DOI: 10.5281/zenodo.17083592
title: Proteomics raw data of P. putida EM2-4 under different growth conditions

description: Proteomics analysis were carried out at the CNB-CSIC proteomic facility Madrid, Spain (http:// proteo. cnb. csic. es/ prote omical) using the iTRAQ protocol (Cuenca et al., 2016; Molina et al., 2019). Cultures were prepared as described for the transcriptomics assays. Cells were harvested by centrifugation (5000 x g for 10 minutes at 4 ºC), washed twice with M9 medium and stored at -80 ºC. For protein extraction, bacterial cell pellets were resuspended in lysis buffer containing 5% (w/v) sodium dodecyl sulfate (SDS; Sigma-Aldrich) and 100 mM triethylammonium bicarbonate (TEAB; Thermo Fisher Scientific). Cell disruption and homogenization were performed by micro-tip probe sonication for 1 min using a UP50H ultrasonic homogenizer (Hielscher Ultrasonics). Lysates were centrifuged at 21,000 × g for 5 min at 4 °C, and the supernatant containing solubilized proteins was collected for further processing. A portion of the extract was subjected to protein precipitation using the methanol/chloroform method and resolubilized in a denaturing buffer composed of 7 M urea, 2 M thiourea, and 100 mM TEAB. Protein disulfide bonds were reduced and alkylated at 37 °C for 60 min in the presence of 5 mM tris(2-carboxyethyl)phosphine (TCEP) and 10 mM chloroacetamide (CAA). Protein concentrations were determined using the Pierce™ 660 nm Protein Assay (Thermo Fisher Scientific).

Aliquots containing 150 μg of protein were diluted 1:5 in 100 mM TEAB and digested overnight at 37 °C with continuous shaking using MS-grade trypsin (Thermo Fisher Scientific) at an enzyme-to-substrate ratio of 1:20 (w/w).

Peptide samples were labelled with the TMT 18plex™ Isobaric Label Reagent Set (Thermo Fisher Scientific, Rockford, IL, USA) following a protocol modified by the manufacturer. Labelling was conducted in 85% 200 mM EPPS buffer (pH 8.5) and 15% anhydrous acetonitrile. Reactions were incubated at 25 °C for 2 h and subsequently quenched with hydroxylamine to a final concentration of 0.3% (w/v) for 15 min. Labelled peptides were pooled, vacuum-dried, and desalted using Sep-Pak C18 cartridges (Waters). Three biological replicates were analyzed per experimental condition.

Peptide mixtures were desalted using StageTips packed with Empore™ C18 material (Sigma-Aldrich). Peptide concentration was quantified with the Qubit™ Fluorometric Quantitation System (Thermo Fisher Scientific). One microgram of each sample was subjected to one-dimensional nanoLC–ESI–MS/MS analysis using an Ultimate 3000 nano-HPLC system (Thermo Fisher Scientific) coupled to an Orbitrap Exploris 240 mass spectrometer (Thermo Fisher Scientific). Peptides were separated on a microPAC™ C18 column (PharmaFluidics) at 45 °C using a 120 min linear gradient from 2% to 95% acetonitrile (0.1% formic acid) at a flow rate of 250 nL/min. The injection volume was 5µL.

Mass spectra were acquired in data-dependent acquisition (DDA) mode with full MS scans from m/z 350 to 1200 at a resolution of 60,000 (m/z 200), AGC target of 300%, and maximum injection time of 40 ms. The top 20 most intense precursor ions were selected for fragmentation by higher-energy collisional dissociation (HCD) with a collision energy of 34. MS/MS scans were acquired at a resolution of 45,000, with an AGC target of 200% and a maximum injection time of 120 ms. Precursor ions with charge states of +1, unassigned, or ≥+6 were excluded. Dynamic exclusion was set to 45 s.

Raw data were processed with Proteome Discoverer v2.5 (Thermo Fisher Scientific). MS/MS spectra were searched using Mascot (v2.7.0 and v2.4.0), MsFragger (v3.1.1), and Sequest HT search engines. The following modifications were considered: fixed carbamidomethylation of cysteine (+57.021 Da), variable oxidation of methionine (+15.995 Da), pyro-glutamate formation from glutamine (−17.027 Da), and TMT tags on peptide N-termini and lysine residues (+229.163 Da). Trypsin specificity was used, allowing up to two missed cleavages. The precursor and fragment mass tolerances were set to 10 ppm and 0.02 Da, respectively. The false discovery rate (FDR) was controlled at 1% for peptide spectral matches (PSMs), peptides, and proteins.

Protein quantification was based on reporter ion intensities summed across all identified peptides. Peptide group abundances were aggregated per sample and normalized to the maximum total signal across all runs to assess differential protein expression.

Peptide identification was restricted to those with a q-value below 0.05. Proteins were considered validated if supported by at least two distinct peptides. The false discovery rate (FDR) for protein identification was estimated to be below 1% using a reversed decoy database approach. Proteins with an abundance change of ≥1.5-fold (log₂ fold change) with a q-value ≤0.05 were considered differentially abundant.