Mstfa

The first dried polar fractions of the cell extracts and the extracellular medium were derivatized and analysed as previously described^{75 (link)}. Derivatization was performed by shaking the samples at 30 °C for 90 min with 10 μL of methoxyamine hydrochloride in pyridine (40 mg/mL), followed by trimethylsilylation at 37 °C for 30 min with 90 μL of N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA, Sigma-Aldrich) containing C8-C30 fatty acid methyl esters (FAMEs) as internal standards (their concentration can be found in Supplementary Table S5). Aliquots of 0.5 μL of derivatized samples were analyzed using a Leco Pegasus IV time-of-flight (TOF) MS (Leco Corporation) coupled to an Agilent 6890 GC (Agilent Technologies) equipped with a 30 m long 0.25 mm id Rtx-5Sil MS column (0.25 μm film thickness) and a Gerstel MPS2 automatic liner exchange system (Gerstel GMBH & Co. KG). The chromatographic gradient used a constant flow of 1 mL/min with following gradient: 50 °C (1 min), 20 °C/min to 330 °C, hold 5 min. Mass spectrometry data was collected using 1525 V detector voltage at m/z 85–500 with 17 spectra/s, electron ionization at − 70 eV and an ion source temperature of 250 °C. QC injections, method blanks and a pooled mixture of all control and treated samples (cells extracts and extracellular medium separately) were included as quality control samples.

To investigate the mechanism by which benzoic acid derivatives enhance the lipid yield of Schizochytrium limacinum SR21, a GC–MS method combined with multivariate analyses was used to indicate the changes in metabolites in the presence of p-ABA at different concentrations. Before GC–MS analysis, sample derivatization was performed according to the method of our previous study [36 (link)], with moderate modifications. The samples obtained as described above and 40 μL of internal standard (methyl heptadecanoic acid in n-hexane, 1 mg/mL) were mixed and dried in a vacuum freeze dryer. Methoxyamine hydrochloride (50 μL) in pyridine (20 mg/mL) was added to the dried sample and incubated at 37 °C for 2 h. Next, the sample was silylated for 2 h at 37 °C by adding 50 μL of N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA, Sigma, Burlington, USA) and vortex mixing for 30 s.
A 1-μL sample was used with splitless injection into the Agilent 7890–5975C GC–MS equipped with a fused silica capillary column (30 m × 0.25 mm × 0.25 μm, DB-5MS, Agilent). The column and ion source temperatures were set according to our previous study. The mass scan range was 50–600 m/z.