Prideapplication/xmlftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD075565/E_coli_K12_PQQ_PQQ4_competition_report.tsvftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD075565/checksum.txtftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD075565/uniprot-download_true_format_fasta_query__28proteome_3AUP000000625_2-2023.05.02-11.18.14.75.fastaftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD075565/E_coli_K12_PQQ_PQQ4_competition.zipprimaryOK200stephan.sieber@tum.deTao WangMass SpectrometryBottom-up proteomicsPyrroloquinoline quinoneProteomicshttps://www.ebi.ac.uk/pride/archive/projects/PXD075565E. coli K12 was treated with PQQ4 in compared to PQQ and competition (PQQ4+PQQ), which was harvested, lysed and protein concentration was confirmed with BCA assay. The samples were clicked to biotin azide using click master mix, cleaned up by using TCEP for reduction and IAA for alkylation, followed by DTT for quench of excessive IAA. All the samples were treated with carboxylate beads, and proteome were precipitated with EtOH, eluted with SDS. The clicked proteome was enriched with streptavidin beads, washed with NP-40, urea and waterPrideNot availableMS data was analyzed using DIA-NN (version 1.8.1) in library-free mode. The DIA-NN was operated with the default settings. The following procedures were used to proceed with data: 1). Rename columns, 2). Add annotations, 3). Log2 Transform of LFQ intensities, 4). Categorical annotations of rows, 5). Remove contaminants, 6). Remove reverse, 7). Filter rows based on valid values (2 for three replicates, 3 for four replicates) with at least in one group, 8). Impute the missing values from normal distribution over the total matrix, 9). Two-sample Student's t-test with 5% FDRProteomicsStephan A. SieberCenter for Functional Protein Assemblies (CPA), Department of Bioscience, TUM School of Natural Sciences, Technical University of Munich (TUM), Ernst-Otto-Fischer-Stra?e 8, 85748 Garching, GermanyPARTIALEscherichia Coli41925033 Wang T, Mühlhofer R, Lei E, Ding W, Klein AS, Zeymer C, Sieber SA. Chemical Proteomics Reveal the Inventory of Pyrroloquinoline Quinone Binding Proteins in Bacteria. J Am Chem Soc. 2026 148(14):15306-15319 10.1021/jacs.6c03427tao.wang@tum.deTechnical University of MunichGermanyPyrroloquinoline quinone (<b>PQQ</b>) is a bacterial redox cofactor enabling enzyme catalysis in various sugar and alcohol dehydrogenases. However, its proposed additional role as a "longevity vitamin" lacks a clear molecular basis and is thus highly debated. Here, we applied chemical proteomics to identify previously unknown classes of <b>PQQ</b>-binding proteins. We designed and synthesized a structurally diverse suite of five <b>PQQ</b> probes equipped with a diazirine photo-cross-linker and an alkyne handle for target identification. The fidelity of the probes was first evaluated for two well-characterized bacterial <b>PQQ</b>-dependent enzymes, demonstrating not only probe binding but also the reconstitution of catalytic activity. We then commenced with proteome profiling of <i>Escherichia coli</i> and <i>Pseudomonas putida</i> cells and unraveled a distinct set of putative <b>PQQ</b>-binding proteins. Recombinant expression of selected hits, including several chaperones, validated <b>PQQ</b> binding. Notably, in some cases, <b>PQQ</b> even formed covalent adducts with selected lysine residues, for instance, in the AAA+ ATPase RuvB involved in DNA remodeling. Overall, our work highlights the utility of <b>PQQ</b> probes to further unravel the complement of cofactor-binding proteins in whole cells. It also provides a basis for future mechanistic studies of <b>PQQ</b> functions beyond redox catalysis.Chemical Proteomics Reveal the Inventory of Pyrroloquinoline Quinone Binding Proteins in Bacteria.Wang Tao T, Mühlhofer Rahel R, Lei E E, Ding Wei W, Klein Andreas S AS, Zeymer Cathleen C, Sieber Stephan A SAfalseChemical proteomics reveal the inventory of pyrroloquinoline quinone binding proteins in bacteriaPyrroloquinoline quinone (PQQ) is a bacterial redox cofactor enabling enzyme catalysis in various sugar and alcohol dehydrogenases. However, its proposed additional role as a "longevity vitamin" lacks a clear molecular basis and is thus highly debated. Here, we applied chemical proteomics to identify so far unknown classes of PQQ-binding proteins. We designed and synthesized a structurally diverse suite of five PQQ probes equipped with a diazirine photocrosslinker and an alkyne handle for target identification. The fidelity of the probes was first evaluated for two well-characterized bacterial PQQ-dependent enzymes, demonstrating not only probe binding but also reconstitution of catalytic activity. We then commenced with proteome profiling of Escherichia coli and Pseudomonas putida cells and unraveled a distinct set of putative PQQ-binding proteins. Recombinant expression of selected hits including several chaperones validated PQQ binding. Notably, in some cases, PQQ even formed covalent adducts with selected lysine residues, for instance in the AAA+ ATPase RuvB involved in DNA remodeling. Overall, our work highlights the utility of PQQ probes to further unravel the complement of cofactor-binding proteins in whole cells. It also provides a basis for future mechanistic studies on PQQ functions beyond redox catalysis.2026-04-272026-03-12PXD075565NEWT:6945NEWT:3555NEWT:241368NEWT:2NEWT:157546NEWT:190802NEWT:35554NEWT:150475NEWT:9417NEWT:347515NEWT:1216979NEWT:307972NEWT:32046NEWT:544496NEWT:5180NEWT:256737NEWT:115104NEWT:1081927NEWT:67825NEWT:13076NEWT:1249668NEWT:317NEWT:1736309NEWT:7227NEWT:7469NEWT:885318NEWT:4081NEWT:876138NEWT:554NEWT:98334NEWT:237561NEWT:10036NEWT:7574NEWT:1351NEWT:7215NEWT:272563NEWT:507601NCBITaxon:79857NCBITaxon:6157NEWT:95648NEWT:746360NEWT:6239NEWT:1589NEWT:470150NEWT:135622NEWT:216257NEWT:6915NEWT:9986NEWT:101510NEWT:4054NEWT:3880NEWT:8782NEWT:1000589NEWT:1902NEWT:85962NEWT:160488NEWT:28104NEWT:317447NEWT:7955NCBITaxon:2NEWT:985076NEWT:7959NEWT:2261NEWT:4565NEWT:1264690NEWT:6192NEWT:28532NCBITaxon:38727NEWT:34305NEWT:59729NCBITaxon:183674NEWT:224308NEWT:626528NEWT:139927NEWT:4558NEWT:209285NEWT:216595NEWT:243230NEWT:8355NEWT:931281NEWT:7029NEWT:1283300NEWT:334747NEWT:61235NCBITaxon:79824NCBITaxon:4563NEWT:5755NEWT:3218NEWT:5759NEWT:1736231NEWT:436486NEWT:6287NEWT:2242NEWT:300641NEWT:4784NEWT:727NEWT:9796NEWT:725NEWT:360106NEWT:260707NEWT:287NEWT:10117NEWT:10239NCBITaxon:6191NEWT:10116NEWT:1280NEWT:1836NEWT:1735272NEWT:83334NEWT:83332NEWT:29760NEWT:260704NEWT:703612NEWT:260705NEWT:80863NEWT:44685NEWT:2697049NEWT:1148NEWT:11676NEWT:55571NEWT:100226NCBITaxon:6073NEWT:4530NEWT:4896NEWT:6279NEWT:1123869NEWT:7370NEWT:83906NEWT:6282NEWT:1134506NEWT:575584NEWT:1773NEWT:38783NEWT:8727NEWT:1895NEWT:1182590NEWT:8726NEWT:10090NEWT:935293NEWT:749200NEWT:4120NEWT:5693NEWT:8724NEWT:51511NEWT:92867NEWT:8723NEWT:990346NEWT:5334NEWT:145953NEWT:257309NEWT:230741NEWT:284812NCBITaxon:10359NCBITaxon:1313NEWT:43330NEWT:242619NEWT:44544NEWT:632957NEWT:373995NEWT:544404NEWT:3702NEWT:129249NEWT:8839NEWT:4232NEWT:990119NEWT:2758385NEWT:4113NEWT:837NEWT:11298NEWT:171101NEWT:196627NEWT:408172NEWT:5691NEWT:408170NEWT:493760NEWT:260710NEWT:627025NEWT:400772NEWT:1097677NEWT:3708NEWT:106592NEWT:1117957NEWT:9913NEWT:1432138NEWT:10312NEWT:4100NEWT:1076NEWT:6763NEWT:803NEWT:8030NEWT:29722NEWT:380394NEWT:1692259NEWT:1639NEWT:188229NEWT:3818NEWT:480NEWT:4909NEWT:180066NEWT:67767NEWT:46835NEWT:135588NEWT:1843183NEWT:95486NEWT:58002NEWT:9103NEWT:4577NEWT:5664NEWT:2157NEWT:146479NEWT:10306NEWT:1911079NEWT:8022NEWT:145943NEWT:3635NEWT:5811NEWT:1480154NEWT:235443NEWT:1274414NEWT:59202NEWT:3197NEWT:9615NEWT:10299NEWT:860688NEWT:884019NEWT:169963NEWT:36329NEWT:1147787NEWT:72407NEWT:9606NEWT:367830NEWT:157295NEWT:410289NEWT:373153NEWT:915099NEWT:74940NEWT:1450511NEWT:470NEWT:84023NEWT:9838NCBITaxon:9615NEWT:1193501NEWT:3055NEWT:6326NEWT:6689NEWT:2762NEWT:5476NEWT:1174673NEWT:562NEWT:33952NEWT:1274432NEWT:1274426NEWT:1423NEWT:4932NEWT:70448NEWT:9825NEWT:1274423NEWT:3603NEWT:698936NEWT:2759NEWT:3847NEWT:39946NEWT:9823NEWT:9940NEWT:573NEWT:9031NEWT:1274420NEWT:709141925033