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MB Sample ID: SA216193

Local Sample ID:Control 04_1_10_1_941
Subject ID:SU002334
Subject Type:Cultured cells
Subject Species:Homo sapiens
Taxonomy ID:9606

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Collection:

Collection ID:CO002327
Collection Summary:The process of extracting metabolites from FaDu cells was carried out in a manner that was similar to that which was utilized for the extraction of proteins. Following the addition of PTX (40 μM), PTX-PLGA NPs (40 μM), and drug-free PLGA NPs to the culture medium, the media was changed to PBS, and the cells were scraped off the plates using cell scrapers. After that, the detached cell pellets were collected by centrifuging them at a speed of 15000 rpm for ten minutes at a temperature of 4 degrees Celsius using a Universal 320R Benchtop Centrifuge (Hettich, Beverly, Massachusetts, United States). The collected cell pellets were first treated with 0.1 % FA in methanol, which was followed by sonication using a probe ultrasonicator Q500 (Terra Universal, Inc., Fullerton, California, USA) for 30 seconds at 30 %amplitude 16. This was done in order to release a wide variety of metabolites. After that, the samples were centrifuged at 15,000 rpm for 10 minutes at 4 degrees Celsius, and the upper phase was transferred into LC vials so that it could be dried further using a Genevac evaporator (SP Industries, Warminster, PA, USA). After the reaction was halted with formic acid (FA) at a concentration of one percent, it was dried using a Genevac evaporator. In preparation for subsequent LC-MS/MS examination, the dried samples were kept at a temperature of 80 degrees Celsius. The version 4.0 of MetaboScape was used for both the processing and the statistical analysis (Bruker Daltonics). Analyte bucketing and identification were accomplished by using the software that was made available to us called T-ReX 2D/3D workflow. The settings that were used were as follows: an intensity threshold of larger than 1000 counts and a peak duration of equal to 7 spectra or higher. Quantification of features was carried out by calculating peak area and for statistical analysis, only characteristics that were present in at least three out of twelve samples for each cell type were taken into account. On import, the spectra of the analyte were averaged, and additional consideration was given to just those features that eluted between 0.3 and 25 minutes and had mz values between 50 and 1000. Using a two-tailed independent Student’s t-test, a comparison was made between each drug treatment condition and DMSO-only treated controls. It was shown that there were significantly differently abundant metabolites between the two groups. As a result of this, volcano plots were developed in order to visualize and display the significance (p-value) of dysregulation of cellular metabolites, along with the fold changes associated with each condition. Metabo analyst, which can be found at https://www.metaboanalyst.ca, was used in order to carry out functional enrichment studies. Analyte metabolite identification was carried out by making use of both MS2 spectra and retention time (RT), although the MS/MS spectra were required as a bare minimum for a valid identification to be made. We conducted annotation using Bruker's version of the Human Metabolome Database (HMDB-4.0) for the set of compounds that satisfied this requirement, either by using MS/MS alone or by utilizing MS/MS plus RT. All of the chosen compounds were then compared to this library to find a match. These putatively matching features were filtered by considering for those each feature with the highest annotation quality score (AQ score) among other putative matches for the same metabolite, i.e., those features exhibiting the best fit across the greatest number of factors such as retention time, MS/MS, m/z values, analyte list, and spectral library matching were ranked first for the associated identifier.
Sample Type:Head and neck Cancer cell line
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