Summary of Study ST002809

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 PR001756. The data can be accessed directly via it's Project DOI: 10.21228/M8QT46 This work is supported by NIH grant, U2C- DK119886.

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Study IDST002809
Study TitleRole of cilia in mitochondrial function
Study Typecultured cells
Study SummaryAutosomal dominant polycystic kidney disease (ADPKD), the most common potentially lethal genetic disease in humans and the fourth leading cause of kidney disease, exhibits features of both a ciliary and metabolic disorder. Our previous research revealed that cells overexpressing Exoc5 with elongated cilia demonstrate enhanced recovery from oxidative stress. To investigate the connection between primary cilia and metabolism, we conducted an unbiased metabolomics screen. Global metabolic profiling was performed on canine MDCK cells (Control, Exoc5 ciliary targeting sequence mutation (CTS-mut), Exoc5 knockdown (KD), Exoc5 overexpression (OE)) and murine cells (Ift88 knockout (KO), Ift88 rescue). Knockdown (KD) or ciliary targeting sequence mutation (CTS-mut) in Exoc5, a central exocyst component, resulted in cilia loss. Similarly, Ift88 knockout (KO) resulted in cilia loss. For each experimental group, we cultivated six independent replicates of Exoc5 OE, KD, CTS-mut, and control MDCK cells, as well as six independent replicates of murine Ift88 KO and rescue cells. Cell pellets were obtained from the cultures, and we analyzed the global metabolic profiles for all 36 cell pellets. The most significant findings from the metabolomics screen indicated defects in tryptophan metabolism. This discovery suggests a potential link between primary cilia function and tryptophan-related metabolic pathways. Further exploration of these findings may shed light on the underlying mechanisms and implications for ADPKD pathogenesis and metabolic disturbances.
Institute
Medical University of South Carolina
DepartmentMedicine
Last NameLipschutz
First NameJosh
Address96 Jonathan Lucas St, Charleston, SC 29425
Emaillipschut@musc.edu
Phone8437927659
Submit Date2023-08-03
Analysis Type DetailLC-MS
Release Date2024-06-01
Release Version1
Josh Lipschutz Josh Lipschutz
https://dx.doi.org/10.21228/M8QT46
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

Select appropriate tab below to view additional metadata details:

Project:

Project ID:PR001756
Project DOI:doi: 10.21228/M8QT46
Project Title:Role of cilia in mitochondrial function
Project Summary:Autosomal dominant polycystic kidney disease (ADPKD), the most common potentially lethal genetic disease in humans and the fourth leading cause of kidney disease, exhibits features of both a ciliary and metabolic disorder. Our previous research revealed that cells overexpressing Exoc5 with elongated cilia demonstrate enhanced recovery from oxidative stress. To investigate the connection between primary cilia and metabolism, we conducted an unbiased metabolomics screen. Global metabolic profiling was performed on canine MDCK cells (Control, Exoc5 ciliary targeting sequence mutation (CTS-mut), Exoc5 knockdown (KD), Exoc5 overexpression (OE)) and murine cells (Ift88 knockout (KO), Ift88 rescue). Knockdown (KD) or ciliary targeting sequence mutation (CTS-mut) in Exoc5, a central exocyst component, resulted in cilia loss. Similarly, Ift88 knockout (KO) resulted in cilia loss. For each experimental group, we cultivated six independent replicates of Exoc5 OE, KD, CTS-mut, and control MDCK cells, as well as six independent replicates of murine Ift88 KO and rescue cells. Cell pellets were obtained from the cultures, and we analyzed the global metabolic profiles for all 36 cell pellets. The most significant findings from the metabolomics screen indicated defects in tryptophan metabolism. This discovery suggests a potential link between primary cilia function and tryptophan-related metabolic pathways. Further exploration of these findings may shed light on the underlying mechanisms and implications for ADPKD pathogenesis and metabolic disturbances.
Institute:Medical University of South Carolina
Department:Medicine
Last Name:Lipschutz
First Name:Josh
Address:96 Jonathan Lucas St, Charleston, SC 29425
Email:lipschut@musc.edu
Phone:843-792-7659

Subject:

Subject ID:SU002916
Subject Type:Mammal
Subject Species:Mus musculus;Canis lupus familiaris
Taxonomy ID:10090;9615

Factors:

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

mb_sample_id local_sample_id Genotype
SA301573MUSC-00285EXOC5_KD
SA301574MUSC-00286EXOC5_KD
SA301575MUSC-00288EXOC5_KD
SA301576MUSC-00283EXOC5_KD
SA301577MUSC-00287EXOC5_KD
SA301578MUSC-00284EXOC5_KD
SA301579MUSC-00278EXOC5_KO
SA301580MUSC-00277EXOC5_KO
SA301581MUSC-00279EXOC5_KO
SA301582MUSC-00280EXOC5_KO
SA301583MUSC-00282EXOC5_KO
SA301584MUSC-00281EXOC5_KO
SA301585MUSC-00294EXOC5_OE
SA301586MUSC-00293EXOC5_OE
SA301587MUSC-00292EXOC5_OE
SA301588MUSC-00289EXOC5_OE
SA301589MUSC-00290EXOC5_OE
SA301590MUSC-00291EXOC5_OE
SA301591MUSC-00304IFT88_KO
SA301592MUSC-00301IFT88_KO
SA301593MUSC-00305IFT88_KO
SA301594MUSC-00303IFT88_KO
SA301595MUSC-00302IFT88_KO
SA301596MUSC-00306IFT88_KO
SA301597MUSC-00307IFT88_KO_WT
SA301598MUSC-00309IFT88_KO_WT
SA301599MUSC-00312IFT88_KO_WT
SA301600MUSC-00311IFT88_KO_WT
SA301601MUSC-00310IFT88_KO_WT
SA301602MUSC-00308IFT88_KO_WT
SA301603MUSC-00295WT
SA301604MUSC-00296WT
SA301605MUSC-00297WT
SA301606MUSC-00298WT
SA301607MUSC-00299WT
SA301608MUSC-00300WT
Showing results 1 to 36 of 36

Collection:

Collection ID:CO002909
Collection Summary:Six independent replicates of Exoc5 OE, KD, CTS-mut, and control MDCK cells were grown in cell culture in 15 cm dishes. The cells were then washed in PBS, scraped, and centrifuged to a pellet and snap frozen in liquid nitrogen. Similarly six independent replicates of murine Ift88 KO and rescue cells were grown in cell culture, washed with PBS, scraped, centrifuged to a pellet and snap frozen in liquid nitrogen.
Sample Type:Cultured kidney cells

Treatment:

Treatment ID:TR002925
Treatment Summary:No treatment

Sample Preparation:

Sampleprep ID:SP002922
Sampleprep Summary:Samples were thawed on ice prior to extraction. Samples were prepared using the automated MicroLab STAR® system from Hamilton Company. Several recovery standards were added prior to the first step in the extraction process for QC purposes. In order to dissociate small molecules bound to or trapped in proteins, lysate was precipitated with methanol under vigorous shaking for 2 min (Glen Mills GenoGrinder 2000) followed by centrifugation. The resulting extract was divided into multiple fractions: two for analysis by two separate reverse phase (RP)/UPLC-MS/MS methods with positive ion mode electrospray ionization (ESI), one for analysis by RP/UPLC-MS/MS with negative ion mode ESI, one for analysis by HILIC/UPLC-MS/MS with negative ion mode ESI, and remaining fractions reserved for backup. Samples were dried under warm nitrogen to remove the organic solvent. The sample extracts were stored sealed at -80oC if not analyzed immediately.

Combined analysis:

Analysis ID AN004566 AN004567 AN004568 AN004569
Analysis type MS MS MS MS
Chromatography type Reversed phase Reversed phase Reversed phase HILIC
Chromatography system Waters Acquity Waters Acquity Waters Acquity Waters Acquity
Column Waters Acquity BEH C18 (100 x 2mm, 1.7um) Waters Acquity BEH C18 (100 x 2mm, 1.7um) Waters Acquity BEH C18 (100 x 2mm, 1.7um) Waters Acquity BEH Amide (150 x 2.1mm, 1.7um)
MS Type ESI ESI ESI ESI
MS instrument type Orbitrap Orbitrap Orbitrap Orbitrap
MS instrument name Thermo Q Exactive Orbitrap Thermo Q Exactive Orbitrap Thermo Q Exactive Orbitrap Thermo Q Exactive Orbitrap
Ion Mode POSITIVE POSITIVE NEGATIVE NEGATIVE
Units Normalized/scaled raw area counts Normalized/scaled raw area counts Normalized/scaled raw area counts Normalized/scaled raw area counts

Chromatography:

Chromatography ID:CH003431
Chromatography Summary:Low pH polar
Instrument Name:Waters Acquity
Column Name:Waters Acquity BEH C18 (100 x 2mm, 1.7um)
Column Temperature:65
Flow Gradient:Linear gradient from 5% B to 80% B over 3.35 minutes.
Flow Rate:0.35 mL/min
Solvent A:0.1% formic acid and 0.05% PFPA in water, pH ~2.5
Solvent B:0.1% formic acid and 0.05% PFPA in methanol, pH ~2.5
Chromatography Type:Reversed phase
  
Chromatography ID:CH003432
Chromatography Summary:Low pH Lipophilic
Instrument Name:Waters Acquity
Column Name:Waters Acquity BEH C18 (100 x 2mm, 1.7um)
Column Temperature:65
Flow Gradient:Linear gradient from 40 % B to 99.5% B over 1.0 minute, hold 99.5% B for 2.4 minutes
Flow Rate:0.60 mL/min
Solvent A:0.1% formic acid and 0.05% PFPA in water, pH ~2.5
Solvent B:0.1% formic acid and 0.05% PFPA in 50% methanol/ 50% acetonitrile, pH ~2.5
Chromatography Type:Reversed phase
  
Chromatography ID:CH003433
Chromatography Summary:High pH
Instrument Name:Waters Acquity
Column Name:Waters Acquity BEH C18 (100 x 2mm, 1.7um)
Column Temperature:65
Flow Gradient:Linear gradient from 0.5 to 70% B over 4.0 minutes, then rapid gradient to 99%B in 0.5 minutes.
Flow Rate:0.35 mL/min
Solvent A:6.5 mM ammonium bicarbonate in water, pH 8
Solvent B:6.5 mM ammonium bicarbonate in 95% methanol/ 5% water
Chromatography Type:Reversed phase
  
Chromatography ID:CH003434
Instrument Name:Waters Acquity
Column Name:Waters Acquity BEH Amide (150 x 2.1mm, 1.7um)
Column Temperature:65
Flow Gradient:Linear gradient from 5% B to 50% B in 3.5 minutes, then linear gradient from 50% B to 95% B in 2 minutes minutes.
Flow Rate:0.50 mL/min
Solvent A:10 mM ammonium formate in 15% water/ 5% methanol/ 80% acetonitrile (effective pH 10.16 with NH4OH)
Solvent B:10 mM ammonium formate in 50% water/ 50% acetonitrile (effective pH 10.60 with NH4OH)
Chromatography Type:HILIC

MS:

MS ID:MS004312
Analysis ID:AN004566
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:Proprietary analytical software for integration and peak picking (Metabolon). Normalized raw area counts for each sample are normalized by Bradford protein concentration. Each metabolite is then rescaled to set the median equal to 1. Lastly, missing values are imputed with the minimum.
Ion Mode:POSITIVE
  
MS ID:MS004313
Analysis ID:AN004567
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:Proprietary analytical software for integration and peak picking (Metabolon). Normalized raw area counts for each sample are normalized by Bradford protein concentration. Each metabolite is then rescaled to set the median equal to 1. Lastly, missing values are imputed with the minimum.
Ion Mode:POSITIVE
  
MS ID:MS004314
Analysis ID:AN004568
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:Proprietary analytical software for integration and peak picking (Metabolon). Normalized raw area counts for each sample are normalized by Bradford protein concentration. Each metabolite is then rescaled to set the median equal to 1. Lastly, missing values are imputed with the minimum.
Ion Mode:NEGATIVE
  
MS ID:MS004315
Analysis ID:AN004569
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:Proprietary analytical software for integration and peak picking (Metabolon). Normalized raw area counts for each sample are normalized by Bradford protein concentration. E.ach metabolite is then rescaled to set the median equal to 1. Lastly, missing values are imputed with the minimum.
Ion Mode:NEGATIVE
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