LC-MS Based Metabolomics Analysis

Liquid chromatography-mass spectrometry based targeted metabolomics was performed according to the protocol described previously (Wang et al., 2014). All chemicals used for LC-MS metabolomics analysis were obtained from Sigma–Aldrich (Taufkirchen, Germany). For metabolomic analysis, the wild type and the mutant cells were collected at 48 and 72 h, respectively, and each sample was prepared with three biological replicates. Due to the large amount of cultivation needed to finish the comparative experiments of 32 TRs, the samples had to be cultivated, prepared, and analyzed in five batches. A separate cultivation and analysis of the wild type as control was conducted for every batch to minimize possible batch difference. Briefly, the cells were collected by centrifugation at 7500 × g for 8 min at 4°C (Eppendorf 5430R, Hamburg, Germany), quenched, and extracted rapidly with 900 μL of 80:20 MeOH/H₂O (-80°C) and then frozen in liquid nitrogen. The samples were then frozen-thawed three times to release metabolites from the cells. The supernatant was collected after centrifugation at 15,000 × g for 5 min at -4°C and then stored at -80°C. The remaining cell pellets were re-suspended in 500 μL of 80:20 MeOH/H₂O (-80°C), and then the above extraction process was repeated. The supernatant from the second extraction was pooled with that from the first extraction and stored at -80°C until the LC-MS analysis was conducted. LC-MS analysis was conducted on an Agilent 1260 series binary HPLC system (Agilent Technologies, Waldbronn, Germany) using a Synergi Hydro-RP (C18) 150 mm × 2.0 mm ID, 4-μm 80-Å particle column (Phenomenex, Torrance, CA, USA), coupled to an Agilent 6410 triple quadrupole mass analyzer equipped with an electrospray ionization (ESI) source. Data was acquired using Agilent Mass Hunter workstation LC/QQQ acquisition software (version B.04.01), and chromatographic peaks were subsequently integrated via Agilent Qualitative Analysis software (version B.04.00). A total of 24 metabolites were selected for LC-MS-based targeted metabolite analysis in this study. All data of metabolomic profiling was first normalized by the internal control and the cell numbers of the samples.

The 24 targeted metabolites include acetyl coenzyme A (AcCOA), adenosine 5′-diphosphate (ADP), adenosine-5′-diphosphoglucose (ADP-GCS), α-ketoglutaric acid (AKG), adenosine 5′-monophosphate (AMP), adenosine 5′-triphosphate (ATP), coenzyme A hydrate (CoA), dihydroxyacetone phosphate (DHAP), D-fructose 1,6-bisphosphate (FBP), D-fructose 6-phosphate (F6P), sodium fumarate dibasic (FUM), DL-glyceraldehyde 3-phosphate (GAP), D-glucose 6-phosphate (G6P), L-glutamic acid (GLU), α-nicotinamide adenine dinucleotide (NAD), reduced α-nicotinamide adenine dinucleotide (NADH), nicotinamide adenine dinucleotide phosphate (NADP), reduced nicotinamide adenine dinucleotide phosphate (NADPH), uridine 5′-diphosphoglucose (UDP-GCS), oxaloacetic acid (OXA), phosphor (enol)pyruvic acid (PEP), D-(-)-3-phosphoglyceric acid (3PG), D-ribose 5-phosphate (R5P), and D-ribulose1,5-bisphosphate (UDP-GCS), uridine 5′-diphosphoglucose (RiBP).