparation of cell pellets/peptides Splenocytes were isolated from naïve mice. Cell sorting of DC subsets was performed using the FACS Aria cytometer. cDC1 were identified as LIVE/DEAD-,CD3-,CD19-,CD11c+,MHCIIhi,XCR1+,SIRP- cells, cDC2s were identified as LIVE/DEAD-,CD3-,CD19-,CD11c+,MHCIIhi,XCR1-,SIRP+ and pDCs were identified as LIVE/DEAD-,CD3-,CD19-,CD11cint ,CD317+ SiglecH+ cells. Cells (10,000 pDC and cDC2 and 2,000 cDC1) were sorted into lysis buffer (RIPA buffer, 50mM TEAB, 5mM TCEP, 0.5% benzonase), snap frozen and sent to Dundee for further processing. Briefly, peptides from frozen cell lysates were prepared using a modified SP3 method ( Hughes C.S., Foehr S., Garfield D.A., Furlong E.E., Steinmetz L.M. and Krijgsveld J. (2014) Ultrasensitive proteome analysis using paramagnetic bead technology. Mol. Syst. Biol. 10, 757 10.15252/msb.20145625) LC-MS Analysis Peptides (equivalent of 200 ng) were injected onto a nanoscale C18 reverse-phase chromatography system (Vanquish, Thermo Scientific) and electrosprayed into an Astral Orbitrap Mass Spectrometer (Thermo Fisher). For liquid chromatography the following buffers were used: buffer A and weak buffer wash (0.1% formic acid in Milli-Q water (v/v) buffer B and strong buffer wash (80% acetonitrile and 0.1% formic acid in Milli-Q water (v/v). Samples were loaded onto a trap column (Pep Map Neo C18, 5µm, 300µm x5 mm) Thermo Scientific) equilibrated in buffer A with the following loading settings, mode: flow control, flow 60 µl/min and loading volume set to automatic. The wash and equilibration settings were as follows equilibration factor 2, trap column, fast wash and equilibration on, zebra wash on, zebra wash cycle 4, equilibration factor automatic, mode combined control, flow 200µl/min, pressure 800 bar. The column (Pep MAP C18, 2µm, 150 µm x15 cm) was equilibrated with 4% buffer and peptides were eluted at a variable flow rate, starting at 1.3 µl/min to 0.8 µl/min from 4% buffer B to 8% buffer B in 0.1 min, then from 8% buffer B to 22.5% buffer B in 13 min, and from 22.5% b to 35% B in 6.90 min. The low rate is increased from 0.8 µl/min to 2 µl/min in 0.4 min with buffer B increasing from 35% to 55%. The column is finally washed at a flow of 2µl/min with 99% buffer B. The column was kept at a constant temperature of 50oC. The data was acquired using an easy spray source operated in positive mode with spray voltage at 2.0 kV, and the ion transfer tube temperature at 280oC. The MS was operated in DIA mode. A scan cycle comprised a full MS scan (m/z range from 380-980), with orbitrap resolution at 2400000, RF lens at 40%, AGC target set to custom, normalised AGC target at 500%, absolute AGC value set to 5.00e6, maximum injection time 5ms and micros-can set to 1. MS survey scan was followed by MS/MS DIA scan events using the following parameters: DIA window type set to auto, isolation window 2, window overlap set to 0, window placement optimisation on, number of scan events 299, collision energy type normalized, HCD collision energy 25%, detector type Astral, scan range 150-2000, normalized AGC target 500%, absolute AGC target 5.000e4, maximum injection time 5 ms, microscan set to 1, loop control time, time 0.6s. Both MS scan and the DIA data are collected in profile mode.PrideNot availableThe data were processed, searched, and quantified using Spectronaut 19 using the directDIA option. The direct DIA data was searched against the UniProt mouse database (SwissProt and Trembl). Protein copy number quantification was performed in the Perseus software package (Tyanova S., Temu T., Sinitcyn P., Carlson A., Hein M.Y., Geiger T. et al. (2016) The perseus computational platform for comprehensive analysis of (prote)omics data. Nat. Methods 13, 731–740 10.1038/nmeth.3901.) Mean copy numbers were estimated using the “proteomic ruler” described in (Wiśniewski et al., 2014). The accuracy of quantification was established using the following guidelines: proteins categorised as high accuracy had more than eight unique and razor peptides and a ratio for unique/unique + razor of ≥0.75; proteins categorised as medium accuracy had at least three unique and razor peptides, and a ratio for unique/unique + razor of ≥0.5; and any proteins below these thresholds were classified as low accuracy. Quantitative proteomic data from pDC, cDC1, and cDC2 subsets were used to estimate relative subcellular composition. Following normalisation and aggregation of protein abundances to the gene level, proteins were assigned to major subcellular compartments based on curated UniProt and Gene Ontology Cellular Component annotations. Each protein was assigned a dominant localisation to avoid over‑representation of multi‑localised proteins. For each sample, compartmental protein abundances were summed and normalised to total cellular protein abundance to calculate proportional subcellular composition. Mean compartmental profiles were calculated across replicates to enable comparison between dendritic cell subsets.ProteomicsDavid K FinlayTrinity College Dublin, IrelandPARTIALMus Musculus (mouse)l.v.sinclair@dundee.ac.uk42307014 Corkish C, O'Shaughnessy S, Lin C, Wugk S, Kenny S, Sinclair L, Cloonan S, Finlay D. Plasmacytoid Dendritic Cells Exhibit High Transferrin Receptor Expression Without Iron Accumulation. Eur J Immunol. 2026 56(6):e70219 10.1002/eji.70219University of DundeeUnited KingdomPlasmacytoid dendritic cells (pDCs) are specialized antiviral sentinels defined by rapid type I interferon (IFN‑I) production, yet their proteomic organization and metabolic requirements remain incompletely understood. We established the steady‑state proteome of murine splenic pDCs directly ex vivo using deep, absolute quantitative mass spectrometry and compared it with conventional dendritic cell subsets and human pDCs. pDCs exhibited a highly conserved proteomic architecture across species, with selective divergence in central carbon metabolism, amino‑acid utilization, and nutrient transporter expression. Notably, pDCs expressed exceptionally high levels of the transferrin receptor (TFRC) in both mice and humans and displayed robust transferrin‑mediated iron uptake relative to other splenic immune populations. Despite this, pDCs did not demonstrate increased total cellular iron or enhanced ferritin‑based storage. Instead, proteome‑wide iron mapping revealed preferential allocation of iron to functional iron-sulfur and heme‑containing proteins, particularly within mitochondrial pathways. Detection of the iron exporter ferroportin indicated coordinated iron import and efflux, establishing sustained iron flux rather than net accumulation. Functional assays showed that iron availability does not constrain TLR9‑induced IFN‑I or TNF production. Together, these data define a conserved iron‑handling program in pDCs characterized by high TFRC expression, balanced iron flux, and targeted redistribution into essential protein systems.Plasmacytoid Dendritic Cells Exhibit High Transferrin Receptor Expression Without Iron Accumulation.Corkish Carrie C, O'Shaughnessy Simon S, Lin Claire C, Wugk Sophia S, Kenny Sarah S, Sinclair Linda L, Cloonan Suzanne S, Finlay David DfalseIron flux in plasmacytoid dendritic cells; potential role for transferrin receptor beyond iron metabolismPlasmacytoid dendritic cells (pDCs) are critical antiviral sentinels known for rapid type I interferon (IFN-I) production. However, their proteomic profile and nutrient dependencies are not well understood. Here, we used absolute quantitative proteomics of ex vivo murine splenic pDCs to characterize their protein landscape. Cross-species comparison with human blood pDCs revealed strong conservation but some differences in metabolic machinery. The transferrin receptor, responsible for transferrin-iron uptake, is the most abundant nutrient transporter in both species with 40,000 copies per murine pDC, and is significantly enriched compared to conventional dendritic cells. Despite pDCs showing constitutive transferrin-iron uptake they have low expression of iron storage proteins and a modest repertoire of iron-dependent enzymes. Also, pDC do not have an increase in iron atoms per cell compared to cDC, which suggests that pDC export excess iron through ferroportin and argue that Tfrc may have iron-independent roles. Indeed, iron chelation did not affect pDC production of type I interferon. These data suggest that Tfrc supports noncanonical functions in pDC biology, possibly related to antigen uptake. This work provides new insights into pDC metabolism and iron biology, advancing understanding of their specialized immune roles. Plasmacytoid dendritic cells (pDCs) are specialized antiviral sentinels defined by rapid type I interferon (IFN‑I) production, yet their metabolic organization and metal‑ion handling remain poorly understood. Here, we establish a high‑confidence, absolute quantitative proteome of murine splenic pDCs directly ex vivo and compare it with conventional dendritic cell subsets and human pDCs. Cross‑species proteomic analysis reveals strong conservation of pDC identity alongside divergence in metabolic pathway usage. pDCs display exceptionally high expression of the transferrin receptor (TFRC) and robust transferrin uptake compared with other immune cells; however, quantitative ironome analysis demonstrates that total cellular iron content is equivalent across dendritic cell subsets. Instead, iron is differentially allocated, with pDCs enriched for iron-sulfur cluster assembly proteins, cDC1s for heme‑binding proteins, and cDC2s for non‑heme iron enzymes. Despite elevated transferrin uptake, pDCs do not accumulate intracellular iron, coincident with expression of the iron exporter ferroportin, suggesting active iron efflux. Functionally, acute chelation or supplementation of extracellular iron does not affect CpG‑A plus IFNα-induced IFNα or TNFα production by pDCs. Together, these data demonstrate that pDCs uncouple surface transferrin receptor abundance from intracellular iron accumulation and effector cytokine production, revealing a distinct organization of iron handling that may support specialized trafficking or sensing functions rather than metabolic iron demand.2026-06-292026-06-12PXD079623NEWT:330879NEWT:377960NEWT:2042546NEWT:259447NEWT:295546NCBITaxon:2719036NEWT:625351NCBITaxon:1280NEWT:112503NEWT:1129NEWT:309807NEWT:59753NEWT:89184NEWT:309800NEWT:281395NEWT:1211601NEWT:876138NEWT:44271NEWT:193516NEWT:1073882NEWT:6819NEWT:475174NEWT:111205NEWT:1117NEWT:498257NEWT:10036NEWT:1590NEWT:10034NEWT:661410NEWT:638632NEWT:224326NEWT:376619NCBITaxon:79857NEWT:1096976NEWT:3765NEWT:1589NEWT:135622NEWT:35786NEWT:1580NEWT:399784NEWT:96731NEWT:656064NEWT:1582NEWT:36630NEWT:383379NEWT:418106NCBITaxon:12721NEWT:10029NEWT:913645NEWT:641809NEWT:1392488NEWT:38815NEWT:317447NEWT:4688NEWT:111225NEWT:7719NEWT:868565NEWT:135674NEWT:79329NEWT:30069NEWT:12637NEWT:59729NEWT:2164133NEWT:295105NEWT:108061NEWT:60711NEWT:224308NEWT:3347NEWT:160621NEWT:212790NEWT:110368NEWT:1310161NEWT:77133NEWT:145481NEWT:1310165NEWT:29058NCBITaxon:79824NEWT:1912919NEWT:410658NEWT:44688NEWT:44689NEWT:498211NEWT:347256NEWT:5518NEWT:398007NEWT:1527468NEWT:498217NEWT:498216NEWT:11320NEWT:246196NEWT:246197NEWT:145458NEWT:44685NEWT:161934NEWT:156587NEWT:1148NEWT:5508NEWT:3329NEWT:419481NEWT:5507NEWT:410661NEWT:1140NCBITaxon:2157NEWT:1143NEWT:1287689NEWT:1094343NEWT:1462472NEWT:1336795NEWT:644042NEWT:1182590NEWT:3712NEWT:3711NEWT:3714NEWT:270643NEWT:2065263NEWT:177437NEWT:10418NEWT:118698NEWT:1616117NEWT:118696NEWT:34865NEWT:52283NEWT:28174NEWT:284812NEWT:8175NEWT:43330NEWT:1603293NEWT:44664NEWT:43346NEWT:3702NEWT:1245466NEWT:3704NEWT:244366NEWT:1246791NEWT:176597NEWT:118694NEWT:2850NEWT:118691NEWT:34871NEWT:33548NEWT:52299NEWT:8165NEWT:96794NEWT:3708NEWT:332648NEWT:44670NEWT:536231NEWT:376219NEWT:219813NEWT:1510NEWT:460519NEWT:1515NEWT:572307NEWT:1432138NEWT:1424507NEWT:1194599NEWT:272844NEWT:1348799NEWT:33995NEWT:55784NEWT:1303443NEWT:9483NEWT:485NEWT:56636NEWT:2709072NEWT:2499525NEWT:2853422NEWT:487NEWT:1679718NEWT:480NEWT:67767NEWT:46835NEWT:109757NEWT:582580NEWT:1185650NEWT:216778NEWT:294607NEWT:1502NEWT:128017NEWT:376686NEWT:237610NEWT:95486NEWT:1883446NEWT:1233435NEWT:118223NEWT:11757NEWT:109760NEWT:29031NEWT:317047NEWT:491NEWT:460523NEWT:235443NEWT:1757312NEWT:85694NEWT:109786NEWT:108458NEWT:5936NEWT:320637NEWT:1078283NEWT:3750NEWT:983964NEWT:11706NEWT:32644NEWT:527796NEWT:499175NEWT:109779NEWT:3745NEWT:1715989NCBITaxon:4751NEWT:3747NEWT:1116234NEWT:1255228NEWT:410289NEWT:373153NEWT:1640278NEWT:472NEWT:940825NEWT:529507NEWT:1071661NEWT:28128NEWT:470NEWT:5911NEWT:748693NCBITaxon:50557NEWT:39251NEWT:29491NEWT:101841NEWT:446NEWT:153481NEWT:2014887NEWT:33952NEWT:445NEWT:153009NEWT:261756NEWT:166313NEWT:63366NEWT:63367NEWT:215402NEWT:1547NEWT:906914NEWT:9031NEWT:27292NEWT:1050722NEWT:3728NEWT:108931NEWT:1293497NEWT:1055524NEWT:150475NEWT:267872NEWT:381666NEWT:172269NEWT:9534NEWT:5180NEWT:256737NEWT:9541NEWT:8694NEWT:33936NEWT:8692NEWT:2903NEWT:185579NEWT:13076NEWT:207340NEWT:1006581NEWT:8673NEWT:33940NEWT:550NEWT:554NEWT:451516NEWT:552NEWT:1325291NEWT:36185NEWT:1054211NEWT:1225786NEWT:771870NEWT:51338NEWT:575412NEWT:28112NEWT:6493NEWT:10832NEWT:6494NEWT:6491NEWT:507601NEWT:520NEWT:186441NEWT:643680NEWT:214092NCBITaxon:6157NEWT:13095NEWT:162425NEWT:104105NEWT:216257NEWT:9986NEWT:8654NEWT:8658NEWT:1268063NEWT:8657NEWT:9983NEWT:8655NEWT:5147NEWT:28108NEWT:28104NEWT:407821NEWT:568703NCBITaxon:2NEWT:568708NEWT:1613NEWT:986NEWT:52641NEWT:28532NEWT:353152NEWT:298176NEWT:353153NEWT:227377NEWT:1774284NEWT:40674NEWT:1194669NEWT:51329NEWT:443906NEWT:519NEWT:2510939NEWT:7394NEWT:6063NEWT:1328388NEWT:105487NEWT:1548728NEWT:667127NEWT:9557NEWT:377586NEWT:300641NEWT:39655NEWT:9554NEWT:38323NEWT:475932NEWT:256318NEWT:206411NCBITaxon:6191NEWT:229533NEWT:214053NEWT:28550NEWT:80863NEWT:90675NEWT:52639NEWT:52638NEWT:57075NEWT:1666905NEWT:8697NEWT:8695NEWT:884204NEWT:510516NEWT:1045775NEWT:1123869NEWT:9544NEWT:9545NEWT:979NEWT:7370NEWT:83906NEWT:99488NEWT:1134506NEWT:255470NEWT:38783NEWT:6426NEWT:33090NEWT:9935NEWT:287889NEWT:305959NEWT:1028307NEWT:13489NEWT:29829NEWT:92867NEWT:92866NCBITaxon:3055NEWT:51750NEWT:202950NEWT:441772NEWT:1293091NEWT:48106NEWT:202956NEWT:295027NCBITaxon:11320NEWT:632957NEWT:9925NCBITaxon:9606NEWT:90690NEWT:1436183NEWT:300273NEWT:4232NEWT:416348NEWT:11298NEWT:4236NEWT:7742NEWT:196627NEWT:200308NEWT:200302NEWT:870435NEWT:9913NEWT:9915NEWT:105841NEWT:2666255NEWT:999810NCBITaxon:5693NEWT:28995NEWT:2144189NEWT:1392998NEWT:380394NEWT:1150621NEWT:226900NEWT:1266738NEWT:231490NEWT:244704NEWT:7725NEWT:9909NEWT:9902NEWT:430615NEWT:563041NEWT:72664NEWT:36599NEWT:41949NEWT:249161NEWT:326423NEWT:452467NEWT:198822NEWT:36111NEWT:326424NEWT:1678078NEWT:749906NEWT:418985NEWT:749907NEWT:150847NEWT:29835NEWT:431947NEWT:69014NEWT:142809NEWT:302409NEWT:130821NEWT:27606NEWT:1519788NEWT:59202NEWT:9975NEWT:8643NEWT:1801631NEWT:1159899NEWT:502780NEWT:860688NEWT:13443NEWT:35231NEWT:8644NEWT:1125630NEWT:5141NEWT:46360NCBITaxon:3044782NEWT:202914NEWT:160235NEWT:502779NEWT:297246NEWT:8639NEWT:1928434NEWT:8637NEWT:1114970NEWT:2096NEWT:285272NEWT:192222NEWT:9963NEWT:1617910NEWT:197911NEWT:197912NEWT:637379NEWT:12107NEWT:278578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