Summary of Study ST000643

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 PR000459. The data can be accessed directly via it's Project DOI: 10.21228/M8QP56 This work is supported by NIH grant, U2C- DK119886.

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Study ID	ST000643
Study Title	Trace 13C-glucose, 13C-glutamine, and 13C-serine in genetically engineered pancreatic cell lines through non-esterified fatty acids (part II)
Study Summary	Trace 13C-glucose, 13C-glutamine, and 13C-serine in genetically engineered pancreatic cell lines through non-esterified fatty acids
Institute	Mayo Clinic
Last Name	Lunt
First Name	Sophia
Address	Michigan State University 410B Biochemistry Building 603 Wilson Road East Lansing, MI 48824
Email	sophia@msu.edu
Phone	517-432-4886
Submit Date	2017-06-23
Analysis Type Detail	LC-MS
Release Date	2019-07-17
Release Version	1

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Sample Preparation:

Sampleprep ID:	SP000673
Sampleprep Summary:	In this proposal, we will interrogate the metabolic pathways that support pancreatic cancer proliferation by performing isotope enrichment analysis on the CRISPR/Cas9 knockout pancreatic cancer cell lines. Each cell line expressing PKM1 or PKM2 will be seeded on 6-well plates so that they are ~70% confluent at the time of labeled media addition. Unlabeled media will be aspirated, and cell will be washed with PBS. Media labeled with 13C-glucose, 13C-glutamine, or 13C-serine labeled media will be added, and cells will be incubated for 1 hour or 24 hours. Polar metabolites and fatty acids will be extracted at the end of the incubation period using methanol, water, and chloroform. The methanol/water fraction containing polar metabolites will be separated from the chloroform fraction containing fatty acids, and each fraction will be dried down under nitrogen. Dried down samples will be sent to the Mayo Clinic Metabolomics Resource Core for analysis of TCA cycle intermediates, amino metabolites, free fatty acids, and sphingolipids. TCA cycle intermediates and amino metabolites (polar metabolites) should become labeled faster than fatty acids and sphingolipids; therefore, polar metabolites are proposed to be analyzed at 1 hour and 24 hours after 13C-label addition. Fatty acids are proposed to be analyzed for the 24 hr time point only, due to slow labeling. Statistical analysis will be performed by the Mayo Clinic Metabolomics Resource Core.

Sampleprep ID:

SP000673

Sampleprep Summary:

In this proposal, we will interrogate the metabolic pathways that support pancreatic cancer proliferation by performing isotope enrichment analysis on the CRISPR/Cas9 knockout pancreatic cancer cell lines. Each cell line expressing PKM1 or PKM2 will be seeded on 6-well plates so that they are ~70% confluent at the time of labeled media addition. Unlabeled media will be aspirated, and cell will be washed with PBS. Media labeled with 13C-glucose, 13C-glutamine, or 13C-serine labeled media will be added, and cells will be incubated for 1 hour or 24 hours. Polar metabolites and fatty acids will be extracted at the end of the incubation period using methanol, water, and chloroform. The methanol/water fraction containing polar metabolites will be separated from the chloroform fraction containing fatty acids, and each fraction will be dried down under nitrogen. Dried down samples will be sent to the Mayo Clinic Metabolomics Resource Core for analysis of TCA cycle intermediates, amino metabolites, free fatty acids, and sphingolipids. TCA cycle intermediates and amino metabolites (polar metabolites) should become labeled faster than fatty acids and sphingolipids; therefore, polar metabolites are proposed to be analyzed at 1 hour and 24 hours after 13C-label addition. Fatty acids are proposed to be analyzed for the 24 hr time point only, due to slow labeling. Statistical analysis will be performed by the Mayo Clinic Metabolomics Resource Core.