The samples were subjected to capillary electrophoresis mass spectrometry with time‐of‐flight (CE‐TOFMS) analysis. Metabolome analysis was conducted with the Basic Scan package of Human Metabolome Technologies (HMT; Tsuruoka, Yamagata, Japan) using CE‐TOFMS based on the methods described previously.7 , 8 CE‐TOFMS analysis was carried out using an Agilent CE capillary electrophoresis system equipped with an Agilent 6,210 TOF mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). The systems were controlled by Agilent G2201AA ChemStation software version B.03.01 for CE (Agilent Technologies). The spectrometer was scanned from m/z 50 to 1,000, and peaks were extracted using MasterHands automatic integration software (Keio University, Tsuruoka, Yamagata, Japan) to obtain peak information including m/z, peak area, and migration time.9 Signal peaks corresponding to isotopomers, adduct ions, and other product ions of known metabolites were excluded, and the remaining peaks were annotated according to the HMT metabolite database based on their m/z values with the migration times. Areas of the annotated peaks were then normalized based on internal standard levels and sample amounts to obtain relative levels of each metabolite. One hundred ten primary metabolites were absolutely quantified based on one‐point calibrations using their respective standard compounds.
Basic scan package
Manufactured by Human Metabolome Technologies
Sourced in Japan
The Basic Scan package is a comprehensive analytical service that utilizes advanced mass spectrometry technology to provide a detailed analysis of the metabolite profile in biological samples. The core function of this package is to detect and identify a wide range of metabolites, allowing for a comprehensive understanding of the metabolic state of the sample.
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Lab products found in correlation
7 protocols using basic scan package
1
Metabolome Analysis by CE-TOFMS
2
Fecal Metabolomic Profiling by CE-TOF-MS
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Metabolomic analysis was conducted using the Basic Scan package from Human Metabolome Technologies, Inc. (HMT; Yamagata, Japan) using CE-TOF-MS. Fecal metabolites were extracted by vigorous shaking with nine volumes of sterilized water containing the internal standard. The mixture was centrifuged, and the upper aqueous layer was centrifugally filtered according to the manufacturer’s instructions. Peaks detected by CE-TOF-MS were extracted using automatic integration software (MasterHands, Keio University, Japan) to obtain peak information, including m/z, migration time (MT), and peak area. The peaks were annotated with putative metabolites from the HMT metabolite database based on their MTs in CE and m/z values determined by TOF-MS. The tolerance range for the peak annotation was configured at ± 0.5 min for MT and ± 10 ppm for m/z. The relative area of each peak was calculated and compared between the two groups based on Welch’s t-test. The concentration of each metabolite was calculated relative to the concentration of the internal standard. Each fecal metabolite concentration in three samples was measured using the standard substances by another CE-TOF-MS experiment, and the levels were calculated based on the relative peak area in the remaining 29 samples.
3
Serum Metabolomic Analysis in Aged Rats
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Serum metabolomic analysis was performed employing the Basic Scan package from Human Metabolome Technologies (HMT) Inc. (Tsuruoka, Japan) and CE-TOF/MS as previously described [81 (link)]. CE-TOF/MS analysis was performed with serum samples from two aged rats after 2 weeks with or without hPD-MSC therapy at 10-day intervals.
4
Metabolomic Analysis of Mouse Plasma
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Pooled plasma samples from 3 mice of each group were prepared, and metabolome analysis was performed using the Basic Scan package from Human Metabolome Technologies using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS) based on methods described previously (67 (link), 68 (link)). Peaks were extracted using the automatic integration software MasterHands (Keio University, Tsuruoka, Yamagata, Japan) to obtain peak information including m/z, peak area, and migration time.
5
Metabolite Extraction and Analysis Protocol
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The cells were washed twice by using 5% mannitol solution and were then incubated with 800 μl of methanol at room temperature to inactivate enzymes. The cell extracts were mixed with 550 μl of Milli-Q water containing internal standard solution (Human Metabolome Technologies (HMT), H3304-1002) and incubated at room temperature for 30 s. The extracted solutions were transferred into microtubes and centrifuged at 2300×g, 4oC for 5 min. The supernatant (800 μl) was transferred to Millipore 5-kDa cutoff filter (UltrafreeMC-PLHCC, HMT), and the filters were centrifuged at 9100×g, 4 °C for 2–5 h until no liquid remained in the filter cup. The extracted sample solutions were completely evaporated and resuspended in 50 μl of Milli-Q water for metabolome analysis at HMT. Metabolome analysis was performed by Basic Scan package of HMT using capillary electrophoresis time-of-flight mass spectrometry (Human Metabolome Technologies, Inc., Tokyo, Japan)
6
Metabolomic Analysis of MKRN1 Knockout Cells
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Metabolomic analysis was performed employing the Basic Scan package from Human Metabolome Technologies (HMT) Inc. (Tsuruoka, Japan) and capillary electrophoresis–time-of-flight mass spectrometry (CE-TOFMS) as described previously52 (link). Metabolites were extracted from MKRN1−/− MEFs and the MEFs of WT littermates as the manufacturer suggested (E-130637; HMT). Briefly, the cells were washed twice using a 5% (w/w) mannitol solution, added to methanol to extract metabolites and treated with internal standards solution 1 (HMT). The extracted solution was centrifuged for 5 min at 2300 × g and 4 °C. The proteins of the upper aqueous layer were removed using a Millipore 5-kDa cutoff filter. The filtrate was subsequently lyophilised and dissolved in 50 µl of Milli-Q water and analysed via CE-TOFMS. Automatic integration software (MasterHands ver. 2.16.0.15 developed at Keio University) was employed to process peaks from CE-TOFMS analysis and to acquire information of m/z, migration time (MT) and a relative peak area. The HMT metabolite database based on the peak MTs and m/z values established using TOFMS were employed to assign all the target metabolite.
7
Metabolomic Analysis of Islet Cells
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The conditioned medium of islets isolated from control and Arid1a-depleted mice were used for metabolome analysis. Samples (80 μL) were mixed with 20 μL of Milli-Q water containing internal standards. Metabolome analysis was performed by Basic Scan package of Human Metabolome Technologies (HMT) using CE-TOF (HMT, Tokyo, Japan). Hierarchical cluster analysis (HCA) and principal component analysis (PCA) were performed by statistical analysis software (HMT). The profiles of peaks with putative metabolites were presented on metabolic pathway maps using VANTED (Visualization and Analysis of Networks containing Experimental Data) software.
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