Summary of Study ST001786

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 PR001135. The data can be accessed directly via it's Project DOI: 10.21228/M80D82 This work is supported by NIH grant, U2C- DK119886.

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This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

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Study IDST001786
Study TitleMulti-omics analysis of glucose-mediated signaling by a moonlighting Gβ protein Asc1/RACK1
Study TypeUntargeted UPLC-MS Metabolomics Analysis
Study SummaryWhile much is known about glucose metabolism in yeast, less is known about the receptors and signaling pathways that indicate glucose availability. We obtained metabolic profiles for wildtype and 16 mutants affecting the yeast glucose sensing pathway, comparing 0.05% glucose vs 10 min after glucose addition to 2%. First, we determined that the G protein-coupled receptor (Gpr1/Gpa2) directs early events in glucose utilization while the transceptors (Snf3/Rgt2) regulate subsequent processes and downstream products of glucose metabolism. Whereas the large G protein transmits the signal from its cognate receptor, Ras2 (but not Ras1) integrates responses from both receptor pathways. Second, we determined the relative contributions of the G protein α (Gpa2) and β (Asc1) subunits to glucose-initiated processes. We determined that Gpa2 is primarily involved in regulating carbohydrate metabolism while Asc1 is primarily involved in amino acid metabolism. Both proteins are involved in regulating purine metabolism. Collectively, our analysis reveals the molecular basis for glucose detection and the earliest events of glucose-dependent signal transduction in yeast.
Institute
University of North Carolina at Chapel Hill
DepartmentNutrition
LaboratoryMetabolomics and Exposome Laboratory, Nutrition Research Institute, UNC Chapel Hill
Last NameSumner
First NameSusan
Address500 Laureate Way, Nutrition Research Institute, UNC Chapel Hill
Emailsusan_sumner@unc.edu
Phone(919) 622-4456
Submit Date2021-05-10
Num Groups28
Total Subjects192
Raw Data AvailableYes
Raw Data File Type(s)raw(Thermo)
Analysis Type DetailLC-MS
Release Date2021-06-11
Release Version1
Susan Sumner Susan Sumner
https://dx.doi.org/10.21228/M80D82
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR001135
Project DOI:doi: 10.21228/M80D82
Project Title:Multi-omics analysis of glucose-mediated signaling by a moonlighting Gβ protein Asc1/RACK1
Project Type:C18 Reversed-Phase Broad Spectrum Metabolomics
Project Summary:While much is known about glucose metabolism in yeast, less is known about the receptors and signaling pathways that indicate glucose availability. We obtained metabolic profiles for wildtype and 16 mutants affecting the yeast glucose sensing pathway, comparing 0.05% glucose vs 10 min after glucose addition to 2%. First, we determined that the G protein-coupled receptor (Gpr1/Gpa2) directs early events in glucose utilization while the transceptors (Snf3/Rgt2) regulate subsequent processes and downstream products of glucose metabolism. Whereas the large G protein transmits the signal from its cognate receptor, Ras2 (but not Ras1) integrates responses from both receptor pathways. Second, we determined the relative contributions of the G protein α (Gpa2) and β (Asc1) subunits to glucose-initiated processes. We determined that Gpa2 is primarily involved in regulating carbohydrate metabolism while Asc1 is primarily involved in amino acid metabolism. Both proteins are involved in regulating purine metabolism. Collectively, our analysis reveals the molecular basis for glucose detection and the earliest events of glucose-dependent signal transduction in yeast.
Institute:University of North Carolina at Chapel Hill
Department:Nutrition
Laboratory:Metabolomics and Exposome Laboratory, Nutrition Research Institute, UNC Chapel Hill
Last Name:Sumner
First Name:Susan
Address:500 Laureate Way, Nutrition Research Institute, UNC Chapel Hill
Email:susan_sumner@unc.edu
Phone:(704) 250-5066

Subject:

Subject ID:SU001863
Subject Type:Yeast
Subject Species:Saccharomyces cerevisiae
Taxonomy ID:4932
Gender:Not applicable
Species Group:Yeast

Factors:

Subject type: Yeast; Subject species: Saccharomyces cerevisiae (Factor headings shown in green)

mb_sample_id local_sample_id Genotype Condition
SA166255asc1_H5_Aasc1 H
SA166256asc1_H4_Aasc1 H
SA166257asc1_H3_Aasc1 H
SA166258asc1_H6_Aasc1 H
SA166259asc1_H1_Aasc1 H
SA166260asc1_L4_Aasc1 L
SA166261asc1_L3_Aasc1 L
SA166262asc1_L2_Aasc1 L
SA166263asc1_L1_Aasc1 L
SA166264asc1_L6_Aasc1 L
SA166265asc1_L5_Aasc1 L
SA166266gpa2_H5_Agpa2 H
SA166267gpa2_H6_Agpa2 H
SA166268gpa2_H4_Agpa2 H
SA166269gpa2_H3_Agpa2 H
SA166270gpa2_H2_Agpa2 H
SA166271gpa2_H1_Agpa2 H
SA166272gpa2_L5_Agpa2 L
SA166273gpa2_L6_Agpa2 L
SA166274gpa2_L4_Agpa2 L
SA166275gpa2_L2_Agpa2 L
SA166276gpa2_L1_Agpa2 L
SA166277gpa2_L3_Agpa2 L
SA166278gpr1_H5_Agpr1 H
SA166279gpr1_H4_Agpr1 H
SA166280gpr1_H6_Agpr1 H
SA166281gpr1_H1_Agpr1 H
SA166282gpr1_H3_Agpr1 H
SA166283gpr1_H2_Agpr1 H
SA166284gpr1_L4_Agpr1 L
SA166285gpr1_L6_Agpr1 L
SA166286gpr1_L5_Agpr1 L
SA166287gpr1_L3_Agpr1 L
SA166288gpr1_L2_Agpr1 L
SA166289gpr1_L1_Agpr1 L
SA166290pde1_H4_Bpde1 H
SA166291pde1_H1_Bpde1 H
SA166292pde1_H2_Bpde1 H
SA166293pde1_H3_Bpde1 H
SA166294pde1_H5_Bpde1 H
SA166295pde1_H6_Bpde1 H
SA166296pde1_L1_Bpde1 L
SA166297pde1_L2_Bpde1 L
SA166298pde1_L3_Bpde1 L
SA166299pde1_L4_Bpde1 L
SA166300pde1_L5_Bpde1 L
SA166301pde1_L6_Bpde1 L
SA166302pde2_H4_Bpde2 H
SA166303pde2_H6_Bpde2 H
SA166304pde2_H5_Bpde2 H
SA166305pde2_H3_Bpde2 H
SA166306pde2_H2_Bpde2 H
SA166307pde2_H1_Bpde2 H
SA166308pde2_L2_Bpde2 L
SA166309pde2_L1_Bpde2 L
SA166310pde2_L3_Bpde2 L
SA166311pde2_L4_Bpde2 L
SA166312pde2_L5_Bpde2 L
SA166313pde2_L6_Bpde2 L
SA166314ras1_H4_Bras1 H
SA166315ras1_H5_Bras1 H
SA166316ras1_H3_Bras1 H
SA166317ras1_H1_Bras1 H
SA166318ras1_H6_Bras1 H
SA166319ras1_H2_Bras1 H
SA166320ras1_L1_Bras1 L
SA166321ras1_L6_Bras1 L
SA166322ras1_L5_Bras1 L
SA166323ras1_L4_Bras1 L
SA166324ras1_L3_Bras1 L
SA166325ras1_L2_Bras1 L
SA166326ras2_H5_Bras2 H
SA166327ras2_H6_Bras2 H
SA166328ras2_H4_Bras2 H
SA166329ras2_H1_Bras2 H
SA166330ras2_H3_Bras2 H
SA166331ras2_H2_Bras2 H
SA166332ras2_L2_Bras2 L
SA166333ras2_L3_Bras2 L
SA166334ras2_L1_Bras2 L
SA166335ras2_L4_Bras2 L
SA166336ras2_L5_Bras2 L
SA166337ras2_L6_Bras2 L
SA166338rgs2_H5_Args2 H
SA166339rgs2_H6_Args2 H
SA166340rgs2_H3_Args2 H
SA166341rgs2_H4_Args2 H
SA166342rgs2_H2_Args2 H
SA166343rgs2_H1_Args2 H
SA166344rgs2_L4_Args2 L
SA166345rgs2_L5_Args2 L
SA166346rgs2_L3_Args2 L
SA166347rgs2_L1_Args2 L
SA166348rgs2_L6_Args2 L
SA166349rgs2_L2_Args2 L
SA166350rgt2_H1_Crgt2 H
SA166351rgt2_H6_Crgt2 H
SA166352rgt2_H4_Crgt2 H
SA166353rgt2_H5_Crgt2 H
SA166354rgt2_H3_Crgt2 H
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Collection:

Collection ID:CO001856
Collection Summary:For metabolomics, each replicate consisting of 3 mL of cell culture was mixed with 45 mL cold pure methanol on dry ice. After 5 min, cells were centrifuged in a precooled rotor (-80 °C). After discarding the supernatant, cell pellets were immediately stored at -80 °C. A small aliquot of each sample was saved to manually determine cell density with a hemocytometer.
Sample Type:Yeast cells
Storage Conditions:-80℃

Treatment:

Treatment ID:TR001876
Treatment Summary:No treatment in this study.

Sample Preparation:

Sampleprep ID:SP001869
Sampleprep Summary:Frozen cell pellets were resuspended with extraction reagent (8:2 methanol-water solution) to 3x108 cell/mL and then transferred into 2 mL ceramic bead MagNalyser tubes. Blank samples were prepared by adding 1300 μL of extraction reagent with no cells to a MagNalyser tube with ceramic beads. Tubes were subjected to homogenization, with Bead Ruptor Elite Bead Mill Homogenizer (OMNI International) at 6.0 m/s for 40 sec in 2 cycles at room temperature. This step was repeated twice. All samples were then centrifuged at 16,000 xg for 10 min at 4 °C. 500 μL of the supernatant was transferred into low-bind 1.7 mL microfuge tubes. Total pools were made by combining an additional 65 μL of the supernatant from each sample and then aliquoting this mixture into low-bind 1.7 mL tubes at a volume of 500 μL. The remaining supernatant was stored at -80 °C for repeat experiments if necessary. For all experimental samples, pooled samples and blanks were dried using a speedvac vacuum concentrator overnight. Dried samples were stored at -80 °C. Before LC-MS analysis, 100 μL of reconstitution buffer (95:5 water:methanol with 500 ng/mL tryptophan d-5) was added to each dried sample. All tubes were vortexed at 5000 rpm for 10 min and then centrifuged at room temperature at 16,000 xg for 4 min. Supernatant was transferred into autosampler vials for LC-MS.
Processing Storage Conditions:4℃
Extract Storage:-80℃

Combined analysis:

Analysis ID AN002897
Analysis type MS
Chromatography type Reversed phase
Chromatography system Thermo Vanquish
Column Waters Acquity BEH HSS T3 (100 x 2.1mm,1.8um)
MS Type ESI
MS instrument type Orbitrap
MS instrument name Thermo Q Exactive HF hybrid Orbitrap
Ion Mode POSITIVE
Units Peak area

Chromatography:

Chromatography ID:CH002148
Chromatography Summary:None
Chromatography Comments:Time(min) Flow Rate %A %B Curve 1. 0 0.4 99.0 1.0 5 2. 1.00 0.4 99.0 1.0 5 3. 16.00 0.4 1.0 99.0 5 4. 19.00 0.4 1.0 99.0 5 5. 19.50 0.4 99.0 1.0 5
Instrument Name:Thermo Vanquish
Column Name:Waters Acquity BEH HSS T3 (100 x 2.1mm,1.8um)
Column Pressure:6000-10000
Column Temperature:8
Flow Rate:0.4 mL/min
Injection Temperature:8
Solvent A:100% water; 0.1% formic acid
Solvent B:100% methanol; 0.1% formic acid
Weak Wash Solvent Name:10:90 Methanol:Water with 0.1% FA solution
Strong Wash Solvent Name:75:25 2-Propanol: Water with 0.1% FA solution
Randomization Order:Yes
Chromatography Type:Reversed phase

MS:

MS ID:MS002689
Analysis ID:AN002897
Instrument Name:Thermo Q Exactive HF hybrid Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:NA
Ion Mode:POSITIVE
Capillary Temperature:275 °C
Capillary Voltage:3.5 KV
Collision Energy:10-35, ramp
Collision Gas:N2
Dry Gas Flow:45
Dry Gas Temp:325°C
Fragmentation Method:CID
Desolvation Gas Flow:45
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