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MB Sample ID: SA086139
| Local Sample ID: | Plasma_08 |
| Subject ID: | SU001284 |
| Subject Type: | Human |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Gender: | Male and female |
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Subject:
| Subject ID: | SU001284 |
| Subject Type: | Human |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Gender: | Male and female |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| Plasma_08 | SA086139 | FL012848 | 4 | GOLD_STAGE_COPD_SEVERITY |
Collection:
| Collection ID: | CO001278 |
| Collection Summary: | Blood is drawn into a 10 ml heparin plasma tube, and immediately sent to the medical center clinical laboratory for further processing. |
| Sample Type: | Blood (plasma) |
| Storage Conditions: | -80℃ |
Treatment:
| Treatment ID: | TR001299 |
| Treatment Summary: | N/A |
Sample Preparation:
| Sampleprep ID: | SP001292 |
| Sampleprep Summary: | Plasma samples were thawed and 100 uL was prepared using methanol precipitation and liquid-liquid extraction as previously described (PMCID PMC4214365, PMCID PMC3734953) . In short, following the addition of standards, 400uL of ice cold MeOH was added to 100uL of plasma to precipitate proteins, then vortexed for 10 seconds, and centrifgued at 0oC for 15 min at 18,000 rpm to pellet precipitated protein. The supernatant was transferred to a glass culture tube and dried under N2. 3.1mL MTBE was added to the dried methanol residue, vortexed for 30 seconds, 750uL of water added to the tube, and vortex 10 seconds. The sample was then spun at 1000 rpm for 10 min at RT to form bilayer. 2.5 mL of MTBE layer was aliquoted and transferred to a new, clean glass culture tube. Then 3.0 mL MTBE was added to remaining water layer of sample, vortexed for 10 seconds, and centrifuged at 200 x g for 10 min at RT to form bilayer. 3mL of MTBE was aliquoted and combined with previous MTBE tube. This MTBE layer was dried under nitrogen at 35oC, and quickly re-suspend in 200uL Methanol, vortexed for 5 seconds and transfered to glass autosampler vial. The aqueous layer was dried under N2, 100uL of water quickly added to minimize oxidation. Then 400uL of ice cold MeOH was added, vortexed for 10 seconds, transfered to a 1.5 ml low retention Fisher microtube and frozen at -80oC for 25 min to crash out any remaining residual proteins. Then centrifuged at 0oC for 15 min at 18,000 xg. The supernatant was transferred to a clean microtube, dried in speed vac at 45oC. The dried supernatant was quickly resuspended in 100uL of 5% ACN in water, vortexed for 30 sec, then transferred to autosampler vials, and stored at -80C prior to MS analysis. |
| Processing Storage Conditions: | Described in summary |
| Extract Storage: | Described in summary |
Combined analysis:
| Analysis ID | AN002029 | AN002030 |
|---|---|---|
| Analysis type | MS | MS |
| Chromatography type | Reversed phase | HILIC |
| Chromatography system | Agilent 6545 | Agilent 6520 |
| Column | Agilent Zorbax RRHD SB-C18 (100 x 2.1mm,1.8um) | Phenomenex Kinetex HILIC 100A (50 x 2.1 mm,2.6um) |
| MS Type | ESI | ESI |
| MS instrument type | QTOF | QTOF |
| MS instrument name | Agilent 6545 QTOF | Agilent 6520 QTOF |
| Ion Mode | POSITIVE | POSITIVE |
| Units | Abundance (Log2) | Abundance (Log2) |
Chromatography:
| Chromatography ID: | CH001470 |
| Chromatography Summary: | Reversed phase samples from the lipid fraction were randomized in the worklist and run randomly in triplicate using an Agilent 1290 series pump with an Agilent Zorbax Rapid Resolution HD (RRHD) SB-C18, 1.8 micron (2.1 × 100 mm) analytical column and an Agilent Zorbax SB-C18, 1.8 micron (2.1 × 5 mm) guard column. The autosampler tray temperature was set at 4 °C, column temperature was set at 60 °C, and the sample injection volume was 8 µL for BAL and 4 µL for plasma. The flow rate was 0.7 mL/min with the following mobile phases: mobile phase A was water with 0.1% formic acid, and mobile phase B was 60:36:4 isopropyl alcohol:acetonitrile:water with 0.1% formic acid. Gradient elution was as follows: 0–0.5 minutes 30–70% B, 0.5–7.42 minutes 70–100% B, 7.42–10.4 minutes 100% B, 10.4–10.5 minutes 100–30% B, 10.5–15.1 minutes 30% B. |
| Instrument Name: | Agilent 6545 |
| Column Name: | Agilent Zorbax RRHD SB-C18 (100 x 2.1mm,1.8um) |
| Column Temperature: | 60 |
| Flow Gradient: | 0-0.5 minutes 30-70% B, 0.5-7.42 minutes 70-100% B, 7.42-10.4 minutes 100% B, 10.4-10.5 minutes 100-30% B, 10.5-15.1 minutes 30% B |
| Flow Rate: | 0.7 mL/min |
| Solvent A: | 100% water; 0.1% formic acid, |
| Solvent B: | 60% isopropanol/36% acetonitrile/4% water; 0.1% formic acid |
| Chromatography Type: | Reversed phase |
| Chromatography ID: | CH001471 |
| Chromatography Summary: | The samples from the aqueous small molecule fraction were analyzed randomly in triplicate using an Agilent 1290 series pump using a Phenomenex Kinetex HILIC, 2.6 µm, 100 Å (2.1 × 50 mm) analytical column and an Agilent Zorbax Eclipse Plus-C8 5 µm (2.1 × 12.5 mm) narrow bore guard column. The autosampler tray temperature was set at 4 °C, column temperature was set at 20 °C, and the sample injection volume was 1 µL for both BAL and plasma. The flow rate of 0.6 mL/min with the following mobile phases: mobile phase A was 50% ACN with pH 5.8 ammonium acetate, and mobile phase B was 90% ACN with pH 5.8 ammonium acetate. Gradient elution was as follows: 0.2 minutes 100% B, 0.2–2.1 minutes 100–90% B, 2.1–8.6 minutes 90–50% B, 8.6–8.7 minutes 50–0% B, 8.7–14.7 minutes 0% B, 14.7–14.8 minutes 0–100% B, 14.8–24.8 minutes 100% B. |
| Instrument Name: | Agilent 6520 |
| Column Name: | Phenomenex Kinetex HILIC 100A (50 x 2.1 mm,2.6um) |
| Column Temperature: | 20 |
| Flow Gradient: | 0.2 minutes 100% B, 0.2-2.1 minutes 100-90% B, 2.1-8.6 minutes 90-50% B, 8.6-8.7 minutes 50-0% B, 8.7-14.7 minutes 0% B, 14.7-14.8 minutes 0-100% B, 14.8-24.8 minutes 100% B. |
| Flow Rate: | 0.6 mL/min |
| Solvent A: | 50% acetonitrile/50% water; ammonium acetate, pH 5.8 |
| Solvent B: | 90% acetonitrile/5% water, pH 5.8 |
| Chromatography Type: | HILIC |
MS:
| MS ID: | MS001882 |
| Analysis ID: | AN002029 |
| Instrument Name: | Agilent 6545 QTOF |
| Instrument Type: | QTOF |
| MS Type: | ESI |
| MS Comments: | Agilent 6545 Quadrupole Time-of-Flight mass spectrometer (QTOF-MS) in positive ionization mode with dual AJS ESI source, mass range 50–1700 m/z, scan rate 2.00, gas temperature 300 °C, gas flow 12.0 L/min, nebulizer 35 psi, sheath gas temperature 275°C, skimmer 65 V, capillary voltage 3500 V, fragmentor 120 V, reference masses 121.050873 and 922.009798 (Agilent reference mix). The analysis was replicated for tandem MS of selected compounds using a scan range 50–1700m/z, and 10, 20, and 40 eV collision energies with a 500 ms/spectra acquisition time, 1.3 m/z (narrow) isolation width, and 0.25 minute delta retention time. |
| Ion Mode: | POSITIVE |
| MS ID: | MS001883 |
| Analysis ID: | AN002030 |
| Instrument Name: | Agilent 6520 QTOF |
| Instrument Type: | QTOF |
| MS Type: | ESI |
| MS Comments: | Agilent 6520 QTOF-MS in positive ionization mode with dual ESI source, mass range 50–1700 m/z, scan rate 2.00, gas temperature 325 °C, gas flow 12.0 L/min, nebulizer 30 psi, skimmer 60 V, capillary voltage 4000 V, fragmentor 120 V, reference masses 121.050873 and 922.009798 (Agilent reference mix). The analysis was replicated for tandem MS of selected compounds using a scan range 50–1700m/z, and 10, 20, and 40 eV collision energies with a 500 ms/spectra acquisition time, 1.3 m/z (narrow) isolation width, and 0.25 minute delta retention time. |
| Ion Mode: | POSITIVE |
