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MB Sample ID: SA227435
| Local Sample ID: | WT_CD40_48hr_4 |
| Subject ID: | SU002408 |
| Subject Type: | Cultured cells |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Genotype Strain: | SCAP fl/fl CD19-Cre (KO) and SCAP+/+ CD19-Cre (WT) |
| Cell Strain Details: | Splenic B cell |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU002408 |
| Subject Type: | Cultured cells |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Genotype Strain: | SCAP fl/fl CD19-Cre (KO) and SCAP+/+ CD19-Cre (WT) |
| Cell Strain Details: | Splenic B cell |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| WT_CD40_48hr_4 | SA227435 | FL027691 | WT | Genotype |
| WT_CD40_48hr_4 | SA227435 | FL027691 | anti-CD40 | TREATMENT |
| WT_CD40_48hr_4 | SA227435 | FL027691 | 48hr | TIME_POINT |
Collection:
| Collection ID: | CO002401 |
| Collection Summary: | Mouse splenic B cells were isolated using STEM CELL B cell isolation kit (Cat:19854) |
| Sample Type: | B cells |
Treatment:
| Treatment ID: | TR002420 |
| Treatment Summary: | Splenic B cells isolated from SCAP+/+ CD19-Cre (WT) and SCAPfl/fl CD19-Cre (KO) mice were stimulated with 10 micro g/ml LPS or 5 micro g/ml anti-CD40 for 24 and 48 hours. Freshly isolated untreated WT and KO B cells were used as untreated control. |
Sample Preparation:
| Sampleprep ID: | SP002414 |
| Sampleprep Summary: | Sample preparation: Proteins were precipitated with methanol under vigorous shaking for 2 min (Glen Mills GenoGrinder 2000) followed by centrifugation. The resulting extract was divided into five fractions: two for analysis by two separate reverse phases (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. |
Combined analysis:
| Analysis ID | AN003789 |
|---|---|
| Analysis type | MS |
| Chromatography type | Reversed phase/HILIC |
| Chromatography system | Waters ACQUITY ultra-performance liquid chromatography (UPLC) |
| Column | Waters UPLC BEH C18 (100 x 2.1mm,1.7um)/Waters Acquity BEH Amide (150 x 2.1mm,1.7um) |
| MS Type | ESI |
| MS instrument type | Orbitrap |
| MS instrument name | Thermo Exactive Plus Orbitrap |
| Ion Mode | UNSPECIFIED |
| Units | Normalized AUC |
Chromatography:
| Chromatography ID: | CH002802 |
| Methods Filename: | B_cell_MS_protocol.docx |
| Instrument Name: | Waters ACQUITY ultra-performance liquid chromatography (UPLC) |
| Column Name: | Waters UPLC BEH C18 (100 x 2.1mm,1.7um)/Waters Acquity BEH Amide (150 x 2.1mm,1.7um) |
| Chromatography Type: | Reversed phase/HILIC |
MS:
| MS ID: | MS003531 |
| Analysis ID: | AN003789 |
| Instrument Name: | Thermo Exactive Plus Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Analysis was performed by Metabolon. Ultrahigh Performance Liquid Chromatography-Tandem Mass Spectroscopy (UPLC-MS/MS): 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 with 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 aforementioned C18 column using methanol, acetonitrile, water, 0.05% PFPA and 0.01% 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. |
| Ion Mode: | UNSPECIFIED |
| Analysis Protocol File: | B_cell_MS_protocol.docx |
