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MB Sample ID: SA185579
| Local Sample ID: | Pooled Human Plasma 8 |
| Subject ID: | SU002061 |
| Subject Type: | Human |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Gender: | Pooled |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU002061 |
| Subject Type: | Human |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Gender: | Pooled |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| Pooled Human Plasma 8 | SA185579 | FL022935 | 8 | Iteration number |
Collection:
| Collection ID: | CO002054 |
| Collection Summary: | A pooled sample of human plasma compiled from hundreds of de-identified consented donors was obtained from the Red Cross of Michigan. |
| Sample Type: | Blood (plasma) |
| Storage Conditions: | -80℃ |
| Additives: | EDTA |
Treatment:
| Treatment ID: | TR002073 |
| Treatment Summary: | All studies were performed on a pooled plasma sample as received. |
Sample Preparation:
| Sampleprep ID: | SP002067 |
| Sampleprep Summary: | Four parts of 1:1:1 methanol:acetonitrile:acetone were added to one part human plasma in 15 mL polypropylene centrifuge tubes and the suspension was vortexed for 20 seconds and allowed to rest on ice for an additional 10 minutes. The extract was centrifuged at 5000 x g for 10 minutes, and the supernatant was divided into 1.5 mL aliquots which were completely dried under a gentle stream of nitrogen gas. Dried samples were reconstituted in varied volumes of 90:10 water:methanol (for RPLC analyses) and 85:15 methanol:water (for HILIC). |
| Sampleprep Protocol Filename: | Mod_vs_Con_Chrom_IDs_Protocol.pdf |
| Processing Storage Conditions: | On ice |
| Extraction Method: | Solvent precipitation with 4 parts 1:1:1 methanol:acetonitrile:acetone to 1 part human plasma |
| Extract Cleanup: | Centrifugation 5000 g for 10 min |
| Extract Storage: | 4℃ |
| Sample Resuspension: | 90:10 water:methanol (RPLC)/85:15 methanol:water (HILIC) volume varied |
Combined analysis:
| Analysis ID | AN002856 |
|---|---|
| Analysis type | MS |
| Chromatography type | HILIC |
| Chromatography system | Agilent 1290 Infinity II |
| Column | Waters Acquity BEH Amide (100 x 2.1mm,1.7um) |
| MS Type | ESI |
| MS instrument type | QTOF |
| MS instrument name | Agilent 6545 |
| Ion Mode | POSITIVE |
| Units | Total Intensity Units |
Chromatography:
| Chromatography ID: | CH002383 |
| Chromatography Summary: | HILIC separations were performed on a Waters Acquity UPLC BEH amide column (2.1 x 100 mm 1.7 m) with a matching Vanguard precolumn. The flow rate was set to 0.3 mL/min and mobile phases consisted of (A) 95:5 acetonitrile/water with 0.125% v/v formic acid and 10 mM ammonium formate and (B) 95:5 water/acetonitrile with 0.125% v/v formic acid and 10 mM ammonium formate. Gradient specified in protocol file. |
| Methods Filename: | 2 mm amide pos MS2 70%H2O 120min_PC6.m |
| Instrument Name: | Agilent 1290 Infinity II |
| Column Name: | Waters Acquity BEH Amide (100 x 2.1mm,1.7um) |
| Column Pressure: | 300 bar |
| Column Temperature: | 55 |
| Flow Rate: | 0.3 mL/min |
| Sampling Cone: | 175 V |
| Solvent A: | 95% acetonitrile/5% water; 0.125% formic acid; 10 mM ammonium formate |
| Solvent B: | 5% acetonitrile/95% water; 0.125% formic acid; 10 mM ammonium formate |
| Analytical Time: | 120 min |
| Capillary Voltage: | 3500 V |
| Weak Wash Solvent Name: | 85:15 acetonitrile:water |
| Target Sample Temperature: | 4 |
| Sample Loop Size: | 20 uL |
| Chromatography Type: | HILIC |
MS:
| MS ID: | MS003005 |
| Analysis ID: | AN002856 |
| Instrument Name: | Agilent 6545 |
| Instrument Type: | QTOF |
| MS Type: | ESI |
| MS Comments: | Instrument settings for positive ionization mode following electrospray ionization were as follows: Sheath gas flow rate, 11 L/min; Drying gas, 8 L/min; Drying gas temperature, 320 ºC; Nebulizer, 35 psi; Capillary voltage, 3500 V; Nozzle voltage, 1000 V; Fragmentor, 175 V; Skimmer, 65 V; Octupole 1 RF Vpp, 750 V; Collision energy, 20; Iterative MS/MS mass error tolerance, ± 20 ppm; Iterative MS/MS retention time exclusion tolerance, ± 0.5 min; Spectrum data type, centroid. Data de-pendent MS/MS parameters: Mass range, 25-1200 m/z; Rate, 2 spectra/s; Max precursor ions per cycle, 3; Absolute precursor threshold, 5000 counts; Relative precursor threshold, 0.001%; Active exclusion enabled after 2 spectra and released after 0.5 min; Isolation width, narrow (~1.3 m/z). Iterative LC-MS/MS data acquired from the modified HILIC separation conditions was loaded into a pre-release version of an in-house software tool, MetIDTracker for feature detection. This software uses MSPepSearch (NIST) for database searching and score assignment. |
| Ion Mode: | POSITIVE |
| Capillary Voltage: | 3500 V |
| Collision Energy: | 20 |
| Collision Gas: | N2 |
| Dry Gas Flow: | 8L/min |
| Dry Gas Temp: | 320 C |
| Fragment Voltage: | 175 V |
| Fragmentation Method: | CID |
| Ionization: | ESI |
| Mass Accuracy: | <20 ppm |
