{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,9,1]],"date-time":"2025-09-01T06:10:05Z","timestamp":1756707005916,"version":"3.44.0"},"reference-count":45,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2025,9,1]],"date-time":"2025-09-01T00:00:00Z","timestamp":1756684800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Microbiol."],"abstract":"<jats:p>Proteins function through complex interaction networks that govern nearly all aspects of cellular physiology. Identifying protein\u2013protein interactions (PPIs) under native conditions remains challenging due to the transient nature of many complexes and technical limitations of conventional approaches. We present TIE-UP-SIN (Targeted Interactome Experiment for Unknown Proteins by Stable Isotope Normalization), a robust and reproducible method for <jats:italic>in vivo<\/jats:italic> identification of PPIs. This approach combines metabolic labeling with <jats:sup>15<\/jats:sup>N isotopes, reversible <jats:italic>in vivo<\/jats:italic> formaldehyde crosslinking, affinity purification, and quantitative mass spectrometry. TIE-UP-SIN is specifically designed to preserve transient or weak interactions during purification and to quantify interaction partners using internal light\/heavy peptide ratios, reducing experimental variability and increasing reproducibility across biological replicates. The method employs a triple-sample design (WT\/WT, Bait\/WT, Bait\/Bait) to distinguish specific from non-specific interactors. Peptide-level L\/H ratios are normalized against sample-specific factors, aggregated at the protein level, and statistically analyzed using moderated testing. This strategy enables reliable detection of differential PPIs across physiological states, even in organisms with limited labeling options. We demonstrate the utility of TIE-UP-SIN by mapping interaction partners of the essential housekeeping sigma factor RpoD (SigA) under control and ethanol stress conditions. Known partners such as RNA polymerase subunits (RpoA, RpoB, RpoC) were robustly enriched, while potential novel candidates, including ClpX and AcpA, were detected at lower abundance. TIE-UP-SIN offers a simple, cost-effective, and modular platform for quantitative interactome analysis and can be adapted to a wide range of bacterial and non-bacterial systems. Compared to established approaches such as label-free IP\u2013MS or proximity-based labeling methods, TIE-UP-SIN is intended as a complementary option. Its combination of specific control, robust quantification, and suitability for low-input material provides an additional tool within the broader proteomics workflow collection.<\/jats:p>","DOI":"10.3389\/fmicb.2025.1657647","type":"journal-article","created":{"date-parts":[[2025,9,1]],"date-time":"2025-09-01T05:30:42Z","timestamp":1756704642000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":0,"title":["TIE-UP-SIN: a novel method for enhanced identification of protein\u2013protein interactions"],"prefix":"10.3389","volume":"16","author":[{"given":"Maximilian","family":"Schedlowski","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Stephan","family":"Michalik","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Tilly","family":"Hoffm\u00fcller","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Marco","family":"Harms","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Leif","family":"Steil","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kristin","family":"Surmann","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Christian","family":"Hentschker","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Manuela Gesell","family":"Salazar","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Uwe","family":"V\u00f6lker","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Alexander","family":"Reder","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1965","published-online":{"date-parts":[[2025,9,1]]},"reference":[{"key":"ref1","doi-asserted-by":"publisher","first-page":"172","DOI":"10.1016\/j.ab.2008.11.038","article-title":"Exploring blocking assays using octet, ProteOn, and Biacore biosensors","volume":"386","author":"Abdiche","year":"2009","journal-title":"Anal. Biochem."},{"key":"ref2","doi-asserted-by":"publisher","first-page":"493","DOI":"10.1038\/s41586-024-07487-w","article-title":"Accurate structure prediction of biomolecular interactions with AlphaFold 3","volume":"630","author":"Abramson","year":"2024","journal-title":"Nature"},{"key":"ref3","doi-asserted-by":"publisher","first-page":"e08504","DOI":"10.7554\/eLife.08504","article-title":"Structure of a bacterial RNA polymerase holoenzyme open promoter complex","volume":"4","author":"Bae","year":"2015","journal-title":"eLife"},{"key":"ref4","doi-asserted-by":"publisher","first-page":"289","DOI":"10.1111\/j.2517-6161.1995.tb02031.x","article-title":"Controlling the false discovery rate: a practical and powerful approach to multiple testing","volume":"57","author":"Benjamini","year":"1995","journal-title":"J. R. Stat. Soc. Ser. B Methodol."},{"key":"ref5","doi-asserted-by":"publisher","first-page":"1077","DOI":"10.3390\/microorganisms11041077","article-title":"Structural analysis of Bacillus subtilis sigma factors","volume":"11","author":"Collins","year":"2023","journal-title":"Microorganisms"},{"key":"ref6","doi-asserted-by":"publisher","first-page":"245","DOI":"10.1038\/340245a0","article-title":"A novel genetic system to detect protein\u2013protein interactions","volume":"340","author":"Fields","year":"1989","journal-title":"Nature"},{"key":"ref7","doi-asserted-by":"publisher","first-page":"141","DOI":"10.1038\/415141a","article-title":"Functional organization of the yeast proteome by systematic analysis of protein complexes","volume":"415","author":"Gavin","year":"2002","journal-title":"Nature"},{"key":"ref8","author":"Gepp","year":"2024"},{"key":"ref9","doi-asserted-by":"publisher","first-page":"645","DOI":"10.1038\/nrm2208","article-title":"Analysis of protein complexes using mass spectrometry","volume":"8","author":"Gingras","year":"2007","journal-title":"Nat. Rev. Mol. Cell Biol."},{"key":"ref10","doi-asserted-by":"publisher","first-page":"49","DOI":"10.1016\/j.tca.2015.04.016","article-title":"Kinetics of depolymerization of paraformaldehyde obtained by thermogravimetric analysis","volume":"609","author":"Grajales","year":"2015","journal-title":"Thermochim. Acta"},{"key":"ref11","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1128\/mr.59.1.1-30.1995","article-title":"The sigma factors of Bacillus subtilis","volume":"59","author":"Haldenwang","year":"1995","journal-title":"Microbiol. Rev."},{"key":"ref12","doi-asserted-by":"publisher","first-page":"e96757","DOI":"10.1371\/journal.pone.0096757","article-title":"Successful conversion of the Bacillus subtilis BirA group II biotin protein ligase into a group I ligase","volume":"9","author":"Henke","year":"2014","journal-title":"PLoS One"},{"key":"ref13","doi-asserted-by":"publisher","first-page":"4032","DOI":"10.1002\/pmic.200700491","article-title":"SPINE: a method for the rapid detection and analysis of protein-protein interactions in vivo","volume":"7","author":"Herzberg","year":"2007","journal-title":"Proteomics"},{"key":"ref14","doi-asserted-by":"publisher","first-page":"2287","DOI":"10.1002\/pro.239","article-title":"The interaction of Bacillus subtilis \u03c3A with RNA polymerase","volume":"18","author":"Johnston","year":"2009","journal-title":"Protein Sci."},{"key":"ref15","doi-asserted-by":"publisher","first-page":"583","DOI":"10.1038\/s41586-021-03819-2","article-title":"Highly accurate protein structure prediction with AlphaFold","volume":"596","author":"Jumper","year":"2021","journal-title":"Nature"},{"key":"ref16","doi-asserted-by":"publisher","first-page":"3259","DOI":"10.1128\/JB.182.11.3259-3265.2000","article-title":"The Clp proteases of Bacillus subtilis are directly involved in degradation of misfolded proteins","volume":"182","author":"Kr\u00fcger","year":"2000","journal-title":"J. Bacteriol."},{"key":"ref17","doi-asserted-by":"publisher","first-page":"6322","DOI":"10.1093\/emboj\/cdg610","article-title":"Transcript cleavage factors GreA and GreB act as transient catalytic components of RNA polymerase","volume":"22","author":"Laptenko","year":"2003","journal-title":"EMBO J."},{"key":"ref18","doi-asserted-by":"publisher","first-page":"299","DOI":"10.1016\/0250-6874(83)85036-7","article-title":"Surface plasmon resonance for gas detection and biosensing","volume":"4","author":"Liedberg","year":"1983","journal-title":"Sensors Actuators"},{"key":"ref19","doi-asserted-by":"publisher","first-page":"1760","DOI":"10.1126\/science.289.5485.1760","article-title":"Printing proteins as microarrays for high-throughput function determination","volume":"289","author":"MacBeath","year":"2000","journal-title":"Science"},{"key":"ref20","doi-asserted-by":"publisher","first-page":"e8664","DOI":"10.1371\/journal.pone.0008664","article-title":"Promoter recognition by a complex of Spx and the C-terminal domain of the RNA polymerase \u03b1 subunit","volume":"5","author":"Nakano","year":"2010","journal-title":"PLoS One"},{"key":"ref21","doi-asserted-by":"publisher","first-page":"4233","DOI":"10.1073\/pnas.0637648100","article-title":"A regulatory protein that interferes with activator-stimulated transcription in bacteria","volume":"100","author":"Nakano","year":"2003","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref22","doi-asserted-by":"publisher","first-page":"15839","DOI":"10.1073\/pnas.0506592102","article-title":"Crystal structure of the Bacillus subtilis anti-alpha, global transcriptional regulator, Spx, in complex with the \u03b1 C-terminal domain of RNA polymerase","volume":"102","author":"Newberry","year":"2005","journal-title":"Proc. Natl. Acad. Sci. U. S. A."},{"key":"ref23","doi-asserted-by":"publisher","first-page":"1103","DOI":"10.1126\/science.1206848","article-title":"Condition-dependent transcriptome reveals high-level regulatory architecture in Bacillus subtilis","volume":"335","author":"Nicolas","year":"2012","journal-title":"Science"},{"key":"ref24","doi-asserted-by":"publisher","first-page":"6591","DOI":"10.1073\/pnas.96.12.6591","article-title":"Accurate quantitation of protein expression and site-specific phosphorylation","volume":"96","author":"Oda","year":"1999","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref25","author":"Pedersen","year":"2024"},{"key":"ref26","doi-asserted-by":"publisher","first-page":"5718","DOI":"10.1128\/jb.181.18.5718-5724.1999","article-title":"Identification of \u03c2B-dependent genes in Bacillus subtilis using a promoter consensus-directed search and oligonucleotide hybridization","volume":"181","author":"Petersohn","year":"1999","journal-title":"J. Bacteriol."},{"key":"ref27","year":"2024"},{"key":"ref28","doi-asserted-by":"publisher","first-page":"e2300294","DOI":"10.1002\/pmic.202300294","article-title":"MassSpecPreppy\u2014an end-to-end solution for automated protein concentration determination and flexible sample digestion for proteomics applications","volume":"24","author":"Reder","year":"2024","journal-title":"Proteomics"},{"key":"ref29","doi-asserted-by":"publisher","first-page":"1328","DOI":"10.1126\/science.1230593","article-title":"Proteomic mapping of mitochondria in living cells via spatially-restricted enzymatic tagging","volume":"339","author":"Rhee","year":"2013","journal-title":"Science"},{"key":"ref30","doi-asserted-by":"publisher","first-page":"e47","DOI":"10.1093\/nar\/gkv007","article-title":"Limma powers differential expression analyses for RNA-sequencing and microarray studies","volume":"43","author":"Ritchie","year":"2015","journal-title":"Nucleic Acids Res."},{"key":"ref31","doi-asserted-by":"publisher","first-page":"801","DOI":"10.1083\/jcb.201112098","article-title":"A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells","volume":"196","author":"Roux","year":"2012","journal-title":"J. Cell Biol."},{"key":"ref32","doi-asserted-by":"publisher","first-page":"54","DOI":"10.1016\/j.pep.2013.08.021","article-title":"Development of the twin-strep-tag\u00ae and its application for purification of recombinant proteins from cell culture supernatants","volume":"92","author":"Schmidt","year":"2013","journal-title":"Protein Expr. Purif."},{"key":"ref33","doi-asserted-by":"publisher","first-page":"706","DOI":"10.1038\/s41586-019-1923-7","article-title":"Improved protein structure prediction using potentials from deep learning","volume":"577","author":"Senior","year":"2020","journal-title":"Nature"},{"key":"ref34","doi-asserted-by":"publisher","first-page":"850","DOI":"10.1021\/ac950914h","article-title":"Mass spectrometric sequencing of proteins from Silver-stained polyacrylamide gels","volume":"68","author":"Shevchenko","year":"1996","journal-title":"Anal. Chem."},{"key":"ref35","doi-asserted-by":"crossref","DOI":"10.1201\/9780429447273","volume-title":"Interactive web-based data visualization with R, Plotly, and shiny","author":"Sievert","year":"2020"},{"key":"ref36","doi-asserted-by":"publisher","first-page":"1225","DOI":"10.1002\/jms.559","article-title":"Chemical cross-linking and mass spectrometry for mapping three-dimensional structures of proteins and protein complexes","volume":"38","author":"Sinz","year":"2003","journal-title":"J. Mass Spectrom."},{"key":"ref37","author":"Slowikowski","year":"2024"},{"key":"ref38","doi-asserted-by":"publisher","first-page":"1315","DOI":"10.1126\/science.4001944","article-title":"Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface","volume":"228","author":"Smith","year":"1985","journal-title":"Science"},{"key":"ref39","doi-asserted-by":"publisher","first-page":"719","DOI":"10.1073\/pnas.58.2.719","article-title":"Energy transfer: a spectroscopic ruler","volume":"58","author":"Stryer","year":"1967","journal-title":"Proc. Natl. Acad. Sci. U. S. A."},{"key":"ref40","doi-asserted-by":"publisher","first-page":"699","DOI":"10.1002\/jms.1415","article-title":"Utility of formaldehyde cross-linking and mass spectrometry in the study of protein-protein interactions","volume":"43","author":"Sutherland","year":"2008","journal-title":"J. Mass Spectrom."},{"key":"ref41","doi-asserted-by":"publisher","first-page":"e0172737","DOI":"10.1371\/journal.pone.0172737","article-title":"Analysis of Spo0M function in Bacillus subtilis","volume":"12","author":"Vega-Cabrera","year":"2017","journal-title":"PLoS One"},{"key":"ref42","doi-asserted-by":"publisher","first-page":"5","DOI":"10.32614\/RJ-2011-002","article-title":"Testthat: get started with testing","volume":"3","author":"Wickham","year":"2011","journal-title":"R J."},{"key":"ref43","doi-asserted-by":"publisher","first-page":"1686","DOI":"10.21105\/joss.01686","article-title":"Welcome to the tidyverse","volume":"4","author":"Wickham","year":"2019","journal-title":"J Open Source Softw."},{"key":"ref44","doi-asserted-by":"publisher","first-page":"131","DOI":"10.1016\/0003-2697(89)90213-3","article-title":"Rapid measurement of binding constants and heats of binding using a new titration calorimeter","volume":"179","author":"Wiseman","year":"1989","journal-title":"Anal. Biochem."},{"key":"ref45","first-page":"1911","article-title":"Spx-RNA polymerase interaction and global transcriptional control during oxidative stress","author":"Zuber","year":"2004"}],"container-title":["Frontiers in Microbiology"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fmicb.2025.1657647\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,9,1]],"date-time":"2025-09-01T05:30:43Z","timestamp":1756704643000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fmicb.2025.1657647\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,9,1]]},"references-count":45,"alternative-id":["10.3389\/fmicb.2025.1657647"],"URL":"https:\/\/doi.org\/10.3389\/fmicb.2025.1657647","relation":{},"ISSN":["1664-302X"],"issn-type":[{"value":"1664-302X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,9,1]]},"article-number":"1657647"}}