Summary of Study ST003996

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR002501. The data can be accessed directly via it's Project DOI: 10.21228/M8C55X This work is supported by NIH grant, U2C- DK119886. See: https://www.metabolomicsworkbench.org/about/howtocite.php

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Study IDST003996
Study TitleMetabolomics analysis of MMTC/57E or MeAIB treated SNAT2-expressing cells relative to SNAT2-knockout cells or those treated with vehicle.
Study SummaryMetabolite analysis of HY15549 cells expressing a dox-inducible SNAT2 cDNA with deletion of endogenous Snat2 treated with MMTC/57E, MeAIB, or vehicle. Analysis was performed after a total of 24-hours. Each independent experiment was performed on an independent batch of cells. For each experiment, cells were plated with or without doxycycline (0.5 micrograms/mL). After approximately 5-6 hours to allow the cells to attached, the cells were then treated with either MMTC/57E, MeAIB, or vehicle for 18-hours. Metabolites were extracted by adding 1 mL of ice cold 80% methanol containing internal, isotope-labeled amino acid standards. Metabolites were concentrated using a SpeedVac until dry. Metabolites were reconstituted into 50 µL of water, vortexed, centrifuged, and transferred to vials for analysis by LCMS. LCMS was performed using a ZIC-pHILIC LC column coupled to a Vanquish LC and a flow gradient consisting of 10 mM ammonium carbonate in water and pure acetonitrile. The LC was coupled to an Exploris 240 mass spectrometer operated in a polarity switching data-dependent Top 5 mode. Full MS scan parameters for both positive and negative mode were set to 67-1000 m/z at a resolution of 120k and ddMS2 were collected at a resolution of 30k.
Institute
University of British Columbia
DepartmentBiochemistry & Molecular Biology
LaboratoryParker laboratory
Last NameParker
First NameSeth
Address950 W 28th Ave, Vancouver, British Columbia, V6H 0B3, Canada
Emailseth.parker@bcchr.ca
Phone6048753121
Submit Date2025-06-18
Raw Data AvailableYes
Raw Data File Type(s)mzML, raw(Thermo)
Analysis Type DetailLC-MS
Release Date2025-07-28
Release Version1
Seth Parker Seth Parker
https://dx.doi.org/10.21228/M8C55X
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

Select appropriate tab below to view additional metadata details:

Project:

Project ID:PR002501
Project DOI:doi: 10.21228/M8C55X
Project Title:Metabolomics analysis of SNAT2-deficient cells: implications for the discovery of selective transporter inhibitors
Project Type:Manuscript
Project Summary:Amino acid uptake by the solute carrier family of transporter proteins is critical to support cell metabolism, and inhibition of transporter activity represents a tractable strategy to restrict nutrient availability to cancer cells. A small molecule inhibitor of the sodium-coupled neutral amino acid transporter 2 (SNAT2), 3-(N-methyl(4-methylphenyl)sulfonamido)-N-(2-trifluoromethylbenzyl)thiophene-2-carboxamide (MMTC/57E), was recently identified and was shown to inhibit cell proliferation when combined with glucose transport inhibitors in breast and pancreatic cancer cell lines. In this study, we use mass spectrometry-based metabolomics and establish cell-based assays for the SNAT2 transporter. We show that SNAT2 knockout cells have significant defects in amino acid availability. Using our established assays, we fail to observe that MMTC/57E inhibits SNAT2 activity likely due to its poor solubility.
Institute:University of British Columbia
Department:Biochemistry & Molecular Biology
Laboratory:Parker laboratory
Last Name:Parker
First Name:Seth
Address:950 W 28th Ave, Vancouver, British Columbia, V6H 0B3, Canada
Email:seth.parker@bcchr.ca
Phone:6048753121

Subject:

Subject ID:SU004133
Subject Type:Mammal
Subject Species:Mus musculus
Taxonomy ID:10090
Gender:Female

Factors:

Subject type: Mammal; Subject species: Mus musculus (Factor headings shown in green)

mb_sample_id local_sample_id Factor Sample source
SA461192KO_DMSO_3SNAT2-KO HY15549
SA461193KO_57E_10microM_2SNAT2-KO HY15549
SA461194KO_MeAIB_10mM_6SNAT2-KO HY15549
SA461195KO_MeAIB_10mM_5SNAT2-KO HY15549
SA461196KO_MeAIB_10mM_4SNAT2-KO HY15549
SA461197KO_MeAIB_10mM_3SNAT2-KO HY15549
SA461198KO_MeAIB_10mM_2SNAT2-KO HY15549
SA461199KO_MeAIB_10mM_1SNAT2-KO HY15549
SA461200KO_DMSO_6SNAT2-KO HY15549
SA461201KO_DMSO_5SNAT2-KO HY15549
SA461202KO_DMSO_4SNAT2-KO HY15549
SA461203KO_57E_10microM_1SNAT2-KO HY15549
SA461204KO_DMSO_2SNAT2-KO HY15549
SA461205KO_57E_10microM_6SNAT2-KO HY15549
SA461206KO_DMSO_1SNAT2-KO HY15549
SA461207KO_57E_10microM_4SNAT2-KO HY15549
SA461208KO_57E_10microM_5SNAT2-KO HY15549
SA461209KO_57E_10microM_3SNAT2-KO HY15549
SA461210KO_control_1SNAT2-KO HY15549
SA461211KO_control_2SNAT2-KO HY15549
SA461212KO_control_3SNAT2-KO HY15549
SA461213KO_control_4SNAT2-KO HY15549
SA461214KO_control_5SNAT2-KO HY15549
SA461215WT_MeAIB_10mM_6SNAT2-WT HY15549
SA461216WT_DMSO_2SNAT2-WT HY15549
SA461217WT_MeAIB_10mM_5SNAT2-WT HY15549
SA461218WT_MeAIB_10mM_4SNAT2-WT HY15549
SA461219WT_MeAIB_10mM_3SNAT2-WT HY15549
SA461220WT_MeAIB_10mM_2SNAT2-WT HY15549
SA461221WT_MeAIB_10mM_1SNAT2-WT HY15549
SA461222WT_DMSO_6SNAT2-WT HY15549
SA461223WT_DMSO_5SNAT2-WT HY15549
SA461224WT_DMSO_4SNAT2-WT HY15549
SA461225WT_DMSO_3SNAT2-WT HY15549
SA461226WT_control_2SNAT2-WT HY15549
SA461227WT_DMSO_1SNAT2-WT HY15549
SA461228WT_control_6SNAT2-WT HY15549
SA461229WT_control_5SNAT2-WT HY15549
SA461230WT_control_4SNAT2-WT HY15549
SA461231WT_control_3SNAT2-WT HY15549
SA461232WT_control_1SNAT2-WT HY15549
SA461233WT_57E_10microM_6SNAT2-WT HY15549
SA461234WT_57E_10microM_5SNAT2-WT HY15549
SA461235WT_57E_10microM_4SNAT2-WT HY15549
SA461236WT_57E_10microM_3SNAT2-WT HY15549
SA461237WT_57E_10microM_1SNAT2-WT HY15549
SA461238WT_57E_10microM_2SNAT2-WT HY15549
Showing results 1 to 47 of 47

Collection:

Collection ID:CO004126
Collection Summary:Metabolites from each well of cultured cells were extracted by adding 1 mL of 80% methanol containing internal isotopic standards and scraping. Supernatant was transferred to a 1.5 mL tube and vortexed for ~10 minutes at 4°C. After centrifugation to pellet insolubles, 900 µL was transferred to a new tube and dried using a SpeedVac before reconstitution in water and LCMS analysis.
Sample Type:Pancreas

Treatment:

Treatment ID:TR004142
Treatment Summary:MMTC/57E at 10 µM or MeAIB at 10 mM. Vehicle for 57E was DMSO and water for MeAIB.

Sample Preparation:

Sampleprep ID:SP004139
Sampleprep Summary:Dried samples were reconstituted in 50 µL of HPLC-grade water. Samples were vortexed for ~10 minutes, then centrifuged at 21,000 x g for 15 min at 4°C. 40 microliters were transferred to LC vials containing glass inserts for analysis.

Combined analysis:

Analysis ID AN006585 AN006586
Chromatography ID CH005000 CH005000
MS ID MS006284 MS006285
Analysis type MS MS
Chromatography type HILIC HILIC
Chromatography system Thermo Vanquish Thermo Vanquish
Column Merck SeQuant ZIC-pHILIC (150 x 2.1 mm, 5 µm) Merck SeQuant ZIC-pHILIC (150 x 2.1 mm, 5 µm)
MS Type ESI ESI
MS instrument type Orbitrap Orbitrap
MS instrument name Thermo Exploris 240 Thermo Exploris 240
Ion Mode POSITIVE NEGATIVE
Units ion counts ion counts

Chromatography:

Chromatography ID:CH005000
Instrument Name:Thermo Vanquish
Column Name:Merck SeQuant ZIC-pHILIC (150 x 2.1 mm, 5 µm)
Column Temperature:25°C
Flow Gradient:80-20%B (0-30 min), 20-20%B (30-40 minute), and 20-80%B (40-40.5 minute); the LC column was re-equilibrated using 80-80%B from 40.5-52 minute before subsequent injections
Flow Rate:100 uL/min
Solvent A:100% Water; 10 mM Ammonium Carbonate, pH 9.0
Solvent B:100% Acetonitrile
Chromatography Type:HILIC

MS:

MS ID:MS006284
Analysis ID:AN006585
Instrument Name:Thermo Exploris 240
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:The LC was coupled to a Thermo Scientific Exploris 240 mass spectrometer operating in heated electrospray ionization mode (HESI) for analysis. The following parameters were set for HESI: spray voltage 3.4 kV (positive) and 2 kV (negative), static spray voltage, sheath gas 25, aux gas 5, sweep gas 0.5, ion transfer tube temperature 320°C, and vaporizer temperature 75°C. The global parameters included an expected peak width of 20 seconds, mild trapping, and a default charge state of 1. A 40-min polarity switching data-dependent Top 5 method was used for positive mode and a data-dependent Top 3 method was used for negative mode. Full MS scan parameters for both positive and negative modes were set as follows: scan range 67-1000 m/z collected in profile mode, Orbitrap resolution 120,000, RF lens 70%, AGC target of 300%, and maximum injection time set to automatic. ddMS2 for positive mode were collected in centroid mode at an Orbitrap resolution of 30,000, isolation window of 1.5 m/z, an AGC target set to standard, a maximum injection time set to automatic, and a normalized collision energy set to 10%, 30%, and 80%. ddMS2 for negative mode were collected in centroid mode at an Orbitrap resolution of 30,000, isolation window of 2 m/z, an AGC target set to standard, a maximum injection time set to automatic, and a normalized collision energy set to 30%. For both positive and negative ddMS2, we applied an intensity threshold of 5e4 and a dynamic exclusion of 5 ppm for 10 seconds, excluding isotopes. A targeted selected ion monitoring (tSIM) scan was also included for pipecolate and P6C/P2C, and the retention time ranges were based on elution of authentic standards (pipecolate) or from positive samples (Aldh7a1-deficient tissues). The tSIM scan for pipecolate was collected from 8-12 minutes in negative mode at an isolation window of 4 m/z (for metabolomics) or 18 m/z (for isotope-tracing experiments, to include m/z shifts), an Orbitrap resolution of 120,000, a RF lens at 70%, an automatic maximum injection time, and collected in profile mode. The tSIM scan for P6C/P2C was collected from 6-10 minutes in positive mode at an isolation window of 4 m/z (for metabolomics) or 18 m/z (for isotope-tracing experiments, to include m/z shifts), an Orbitrap resolution of 120,000, a RF lens at 70%, an automatic maximum injection time, and collected in profile mode.
Ion Mode:POSITIVE
  
MS ID:MS006285
Analysis ID:AN006586
Instrument Name:Thermo Exploris 240
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:The LC was coupled to a Thermo Scientific Exploris 240 mass spectrometer operating in heated electrospray ionization mode (HESI) for analysis. The following parameters were set for HESI: spray voltage 3.4 kV (positive) and 2 kV (negative), static spray voltage, sheath gas 25, aux gas 5, sweep gas 0.5, ion transfer tube temperature 320°C, and vaporizer temperature 75°C. The global parameters included an expected peak width of 20 seconds, mild trapping, and a default charge state of 1. A 40-min polarity switching data-dependent Top 5 method was used for positive mode and a data-dependent Top 3 method was used for negative mode. Full MS scan parameters for both positive and negative modes were set as follows: scan range 67-1000 m/z collected in profile mode, Orbitrap resolution 120,000, RF lens 70%, AGC target of 300%, and maximum injection time set to automatic. ddMS2 for positive mode were collected in centroid mode at an Orbitrap resolution of 30,000, isolation window of 1.5 m/z, an AGC target set to standard, a maximum injection time set to automatic, and a normalized collision energy set to 10%, 30%, and 80%. ddMS2 for negative mode were collected in centroid mode at an Orbitrap resolution of 30,000, isolation window of 2 m/z, an AGC target set to standard, a maximum injection time set to automatic, and a normalized collision energy set to 30%. For both positive and negative ddMS2, we applied an intensity threshold of 5e4 and a dynamic exclusion of 5 ppm for 10 seconds, excluding isotopes. A targeted selected ion monitoring (tSIM) scan was also included for pipecolate and P6C/P2C, and the retention time ranges were based on elution of authentic standards (pipecolate) or from positive samples (Aldh7a1-deficient tissues). The tSIM scan for pipecolate was collected from 8-12 minutes in negative mode at an isolation window of 4 m/z (for metabolomics) or 18 m/z (for isotope-tracing experiments, to include m/z shifts), an Orbitrap resolution of 120,000, a RF lens at 70%, an automatic maximum injection time, and collected in profile mode. The tSIM scan for P6C/P2C was collected from 6-10 minutes in positive mode at an isolation window of 4 m/z (for metabolomics) or 18 m/z (for isotope-tracing experiments, to include m/z shifts), an Orbitrap resolution of 120,000, a RF lens at 70%, an automatic maximum injection time, and collected in profile mode.
Ion Mode:NEGATIVE
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