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MB Sample ID: SA083354
| Local Sample ID: | 4-Synechocystis_6803-cell-6a-1 |
| Subject ID: | SU001265 |
| Subject Type: | Bacteria |
| Subject Species: | Synechocystis sp. PCC 6803 |
| Taxonomy ID: | 1148 |
| Genotype Strain: | NCBI:txid1148 |
| Cell Biosource Or Supplier: | ATCC |
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Collection:
| Collection ID: | CO001259 |
| Collection Summary: | For each metabolomics time-point, a 10 mL culture were rapidly sampled via sterile on-reactor syringes into a pre-weighed centrifuge tube, quenched in -4°C 1X PBS, spun at 3,000g for 5 min., decanted, frozen in liquid nitrogen, and lyophilized at -50°C. The workflow from sampling to centrifugation took < 2 minutes; lyophilized samples were stored at -80°C for < 1 month prior to extraction. A biphasic extraction from lyophilized cell pellets was performed via a 2:1:1.6 MTBE:MeOH:H2O biphasic extraction, modified from the protocol developed by Salem et al. (Salem et al., 2016) resulting in a top layer of MTBE with non-polar soluble metabolites, a lower layer of MeOH:H2O with polar and semi-polar soluble metabolites, and an insoluble pellet. Each liquid layer was transferred to a fresh glass vial and dried under nitrogen gas overnight. The MTBE layer was resuspended in 1:1 toluene:MeOH and analyzed via Q-TOF-MS with a UPLC Phenyl Hexyl column (“RP-MS”). The MeOH:H2O layer was resuspended in 1:1 H2O:MeOH, split evenly and subjected to either i) derivatization in methoxyamine HCl and MSTFA followed by GC-MS analysis, or ii) targeted SRM analysis on a tandem quadrupole-MS equipped with a HILIC column. The insoluble pellet was hydrolyzed with a hydrochloric acid (HCl) based on previously published protocols (Fountoulakis and Lahm, 1998) (Huang, Kaiser and Benner, 2012) to analyze individual amino acids, nucleoside, and carbohydrate content of the insoluble polymers utilizing MTBSTFA derivatization for insoluble amino acids. Of the soluble phases, 10 µL were removed from each sample and pooled to create a QC sample, mixed, and aliquoted into thirteen vials. A QC sample was run after every sixth injection. |
| Sample Type: | Bacterial cells |
