The lipid extracts were dried by nitrogen gas, and re-dissolved with 400 µL of acetonitrile/2-propanol (1:9) with 0.1% formic acid and 10 mM ammonium formate. The sample vials were stored in −20 °C until the analysis by Bruker LC/MS Ion-Trap. Reverse phase HPLC gradient was operated in an Acclaim RSLC 120 C18 column at a flow rate of 0.2 mL/min at 55 °C. The gradient contained solution A: acetonitrile:water (60:40), 10 mM ammonium formate, 0.1% formic acid, and solution B: isopropanol: acetonitrile (90:10), 10 mM ammonium formate, 0.1% formic acid. The acquired mass spectrometry (MS) data were further analyzed by Bruker DataAnalysis (ver.4.1). The extract ion current (XIC) of each phospholipid species was quantitated by their relativity of XIC to internal standard. Each type of total phospholipid is the sum of all quantitated species of its type. The percentage of each phospholipid was the ratio of XIC of each phospholipid species to total XIC of its type.
Dataanalysis ver 4.1
Manufactured by Bruker
DataAnalysis (ver.4.1) is a software application developed by Bruker for data analysis. It provides tools for processing and visualizing data from various analytical techniques, including mass spectrometry, nuclear magnetic resonance (NMR) spectroscopy, and X-ray diffraction. The software offers a range of features to support scientific research and data interpretation.
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4 protocols using dataanalysis ver 4.1
1
Quantitation of Phospholipid Species
2
Lipid Profiling by LC-MS/MS
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The extracted total lipids were re-dissolved in 200 μL of Acetonitrile/Isopropanol/H2O (63:30:5). 20 μL of the samples were analyzed by LC/MS Ion-trap (Bruker). There are two different HPLC mobile phases, including solution A: Acetonitrile:H2O (60:40), 10 mM ammonium formate, 0.1% formic acid and solution B: Isopropanol:Acetonitrile (90:10), 10 mM ammonium formate, 0.1% formic acid. Gradient was from 60% solution A to 100% solution B in 25 min and preserved in 100% solution B until 40 min and then backed to 60% solution A in an Acclaim RSLC 120 C18 2.1 mm × 100 mm × 2.2 μm column (Thermo, USA) at a flow rate of 0.2 mL/min at 55 °C. Then, we calculated the integrated area of the extract ion current (XIC) in Bruker DataAnalysis (ver.4.1). All experiments were in triplicate and the standard deviations of the triplicated experiments were plotted as the error bars of the figures.
3
Quantitative Lipidomics Analysis of Zebrafish
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The extracted total lipids from zebrafish’s tissues were dried under nitrogen gas and re-dissolved in 400 μl of acetonitrile/2-propanol/H2O (65:30:5) immediately. The samples were analyzed by LC/MS Ion-Trap (Bruker Corporation). A total of 50 μl dissolved sample was injected through the autosampler. HPLC mobile phases contained solution A: ACN:H2O (60:40), 10 mM ammonium formate, 0.1% formic acid and solution B: IPA:ACN (90:10), 10 mM ammonium formate, 0.1% formic acid[34 (link)]. Gradient was from 60% solution A to 100% solution B in 25 min and maintained 100% solution B until 45 min in an Acclaim RSLC 120 C18 2.1 mm x 100 mm 2.2 μm column (Thermo) at a flow rate of 0.2 mL/min at 55 °C. Data were further analyzed by Bruker DataAnalysis (ver.4.1). The extract ion current (XIC) of each cardiolipin species was quantitated by their relativity of XIC to internal standard. Standard curve of XIC detector response versus content of cardiolipin standard is provided in S1 Fig .
4
Cardiolipin Analysis via LC/MS
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The extracted total lipid was dried under nitrogen gas and immediately re-dissolved with acetonitrile/2-propanol/H2O (65:30:5). The samples were capped and stored in −20 °C to prevent evaporation until LC/MS Ion-Trap analysis (Bruker Corporation). Reverse phase HPLC contained solution A: ACN:H2O (60:40), 10 mM ammonium formate, 0.1% formic acid and solution B: IPA:ACN (90:10), 10 mM ammonium formate, 0.1% formic acid for the elution gradient from 60% solution A/40% solution B to 100% solution B in 25 min and maintained 100% solution B until 45 min. The elution was in an Acclaim RSLC 120 C18 2.1mm × 100 mm 2.2 μm column (Thermo) at a flow rate of 0.2 mL/min at 55 °C. Data were further analyzed by Bruker DataAnalysis (ver.4.1). The extract ion current (XIC) of each cardiolipin species was quantitated by their area ratio of XIC to internal standard. The total CL is the sum of all quantitated cardiolipin species. The percentage of CL was the ratio of XIC of each cardiolipin species to total XIC. Standard deviations were calculated by the STDEV function in Microsoft Excel for the error bars of the histograms and t-tests are applied to all triplicated data
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