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MB Sample ID: SA416148
| Local Sample ID: | FP-R2_Gifu_48h |
| Subject ID: | SU003933 |
| Subject Type: | Bacteria |
| Subject Species: | Dorea longicatena |
| Taxonomy ID: | 88431 |
| Genotype Strain: | Flavonifractor plautii, Dorea longicatena, Turicibacter sanguinis, Clostridium bolteae, Clostridium symbiosum, Clostridium innocuum, Clostridium clostridioforme, Streptococcus parasanguinis |
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Subject:
| Subject ID: | SU003933 |
| Subject Type: | Bacteria |
| Subject Species: | Dorea longicatena |
| Taxonomy ID: | 88431 |
| Genotype Strain: | Flavonifractor plautii, Dorea longicatena, Turicibacter sanguinis, Clostridium bolteae, Clostridium symbiosum, Clostridium innocuum, Clostridium clostridioforme, Streptococcus parasanguinis |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| FP-R2_Gifu_48h | SA416148 | FL049600 | Culture | Sample source |
| FP-R2_Gifu_48h | SA416148 | FL049600 | Flavonifractor plautii | Species |
| FP-R2_Gifu_48h | SA416148 | FL049600 | Gifu | Media |
| FP-R2_Gifu_48h | SA416148 | FL049600 | R2 | Replicate |
Collection:
| Collection ID: | CO003926 |
| Collection Summary: | All strains were cultured anaerobically at 37°C in different media types according to the experiment being performed. The media compositions used in this study include BHI-R, CHG-R, Gifu-R, and Mega-R, each formulated with specific components to support microbial growth. BHI-R was prepared by dissolving 18.5 g of Brain Heart Infusion (BHI) and 5 g of L-arginine in 500 mL of Milli-Q water, followed by sterilization via autoclaving (10 lbs, 115°C, 15 minutes). CHG-R was based on CHG media, which contained 18.5 g of BHI, 0.5 g each of D-(+)-cellobiose, D-(+)-maltose monohydrate, and D-(-)-fructose, and 0.25 g of cysteine in 500 mL of Milli-Q water. After filter sterilization, additional supplements, including 5 mL each of Vitamin K + Hematin (1%), trace mineral supplement, and vitamin supplement, were added in an anaerobic chamber. Gifu-R was derived from commercially available Gifu Anaerobic Media (GAM) (Himedia, Cat # M1801), prepared by suspending 59 g of GAM in 1000 mL of purified/distilled water and autoclaving under the same conditions as BHI-R. Mega-R was formulated with a complex mixture of components, including 5 g of Trypticase Peptone (BBL), 2.5 g each of Yeast Extract (Bacto) and Meat Extract, and multiple carbohydrate sources such as 1 g of D-(+)-glucose and 0.5 g each of D-(+)-cellobiose, D-(+)-maltose monohydrate, and D-(-)-fructose, all dissolved in 500 mL of Milli-Q water. Additional components included 50 mL of 1 M potassium phosphate buffer (pH 7.2), 20 mL of TYG salts solution, 1 mL of 25% Tween 80, and various supplements such as SCFA, CaCl₂, FeSO₄, and resazurin. Like CHG-R, Mega-R was supplemented with 5 mL each of Vitamin K + Hematin (1%), trace mineral supplement, and vitamin supplement. For CHG-R, Gifu-R, and Mega-R, an additional 5 g of L-arginine was incorporated into 500 mL of each respective medium to create their corresponding arginine-enriched formulations. All cultures were grown in an anaerobic chamber (Coy Laboratory Products) with an atmosphere of 5% CO2, 5% H2, and 90% N2 at 37°C. The isolates were streaked onto CHG plates and incubated for 48 hours. Once colonies were visible, a single colony from each plate was picked and processed for 16S rRNA Sanger sequencing using primer sequences 27F: AGAGTTTGATCMTGGCTCAG and 1492R: GGTTACCTTGTTACGACTT. Once the identity of the bacterial strains was confirmed, a single colony from each strain was inoculated in triplicates in 6 mL of the respective media types (CHG, Gifu, Mega) and incubated overnight. The overnight cultures were inoculated into fresh media and normalized to the OD600 of 0.05. At each time point after inoculation (6h, 24h, 48 h), 200 μL of the bacterial culture were collected. |
| Sample Type: | Bacterial cells |
Treatment:
| Treatment ID: | TR003942 |
| Treatment Summary: | NA |
Sample Preparation:
| Sampleprep ID: | SP003939 |
| Sampleprep Summary: | LC–MS samples were prepared from bacterial cultures for each profiling method as follows: - HILIC-pos: Bacterial cultures (10 μL) were extracted with the addition of nine volumes of 74.9:24.9:0.2 v/v/v acetonitrile/methanol/formic acid containing stable isotope-labeled internal standards (valine-d8, Isotec; and phenylalanine-d8, Cambridge Isotope Laboratories). The samples were centrifuged (10 min, 9,000g, 4°C), and the supernatants (10 μL) injected directly onto column. - C8-pos: Bacterial cultures (10 μL) using 190 μL isopropanol containing 1-dodecanoyl-2-tridecanoyl-sn-glycero-3-phosphocholine as an internal standard (Avanti Polar Lipids; Alabaster, AL). After centrifugation (10 min, 9,000g, ambient temperature), supernatants (2 μL) were injected directly onto column. - C18-neg: Bacterial cultures (30 μL) were extracted using 90 μl methanol containing 15R-15-methyl Prostaglandin A2,15R-15-methyl Prostaglandin F2α, 15S-15-methyl Prostaglandin D2, 15S-15-methyl Prostaglandin E1, and 15S-15-methyl Prostaglandin E2 as internal standards (Cayman Chemical Co.) and centrifuged (10 min, 9,000g, 4°C). The supernatants (10 μL) were injected onto column. - HILIC-neg: Bacterial cultures (30 μL) were extracted with the addition of four volumes of 80% methanol containing inosine-15N4, thymine-d4 and glycocholate-d4 internal standards (Cambridge Isotope Laboratories). The samples were centrifuged (10 min, 9,000g, 4°C) and the supernatants 10 μL) were injected directly onto column. |
Combined analysis:
| Analysis ID | AN006244 | AN006245 | AN006246 | AN006247 |
|---|---|---|---|---|
| Chromatography ID | CH004735 | CH004736 | CH004737 | CH004738 |
| MS ID | MS005947 | MS005948 | MS005949 | MS005950 |
| Analysis type | MS | MS | MS | MS |
| Chromatography type | HILIC | Reversed phase | HILIC | Reversed phase |
| Chromatography system | Shimadzu Nexera X2 | Shimadzu Nexera X2 | Shimadzu Nexera X2 | Shimadzu Nexera X2 |
| Column | Waters Atlantis HILIC (150 x 2 mm, 3 µm) | Waters Acquity BEH C8 (100 x 2.1 mm, 1.7 µm) | Phenomenex Luna NH2 (150 x 2.1 mm, 3 µm) | Waters ACQUITY UPLC BEH C18 (150 x 1.7 mm,2.1 µm) |
| MS Type | ESI | ESI | ESI | ESI |
| MS instrument type | IDX | IDX | Orbitrap | IDX |
| MS instrument name | Thermo Q Exactive Plus Orbitrap | Thermo Q Exactive Plus Orbitrap | Thermo Q Exactive Plus Orbitrap | Thermo Q Exactive Orbitrap |
| Ion Mode | POSITIVE | POSITIVE | NEGATIVE | NEGATIVE |
| Units | Abudances | Abudances | A | Abudances |
Chromatography:
| Chromatography ID: | CH004735 |
| Instrument Name: | Shimadzu Nexera X2 |
| Column Name: | Waters Atlantis HILIC (150 x 2 mm, 3 µm) |
| Column Temperature: | 30°C |
| Flow Gradient: | Isocratically with 5% mobile phase A for 1 minute followed by a linear gradient to 40% mobile phase B over 10 minutes |
| Flow Rate: | 250 µL/min |
| Solvent A: | 100% Water; 10 mM Ammonium formate; 0.1% Formic acid |
| Solvent B: | 100% Acetonitrile; 0.1% Formic acid |
| Chromatography Type: | HILIC |
| Chromatography ID: | CH004736 |
| Instrument Name: | Shimadzu Nexera X2 |
| Column Name: | Waters Acquity BEH C8 (100 x 2.1 mm, 1.7 µm) |
| Column Temperature: | 40°C |
| Flow Gradient: | The column was eluted at a flow rate of 450 µL/min isocratically for 1 minute at 80% mobile phase A, followed by a linear gradient to 80% mobile-phase B over 2 minutes, a linear gradient to 100% mobile phase B over 7 minutes, and then 3 minutes at 100% mobile-phase B. |
| Flow Rate: | 450 µL/min |
| Solvent A: | 95% Water/5% Methanol; 10 mM Ammonium acetate; 0.1% Acetic acid |
| Solvent B: | 100% Methanol; 0.1% Acetic acid |
| Chromatography Type: | Reversed phase |
| Chromatography ID: | CH004737 |
| Instrument Name: | Shimadzu Nexera X2 |
| Column Name: | Phenomenex Luna NH2 (150 x 2.1 mm, 3 µm) |
| Column Temperature: | 30°C |
| Flow Gradient: | The column was eluted with initial conditions of 10% mobile phase A and 90% mobile phase B followed by a 10 min linear gradient to 100% mobile phase A. |
| Flow Rate: | 400 µL/min |
| Solvent A: | 100% water; 20 mM ammonium acetate; 20 mM ammonium hydroxide |
| Solvent B: | 75% acetonitrile/25% methanol; 10 mM ammonium hydroxide |
| Chromatography Type: | HILIC |
| Chromatography ID: | CH004738 |
| Instrument Name: | Shimadzu Nexera X2 |
| Column Name: | Waters ACQUITY UPLC BEH C18 (150 x 1.7 mm,2.1 µm) |
| Column Temperature: | 45°C |
| Flow Gradient: | The column was eluted isocratically at a flow rate of 450 µL/min with 20% mobile phase A for 3 minutes followed by a linear gradient to 100% mobile phase B over 12 minutes. |
| Flow Rate: | 450 µL/min |
| Solvent A: | 100% Water; 0.01% Formic acid |
| Solvent B: | 100% Acetonitrile; 0.01% Acetic acid |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS005947 |
| Analysis ID: | AN006244 |
| Instrument Name: | Thermo Q Exactive Plus Orbitrap |
| Instrument Type: | IDX |
| MS Type: | ESI |
| MS Comments: | Raw data were processed using TraceFinder 3.3 software (Thermo Fisher Scientific; Waltham, MA) and Progenesis QI (Nonlinear Dynamics; Newcastle upon Tyne, UK). Metabolite identities were confirmed using authentic reference standards or reference samples. |
| Ion Mode: | POSITIVE |
| MS ID: | MS005948 |
| Analysis ID: | AN006245 |
| Instrument Name: | Thermo Q Exactive Plus Orbitrap |
| Instrument Type: | IDX |
| MS Type: | ESI |
| MS Comments: | Raw data were processed using TraceFinder 3.3 software (Thermo Fisher Scientific; Waltham, MA) and Progenesis QI (Nonlinear Dynamics; Newcastle upon Tyne, UK). Metabolite identities were confirmed using authentic reference standards or reference samples. |
| Ion Mode: | POSITIVE |
| MS ID: | MS005949 |
| Analysis ID: | AN006246 |
| Instrument Name: | Thermo Q Exactive Plus Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Raw data were processed using TraceFinder 3.3 software (Thermo Fisher Scientific; Waltham, MA) and Progenesis QI (Nonlinear Dynamics; Newcastle upon Tyne, UK). Metabolite identities were confirmed using authentic reference standards or reference samples. |
| Ion Mode: | NEGATIVE |
| MS ID: | MS005950 |
| Analysis ID: | AN006247 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | IDX |
| MS Type: | ESI |
| MS Comments: | Raw data were processed using TraceFinder 3.3 software (Thermo Fisher Scientific; Waltham, MA) and Progenesis QI (Nonlinear Dynamics; Newcastle upon Tyne, UK). Metabolite identities were confirmed using authentic reference standards or reference samples. |
| Ion Mode: | NEGATIVE |
