MetaboLightsapplication/xmlftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15927/m_MTBLS15927_LC-MS_positive_reverse-phase_v2_maf.tsvftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15927/s_MTBLS15927.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15927/i_Investigation.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15927/a_MTBLS15927_LC-MS_positive_reverse-phase.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15927/FILES/RAW_FILES/20231019_fAA_samples_Data_20231019_fAA_samples.wiff.scanftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15927/FILES/20231019_fAA_samples_Data_20231019_fAA_samples.wiffftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15927/FILES/RAW_FILES/20231019_fAA_samples_Data_20231019_fAA_samples.wiffftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15927/FILES/20231019_fAA_samples_Data_20231019_fAA_samples.wiff.scanprimaryOK200ftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15927<p>Raw data were processed using Skyline software and Excel.</p>MetaboLightsPublicLiquid Chromatography MS - positive - reverse-phase<p>Standards and samples were analyzed using a Sciex 6500+ QTrap Mass Spectrometer interfaced with an Eksigent M5 microLC using water with 0.1% formic acid as mobile phase A and acetonitrile with 0.1% formic acid as mobile phase B. The LC system was plumbed with an Acquity UPLC BEH HILIC column, 130 angstrom, 1.7 µm, 2.1 mm x 100mm, and run at a flow rate of 40 µL/min. Samples were run at a gradient of 100% B for 5 min, followed by a decrease to 10% B over a further 7.5 min after which the %B was increased to %100 over 30 seconds followed by a further five min at 100% B.</p>Decreasing peptide deformylase activity is a beneficial strategy for increasing formaldehyde resistance in Methylobacterium extorquens.University of MinnesotaAndrew Rajczewskiprotein digestmass spectrometry assay<p>Following protein digestion, samples were dried down and reconstituted in acetonitrile for analysis via LC-MS.</p>Methylobacterium extorquenshttps://www.ebi.ac.uk/metabolights/MTBLS15927Jannell Bazurto. University of Minnesota. jbazurto@umn.edu.<p>Raw data were not transformed for this analysis but analyzed in Skyline software.</p>Formaldehyde treatmentRF treatmentBiological replicaterajcz001@umn.edu<p>Proteins were extracted from 10 mL of M. extorquens culture. Cells were harvested by centrifugation for 5 min at max speed and washed in 1 mL of MP. Cell pellets were stored at -80 °C before proceeding to the next steps. Cells were resuspended in 1 mL TRIzol reagent and lysed by bead beating. Cell suspensions were transferred to 2 mL screw cap tubes containing 0.1 mm beads and a single 3.2 mm bead. Samples were beat for 5 cycles of max RPM for 1 min and iced for 1 min. After incubation at room temperature for 5 min, samples were centrifuged for 1 min at max speed. 200 µL of chloroform was added to each supernatant, and samples were shaken vigorously by hand for 15 sec, incubated for 3 min at room temperature, and then centrifuged at 12,000 x g for 15 min at 4 °C. The upper aqueous phase was discarded. DNA was precipitated by the addition of 300 µL ethanol. Samples were mixed by inversion, incubated at room temperature for 3 min, then centrifuged at 4,000 x g for 10 min. Supernatants were transferred to a new tube with 1.25 mL isopropanol to precipitate proteins. Samples were incubated at room temperature for 10 min and then centrifuged at 12,000 x g for 15 min. Supernatants were discarded, and protein pellets were washed 3 times in 1 mL of 0.3 M guanidine HCL in 95% ethanol and once more in 100% ethanol. Pellets were allowed to air dry for 10 min. Proteins were resuspended in 200 µL of 1 M NaOH and incubated at 50°C until fully dissolved. The buffer was exchanged using PD MiniTrap G-25 columns and eluting with phosphate buffer. Protein concentrations were measured via BCA assay, and samples were stored at -80 °C. Purified proteins were digested by S. griseus protease in a 10:1 mass ratio. Samples were incubated at 37 °C for 16 hr, vacuum dried, and stored at -20 °C before submitting to University of Minnesota CMSP for MS analysis.&nbsp;</p>MetabolomicsCenter for Metabolomics and ProteomicsMetabolomicsEksigent M5 Micro-LCSkylineformaldehydetargeted analysisSciex 6500+ Q Trapprotein digestBacteriametabolic processMethylobacterium extorquensexperimental sampleCenter for Metabolomics and ProteomicsMetabolomicsEksigent M5 Micro-LCSkylineformaldehydetargeted analysisSciex 6500+ Q Trapprotein digestBacteriametabolic processMethylobacterium extorquensexperimental sample<p>AB Sciex 6500+ Q Trap run in positive mode, transitions were monitored for formylmethionine (178 to 84.1 m/z, 178 to 61.1 m/z, 178 to 56.1 m/z).</p>N-formyl-L-methioninefalseDecreasing peptide deformylase activity is a beneficial strategy for increasing formaldehyde resistance in Methylobacterium extorquensFormaldehyde is a highly toxic metabolite that can cause extensive damage to DNA and proteins, and strategies to mitigate formaldehyde toxicity are poorly understood. Methylotrophic bacteria, such as Methylobacterium extorquens, thrive on one-carbon compounds as sole sources of carbon and energy. These organisms are excellent models for discovering formaldehyde stress response systems because formaldehyde is an obligate intermediate in their central carbon metabolism. Here, we characterize an evolved def allele (defevo) that increases formaldehyde resistance in M. extorquens. The def gene encodes peptide deformylase (PDF, EC:3.5.1.88), an enzyme that contributes to protein processing by removing the formyl group from N-formylmethionine (fMet) on nascent peptides. The defevo allele has a single missense mutation that decreases PDF activity both in vitro and in vivo. Transcriptomic analysis of the defevo strain indicates there are pleiotropic effects of this mutation and a differential response to formaldehyde stress. We investigate possible mechanisms for the defevo mutant’s increased resistance to formaldehyde, including mitigation of formaldehyde-induced protein stress and altered membrane physiology. We find that the defevo allele selectively alleviates exogenous, but not endogenous, formaldehyde stress and identify a tradeoff in heat shock resistance. This study reports the first observation of lowered PDF activity benefiting a cellular physiological phenotype. Our work indicates that altered protein metabolism can mitigate the toxic effects of formaldehyde and furthers our understanding of the strategies that can protect cells from formaldehyde-induced damage.2026-10-052026-10-05MTBLS15927MTBLC49298MTBLC49299MTBLC49300CHEBI:49298CHEBI:49299CHEBI:49300