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MB Sample ID: SA345820
| Local Sample ID: | NINR-00417 |
| Subject ID: | SU003297 |
| Subject Type: | Human |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU003297 |
| Subject Type: | Human |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| NINR-00417 | SA345820 | FL040587 | CSF | Sample source |
| NINR-00417 | SA345820 | FL040587 | Healthy volunteer | MECFS_STATUS |
| NINR-00417 | SA345820 | FL040587 | M | SEX |
Collection:
| Collection ID: | CO003290 |
| Collection Summary: | Metabolomics on cerebrospinal fluid samples was performed using Metabolon’s Ultrahigh Performance Liquid Chromatography-Tandem Mass Spectroscopy (UPLC-MS/MS). Samples were prepared using the automated MicroLab STAR® system from Hamilton Company. Several recovery standards were added prior to the first step in the extraction process for QC purposes. To remove protein, dissociate small molecules were bound to protein or trapped in the precipitated protein matrix, and to recover chemically diverse metabolites, proteins were precipitated with methanol under vigorous shaking for two minutes (Glen Mills GenoGrinder 2000) followed by centrifugation. The resulting extract was divided into five fractions: two for analysis by two separate reverse phase (RP)/UPLC-MS/MS methods with positive ion mode electrospray ionization (ESI), one for analysis by RP/UPLC-MS/MS with negative ion mode ESI, one for analysis by HILIC/UPLC-MS/MS with negative ion mode ESI, and one sample was reserved for backup. Samples were placed briefly on a TurboVap® (Zymark) to remove the organic solvent. The sample extracts were stored overnight under nitrogen before preparation for analysis. |
| Sample Type: | Cerebrospinal fluid |
Treatment:
| Treatment ID: | TR003306 |
| Treatment Summary: | No treatment |
Sample Preparation:
| Sampleprep ID: | SP003304 |
| Sampleprep Summary: | All methods utilized a Waters ACQUITY ultra-performance liquid chromatography (UPLC) and a Thermo Scientific Q-Exactive high resolution/accurate mass spectrometer interfaced with a heated electrospray ionization (HESI-II) source and Orbitrap mass analyzer operated at 35,000 mass resolution. The sample extract was dried then reconstituted in solvents compatible to each of the four methods. Each reconstitution solvent contained a series of standards at fixed concentrations to ensure injection and chromatographic consistency. One aliquot was analyzed using acidic positive ion conditions, chromatographically optimized for more hydrophilic compounds. In this method, the extract was gradient eluted from a C18 column (Waters UPLC BEH C18-2.1x100 mm, 1.7 µm) using water and methanol, containing 0.05% perfluoropentanoic acid (PFPA) and 0.1% formic acid (FA). Another aliquot was also analyzed using acidic positive ion conditions; however, it was chromatographically optimized for more hydrophobic compounds. In this method, the extract was gradient eluted from the same afore-mentioned C18 column using methanol, acetonitrile, water, 0.05% PFPA and 0.1% FA and was operated at an overall higher organic content. Another aliquot was analyzed using basic negative ion optimized conditions using a separate dedicated C18 column. The basic extracts were gradient eluted from the column using methanol and water, however, with 6.5mM Ammonium Bicarbonate at pH 8. The fourth aliquot was analyzed via negative ionization following elution from a HILIC column (Waters UPLC BEH Amide 2.1x150 mm, 1.7 µm) using a gradient consisting of water and acetonitrile with 10mM Ammonium Formate, pH 10.8. The MS analysis alternated between MS and data-dependent MSn scans using dynamic exclusion. The scan range varied slightly between methods but covered 70-1000 m/z. |
Combined analysis:
| Analysis ID | AN005217 | AN005218 | AN005219 | AN005220 |
|---|---|---|---|---|
| Analysis type | MS | MS | MS | MS |
| Chromatography type | Reversed phase | Reversed phase | Reversed phase | HILIC |
| Chromatography system | Waters Acquity | Waters Acquity | Waters Acquity | Waters Acquity |
| Column | Waters ACQUITY UPLC BEH C18 (100 x 2.1mm,1.7um) | Waters ACQUITY UPLC BEH Amide (100 x 2.1mm,1.7um) | Waters ACQUITY UPLC BEH C18 (100 x 2.1mm,1.7um) | Waters Acquity BEH Amide (150 x 2.1mm, 1.7um) |
| MS Type | Other | Other | Other | Other |
| MS instrument type | Orbitrap | Orbitrap | Orbitrap | Orbitrap |
| MS instrument name | Thermo Q Exactive Orbitrap | Thermo Q Exactive Orbitrap | Thermo Q Exactive Orbitrap | Thermo Q Exactive Orbitrap |
| Ion Mode | POSITIVE | POSITIVE | NEGATIVE | NEGATIVE |
| Units | Peak area | Peak area | Peak area | Peak area |
Chromatography:
| Chromatography ID: | CH003945 |
| Chromatography Summary: | Low pH polar (LC/MS Pos early) |
| Instrument Name: | Waters Acquity |
| Column Name: | Waters ACQUITY UPLC BEH C18 (100 x 2.1mm,1.7um) |
| Column Temperature: | 40-50 |
| Flow Gradient: | Linear gradient from 5% B to 80% B over 3.35 minutes |
| Flow Rate: | 0.35 mL/min |
| Solvent A: | 100% water; 0.1% formic acid; 0.05% PFPA, pH ~2.5 |
| Solvent B: | 100% methanol; 0.1% formic acid; 0.05% PFPA, pH ~2.5 |
| Chromatography Type: | Reversed phase |
| Chromatography ID: | CH003946 |
| Chromatography Summary: | Low pH Lipophilic (LC/MS Pos late) |
| Instrument Name: | Waters Acquity |
| Column Name: | Waters ACQUITY UPLC BEH Amide (100 x 2.1mm,1.7um) |
| Column Temperature: | 40-50 |
| Flow Gradient: | Linear gradient from 40% B to 99.5% B over 1.0 minute, hold 99.5% B for 2.4 minutes. |
| Flow Rate: | 0.60 mL/min |
| Solvent A: | 100% water; 0.1% formic acid; 0.05% PFPA, pH ~2.5 |
| Solvent B: | 50% methanol/50% acetonitrile; 0.1% formic acid; 0.05% PFPA, pH ~2.5 |
| Chromatography Type: | Reversed phase |
| Chromatography ID: | CH003947 |
| Chromatography Summary: | High pH (LC/MS Neg) |
| Instrument Name: | Waters Acquity |
| Column Name: | Waters ACQUITY UPLC BEH C18 (100 x 2.1mm,1.7um) |
| Column Temperature: | 40-50 |
| Flow Gradient: | Linear gradient from 0.5 to 70% B over 4.0 minutes, then rapid gradient to 99% B in 0.5 minutes. |
| Flow Rate: | 0.35 mL/min |
| Solvent A: | 100% water; 6.5 mM ammonium bicarbonate, pH 8 |
| Solvent B: | 95% methanol/5% water; 6.5 mM ammonium bicarbonate |
| Chromatography Type: | Reversed phase |
| Chromatography ID: | CH003948 |
| Chromatography Summary: | HILIC (LC/MS Polar Neg) |
| Instrument Name: | Waters Acquity |
| Column Name: | Waters Acquity BEH Amide (150 x 2.1mm, 1.7um) |
| Column Temperature: | 40-50 |
| Flow Gradient: | Linear gradient from 5% B to 50% B in 3.5 minutes, then linear gradient from 50% B to 95% B in 2 minutes. |
| Flow Rate: | 0.50 mL/min |
| Solvent A: | 15% water/5% methanol/80% acetonitrile; 10 mM ammonium formate, (effective pH 10.16 with NH4OH) |
| Solvent B: | 50% water/50% acetonitrile; 10 mM ammonium formate, (effective pH 10.60 with NH4OH) |
| Chromatography Type: | HILIC |
MS:
| MS ID: | MS004950 |
| Analysis ID: | AN005217 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | Other |
| MS Comments: | Metabolon (LC/MS Pos early) |
| Ion Mode: | POSITIVE |
| MS ID: | MS004951 |
| Analysis ID: | AN005218 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | Other |
| MS Comments: | Metabolon (LC/MS Pos late) |
| Ion Mode: | POSITIVE |
| MS ID: | MS004952 |
| Analysis ID: | AN005219 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | Other |
| MS Comments: | Metabolon (LC/MS Neg) |
| Ion Mode: | NEGATIVE |
| MS ID: | MS004953 |
| Analysis ID: | AN005220 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | Other |
| MS Comments: | Metabolon (LC/MS Polar) |
| Ion Mode: | NEGATIVE |
