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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 |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU002966 |
| Subject Type: | Bacteria |
| Subject Species: | Staphylococcus aureus/ Acinetobacter baumannii/ Enterococcus faecium/ Pseudomonas aeruginosa |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| N_AB-A-5 | SA308666 | FL037171 | Acinetobacter baumannii | Species |
Collection:
| Collection ID: | CO002959 |
| Collection Summary: | All work with microorganisms was performed under Biosafety Level 2 (BSL-2) conditions. Bacteria were streaked onto agar plates from stocks and incubated overnight at 37°C. Single colonies were collected from the agar plates and suspended in sterile deionized (DI) water to a turbidity of 2.0-2.05 McFarlands (equivalent to ca. 6.0 x 108 CFU/mL). Five biological replicates were prepared for each strain. Tryptic Soy Broth was inoculated at a 1:10 dilution (5 mL total volume) and incubated overnight at 37°C with shaking (180 rpm). The cultures were then centrifuged at 2700 rpm for 10 min at 4 °C, after which the broth was discarded. The pelleted bacteria were washed and resuspended in 2 mL of sterile water. |
| Sample Type: | Bacterial cells |
Treatment:
| Treatment ID: | TR002975 |
| Treatment Summary: | No treatment |
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℃ |
Combined analysis:
| Analysis ID | AN004675 | AN004676 |
|---|---|---|
| Analysis type | MS | MS |
| Chromatography type | HILIC | HILIC |
| Chromatography system | Waters Acquity | Waters Acquity |
| Column | Waters ACQUITY UPLC BEH Amide (100 x 2.1mm,1.7um) | Waters ACQUITY UPLC BEH Amide (100 x 2.1mm,1.7um) |
| MS Type | ESI | ESI |
| MS instrument type | QTOF | QTOF |
| MS instrument name | Waters Synapt G2 XS QTOF | Waters Synapt G2 XS QTOF |
| Ion Mode | POSITIVE | NEGATIVE |
| Units | internsity | intensity |
Chromatography:
| Chromatography ID: | CH003518 |
| Instrument Name: | Waters Acquity |
| Column Name: | Waters ACQUITY UPLC BEH Amide (100 x 2.1mm,1.7um) |
| Column Temperature: | 45 |
| Flow Gradient: | 0-2 min at 100% B, 2-7.7 min from 100% to 70% B, 7.7-9.5 min from 70% to 40% B, 9.5-10.25 min from 40% to 30% B, 10.25-12.75 min from 30% to 100% B, and 12.75-17 min to re-equilibrate to 100% B |
| Flow Rate: | 0.4 mL/min |
| Solvent A: | 100% water; 10 mM ammonium formate; 0.125% formic acid |
| Solvent B: | 95% ACN/5% water; 10 mM ammonium formate; 0.125% formic acid |
| Chromatography Type: | HILIC |
MS:
| MS ID: | MS004422 |
| Analysis ID: | AN004675 |
| Instrument Name: | Waters Synapt G2 XS QTOF |
| Instrument Type: | QTOF |
| MS Type: | ESI |
| MS Comments: | The UPLC was connected to the electrospray ionization source of the traveling wave ion mobility-mass spectrometer (Waters Synapt XS) and samples were injected at 5 uL. Prior to acquisition of sample data, data was acquired for a mixture of CCS calibrants using direct infusion. Randomized sample queues were analyzed in both positive and negative ionization modes. A pooled mixture of all samples was used as a quality control (QC). Data was collected across the entire 17 min chromatographic method using data-independent MS/MS acquisition. Leucine enkephalin was monitored for post-acquisition lockmass correction. Capillary +3 kV; Sampling Cone 30 V; Sampling Cone 25 V; Source Offset 40 V; Source Temp 150 ºC; Desolvation Temp 400 ºC; Cone gas flow 50 L/h; Desolvation gas flow 650 L/h; Nebulizer gas flow 7 Bar. Mass Range 50-1200 m/z. |
| Ion Mode: | POSITIVE |
| MS ID: | MS004423 |
| Analysis ID: | AN004676 |
| Instrument Name: | Waters Synapt G2 XS QTOF |
| Instrument Type: | QTOF |
| MS Type: | ESI |
| MS Comments: | The UPLC was connected to the electrospray ionization source of the traveling wave ion mobility-mass spectrometer (Waters Synapt XS) and samples were injected at 5 uL. Prior to acquisition of sample data, data was acquired for a mixture of CCS calibrants using direct infusion. Randomized sample queues were analyzed in both positive and negative ionization modes. A pooled mixture of all samples was used as a quality control (QC). Data was collected across the entire 17 min chromatographic method using data-independent MS/MS acquisition. Leucine enkephalin was monitored for post-acquisition lockmass correction. Capillary -2 kV; Sampling Cone 30 V; Sampling Cone 25 V; Source Offset 40 V; Source Temp 150 ºC; Desolvation Temp 400 ºC; Cone gas flow 50 L/h; Desolvation gas flow 650 L/h; Nebulizer gas flow 7 Bar. Mass Range 50-1200 m/z. |
| Ion Mode: | NEGATIVE |
