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MB Sample ID: SA231952
| Local Sample ID: | VT_181119_M338_091 |
| Subject ID: | SU002419 |
| Subject Type: | Human |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU002419 |
| Subject Type: | Human |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| VT_181119_M338_091 | SA231952 | FL029045 | mother | Status |
Collection:
| Collection ID: | CO002412 |
| Collection Summary: | The Autism Genetic Resource Exchange (AGRE) Consortium has the collection of WGS, biospecimen and phenotype data of families containing at least one individual diagnosed with Autism Spectrum Disorder (ASD) by the Autism Diagnostic Interview-Revised (ADI-R) and the Autism Diagnostic Observation Schedule (ADOS). For identifying ASD patients, we used AGRE’s “derived affected status” information marked with “Autism”, “Not Quite Autism (NQA)“, or “ASD”. Patients with known genetic causes of ASD (e.g., Fragile X, Trisomy 21, 15q deletion, and 22q duplication) or syndromes with overlapping ASD-features (e.g., Sotos syndrome) were excluded. We used plasma samples from individuals with both WGS and detailed phenotype information were available. |
| Sample Type: | Blood (plasma) |
Treatment:
| Treatment ID: | TR002431 |
| Treatment Summary: | Plasma samples in 50μL aliquot were mixed with acetonitrile containing 14 stable isotope internal standards at a 2:1 ratio to precipitate proteins. Samples were then equilibrated on ice for 30 minutes and centrifuged for 10 minutes at 13,400 rpm at 4°C. The supernatant was transferred to autosampler vials and kept in a refrigerated autosampler until analysis. Each extract was analyzed in triplicate using a dual column chromatography scheme that includes hydrophilic interaction liquid chromatography (HILIC; XBridge BEH Amide XP HILIC column; Waters, Waltham, MA, 50x2.1mm, 2.5μm) and reversed phase liquid chromatography (RPLC; C18 column; Higgins Analytical, Mountain View, CA, 50x2.1mm, 2.6 μm). |
Sample Preparation:
| Sampleprep ID: | SP002425 |
| Sampleprep Summary: | Samples are prepared for metabolomics analysis using established methods (Johnson et al. (2010). Analyst; Go et al. (2015). Tox Sci). Prior to analysis, plasma aliquots were removed from storage at -80°C and thawed on ice. Each cryotube is then vortexed briefly to ensure homogeneity, and 50 μL transferred to a clean microfuge tube. Immediately after, the plasma is treated with 100 μL of ice-cold LC-MS grade acetonitrile (Sigma Aldrich) containing 2.5 μL of internal standard solution with eight stable isotopic chemicals selected to cover a range of chemical properties. Following addition of acetonitrile, plasma is then equilibrated for 30 min on ice, upon which precipitated proteins are removed by centrifuge (16.1 ×g at 4°C for 10 min). The resulting supernatant (100 μL) is removed, added to a low volume autosampler vial and maintained at 4°C until analysis (<22 h). |
| Sampleprep Protocol Filename: | EmoryUniversity_HRM_sample_preparation_082016_01.pdf |
Combined analysis:
| Analysis ID | AN003806 | AN003807 |
|---|---|---|
| Analysis type | MS | MS |
| Chromatography type | HILIC | Reversed phase |
| Chromatography system | Dionex UltiMate 3000 | Dionex UltiMate 3000 |
| Column | Waters XBridge BEH Amide XP HILIC (50 x 2.1mm,2.5um) Product #186006089; Thermo Accucore HILIC guard with holder,Product # 17526-012105 | Higgins endcapped C18 stainless steel (50 x 2.1mm,3um),Product #TS-0521-C183; Thermo Accucore C18 guard with holder,Product #17126-014005 |
| MS Type | ESI | ESI |
| MS instrument type | Orbitrap | Orbitrap |
| MS instrument name | Thermo Q Exactive HF hybrid Orbitrap | Thermo Q Exactive HF hybrid Orbitrap |
| Ion Mode | POSITIVE | NEGATIVE |
| Units | peak area | peak area |
Chromatography:
| Chromatography ID: | CH002816 |
| Chromatography Summary: | The HILIC column is operated parallel to reverse phase column for simultaneous analytical separation and column flushing through the use of a dual head HPLC pump equipped with 10- port and 6-port switching valves. During operation of HILIC separation method, the MS is operated in positive ion mode and 10 μL of sample is injected onto the HILIC column while the reverse phase column is flushing with wash solution. Flow rate is maintained at 0.35 mL/min until 1.5 min, increased to 0.4 mL/min at 4 min and held for 1 min. Solvent A is 100% LC-MS grade water, solvent B is 100% LC-MS grade acetonitrile and solvent C is 2% formic acid (v/v) in LC-MS grade water. Initial mobile phase conditions are 22.5% A, 75% B, 2.5% C hold for 1.5 min, with linear gradient to 77.5% A, 20% B, 2.5% C at 4 min, hold for 1 min, resulting in a total analytical run time of 5 min. During the flushing phase (reverse phase analytical separation), the HILIC column is equilibrated with a wash solution of 77.5% A, 20% B, 2.5% C. |
| Methods Filename: | EmoryUniversity_HRM-QEHF_chromatography_5min_092017_v1.pdf |
| Instrument Name: | Dionex UltiMate 3000 |
| Column Name: | Waters XBridge BEH Amide XP HILIC (50 x 2.1mm,2.5um) Product #186006089; Thermo Accucore HILIC guard with holder,Product # 17526-012105 |
| Column Temperature: | 60 |
| Solvent A: | 100% water |
| Solvent B: | 100% acetonitrile |
| Chromatography Type: | HILIC |
| Chromatography ID: | CH002817 |
| Chromatography Summary: | The C18 column is operated parallel to the HILIC column for simultaneous analytical separation and column flushing through the use of a dual head HPLC pump equipped with 10-port and 6- port switching valves. During operation of the C18 method, the MS is operated in negative ion mode and 10 μL of sample is injected onto the C18 column while the HILIC column is flushing with wash solution. Flow rate is maintained at 0.4 mL/min until 1.5 min, increased to 0.5 mL/min at 2 min and held for 3 min. Solvent A is 100% LC-MS grade water, solvent B is 100% LC-MS grade acetonitrile and solvent C is 10mM ammonium acetate in LC-MS grade water. Initial mobile phase conditions are 60% A, 35% B, 5% C hold for 0.5 min, with linear gradient to 0% A, 95% B, 5% C at 1.5 min, hold for 3.5 min, resulting in a total analytical run time of 5 min. During the flushing phase (HILIC analytical separation), the C18 column is equilibrated with a wash solution of 0% A, 95% B, 5% C until 2.5 min, followed by an equilibration solution of 60% A, 35% B, 5% C for 2.5 min. |
| Methods Filename: | EmoryUniversity_HRM-QEHF_chromatography_5min_092017_v1.pdf |
| Instrument Name: | Dionex UltiMate 3000 |
| Column Name: | Higgins endcapped C18 stainless steel (50 x 2.1mm,3um),Product #TS-0521-C183; Thermo Accucore C18 guard with holder,Product #17126-014005 |
| Column Temperature: | 60 |
| Solvent A: | 100% water |
| Solvent B: | 100% acetonitrile |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS003548 |
| Analysis ID: | AN003806 |
| Instrument Name: | Thermo Q Exactive HF hybrid Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | No comment |
| Ion Mode: | POSITIVE |
| Acquisition Parameters File: | EmoryUniversity_HRM_DataAnalysis-MS_092017_v1.pdf |
| Analysis Protocol File: | EmoryUniversity_HRM_QEHF-MassSpec_092017_v1.pdf |
| MS ID: | MS003549 |
| Analysis ID: | AN003807 |
| Instrument Name: | Thermo Q Exactive HF hybrid Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | No comment |
| Ion Mode: | NEGATIVE |
| Acquisition Parameters File: | EmoryUniversity_HRM_DataAnalysis-MS_092017_v1.pdf |
| Analysis Protocol File: | EmoryUniversity_HRM_QEHF-MassSpec_092017_v1.pdf |
