Characterization of a soluble library of the Pseudomonas aeruginosa PAO1 membrane proteome with emphasis on c-di-GMP turnover enzymes.

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The extraction of proteins from the native membrane environment is a critical step in the preparation of membrane proteins that might affect the stability of protein complexes. In this work, we used the amphiphilic diisobutylene/maleic acid copolymer to extract the membrane proteome of the opportunistic pathogen Pseudomonas aeruginosa, thereby creating a soluble membrane-protein library within a native-like lipid-bilayer environment. Size fractionation of nanodisc-embedded proteins and subsequent mass spectrometry enabled the identification of 3358 proteins. The native membrane-protein library showed a very good overall coverage compared to previous proteome data. The pattern of size fractionation indicated that protein complexes were preserved in the library. More than 20 previously described complexes, e.g. the SecYEG and Pili complexes, were identified and analyzed for coelution. Although the mass-spectrometric dataset alone did not reveal new protein complexes, combining pulldown assays with mass spectrometry was successful in identifying new protein interactions in the native membrane-protein library. Thus, we identified several candidate proteins for interactions with the membrane phosphodiesterase NbdA, a member of the c-di-GMP network. We confirmed the candidate proteins CzcR, PA4200, SadC, and PilB as novel interaction partners of NbdA using the bacterial adenylate cyclase two-hybrid assay. Taken together, this work demonstrates the usefulness of the native membrane-protein library of P. aeruginosa for the investigation of protein interactions and membrane-protein complexes. Data are available via ProteomeXchange with identifiers PXD039702 and PXD039700.", "claims": [{"id": "claim-method", "statement": "Two sample t-tests with permutation-based FDR control were carried out in Perseus.", "kind": "methodological", "target": null, "evidence": [{"asset": "source:pmid:37441524", "locator": "sections[12] \"MS data analysis for pulldown\" paragraph 2", "quote": "Two sample t-tests with permutation-based FDR control were carried out in Perseus."}], "state": "complete"}, {"id": "claim-data-access", "statement": "The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (Perez-Riverol et al. 2022) partner repository with the dataset identifier PXD039702 (nanodisc library) and PXD039700 (pulldown).", "kind": "methodological", "target": null, "evidence": [{"asset": "source:pmid:37441524", "locator": "sections[28] \"Data availability\" paragraph 2", "quote": "The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (Perez-Riverol et al. 2022) partner repository with the dataset identifier PXD039702 (nanodisc library) and PXD039700 (pulldown)."}], "state": "complete"}, {"id": "claim-target-result", "statement": "Combining pulldown assays with mass spectrometry successfully identified new protein interactions in the native membrane-protein library, including four NbdA interaction partners validated by the bacterial adenylate cyclase two-hybrid assay.", "kind": "qualitative", "target": null, "evidence": [{"asset": "source:pmid:37441524", "locator": "sections[0] \"Abstract\" paragraph 1", "quote": "Although the mass-spectrometric dataset alone did not reveal new protein complexes, combining pulldown assays with mass spectrometry was successful in identifying new protein interactions in the native membrane-protein library."}, {"asset": "source:pmid:37441524", "locator": "sections[0] \"Abstract\" paragraph 1", "quote": "We confirmed the candidate proteins CzcR, PA4200, SadC, and PilB as novel interaction partners of NbdA using the bacterial adenylate cyclase two-hybrid assay."}], "state": "complete"}], "gaps": []}, "experiment": {"state": "complete", "objective": "Combining pulldown assays with mass spectrometry successfully identified new protein interactions in the native membrane-protein library, including four NbdA interaction partners validated by the bacterial adenylate cyclase two-hybrid assay.", "steps": [{"id": "read_published_estimate", "order": 1, "name": "Read the published estimate from the source", "kind": "other", "state": "complete", "blocked_on": null, "description": "The claim, method and evidence span published with the source article.", "duration_hours": null, "parameters": {}, "inputs": ["source:pmid:37441524"], "outputs": ["the published estimate and its evidence"], "instruments": [], "reagents": []}, {"id": "obtain_source_data", "order": 2, "name": "Obtain the source data from the named repository", "kind": "data-preparation", "state": "complete", "blocked_on": null, "description": "The GEO series accession resolves and its sample accessions are listed. 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