Mstfa

Derivatization was carried out as described with modifications
[19 (link)]. The dried cell extracts were dissolved in 20 μl of methoxyamine hydrochloride solution (Sigma, 40 mg/ml in pyridine (Roth)) and incubated for 90 min at 30°C with constant shaking followed by the addition of 80 μl of N-methyl-N-[trimethylsilyl]trifluoroacetamide (MSTFA; Machery-Nagel, Dueren, Germany) and incubation at 37°C for 45 min. The extracts were centrifuged for 10 min at 10,000 × g, and aliquots of 30 μl were transferred into glass vials (Th. Geyer, Berlin, Germany) for gas chromatography-mass spectrometry (GC-MS) measurement.

Quantitative composition of the PHAs was determined by gas chromatography (Trace GC Ultra, Thermo Scientific) coupled to a mass spectrum analysis (ISQ, Thermo Scientific) of the extracted polyester. The extraction of the PHAs from dried cells was performed by methanolysis using (per 10 mg CDW) 2 mL chloroform and 2 mL methanol containing 15% sulfuric acid and 0.5 mg/mL 3-methylbenzoic acid as internal standard and then incubated at 100°C for 4 h (de Eugenio et al., 2010 (link)). Derivatization of the samples was performed using N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA, Macherey-Nagel, Düren) at 85°C for 1 h. For analysis, 1 μL of derivatized sample was injected by a split-splitless injector at a 1:50 split ratio into the gas chromatograph with a 1,4-bis(dimethylsiloxy)phenylene dimethyl polysiloxane separation column (30 m with 0.25 mm inner diameter, film thickness 0.25, Rxi-5Sil MS, Restek) at a flow rate of 0.9 mL/min with Helium as inert carrier gas. Electron ionization (EI) mass spectra were recorded in full scan mode (m/z 40–550). PHA monomers were identified by their specific mass spectra. The most commonly produced P. putida PHAs, 3-hydroxydecanoic acid and 3-hydroxydodecanoic acid, were commercially obtained as standards for sample quantification (Abcam plc, UK). The standards were treated like all other samples.

The samples were extracted for metabolite profiling (LC–MS and GC–MS) as described in Weckwerth et al. (2004) (link). Briefly tissue was grounded using RETCH-mill (Retsch Gmbh, 42787 Haan, Germany) with pre-chilled holders and grinding beads. Forty milligram frozen dried powder were extracted in a pre-chilled methanol/chloroform/water extraction solution (2.5/1/1 v/v/v). Internal standards, i.e., 0.2 mg/mL ribitol in water, 1 mg/mL ampicillin in water and 1 mg/mL corticosterone in methanol were subsequently added. The mixture was then briefly vortexed and ultra-sonicated for 10 min to release the cell components. Subsequently, samples were centrifuged for 10 min at 14000 RPM (micro centrifuge 5417R). The supernatant was mixed with equal volumes (300 μl) of chloroform and Millipore Direct-Q3 UV system purified water, vortexed, and then centrifuged at 14,000 RPM for 5 mins. Finally, 1 mL of water/methanol phase was transferred to UPLC vials for LC–MS and 75 μL were derivatized for GC–MS analysis with micro filtered retention time standard alkane mixture (0.029% v/v n-dodecane, n-pentadecane, n-nonadecane, n-docosane, n-octacosane, n-dotracontane, and n-hexatriacontane dissolved in pyridine (0.0075% H₂O), purchased from Sigma–Aldrich (Jerusalem, Israel) and MSTFA, N-methyl-N-[trimethylsilyl] trifluoroacetamide purchased from Macherey-Nagel GmbH & Co. KG, Düren, Germany.