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MB Sample ID: SA308666
| Local Sample ID: | N_AB-A-5 |
| Subject ID: | SU002966 |
| Subject Type: | Bacteria |
| Subject Species: | Staphylococcus aureus/ Acinetobacter baumannii/ Enterococcus faecium/ Pseudomonas aeruginosa |
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Sample Preparation:
| Sampleprep ID: | SP002972 |
| Sampleprep Summary: | Prior to extraction, the suspended bacteria were normalized by turbidity to obtain equivalent amounts of bacteria. The suspensions were then aliquoted at 0.5 mL into 8 mL glass culture tubes (for biphasic extraction) or 2 mL polypropylene microcentrifuge tubes (for single-phase extraction) and pelleted by centrifugation. Before extraction solvents were added, stable isotope labeled internal standards of lipids and metabolites were added for recovery and quantitation purposes. The metabolite internal standards (Cambridge Isotope Laboratories) included 13C5-hypoxanthine (final concentration, 1 µg/mL), 13C6-sucrose (5 µg/mL), and 13C5-L-glutamine (10 µg/mL). The lipid internal standards (Avanti Polar Lipids) included phosphatidylethanolamine (PE) 15:0/d7-18:1 (final concentration, 37.5 ng/mL), diacylglycerol (DG) 15:0/d7-18:1 (100 ng/mL), and phosphatidylglycerol (PG) 15:0/d7-18:1 (12.5 ng/mL). For the biphasic Bligh and Dyer (B&D) extraction, the pelleted bacteria were reconstituted with 0.5 mL of HPLC grade H2O and sonicated for 30 min at 4 °C. A chilled solution of 1:2 CHCl3/MeOH (2 mL) was added to the sample and vortexed for 5 min, followed by the addition of 0.5 mL CHCl3 and 0.5 mL H2O to induce phase separation. After an additional 1 min of vortexing, the samples were centrifuged for 10 min at 3500 rpm and 4 °C. The lower organic layer and the upper aqueous layer of the biphasic solution were collected into separate glass tubes and dried under vacuum. Both dried extracts were reconstituted in 200 µL of 2:2:1 ACN/MeOH/H2O and stored at -80°C or directly diluted for LC-IM-MS analysis. A single-phase extraction solvent system based on butanol, acetonitrile and water (BAW) was evaluated for the recovery of both lipids and metabolites. We tested three compositions of the BAW extraction solution: 30% butanol/70% acetonitrile (30% Bu), 45% butanol/55% acetonitrile (45% Bu), and 60% butanol/40% acetonitrile (60% Bu), with H2O constant at 20% for all three compositions. For the extraction, 1 mL of chilled, pre-mixed extraction solution was added to pelleted bacteria. The samples were vortexed and sonicated in an ice bath in alternating 5 min intervals for a total of 30 min. The samples were then chilled at 4 °C for 10 min, and then centrifuged at 3500 rpm and 4 °C for 10 min. The supernatants were collected into fresh 2 mL microcentrifuge tubes and dried under vacuum. The dried single-phase extracts were reconstituted in 200 µL of 2:2:1 ACN/MeOH/H2O and stored at -80 °C freezer or diluted for LC-IM-MS analysis. |
| Processing Storage Conditions: | On ice |
| Extract Storage: | -80℃ |
