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Protein extraction and sample preparation were performed as described in Hollstein et al. (2025). In brief, biotinylated proteins were enriched from denatured cell lysates by affinity purification with Strep-Tactin® Sepharose® (iba Lifescience GmbH, Germany). Biotinylated proteins were released from the resin by adding a buffer containing 50 mM biotin for competitive elution. After chloroform methanol precipitation of the eluted proteins, SDS-PAGE and in gel digestion with trypsin were applied to digest the proteins (Shevchenko et al., 1996; Wessel & Flügge, 1984). Peptides were desalted with the C18 stage tip purification protocol according to Rappsilber et al. (2003). Samples containing Strep-Tactin® Sepharose® enriched proteins were not fractionated, whereas the complex input controls were PAGE-separated into four fractions (SmDOC1-BioID) or five fractions (SmDOC2-BioID) for in-gel trypsin digestion and LC-MS analysis. Dried peptide samples were reconstituted in 20 µl LC-MS sample buffer (2 % acetonitrile, 0.1 % formic acid). 2 to 8 µl of each sample were subjected to reverse phase liquid chromatography for peptide separation using an RSLCnano Ultimate 3000 system (Thermo Fisher Scientific): Peptides were loaded on an Acclaim PepMap 100 pre-column (100 µm x 2 cm, C18, 5 µm, 100 Å; Thermo Fisher Scientific) with 0.07 % trifluoroacetic acid at a flow rate of 20 µL/min for 3 min. Analytical separation of peptides was done on an Acclaim PepMap RSLC column (75 µm x 50 cm, C18, 2 µm, 100 Å; Thermo Fisher Scientific) at a flow rate of 300 nL/min. The solvent composition was gradually changed within 94 min from 96 % solvent A (0.1 % formic acid) and 4 % solvent B (80 % acetonitrile, 0.1 % formic acid) to 10 % solvent B within 2 minutes, to 30 % solvent B within the next 58 min, to 45 % solvent B within the following 22 min, and to 90 % solvent B within the last 12 min of the gradient. All solvents and acids had Optima grade for LC-MS (Fisher Chemical). Eluting peptides were on-line ionized by nano-electrospray (nESI) using the Nanospray Flex Ion Source (Thermo Fisher Scientific) at 1.5 kV (liquid junction) and transferred into a Q Exactive HF mass spectrometer (Thermo Fisher Scientific). Full scans in a mass range of 300 to 1650 m/z were recorded at a resolution of 30,000 followed by data-dependent top 10 HCD fragmentation at a resolution of 15,000 (dynamic exclusion enabled). LC-MS method programming and data acquisition was performed with the XCalibur 4.0 software (Thermo Fisher Scientific). Hollstein, L. S., Groth, S., Schmitt, K., Valerius, O., & Pöggeler, S. (2025). In vivo proximity-labeling with miniTurboID to screen for protein-protein interactions in the filamentous ascomycete Sordaria macrospora. MethodsX, 14, 103351.  Wessel, D., & Flügge, U. I. (1984). A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Analytical Biochemistry, 138(1), 141–143.  Shevchenko, A., Wilm, M., Vorm, O., & Mann, M. (1996). Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Analytical Chemistry, 68(5), 850–858.  Rappsilber, J., Ishihama, Y., & Mann, M. (2003). Stop and go extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics. Analytical Chemistry, 75(3), 663–670."],"repository":["Pride"],"quantification_method":[""],"modification":[""],"data_protocol":["The MaxQuant software (v1.6.10.43; Cox & Mann, 2008) was configured with standard parameters for protein identification and label free relative quantification using the MaxLFQ algorithm. The S. macrospora genome v04-derived protein database in FASTA format (Breuer et al., 2024) was used for the database search. Biotinylation of lysine and phosphorylation (at serine, threonine, or tyrosine) were considered as variable modifications. The number of maximum missed cleavage sites was set to three and trypsin/P was used for digestion. The input controls were configured in a different parameter group than the eluates of the biotin affinity purification, with the option “Separate LFQ in parameter groups” enabled. The proteinGroups.txt output file from MaxQuant was processed in Perseus (v1.6.15.0; Tyanova et al., 2016).  Cox, J., & Mann, M. (2008). Maxquant enables high peptide identification rates, individualized p.P.B.-range mass accuracies and proteome-wide protein quantification. Nature Biotechnology, 26(12), 1367–1372.  Breuer, J., Busche, T., Kalinowski, J., & Nowrousian, M. (2024). Histone binding of ASF1 is required for fruiting body development but not for genome stability in the filamentous fungus Sordaria macrospora. MBio, 15(1), e0289623.  Tyanova, S., Temu, T., Sinitcyn, P., Carlson, A., Hein, M. Y., Geiger, T., Mann, M., & Cox, J. (2016). The Perseus computational platform for comprehensive analysis of (prote)omics data. Nature Methods, 13(9), 731–740."],"omics_type":["Proteomics"],"labhead":["Dr. Prof. Stefanie Pöggeler"],"instrument_platform":[""],"submission_type":["PARTIAL"],"labhead_affiliation":["Georg-August Universität Göttingen, Institute of Microbiology and Genetics, Department of Genetics of Eukaryotic Microorganisms"],"species":["Sordaria Macrospora"],"publication":["10.1371/JOURNAL.PBIO.3003969"],"submitter_mail":["ovaleri@gwdg.de"],"submitter_affiliation":["Georg-August-Universität Göttingen"],"submitter_country":["Germany"],"additional_accession":[]},"is_claimable":false,"name":"Proxiomes of the greenbeard proteins SmDOC1 and SmDOC2 in the filamentous fungus Sordaria macrospora","description":"In this study, we used the in vivo proximity labeling biotin identification (BioID; Roux et al., 2012) method to capture proteins of the microenvironment of the greenbeard determinant of communication (DOC) proteins SmDOC1 and SmDOC2 in the filamentous fungus Sordaria macrospora. The genes Smdoc1 (SMAC_06903) and Smdoc2 (SMAC_06902) were fused to the TurboID ligase via a GGGGS x 2 Linker (Branon et al., 2018) and integrated at their native chromosomal locus. To ensure authentic expression regulation, the Smdoc-TurboID fusion genes were under the control of their native 5’ regions. The homologous doc genes of the closely related fungus Neurospora crassa were shown to be involved in long distance kind discrimination, regulation hyphal fusion upon compatible allorecognition (Heller et al., 2016). The S. macrospora Smdoc1/2 are required for proper sexual development. Our SmDOC1-BioID experimental setup comprises five biological replicates of the strain expressing the Smdoc1-TurboID fusion gene from its native locus and a control strain expressing an egfp-TurboID fusion gene under control of the 1 kb 5’ Smdoc1 promoter region, which was ectopically integrated into the S. macrospora wild type. Our SmDOC2-BioID experimental setup consists of four biological replicates of three strains: 1) a strain expressing the free egfp-TurboID fusion gene under control of the 1 kb 5’ Smdoc1 promoter region, ectopically integrated into the S. macrospora wild type; 2) a strain expressing the Smdoc2-TurboID fusion gene from its native locus; 3) a strain expressing the Smdoc2-TurboID fusion gene from its native locus in the Δmak2 deletion strain background which lacks the MAP kinase mak2 of the pheromone response pathway (Schmidt et al., 2020). To account for possible expression artefacts among strains used in the SmDOC1- and SmDOC2-BioID experiments, we performed additional input controls of the crude protein extract prior to biotin affinity purification.  Roux, K. J., Kim, D. in, Raida, M., & Burke, B. (2012). A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells. The Journal of Cell Biology, 196(6), 801–810. Branon, T. C., Bosch, J. A., Sanchez, A. D., Udeshi, N. D., Svinkina, T., Carr, S. A., Feldman, J. L., Perrimon, N., & Ting, A. Y. (2018). Efficient proximity labeling in living cells and organisms with TurboID. Nature Biotechnology, 36(9), 880–887.  Heller, J., Zhao, J., Rosenfield, G., Kowbel, D. J., Gladieux, P., & Glass, N. L. (2016). Characterization of Greenbeard Genes Involved in Long-Distance Kind Discrimination in a Microbial Eukaryote. PLOS Biology, 14(4), e1002431. Schmidt, S., Märker, R., Ramšak, B., Beier-Rosberger, A. M., Teichert, I., & Kück, U. (2020). Crosstalk Between Pheromone Signaling and NADPH Oxidase Complexes Coordinates Fungal Developmental Processes. Frontiers in Microbiology, 11, 1722.","dates":{"publication":"2026-08-19","submission":"2025-10-16"},"accession":"PXD069539","cross_references":{"TAXONOMY":["NEWT:330879","NEWT:377960","NEWT:2042546","NEWT:259447","NEWT:295546","NCBITaxon:2719036","NCBITaxon:1280","NEWT:112503","NEWT:1129","NEWT:309807","NEWT:59753","NEWT:89184","NEWT:309800","NEWT:281395","NEWT:1211601","NEWT:876138","NEWT:44271","NEWT:193516","NEWT:1073882","NEWT:6819","NEWT:475174","NEWT:111205","NEWT:1117","NEWT:498257","NEWT:10036","NEWT:1590","NEWT:10034","NEWT:661410","NEWT:638632","NEWT:224326","NEWT:376619","NCBITaxon:79857","NEWT:1096976","NEWT:3765","NEWT:1589","NEWT:135622","NEWT:67352","NEWT:35786","NEWT:1580","NEWT:399784","NEWT:96731","NEWT:656064","NEWT:36630","NEWT:383379","NEWT:418106","NCBITaxon:12721","NEWT:10029","NEWT:913645","NEWT:641809","NEWT:1392488","NEWT:38815","NEWT:317447","NEWT:4688","NEWT:111225","NEWT:7719","NEWT:868565","NEWT:135674","NEWT:79329","NEWT:30069","NEWT:12637","NEWT:59729","NEWT:2164133","NEWT:295105","NEWT:108061","NEWT:60711","NEWT:224308","NEWT:3347","NEWT:160621","NEWT:212790","NEWT:110368","NEWT:1310161","NEWT:77133","NEWT:145481","NEWT:1310165","NEWT:29058","NCBITaxon:79824","NEWT:1912919","NEWT:410658","NEWT:44688","NEWT:44689","NEWT:498211","NEWT:347256","NEWT:5518","NEWT:398007","NEWT:1527468","NEWT:498217","NEWT:498216","NEWT:11320","NEWT:246196","NEWT:246197","NEWT:145458","NEWT:44685","NEWT:161934","NEWT:156587","NEWT:1148","NEWT:5508","NEWT:3329","NEWT:419481","NEWT:5507","NEWT:410661","NEWT:1140","NCBITaxon:2157","NEWT:1143","NEWT:1287689","NEWT:1094343","NEWT:1462472","NEWT:1336795","NEWT:644042","NEWT:1182590","NEWT:3712","NEWT:3711","NEWT:3714","NEWT:270643","NEWT:2065263","NEWT:177437","NEWT:10418","NEWT:118698","NEWT:1616117","NEWT:118696","NEWT:34865","NEWT:52283","NEWT:28174","NEWT:284812","NEWT:8175","NEWT:43330","NEWT:980415","NEWT:1603293","NEWT:44664","NEWT:43346","NEWT:3702","NEWT:1245466","NEWT:3704","NEWT:244366","NEWT:1246791","NEWT:118694","NEWT:2850","NEWT:118691","NEWT:34871","NEWT:33548","NEWT:52299","NEWT:8165","NEWT:96794","NEWT:3708","NEWT:332648","NEWT:44670","NEWT:536231","NEWT:376219","NEWT:219813","NEWT:1510","NEWT:460519","NEWT:1515","NEWT:572307","NEWT:1432138","NEWT:1424507","NEWT:1194599","NEWT:272844","NEWT:1348799","NEWT:33995","NEWT:55784","NEWT:1303443","NEWT:9483","NEWT:485","NEWT:56636","NEWT:2709072","NEWT:2499525","NEWT:2853422","NEWT:1679718","NEWT:480","NEWT:67767","NEWT:46835","NEWT:109757","NEWT:582580","NEWT:1185650","NEWT:216778","NEWT:294607","NEWT:1502","NEWT:128017","NEWT:376686","NEWT:237610","NEWT:95486","NEWT:1883446","NEWT:1233435","NEWT:118223","NEWT:109760","NEWT:29031","NEWT:317047","NEWT:491","NEWT:460523","NEWT:235443","NEWT:1757312","NEWT:85694","NEWT:109786","NEWT:108458","NEWT:5936","NEWT:320637","NEWT:1078283","NEWT:3750","NEWT:983964","NEWT:11706","NEWT:32644","NEWT:527796","NEWT:499175","NEWT:109779","NEWT:3745","NEWT:1715989","NCBITaxon:4751","NEWT:3747","NEWT:1116234","NEWT:1255228","NEWT:410289","NEWT:373153","NEWT:1640278","NEWT:472","NEWT:940825","NEWT:529507","NEWT:1071661","NEWT:470","NEWT:5911","NEWT:748693","NCBITaxon:50557","NEWT:39251","NEWT:29491","NEWT:101841","NEWT:446","NEWT:153481","NEWT:2014887","NEWT:33952","NEWT:445","NEWT:153009","NEWT:261756","NEWT:166313","NEWT:63366","NEWT:63367","NEWT:215402","NEWT:1547","NEWT:906914","NEWT:9031","NEWT:27292","NEWT:1050722","NEWT:108931","NEWT:1293497","NEWT:1055524","NEWT:150475","NEWT:267872","NEWT:381666","NEWT:172269","NEWT:9534","NEWT:5180","NEWT:256737","NEWT:9541","NEWT:8694","NEWT:33936","NEWT:8692","NEWT:2903","NEWT:185579","NEWT:13076","NEWT:207340","NEWT:1006581","NEWT:8673","NEWT:33940","NEWT:550","NEWT:554","NEWT:451516","NEWT:552","NEWT:1325291","NEWT:36185","NEWT:1054211","NEWT:1225786","NEWT:771870","NEWT:51338","NEWT:575412","NEWT:28112","NEWT:6493","NEWT:10832","NEWT:6494","NEWT:6491","NEWT:507601","NEWT:520","NEWT:186441","NEWT:643680","NEWT:214092","NCBITaxon:6157","NEWT:13095","NEWT:162425","NEWT:104105","NEWT:216257","NEWT:9986","NEWT:8654","NEWT:8658","NEWT:1268063","NEWT:8657","NEWT:9983","NEWT:8655","NEWT:5147","NEWT:28108","NEWT:28104","NEWT:407821","NEWT:568703","NCBITaxon:2","NEWT:568708","NEWT:986","NEWT:52641","NEWT:28532","NEWT:353152","NEWT:298176","NEWT:227377","NEWT:1774284","NEWT:40674","NEWT:1194669","NEWT:51329","NEWT:443906","NEWT:519","NEWT:2510939","NEWT:7394","NEWT:6063","NEWT:1328388","NEWT:105487","NEWT:1548728","NEWT:667127","NEWT:9557","NEWT:377586","NEWT:7375","NEWT:300641","NEWT:39655","NEWT:9554","NEWT:38323","NEWT:475932","NEWT:256318","NEWT:206411","NCBITaxon:6191","NEWT:229533","NEWT:2925","NEWT:214053","NEWT:28550","NEWT:80863","NEWT:90675","NEWT:52639","NEWT:52638","NEWT:57075","NEWT:1666905","NEWT:8697","NEWT:8695","NEWT:884204","NEWT:510516","NEWT:1045775","NEWT:1123869","NEWT:9544","NEWT:9545","NEWT:979","NEWT:7370","NEWT:83906","NEWT:99488","NEWT:1134506","NEWT:255470","NEWT:38783","NEWT:6426","NEWT:33090","NEWT:9935","NEWT:287889","NEWT: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