Prideapplication/xmlftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD075443/report.pg_matrix.tsvftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD075443/report.stats.tsvftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD075443/combined_peptide.tsvftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD075443/combined_ion.tsvftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD075443/combined_site_M_15.9949.tsvftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD075443/combined_modified_peptide.tsvftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD075443/report.pr_matrix.tsvftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD075443/combined_protein.tsvftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD075443/report.protein_description.tsvftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD075443/combined_site_C_57.0215.tsvftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD075443/report.gg_matrix.tsvftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD075443/report.unique_genes_matrix.tsvftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD075443/report.log.txtftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD075443/checksum.txtftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD075443/report.manifest.txtftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD075443/report_trends.pdfftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD075443/report_runs.pdfftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD075443/fragger.paramsftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD075443/report.parquetftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD075443/fragpipe.workflowftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD075443/D11_Lysate_KO_G1_1_42373.d.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD075443/D11_Lysate_WT_RD12_1_42204.d.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD075443/D11_Lysate_WT_RD12_1_42372.d.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD075443/D11_Lysate_KO_G1_1_42205.d.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD075443/report-lib.parquetftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD075443/fragpipe-files.fp-manifestftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD075443/report.site_report.parquetprimaryOK200davies@sund.ku.dkLuke GamonData-dependent acquisitionMass SpectrometrydiaPASEFBottom-up proteomicsData-independent acquisitionHumanIpschttps://www.ebi.ac.uk/pride/archive/projects/PXD075443Whole BodyProteomic sample preparation and mass spectrometry analysis Five micrograms of cell lysate from WT and SULF1/2 DKO samples collected at D11 of differentiation were subjected to SP3 bead-based protein cleanup prior to reduction, alkylation, and overnight proteolytic digestion with trypsin/Lys-C. Cleanup was performed as described by Batth et al., 2019 (REF Batth et al., 2019), with minor modifications. Briefly, samples (50 µL) were precipitated onto magnetic beads (Sera-Mag, GE Healthcare) by addition of acetonitrile (120 µL), washed (800 µL; 1 × 70% ethanol, 2 × 100% acetonitrile), and resuspended in 50 mM triethylammonium bicarbonate (TEAB; 50 µL). Proteins were reduced and alkylated for 10 min at 70 °C using tris(2-carboxyethyl)phosphine (TCEP; 10 mM) and chloroacetamide (CAA; 40 mM), followed by incubation with Lys-C (0.05 µg) for 1 h at 37 °C and overnight digestion with trypsin (0.1 µg) at 37 °C. Digested peptides were acidified with 10% trifluoroacetic acid (TFA) and subjected to StageTip solid-phase extraction using C18 discs (Affinisep). Peptides were analyzed on a timsTOF Pro mass spectrometer (Bruker) coupled online to a Dionex UltiMate 3000 nanoLC system (Thermo Fisher Scientific).PridePeptide separation was performed on a 15 cm × 75 µm C18 nanoflow column (Aurora, IonOpticks) at a flow rate of 400 nL min⁻¹ using a 22 min gradient of 0.1% formic acid in water (solvent A) and 99.9% acetonitrile/0.1% formic acid (solvent B). The mass spectrometer was operated in parallel accumulation-serial fragmentation mode using either data-dependent (DDA-PASEF) or data-independent (DIA-PASEF) acquisition, with a cycle time of 0.63 s and a TIMS ramp time of 100 ms. The MS scan range was set to 100-1700 m/z. For DIA-PASEF, isolation windows and collision energies were set to default values, with a base of 0.85 1/K₀ (V s cm⁻²) at 20 eV and 1.30 1/K₀ (V s cm⁻²) at 59 eV. Fragmentation spectra were searched against a FASTA database containing the human proteome and common contaminants using MSFragger (v4.3) implemented in FragPipe (v23.1) for DDA data, or DIA-NN (v2.2.0 Academia) for DIA data. Default settings were used with minor modifications (peptide length 7-50 for DDA and 7-35 for DIA; two missed cleavages for DDA and one missed cleavage for DIA). Fixed modifications included cysteine alkylation (+57), and variable modifications included N-terminal acetylation (+42) and methionine oxidation (+16). MS/MS spectral annotation was performed using FragPipe-PDV.ProteomicsMichael DaviesPARTIALDepartment of Biomedical Sciences, University of Copenhagen, DenmarkHomo Sapiens (human)lgamon@sund.ku.dkNot availableThe University of CopenhagenDenmarkfalseHeparan sulfate Sulfatases are essential for the patterning of human stem cell-derived midbrain dopaminergic neuronsMidbrain dopaminergic neurons (mDA) are selectively lost in Parkinson’s disease (PD), driving sustained efforts to generate bona fide mDA neurons from human-induced pluripotent stem cells (iPSCs) for replacement therapy. While morphogen gradients and transcription factors have been extensively studied, extracellular regulators remain largely overlooked. Here, we identify the heparan sulfate-modifying enzymes SULF1 and SULF2 as essential for establishing mDA neuron identity in vitro. Using CRISPR/Cas9-engineered iPSCs, we show that loss of SULF1/2 increases 6-O-sulfation of heparan sulfate chains and disrupts anterior-posterior and dorsoventral patterning in cells exposed to a midbrain differentiation protocol. Double-knockout cells fail to acquire midbrain fate and instead adopt caudal and neural crest-like identities, as revealed by single-nucleus RNA sequencing. Mechanistically, we find enhanced FGF signaling and demonstrate that FGF inhibition redirects cells toward midbrain progenitors, without fully restoring ventral identity. These findings establish a critical role for SULF1/2 in human mDA neuron development and uncover a previously unrecognized layer of extracellular control over neuronal patterning, opening for novel strategies to refine differentiation protocols for PD and beyond.2026-07-162026-03-10PXD075443NEWT:6945NEWT:3555NEWT:241368NEWT:2NEWT:157546NEWT:190802NEWT:35554NEWT:9778NEWT:150475NEWT:9417NEWT:7111NEWT:347515NEWT:1216979NEWT:307972NEWT:32046NEWT:544496NEWT:5180NEWT:256737NEWT:2042546NEWT:115104NEWT:1081927NEWT:67825NEWT:185579NEWT:43179NEWT:13076NEWT:1249668NEWT:376741NEWT:317NEWT:55153NCBITaxon:10407NEWT:1736309NEWT:7227NEWT:7469NEWT:885318NEWT:1211601NEWT:415540NEWT:876138NEWT:4081NEWT:554NEWT:98334NEWT:426428NEWT:237561NEWT:6928NEWT:10036NEWT:7574NEWT:1351NEWT:7215NEWT:29204NEWT:272563NEWT:507601NCBITaxon:79857NCBITaxon:6157NEWT:95648NEWT:3885NEWT:746360NEWT:6239NEWT:3888NEWT:1589NEWT:470150NEWT:135622NEWT:216257NEWT:6915NEWT:9986NEWT:101510NEWT:4054NEWT:3880NEWT:272559NEWT:226186NEWT:3641NEWT:383379NEWT:8782NEWT:1263854NEWT:1000589NEWT:435590NEWT:1902NEWT:85962NEWT:160488NEWT:28104NEWT:317447NEWT:7955NCBITaxon:2NEWT:985076NEWT:7959NEWT:2261NEWT:4565NEWT:1264690NEWT:515619NEWT:6192NEWT:28532NCBITaxon:38727NEWT:34305NEWT:59729NCBITaxon:183674NEWT:224308NEWT:626528NEWT:139927NEWT:4558NEWT:209285NEWT:216595NEWT:243230NEWT:8355NEWT:1283NEWT:931281NEWT:4550NEWT:1000561NEWT:9685NEWT:7029NEWT:1283300NEWT:6183NEWT:6063NEWT:334747NEWT:61235NCBITaxon:79824NEWT:4787NCBITaxon:4563NEWT:5755NEWT:3218NEWT:5759NEWT:1736231NEWT:436486NEWT:6287NEWT:2242NEWT:300641NEWT:4784NEWT:727NEWT:9796NEWT:725NEWT:360106NEWT:260707NEWT:287NEWT:10117NEWT:10239NCBITaxon:6191NEWT:10116NEWT:1280NEWT:1836NEWT:1735272NEWT:83334NEWT:185431NEWT:83332NEWT:29760NEWT:260704NEWT:703612NEWT:260705NEWT:80863NEWT:44685NEWT:2697049NEWT:1148NEWT:11676NEWT:55571NEWT:100226NCBITaxon:6073NEWT:4530NEWT:4896NEWT:6279NEWT:1123869NEWT:7370NEWT:75058NEWT:83906NEWT:607699NEWT:6282NEWT:1094343NEWT:208964NEWT:1134506NEWT:575584NEWT:296543NEWT:1773NEWT:38783NEWT:8727NEWT:1895NEWT:4006NEWT:1182590NEWT:8726NEWT:6669NEWT:10090NEWT:935293NEWT:64152NEWT:749200NEWT:4120NEWT:51515NEWT:5693NEWT:8724NEWT:51511NEWT:92867NEWT:8723NEWT:990346NEWT:5334NEWT:145953NEWT:257309NEWT:100816NEWT:230741NEWT:284812NCBITaxon:10359NCBITaxon:1313NEWT:43330NEWT:242619NEWT:44544NEWT:632957NEWT:373995NEWT:5689NEWT:645463NEWT:544404NEWT:3702NEWT:129249NEWT:9925NEWT:8839NEWT:4232NEWT:990119NEWT:2758385NEWT:4113NEWT:837NEWT:11298NEWT:171101NEWT:421932NEWT:196627NEWT:408172NEWT:5691NEWT:408170NEWT:493760NEWT:260710NEWT:627025NEWT:400772NEWT:1097677NEWT:3708NEWT:128161NEWT:106592NEWT:536231NEWT:61674NEWT:1117957NEWT:9913NEWT:1432138NEWT:10312NEWT:1424507NEWT:4100NEWT:1076NEWT:6763NEWT:803NEWT:8030NEWT:29722NEWT:380394NEWT:1692259NEWT:1639NEWT:188229NEWT:3818NEWT:480NEWT:4909NEWT:180066NEWT:67767NEWT:46835NEWT:135588NEWT:1843183NEWT:95486NEWT:58002NEWT:9103NEWT:4577NEWT:1416333NEWT:5664NEWT:2157NEWT:146479NEWT:10306NCBITaxon:2759NEWT:1911079NEWT:8022NEWT:145943NCBITaxon:4932NEWT:595536NEWT:3635NEWT:5811NEWT:235443NEWT:1480154NEWT:1274414NEWT:27606NEWT:59202NEWT:9975NEWT:3197NEWT:9615NEWT:10299NEWT:860688NEWT:411483NEWT:884019NEWT:411490NEWT:169963NEWT:36329NEWT:1147787NCBITaxon:3044782NEWT:72407NEWT:476272NEWT:349741NEWT:9606NEWT:367830NEWT:157295NEWT:641501NEWT:178616NEWT:410289NEWT:373153NEWT:915099NEWT:74940NEWT:9721NEWT:1450511NEWT:360094NEWT:470NEWT:411901NEWT:1313NEWT:411469NEWT:84023NEWT:9838NCBITaxon:9615NCBITaxon:5811NEWT:58334NEWT:411464NEWT:1193501NEWT:3055NEWT:6326NEWT:6689NEWT:411460NEWT:2762NEWT:5476NEWT:1174673NEWT:562NEWT:411470NEWT:33952NEWT:2094720NEWT:1274432NEWT:1274426NEWT:1423NEWT:4932NEWT:70448NEWT:9825NEWT:1274423NEWT:3603NEWT:698936NEWT:2759NEWT:3847NEWT:39946NEWT:9823NEWT:178876NEWT:9940NEWT:327160NEWT:573NEWT:521001NEWT:9031NEWT:1274420NEWT:7091NEWT:5784580000-0001-7894-8035