Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA036348

Local Sample ID:	ms6129-23
Subject ID:	SU000666
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Species Group:	Human

Select appropriate tab below to view additional metadata details:

Subject:

Subject ID:	SU000666
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Species Group:	Human

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
ms6129-23	SA036348	FL007841	PHGDH B34 - Dox (PKM2)	experiment
ms6129-23	SA036348	FL007841	No label	tracer
ms6129-23	SA036348	FL007841	-	time
ms6129-23	SA036348	FL007841	PHGDH	groupID

Collection:

Collection ID:	CO000660
Collection Summary:	Pancreatic ductal adenocarcinoma cell line with PHGDH, PSAT, and PSPH KO. These cells have been incubated with no label, 13C-glucose or 13C-glutamine label for 48 hours.
Sample Type:	Pancreas

Treatment:

Treatment ID:	TR000680
Treatment Summary:	Pancreatic cancer cells exhibit altered metabolism, which is mediated in part by expressing the proliferation-supportive M2 isoform of pyruvate kinase (PK). Unlike normal embryonic cells that stop proliferating when forced to express the proliferation-incompatible M1 isoform of PK, pancreatic cancer cells can proliferate just as rapidly with either the M1 or M2 isoform. During forced PKM1 expression, pancreatic cancer cells upregulate their serine biosynthesis pathway. The three enzymes in the serine biosynthesis pathway include phosphoglycerate dehydrogenase (PHGDH), phosphoserine aminotransferase (PSAT), and phosphoserine phosphatase (PSPH). Each of the genes encoding the three enzymes in the serine biosynthesis pathway have successfully been knocked out from pancreatic cancer cells using the CRISPR/Cas9 system in our laboratory, with multiple confirmed clones for each knockout ready for metabolic characterization.

Treatment ID:

TR000680

Treatment Summary:

Pancreatic cancer cells exhibit altered metabolism, which is mediated in part by expressing the proliferation-supportive M2 isoform of pyruvate kinase (PK). Unlike normal embryonic cells that stop proliferating when forced to express the proliferation-incompatible M1 isoform of PK, pancreatic cancer cells can proliferate just as rapidly with either the M1 or M2 isoform. During forced PKM1 expression, pancreatic cancer cells upregulate their serine biosynthesis pathway. The three enzymes in the serine biosynthesis pathway include phosphoglycerate dehydrogenase (PHGDH), phosphoserine aminotransferase (PSAT), and phosphoserine phosphatase (PSPH). Each of the genes encoding the three enzymes in the serine biosynthesis pathway have successfully been knocked out from pancreatic cancer cells using the CRISPR/Cas9 system in our laboratory, with multiple confirmed clones for each knockout ready for metabolic characterization.

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.

Combined analysis:

Analysis ID	AN000975
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Waters Acquity
Column	Waters Acquity BEH C18 (150 x 2.1mm,1.7um)
MS Type	ESI
MS instrument type	QTOF
MS instrument name	Agilent 6550 QTOF
Ion Mode	NEGATIVE
Units	% enrichment: MPE

Chromatography:

Chromatography ID:	CH000700
Instrument Name:	Waters Acquity
Column Name:	Waters Acquity BEH C18 (150 x 2.1mm,1.7um)
Chromatography Type:	Reversed phase

MS:

MS ID:	MS000870
Analysis ID:	AN000975
Instrument Name:	Agilent 6550 QTOF
Instrument Type:	QTOF
MS Type:	ESI
Ion Mode:	NEGATIVE