Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA216203

Local Sample ID:	DF nP 03_2_17_1_956
Subject ID:	SU002334
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606

Select appropriate tab below to view additional metadata details:

Subject:

Subject ID:	SU002334
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
DF nP 03_2_17_1_956	SA216203	FL026085	Drug Free PLGA Nanoparticle	Treatment

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

Treatment:

Treatment ID:	TR002346
Treatment Summary:	The extraction of metabolites from FaDu cells was carried out following a similar procedure used in protein extraction. After treatment with PTX (40μM), PTX-PLGA NPs (40 μM), drug-free PLGA NPs and culture media, PBS replaced media and the cells were collected from the dishes using cell scrapers. The detached cell pellets were then collected by centrifugation at 15000 rpm for 10 min at 4oC using Universal 320R Benchtop Centrifuge (Hettich, Beverly, MA, USA) to remove excess PBS. To release a broad range of metabolites, the collected cell pellets were first treated with 0.1% FA in methanol followed by sonication using probe ultrasonicator Q500 (Terra Universal, Inc, Fullerton, CA, USA)for 30 sec at 30% amplitude 16. The samples were then centrifuged at 15,000 rpm for 10 minutes at 4oC, and the upper phase was transferred into LC vials for further drying using a Genevac evaporator (SP Industries, Warminster, PA, USA). The reaction was stopped with 1% formic acid (FA) followed by drying using a Genevac evaporator. Dried samples were stored at −80 °C for further LC-MS/MS analysis.

Treatment ID:

TR002346

Treatment Summary:

The extraction of metabolites from FaDu cells was carried out following a similar procedure used in protein extraction. After treatment with PTX (40μM), PTX-PLGA NPs (40 μM), drug-free PLGA NPs and culture media, PBS replaced media and the cells were collected from the dishes using cell scrapers. The detached cell pellets were then collected by centrifugation at 15000 rpm for 10 min at 4oC using Universal 320R Benchtop Centrifuge (Hettich, Beverly, MA, USA) to remove excess PBS. To release a broad range of metabolites, the collected cell pellets were first treated with 0.1% FA in methanol followed by sonication using probe ultrasonicator Q500 (Terra Universal, Inc, Fullerton, CA, USA)for 30 sec at 30% amplitude 16. The samples were then centrifuged at 15,000 rpm for 10 minutes at 4oC, and the upper phase was transferred into LC vials for further drying using a Genevac evaporator (SP Industries, Warminster, PA, USA). The reaction was stopped with 1% formic acid (FA) followed by drying using a Genevac evaporator. Dried samples were stored at −80 °C for further LC-MS/MS analysis.

Sample Preparation:

Sampleprep ID:	SP002340
Sampleprep Summary:	After treatment with PTX (40μM), PTX-PLGA NPs (40 μM), drug-free PLGA NPs and culture media , PBS replaced media and the cells were collected from the dishes using cell scrapers. The detached cell pellets were then collected by centrifugation at 15000 rpm for 10 min at 4oC using Universal 320R Benchtop Centrifuge (Hettich, Beverly, MA, USA) to remove excess PBS. To release a broad range of metabolites, the collected cell pellets were first treated with 0.1% FA in methanol followed by sonication using probe ultrasonicator Q500 (Terra Universal, Inc, Fullerton, CA, USA)for 30 sec at 30% amplitude 16. The samples were then centrifuged at 15,000 rpm for 10 minutes at 4oC, and the upper phase was transferred into LC vials for further drying using a Genevac evaporator (SP Industries, Warminster, PA, USA). The reaction was stopped with 1% formic acid (FA) followed by drying using a Genevac evaporator. Dried samples were stored at −80 °C for further LC-MS/MS analysis.

Sampleprep ID:

SP002340

Sampleprep Summary:

After treatment with PTX (40μM), PTX-PLGA NPs (40 μM), drug-free PLGA NPs and culture media , PBS replaced media and the cells were collected from the dishes using cell scrapers. The detached cell pellets were then collected by centrifugation at 15000 rpm for 10 min at 4oC using Universal 320R Benchtop Centrifuge (Hettich, Beverly, MA, USA) to remove excess PBS. To release a broad range of metabolites, the collected cell pellets were first treated with 0.1% FA in methanol followed by sonication using probe ultrasonicator Q500 (Terra Universal, Inc, Fullerton, CA, USA)for 30 sec at 30% amplitude 16. The samples were then centrifuged at 15,000 rpm for 10 minutes at 4oC, and the upper phase was transferred into LC vials for further drying using a Genevac evaporator (SP Industries, Warminster, PA, USA). The reaction was stopped with 1% formic acid (FA) followed by drying using a Genevac evaporator. Dried samples were stored at −80 °C for further LC-MS/MS analysis.

Combined analysis:

Analysis ID	AN003674
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Bruker Elute
Column	Hamilton Intensity Solo 2 C18
MS Type	ESI
MS instrument type	QTOF
MS instrument name	Bruker timsTOF
Ion Mode	POSITIVE
Units	AU

Chromatography:

Chromatography ID:	CH002724
Instrument Name:	Bruker Elute
Column Name:	Hamilton Intensity Solo 2 C18
Column Temperature:	35
Flow Gradient:	1%B to 99%B in 15 min
Flow Rate:	250 uL/min
Solvent A:	100% water; 0.1% formic acid
Solvent B:	100% acetonitrile; 0.1% formic acid
Chromatography Type:	Reversed phase

MS:

MS ID:	MS003425
Analysis ID:	AN003674
Instrument Name:	Bruker timsTOF
Instrument Type:	QTOF
MS Type:	ESI
MS Comments:	Processing and statistical analysis were performed using MetaboScape® 4.0 (Bruker Daltonics). Analyte bucketing and identification were done using the software provided available T-ReX 2D/3D workflow with the following parameters: intensity threshold greater than 1000 counts and peak length equal to 7 spectra or greater. Feature quantitation was, performed using peak area and , for features present in at least 3 (of 12) samples (per cell type) were considered for statistical analysis. Analyte MS2 spectra were averaged on import and only features eluting between 0.3 and 25 min with mz between 50 and 1000 were considered further.
Ion Mode:	POSITIVE