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InnsMass SpectrometrydiaPASEFBrooke-spiegler SyndromeNot availableScpTamCcsSkin tumorhttps://www.ebi.ac.uk/pride/archive/projects/PXD073250MonocyteDendritic CellEpithelial CellKeratinocyteSkinMacrophagePatient tumor samples were dissociated with scalpels and digested with trypsin for 40 min at 37°C with agitation. Further digestion was performed with 1 mg/mL collagenase at 4°C and agitation overnight. A single cell suspension was obtained after passing through a 40 μm filter and the resulting suspension was labelled with CD45/CD74/CD200 antibody mix and DAPI (4′,6-diamidino-2-phenylindole) viability stain. Single cells were FACS sorted into wells of a 96-well plate containing 5 μL of solidified hexadecane and 1μL digestion buffer (0.2% DDM, 100 mM TEAB, 5ng/μL trypsin, 5ng/μL LysC). Plates were incubated at 45 °C for 2 h followed with quenching by addition of 30 μL 0.1% FA, 1% DMSO in water. Hexadecane was solidified on ice and plates were centrifuged at 500 g for 1 min at 4°C into prepared Evotips. We analysed single cell samples with a Bruker timsTOF HT mass spectrometer in line with an EvoSep One liquid chromatography system (LC) operating the Whisper-Zoom 40 SPD method. The EvoSep LC injected samples onto a 15 cm Aurora Elite C18 column with integrated captive spray emitter (IonOpticks) at 50°C. Buffer A was 0.1% formic acid in HPLC water, buffer B was 0.1% formic acid in acetonitrile. The Bruker timsTOF HT MS was operated in diaPASEF (data-independent acquisition, parallel accumulation–serial fragmentation) mode with mass and ion mobility (IM) ranges of 300-1200 m/z and 0.6-1.45 1/K0. Four variable width IM-m/z windows each with two frames and no overlap were used. TIMS ramp and accumulation times were 200 ms, total cycle time was ~1.03 seconds. Collision energy was applied in a linear fashion, where ion mobility = 0.6-1.6 1/K0, and collision energy = 20 - 59 eV.PrideMS data files were searched in DIA-NN v2.0.1 against a Uniprot Homo sapiens reference proteome (UP000005640 SwissProt with isoforms, accessed: 24/06/2025) and a modified contaminant FASTA database at 1% FDR. MS1 and MS2 mass accuracy were fixed to 15 ppm. Precursor range was m/z 300-1200, charge +2,+3,+4, maximum missed cleavages and variable modifications set to 2, with oxidation of methionine and N-terminal acetylation included as variable modifications. For the preliminary hexadecane versus C18 searches, settings included no variable modifications and 1 missed cleavage. The in silico digest and spectral library prediction tool was used to search bulk samples, and generate an empirical DIA-spectral library, which was then employed to search single cells.ProteomicsJoseph InnsNewcastle University translational and clinical research institute United KingdomPARTIALHomo Sapiens (human)joe.inns.ac@gmail.com10.26508/LSA.202603759RNewcastle UniversityUnited KingdomfalseDemocratised single cell proteomics uncovers tumor associated macrophage signature in CYLD cutaneous syndrome skin tumorsSingle-cell proteomics (SCP) reveals heterogeneity and biological insights inaccessible to bulk analysis. Key limitations are cost, sample loss during processing, and accessibility to state-of-the-art instrumentation. We describe a label free SCP methodology in human tissue, combining fluorescence activated cell sorting (FACS), oil-immersion cell handling, mass-spectrometry, and neural-network derived spectral libraries which aims to improve user uptake. We tested this methodology in a skin tumor syndrome, CYLD cutaneous syndrome (CCS), assessing tumor heterogeneity. Using a Bruker timsTOF HT platform we quantified > 4000 proteins, averaging ~700 per cell, through a cost-effective pipeline without specialised liquid handling infrastructure. By utilising pre-existing bioinformatic tools from the scRNA-seq field we implemented a robust analysis methodology, discriminating between macrophages, dendritic cells and tumor keratinocytes, in an unbiased analysis of 419 CCS tumor cells. We confirmed the biological accuracy of cell annotations by cross referencing with each cell's FACS markers. Furthermore, we identified novel CCS tumor biology in a tumor associated macrophage population with a tumor microenvironment remodelling signature. Our findings demonstrate an accessible SCP technology capable of yielding novel biological discoveries in clinical tissue.2026-06-182026-01-19PXD073250NEWT:6945NEWT:184922NEWT:6703NEWT:3555NEWT:71647NEWT:2NEWT:157546NEWT:474186NEWT:35554NEWT:38942NEWT:307972NEWT:32046NEWT:1496NEWT:544496NEWT:2102NEWT:2042546NEWT:259447NEWT:45351NEWT:43179NEWT:4513NEWT:180454NEWT:5722NEWT:1247NEWT:6938NEWT:1129NEWT:55153NCBITaxon:10407NEWT:1736309NEWT:309800NEWT:281395NEWT:10360NEWT:1211601NEWT:876138NEWT:47664NEWT:317548NEWT:3654NEWT:237561NEWT:5833NEWT:6928NEWT:10036NEWT:36745NEWT:498019NEWT:1351NEWT:1438992NEWT:1352NEWT:2649997NEWT:272563NEWT:224326NEWT:1333499NCBITaxon:79857NEWT:1096976NEWT:82688NEWT:95648NEWT:3885NEWT:3888NEWT:5821NEWT:1589NEWT:135622NCBITaxon:4896NEWT:6915NEWT:3649NEWT:101510NEWT:28903NEWT:3880NEWT:272559NEWT:28909NEWT:515849NEWT:3641NEWT:383379NEWT:466585NEWT:10029NEWT:913645NEWT:1000589NEWT:85963NEWT:85962NEWT:143361NEWT:317447NEWT:4688NEWT:7955NEWT:7959NEWT:2261NEWT:31156NEWT:398580NEWT:4442NEWT:4565NEWT:1264690NEWT:515619NEWT:192875NEWT:34305NEWT:59729NEWT:2164133NCBITaxon:183674NEWT:224308NEWT:167387NEWT:84645NEWT:44586NEWT:626528NEWT:3347NEWT:139927NEWT:4558NEWT:209285NEWT:5888NEWT:211586NEWT:747078NEWT:1282NEWT:1283NEWT:931281NEWT:4550NEWT:1000561NEWT:294381NEWT:197NEWT:1390363NEWT:77133NEWT:288705NEWT:29176NEWT:145481NCBITaxon:79824NEWT:4787NCBITaxon:4563NEWT:5755NEWT:44689NEWT:3218NEWT:5759NEWT:1736231NEWT:556182NEWT:1270NEWT:374990NEWT:1392NEWT:498217NEWT:156471NEWT:2242NEWT:4784NEWT:11320NEWT:360106NEWT:156476NEWT:286NEWT:391619NEWT:360104NEWT:246196NEWT:287NEWT:246197NEWT:10117NEWT:10239NEWT:10116NEWT:1280NEWT:1735272NEWT:83334NEWT:83332NEWT:44685NEWT:317513NEWT:161934NEWT:1148NEWT:580240NEWT:5508NEWT:294128NEWT:5507NEWT:11676NEWT:55571NEWT:35500NEWT:1140NEWT:100226NEWT:4530NEWT:1143NEWT:4896NEWT:75058NEWT:13616NEWT:1390NEWT:1094343NEWT:1336795NEWT:644042NEWT:296543NEWT:316435NEWT:42157NEWT:1773NEWT:1895NEWT:1182590NEWT:3712NEWT:82380NEWT:1034304NEWT:105023NEWT:866628NEWT:935293NEWT:64152NEWT:4924NEWT:749200NEWT:375146NEWT:9378NEWT:990346NEWT:145953NEWT:257309NEWT:100816NEWT:263NEWT:230741NEWT:52283NEWT:284812NEWT:8175NCBITaxon:1313NEWT:43330NEWT:1603293NEWT:408169NEWT:44544NEWT:47946NEWT:4911NEWT:645463NEWT:3702NEWT:129249NEWT:1077286NEWT:243277NEWT:990119NEWT:2850NEWT:408172NEWT:227321NEWT:408170NEWT:493760NEWT:106590NEWT:260710NEWT:257313NEWT:3827NEWT:400772NEWT:3708NEWT:128161NEWT:332648NEWT:930945NEWT:106592NEWT:536231NEWT:46704NEWT:1436733NEWT:460519NEWT:1187947NEWT:572307NEWT:1432138NEWT:269796NEWT:10312NEWT:1424507NCBITaxon:1773NEWT:1194599NEWT:54571NEWT:9598NEWT:8030NEWT:9483NEWT:1513458NCBITaxon:40559NEWT:1639NEWT:188229NEWT:3818NEWT:480NEWT:4909NEWT:67767NEWT:759272NEWT:432359NEWT:46835NEWT:1182263NEWT:109757NEWT:943146NEWT:2711NEWT:300852NEWT:1502NEWT:376686NEWT:95486NEWT:9103NEWT:508771NEWT:1883446NEWT:29159NEWT:253NEWT:10306NCBITaxon:2759NEWT:1233435NEWT:93061NEWT:8022NEWT:145943NCBITaxon:4932NEWT:595536NEWT:240906NEWT:593117NEWT:89920NEWT:29158NEWT:3635NEWT:5811NEWT:235443NEWT:180923NEWT:108458NEWT:272623NEWT:272624NEWT:411483NEWT:884019NEWT:198215NEWT:411490NEWT:983964NEWT:118499NEWT:169963NEWT:32644NEWT:527796NEWT:225117NEWT:746128NEWT:499175NEWT:109779NEWT:43665NEWT:1266464NEWT:1715989NEWT:476272NEWT:3747NEWT:195051NEWT:367830NEWT:1255228NEWT:178616NEWT:649908NEWT:410289NEWT:373153NEWT:375451NEWT:352472NEWT:357NEWT:1071661NEWT:360094NEWT:470NEWT:41364NEWT:1313NEWT:411469NEWT:84023NEWT:559292NEWT:39491NCBITaxon:5811NEWT:29491NEWT:411464NEWT:411460NEWT:2014887NEWT:2762NEWT:1174673NEWT:1328426NEWT:562NEWT:411470NEWT:33952NEWT:2094720NCBITaxon:2697049NEWT:571256NCBITaxon:138NEWT:40483NEWT:28038NEWT:1663NEWT:1423NEWT:4932NEWT:3603NEWT:2759NEWT:3847NEWT:38293NEWT:1428NEWT:327159NEWT:178876NEWT:1660NEWT:327160NEWT:573NEWT:9031NEWT:1872122NEWT:7091NEWT:108931NEWT:241368NEWT:1055524NEWT:42528NEWT:190802NEWT:9778NEWT:150475NEWT:303NEWT:9417NEWT:7111NEWT:347515NEWT:1216979NEWT:7237NEWT:9534NEWT:5180NEWT:256737NEWT:9541NEWT:115104NEWT:1121114NEWT:663NEWT:1081927NEWT:1238993NEWT:67825NEWT:187878NEWT:185579NEWT:941442NEWT:220668NEWT:13076NEWT:1821314NEWT:1249668NEWT:7108NEWT:317NEWT:5286NEWT:7227NEWT:7469NEWT:885318NEWT:9402NEWT:9644NEWT:415540NEWT:550NEWT:675060NEWT:4081NEWT:334542NEWT:273068NEWT:554NEWT:27592NEWT:98334NEWT:426428NEWT:451516NEWT:63459NEWT:1276815NEWT:36185NEWT:588858NEWT:1225786NEWT:9639NCBITaxon:11071NEWT:242231NEWT:7574NEWT:1715256NEWT:197221NEWT:7215NEWT:575412NEWT:929793NEWT:29204NEWT:28112NEWT:2172103NEWT:6494NEWT:7460NEWT:6491NEWT:507601NEWT:643680NCBITaxon:6157NEWT:42752NEWT:746360NEWT:6239NEWT:89764NEWT:470150NEWT:162425NEWT:216257NEWT:102169NEWT:543734NEWT:9986NEWT:4054NEWT:73239NEWT:226186NEWT:1268063NEWT:8782NEWT:1263854NEWT:118503NEWT:2059687NEWT:115357NEWT:435590NEWT:1902NEWT:160488NEWT:28104NEWT:1908NEWT:13164NEWT:216129NCBITaxon:2NEWT:985076NEWT:1215323NEWT:2664179NEWT:986NEWT:52641NEWT:7038NEWT:7159NEWT:6192NEWT:28532NCBITaxon:38727NEWT:353152NEWT:2829NEWT:366581NEWT:216599NEWT:216595NEWT:1194669NEWT:51329NEWT:40559NEWT:243230NEWT:8355NEWT:633NEWT:9685NEWT:7029NEWT: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