Abstract:Detailed molecular analysis is of increasing importance in research into the regulation of biochemical pathways,
organismal growth and disease. Lipidomics in particular is increasingly sought after as it provides insight into molecular
species involved in energy storage, signalling and fundamental cellular structures. This has led to the use of a range of
tools and techniques to acquire lipidomics data.
31P NMR for lipidomics offers well-resolved head group/lipid class
analysis, structural data that can be use… Show more
“…We developed novel procedures for handling cultures for lipidomics as established procedures for handling this cell type for proteomics were not suitable for handling the lipid fraction. The lipids were extracted ( n = 6 biological replicates) in a similar manner to previous work [ 14 , [30] , [31] , [32] ]. Phosphorus ( 31 P) NMR was then used to measure the relative abundance of lipid classes [ 30 , 31 ].…”
Section: Resultsmentioning
confidence: 99%
“…The lipids were extracted ( n = 6 biological replicates) in a similar manner to previous work [ 14 , [30] , [31] , [32] ]. Phosphorus ( 31 P) NMR was then used to measure the relative abundance of lipid classes [ 30 , 31 ]. Fig.…”
Section: Resultsmentioning
confidence: 99%
“…Lipid films were dissolved in the CUBO solvent system [ 30 , 31 ] (450 μL, 23–26 mg isolate/sample). Data acquisition was similar to published work [ 13 , 14 ], but using a Bruker 400 MHz Avance III HD spectrometer equipped with a 5 mm BBO S1 (smart) probe operating at 298 K. 31 P NMR spectra were acquired at 161·98 MHz using inverse gated proton decoupling, with 2048 scans per sample and a spectral width of 19·99 ppm.…”
Section: Methodsmentioning
confidence: 99%
“…Spectra were processed and analysed using TopSpin 3.2. The dcon function was used to deconvolute spectra in order to determine the integration of each resonance, in a similar manner to previous studies [ 30 , 31 ]. The integration of each resonance was divided by the total integration for that spectrum, and assigned according to known shifts [ 14 ].…”
Section: Methodsmentioning
confidence: 99%
“…We cultivated populations of D. quadricauda , synchronised them and extracted the lipid fraction at defined points in the cell cycle. We developed a novel method for preparing cells that is compatible with established procedures for extracting lipids from biological samples [ 14 , 30 , 31 ]. Lipid class abundance was measured using 31 P Nuclear Magnetic Resonance Spectroscopy (NMR) and the fatty acid composition of lipid class members determined using high resolution mass spectrometry (HRMS).…”
The structural challenges faced by eukaryotic cells through the cell cycle are key for understanding cell viability and proliferation. We tested the hypothesis that the biosynthesis of structural lipids is linked to the cell cycle. If true, this would suggest that the cell's structure is important for progress through and perhaps even control of the cell cycle. Lipidomics (
31
P NMR and MS), proteomics (Western immunoblotting) and transcriptomics (RT-qPCR) techniques were used to profile the lipid fraction and characterise aspects of its metabolism at seven stages of the cell cycle of the model eukaryote,
Desmodesmus quadricauda
. We found considerable, transient increases in the abundance of phosphatidylethanolamine during the G
1
phase (+35%, ethanolamine phosphate cytidylyltransferase increased 2·5×) and phosphatidylglycerol (+100%, phosphatidylglycerol synthase increased 22×) over the G
1
/pre-replication phase boundary. The relative abundance of phosphatidylcholine fell by ~35% during the G
1
.
N
-Methyl transferases for the conversion of phosphatidylethanolamine into phosphatidylcholine were not found in the
de novo
transcriptome profile, though a choline phosphate transferase was found, suggesting that the Kennedy pathway is the principal route for the synthesis of PC. The fatty acid profiles of the four most abundant lipids suggested that these lipids were not generally converted between one another. This study shows for the first time that there are considerable changes in the biosynthesis of the three most abundant phospholipid classes in the normal cell cycle of
D. quadricauda
, by margins large enough to elicit changes to the physical properties of membranes.
“…We developed novel procedures for handling cultures for lipidomics as established procedures for handling this cell type for proteomics were not suitable for handling the lipid fraction. The lipids were extracted ( n = 6 biological replicates) in a similar manner to previous work [ 14 , [30] , [31] , [32] ]. Phosphorus ( 31 P) NMR was then used to measure the relative abundance of lipid classes [ 30 , 31 ].…”
Section: Resultsmentioning
confidence: 99%
“…The lipids were extracted ( n = 6 biological replicates) in a similar manner to previous work [ 14 , [30] , [31] , [32] ]. Phosphorus ( 31 P) NMR was then used to measure the relative abundance of lipid classes [ 30 , 31 ]. Fig.…”
Section: Resultsmentioning
confidence: 99%
“…Lipid films were dissolved in the CUBO solvent system [ 30 , 31 ] (450 μL, 23–26 mg isolate/sample). Data acquisition was similar to published work [ 13 , 14 ], but using a Bruker 400 MHz Avance III HD spectrometer equipped with a 5 mm BBO S1 (smart) probe operating at 298 K. 31 P NMR spectra were acquired at 161·98 MHz using inverse gated proton decoupling, with 2048 scans per sample and a spectral width of 19·99 ppm.…”
Section: Methodsmentioning
confidence: 99%
“…Spectra were processed and analysed using TopSpin 3.2. The dcon function was used to deconvolute spectra in order to determine the integration of each resonance, in a similar manner to previous studies [ 30 , 31 ]. The integration of each resonance was divided by the total integration for that spectrum, and assigned according to known shifts [ 14 ].…”
Section: Methodsmentioning
confidence: 99%
“…We cultivated populations of D. quadricauda , synchronised them and extracted the lipid fraction at defined points in the cell cycle. We developed a novel method for preparing cells that is compatible with established procedures for extracting lipids from biological samples [ 14 , 30 , 31 ]. Lipid class abundance was measured using 31 P Nuclear Magnetic Resonance Spectroscopy (NMR) and the fatty acid composition of lipid class members determined using high resolution mass spectrometry (HRMS).…”
The structural challenges faced by eukaryotic cells through the cell cycle are key for understanding cell viability and proliferation. We tested the hypothesis that the biosynthesis of structural lipids is linked to the cell cycle. If true, this would suggest that the cell's structure is important for progress through and perhaps even control of the cell cycle. Lipidomics (
31
P NMR and MS), proteomics (Western immunoblotting) and transcriptomics (RT-qPCR) techniques were used to profile the lipid fraction and characterise aspects of its metabolism at seven stages of the cell cycle of the model eukaryote,
Desmodesmus quadricauda
. We found considerable, transient increases in the abundance of phosphatidylethanolamine during the G
1
phase (+35%, ethanolamine phosphate cytidylyltransferase increased 2·5×) and phosphatidylglycerol (+100%, phosphatidylglycerol synthase increased 22×) over the G
1
/pre-replication phase boundary. The relative abundance of phosphatidylcholine fell by ~35% during the G
1
.
N
-Methyl transferases for the conversion of phosphatidylethanolamine into phosphatidylcholine were not found in the
de novo
transcriptome profile, though a choline phosphate transferase was found, suggesting that the Kennedy pathway is the principal route for the synthesis of PC. The fatty acid profiles of the four most abundant lipids suggested that these lipids were not generally converted between one another. This study shows for the first time that there are considerable changes in the biosynthesis of the three most abundant phospholipid classes in the normal cell cycle of
D. quadricauda
, by margins large enough to elicit changes to the physical properties of membranes.
The structural challenges faced by eukaryotic cells through the cell cycle are key for understanding cell viability and proliferation. In this study, we tested the hypothesis that the biosynthesis of structural lipids is linked to the cell cycle. If true, this would suggest that the cell's structure would form part the control of the cell cycle. Lipidomics (31P NMR and MS), proteomics (Western immunoblotting) and transcriptomics (RT-qPCR) techniques were used to profile the lipid fraction and characterise aspects of its metabolism at seven stages of the cell cycle of the model eukaryote, Desmodesmus quadricauda. We found considerable, transient increases in the abundance of phosphatidylethanolamine during the G1 phase (+35%, ethanolamine phosphate cytidylyltransferase increased 2.5x) and phosphatidylglycerol over the G1/pre-replication phase boundary (+100%, phosphatidylglycerol synthase increased 22x). The relative abundance of phosphatidylcholine fell by ~35% during the G1. N Methyl transferases for the conversion of phosphatidylethanolamine into phosphatidylcholine were not found in the de novo transcriptome profile, though a choline phosphate transferase was found, suggesting that the Kennedy pathway is the principal route for the synthesis of PC. The fatty acid profiles of the four most abundant lipids suggested that these lipids were not generally converted between one another. The relative abundance of both phosphatidylinositol and its synthase remained constant despite an eightfold increase in cell volume. We conclude that the biosynthesis of the three most abundant structural phospholipids is linked to the cell cycle in D. quadricauda.
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