#METABOLOMICS WORKBENCH tlhowellbray_20221012_062212 DATATRACK_ID:3505 STUDY_ID:ST002478 ANALYSIS_ID:AN004047 PROJECT_ID:PR001601
VERSION             	1
CREATED_ON             	February 10, 2023, 7:14 am
#PROJECT
PR:PROJECT_TITLE                 	The effect of prions on cellular metabolism: The metabolic impact of the [RNQ+]
PR:PROJECT_TITLE                 	prion and the native role of Rnq1p
PR:PROJECT_SUMMARY               	Within the field of amyloid and prion disease there is a need for a more
PR:PROJECT_SUMMARY               	comprehensive understanding of the fundamentals of disease biology. In order to
PR:PROJECT_SUMMARY               	facilitate the progression treatment and underpin comprehension of toxicity,
PR:PROJECT_SUMMARY               	fundamental understanding of the disruption to normal cellular biochemistry and
PR:PROJECT_SUMMARY               	trafficking is needed. Here, by removing the complex biochemistry of the brain,
PR:PROJECT_SUMMARY               	we have utilised known prion forming strains of Saccharomyces cerevisiae
PR:PROJECT_SUMMARY               	carrying different conformational variants of the Rnq1p to obtain Liquid
PR:PROJECT_SUMMARY               	Chromatography-Mass Spectrometry (LC-MS) metabolic profiles and identify key
PR:PROJECT_SUMMARY               	perturbations of prion presence. These studies reveal that prion containing
PR:PROJECT_SUMMARY               	[RNQ+] cells display a significant reduction in amino acid biosynthesis and
PR:PROJECT_SUMMARY               	distinct perturbations in sphingolipid metabolism, with significant
PR:PROJECT_SUMMARY               	downregulation in metabolites within these pathways. Moreover, that native Rnq1p
PR:PROJECT_SUMMARY               	downregulates ubiquinone biosynthesis pathways within cells, suggesting that
PR:PROJECT_SUMMARY               	Rnq1p may play a lipid/mevalonate-based cytoprotective role as a regulator of
PR:PROJECT_SUMMARY               	ubiquinone production. These findings contribute to the understanding of how
PR:PROJECT_SUMMARY               	prion proteins interact in vivo in both their prion and non-prion confirmations
PR:PROJECT_SUMMARY               	and indicate potential targets for the mitigation of these effects. . We
PR:PROJECT_SUMMARY               	demonstrate specific sphingolipid centred metabolic disruptions due to prion
PR:PROJECT_SUMMARY               	presence and give insight into a potential cytoprotective role of the native
PR:PROJECT_SUMMARY               	Rnq1 protein. This provides evidence of metabolic similarities between yeast and
PR:PROJECT_SUMMARY               	mammalian cells as a consequence of prion presence and establishes the
PR:PROJECT_SUMMARY               	application of metabolomics as a tool to investigate prion/amyloid-based
PR:PROJECT_SUMMARY               	phenomena.
PR:INSTITUTE                     	Canterbury Christ Church University
PR:LAST_NAME                     	Howell-Bray
PR:FIRST_NAME                    	Tyler
PR:ADDRESS                       	46 Canterbury Road, Kent
PR:EMAIL                         	t.l.howellbray@gmail.com
PR:PHONE                         	07841631495
#STUDY
ST:STUDY_TITLE                   	The effect of prions on cellular metabolism: The metabolic impact of the [RNQ+]
ST:STUDY_TITLE                   	prion and the native role of Rnq1p
ST:STUDY_SUMMARY                 	Within the field of amyloid and prion disease there is a need for a more
ST:STUDY_SUMMARY                 	comprehensive understanding of the fundamentals of disease biology. In order to
ST:STUDY_SUMMARY                 	facilitate the progression treatment and underpin comprehension of toxicity,
ST:STUDY_SUMMARY                 	fundamental understanding of the disruption to normal cellular biochemistry and
ST:STUDY_SUMMARY                 	trafficking is needed. Here, by removing the complex biochemistry of the brain,
ST:STUDY_SUMMARY                 	we have utilised known prion forming strains of Saccharomyces cerevisiae
ST:STUDY_SUMMARY                 	carrying different conformational variants of the Rnq1p to obtain Liquid
ST:STUDY_SUMMARY                 	Chromatography-Mass Spectrometry (LC-MS) metabolic profiles and identify key
ST:STUDY_SUMMARY                 	perturbations of prion presence. These studies reveal that prion containing
ST:STUDY_SUMMARY                 	[RNQ+] cells display a significant reduction in amino acid biosynthesis and
ST:STUDY_SUMMARY                 	distinct perturbations in sphingolipid metabolism, with significant
ST:STUDY_SUMMARY                 	downregulation in metabolites within these pathways. Moreover, that native Rnq1p
ST:STUDY_SUMMARY                 	downregulates ubiquinone biosynthesis pathways within cells, suggesting that
ST:STUDY_SUMMARY                 	Rnq1p may play a lipid/mevalonate-based cytoprotective role as a regulator of
ST:STUDY_SUMMARY                 	ubiquinone production. These findings contribute to the understanding of how
ST:STUDY_SUMMARY                 	prion proteins interact in vivo in both their prion and non-prion confirmations
ST:STUDY_SUMMARY                 	and indicate potential targets for the mitigation of these effects. . We
ST:STUDY_SUMMARY                 	demonstrate specific sphingolipid centred metabolic disruptions due to prion
ST:STUDY_SUMMARY                 	presence and give insight into a potential cytoprotective role of the native
ST:STUDY_SUMMARY                 	Rnq1 protein. This provides evidence of metabolic similarities between yeast and
ST:STUDY_SUMMARY                 	mammalian cells as a consequence of prion presence and establishes the
ST:STUDY_SUMMARY                 	application of metabolomics as a tool to investigate prion/amyloid-based
ST:STUDY_SUMMARY                 	phenomena.
ST:INSTITUTE                     	Canterbury Christ Church University
ST:LAST_NAME                     	Howell-Bray
ST:FIRST_NAME                    	Tyler
ST:ADDRESS                       	46 Canterbury Road, Kent
ST:EMAIL                         	t.l.howellbray@gmail.com
ST:PHONE                         	07841631495
#SUBJECT
SU:SUBJECT_TYPE                  	Yeast
SU:SUBJECT_SPECIES               	Saccharomyces cerevisiae
#SUBJECT_SAMPLE_FACTORS:         	SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data
SUBJECT_SAMPLE_FACTORS           	-	DeltaNIM1	Treatment:none | Ion mode:negative	Genotype=Deltarnq-knockout; RAW_FILE_NAME=DeltaNIM1 .mzML
SUBJECT_SAMPLE_FACTORS           	-	DeltaNIM2	Treatment:none | Ion mode:negative	Genotype=Deltarnq-knockout; RAW_FILE_NAME=DeltaNIM2 .mzML
SUBJECT_SAMPLE_FACTORS           	-	DeltaNIM3	Treatment:none | Ion mode:negative	Genotype=Deltarnq-knockout; RAW_FILE_NAME=DeltaNIM3 .mzML
SUBJECT_SAMPLE_FACTORS           	-	DeltaNIM4	Treatment:none | Ion mode:negative	Genotype=Deltarnq-knockout; RAW_FILE_NAME=DeltaNIM4 .mzML
SUBJECT_SAMPLE_FACTORS           	-	DeltaNIM5	Treatment:none | Ion mode:negative	Genotype=Deltarnq-knockout; RAW_FILE_NAME=DeltaNIM5 .mzML
SUBJECT_SAMPLE_FACTORS           	-	DeltaNIM6	Treatment:none | Ion mode:negative	Genotype=Deltarnq-knockout; RAW_FILE_NAME=DeltaNIM6 .mzML
SUBJECT_SAMPLE_FACTORS           	-	rnq-NIM1	Treatment:none | Ion mode:negative	Genotype=74-D694; RAW_FILE_NAME=rnq-NIM1 .mzML
SUBJECT_SAMPLE_FACTORS           	-	rnq-NIM2	Treatment:none | Ion mode:negative	Genotype=74-D694; RAW_FILE_NAME=rnq-NIM2 .mzML
SUBJECT_SAMPLE_FACTORS           	-	rnq-NIM3	Treatment:none | Ion mode:negative	Genotype=74-D694; RAW_FILE_NAME=rnq-NIM3 .mzML
SUBJECT_SAMPLE_FACTORS           	-	rnq-NIM4	Treatment:none | Ion mode:negative	Genotype=74-D694; RAW_FILE_NAME=rnq-NIM4 .mzML
SUBJECT_SAMPLE_FACTORS           	-	rnq-NIM5	Treatment:none | Ion mode:negative	Genotype=74-D694; RAW_FILE_NAME=rnq-NIM5 .mzML
SUBJECT_SAMPLE_FACTORS           	-	rnq-StressNIM1	Treatment:0.2mM H202 | Ion mode:negative	Genotype=74-D694; RAW_FILE_NAME=rnq-StressNIM1 .mzML
SUBJECT_SAMPLE_FACTORS           	-	rnq-StressNIM2	Treatment:0.2mM H202 | Ion mode:negative	Genotype=74-D694; RAW_FILE_NAME=rnq-StressNIM2 .mzML
SUBJECT_SAMPLE_FACTORS           	-	rnq-StressNIM3	Treatment:0.2mM H202 | Ion mode:negative	Genotype=74-D694; RAW_FILE_NAME=rnq-StressNIM3 .mzML
SUBJECT_SAMPLE_FACTORS           	-	rnq-StressNIM4	Treatment:0.2mM H202 | Ion mode:negative	Genotype=74-D694; RAW_FILE_NAME=rnq-StressNIM4 .mzML
SUBJECT_SAMPLE_FACTORS           	-	rnq-StressNIM5	Treatment:0.2mM H202 | Ion mode:negative	Genotype=74-D694; RAW_FILE_NAME=rnq-StressNIM5 .mzML
SUBJECT_SAMPLE_FACTORS           	-	rnq-StressNIM6	Treatment:0.2mM H202 | Ion mode:negative	Genotype=74-D694; RAW_FILE_NAME=rnq-StressNIM6 .mzML
SUBJECT_SAMPLE_FACTORS           	-	RNQ+NIM1	Treatment:none | Ion mode:Positive	Genotype=74-D694; RAW_FILE_NAME=RNQ+NIM1 .mzML
SUBJECT_SAMPLE_FACTORS           	-	RNQ+NIM2	Treatment:none | Ion mode:Positive	Genotype=74-D694; RAW_FILE_NAME=RNQ+NIM2 .mzML
SUBJECT_SAMPLE_FACTORS           	-	RNQ+NIM3	Treatment:none | Ion mode:Positive	Genotype=74-D694; RAW_FILE_NAME=RNQ+NIM3 .mzML
SUBJECT_SAMPLE_FACTORS           	-	RNQ+NIM4	Treatment:none | Ion mode:Positive	Genotype=74-D694; RAW_FILE_NAME=RNQ+NIM4 .mzML
SUBJECT_SAMPLE_FACTORS           	-	RNQ+NIM5	Treatment:none | Ion mode:Positive	Genotype=74-D694; RAW_FILE_NAME=RNQ+NIM5 .mzML
SUBJECT_SAMPLE_FACTORS           	-	RNQ+NIM6	Treatment:none | Ion mode:Positive	Genotype=74-D694; RAW_FILE_NAME=RNQ+NIM6 .mzML
SUBJECT_SAMPLE_FACTORS           	-	RNQ+StressNIM1	Treatment:0.2mM H202 | Ion mode:Positive	Genotype=74-D694; RAW_FILE_NAME=RNQ+StressNIM1 .mzML
SUBJECT_SAMPLE_FACTORS           	-	RNQ+StressNIM2	Treatment:0.2mM H202 | Ion mode:Positive	Genotype=74-D694; RAW_FILE_NAME=RNQ+StressNIM2 .mzML
SUBJECT_SAMPLE_FACTORS           	-	RNQ+StressNIM3	Treatment:0.2mM H202 | Ion mode:Positive	Genotype=74-D694; RAW_FILE_NAME=RNQ+StressNIM3 .mzML
SUBJECT_SAMPLE_FACTORS           	-	RNQ+StressNIM4	Treatment:0.2mM H202 | Ion mode:Positive	Genotype=74-D694; RAW_FILE_NAME=RNQ+StressNIM4 .mzML
SUBJECT_SAMPLE_FACTORS           	-	RNQ+StressNIM5	Treatment:0.2mM H202 | Ion mode:Positive	Genotype=74-D694; RAW_FILE_NAME=RNQ+StressNIM5 .mzML
SUBJECT_SAMPLE_FACTORS           	-	RNQ+StressNIM6	Treatment:0.2mM H202 | Ion mode:Positive	Genotype=74-D694; RAW_FILE_NAME=RNQ+StressNIM6 .mzML
SUBJECT_SAMPLE_FACTORS           	-	DeltaPIM1	Treatment:none | Ion mode:negative	Genotype=Deltarnq-knockout; RAW_FILE_NAME=DeltaPIM1 .mzML
SUBJECT_SAMPLE_FACTORS           	-	DeltaPIM2	Treatment:none | Ion mode:negative	Genotype=Deltarnq-knockout; RAW_FILE_NAME=DeltaPIM2 .mzML
SUBJECT_SAMPLE_FACTORS           	-	DeltaPIM3	Treatment:none | Ion mode:negative	Genotype=Deltarnq-knockout; RAW_FILE_NAME=DeltaPIM3 .mzML
SUBJECT_SAMPLE_FACTORS           	-	DeltaPIM4	Treatment:none | Ion mode:negative	Genotype=Deltarnq-knockout; RAW_FILE_NAME=DeltaPIM4 .mzML
SUBJECT_SAMPLE_FACTORS           	-	DeltaPIM5	Treatment:none | Ion mode:negative	Genotype=Deltarnq-knockout; RAW_FILE_NAME=DeltaPIM5 .mzML
SUBJECT_SAMPLE_FACTORS           	-	DeltaPIM6	Treatment:none | Ion mode:negative	Genotype=Deltarnq-knockout; RAW_FILE_NAME=DeltaPIM6 .mzML
SUBJECT_SAMPLE_FACTORS           	-	rnq-PIM1	Treatment:none | Ion mode:negative	Genotype=74-D694; RAW_FILE_NAME=rnq-PIM1 .mzML
SUBJECT_SAMPLE_FACTORS           	-	rnq-PIM2	Treatment:none | Ion mode:negative	Genotype=74-D694; RAW_FILE_NAME=rnq-PIM2 .mzML
SUBJECT_SAMPLE_FACTORS           	-	rnq-PIM3	Treatment:none | Ion mode:negative	Genotype=74-D694; RAW_FILE_NAME=rnq-PIM3 .mzML
SUBJECT_SAMPLE_FACTORS           	-	rnq-PIM4	Treatment:none | Ion mode:negative	Genotype=74-D694; RAW_FILE_NAME=rnq-PIM4 .mzML
SUBJECT_SAMPLE_FACTORS           	-	rnq-PIM5	Treatment:none | Ion mode:negative	Genotype=74-D694; RAW_FILE_NAME=rnq-PIM5 .mzML
SUBJECT_SAMPLE_FACTORS           	-	rnq-PIM6	Treatment:none | Ion mode:negative	Genotype=74-D695; RAW_FILE_NAME=rnq-PIM6 .mzML
SUBJECT_SAMPLE_FACTORS           	-	rnq-StressPIM1	Treatment:0.2mM H202 | Ion mode:negative	Genotype=74-D694; RAW_FILE_NAME=rnq-StressPIM1 .mzML
SUBJECT_SAMPLE_FACTORS           	-	rnq-StressPIM2	Treatment:0.2mM H202 | Ion mode:negative	Genotype=74-D694; RAW_FILE_NAME=rnq-StressPIM2 .mzML
SUBJECT_SAMPLE_FACTORS           	-	rnq-StressPIM3	Treatment:0.2mM H202 | Ion mode:negative	Genotype=74-D694; RAW_FILE_NAME=rnq-StressPIM3 .mzML
SUBJECT_SAMPLE_FACTORS           	-	rnq-StressPIM4	Treatment:0.2mM H202 | Ion mode:negative	Genotype=74-D694; RAW_FILE_NAME=rnq-StressPIM4 .mzML
SUBJECT_SAMPLE_FACTORS           	-	rnq-StressPIM5	Treatment:0.2mM H202 | Ion mode:negative	Genotype=74-D694; RAW_FILE_NAME=rnq-StressPIM5 .mzML
SUBJECT_SAMPLE_FACTORS           	-	rnq-StressPIM6	Treatment:0.2mM H202 | Ion mode:negative	Genotype=74-D694; RAW_FILE_NAME=rnq-StressPIM6 .mzML
SUBJECT_SAMPLE_FACTORS           	-	RNQ+PIM1	Treatment:none | Ion mode:Positive	Genotype=74-D694; RAW_FILE_NAME=RNQ+PIM1 .mzML
SUBJECT_SAMPLE_FACTORS           	-	RNQ+PIM2	Treatment:none | Ion mode:Positive	Genotype=74-D694; RAW_FILE_NAME=RNQ+PIM2 .mzML
SUBJECT_SAMPLE_FACTORS           	-	RNQ+PIM3	Treatment:none | Ion mode:Positive	Genotype=74-D694; RAW_FILE_NAME=RNQ+PIM3 .mzML
SUBJECT_SAMPLE_FACTORS           	-	RNQ+PIM4	Treatment:none | Ion mode:Positive	Genotype=74-D694; RAW_FILE_NAME=RNQ+PIM4 .mzML
SUBJECT_SAMPLE_FACTORS           	-	RNQ+PIM5	Treatment:none | Ion mode:Positive	Genotype=74-D694; RAW_FILE_NAME=RNQ+PIM5 .mzML
SUBJECT_SAMPLE_FACTORS           	-	RNQ+PIM6	Treatment:none | Ion mode:Positive	Genotype=74-D694; RAW_FILE_NAME=RNQ+PIM6 .mzML
SUBJECT_SAMPLE_FACTORS           	-	RNQ+StressPIM1	Treatment:0.2mM H202 | Ion mode:Positive	Genotype=74-D694; RAW_FILE_NAME=RNQ+StressPIM1 .mzML
SUBJECT_SAMPLE_FACTORS           	-	RNQ+StressPIM2	Treatment:0.2mM H202 | Ion mode:Positive	Genotype=74-D694; RAW_FILE_NAME=RNQ+StressPIM2 .mzML
SUBJECT_SAMPLE_FACTORS           	-	RNQ+StressPIM3	Treatment:0.2mM H202 | Ion mode:Positive	Genotype=74-D694; RAW_FILE_NAME=RNQ+StressPIM3 .mzML
SUBJECT_SAMPLE_FACTORS           	-	RNQ+StressPIM4	Treatment:0.2mM H202 | Ion mode:Positive	Genotype=74-D694; RAW_FILE_NAME=RNQ+StressPIM4 .mzML
SUBJECT_SAMPLE_FACTORS           	-	RNQ+StressPIM5	Treatment:0.2mM H202 | Ion mode:Positive	Genotype=74-D694; RAW_FILE_NAME=RNQ+StressPIM5 .mzML
SUBJECT_SAMPLE_FACTORS           	-	RNQ+StressPIM6	Treatment:0.2mM H202 | Ion mode:Positive	Genotype=74-D694; RAW_FILE_NAME=RNQ+StressPIM6 .mzML
#COLLECTION
CO:COLLECTION_SUMMARY            	Strain and cultivation conditions The S. cerevisiae strain used in this study
CO:COLLECTION_SUMMARY            	were derivatives of 74-D694 (MATa ade1-14(UGA) trp1-289(UAG) ura3-52 his3-∆200
CO:COLLECTION_SUMMARY            	leu2-3, 112) (Chernoff et al. 1993). Yeast harbouring [RNQ+] and knockout strain
CO:COLLECTION_SUMMARY            	Δrnq were kind gifts from the Kent Fungal Group. Yeast were grown at 30°C with
CO:COLLECTION_SUMMARY            	shaking at 180 rpm in synthetic complete (SC) media (2 % (w/v) glucose, 0.17 %
CO:COLLECTION_SUMMARY            	Yeast Nitrogen Base (without amino acids, without ammonium sulphate), 0.5%
CO:COLLECTION_SUMMARY            	ammonium sulphate, the appropriate concentration of yeast synthetic complete
CO:COLLECTION_SUMMARY            	supplement mixture or synthetic complete drop-out media supplement). Transient
CO:COLLECTION_SUMMARY            	growth on SC media containing 3mM guanidine hydrochloride (GdnHCl) was used as a
CO:COLLECTION_SUMMARY            	curing agent in the media of S. cerevisiae cells that required a [prion-]
CO:COLLECTION_SUMMARY            	status. Mild oxidative stresses were achieved by the addition of H2O2 (final
CO:COLLECTION_SUMMARY            	concentration 0.2 mM) to the appropriate culture mediums. Cultures were grown
CO:COLLECTION_SUMMARY            	using the filter culture method (as described by rabinowiz 2008)
CO:SAMPLE_TYPE                   	Yeast cells
CO:STORAGE_CONDITIONS            	-80℃
#TREATMENT
TR:TREATMENT_SUMMARY             	Mild oxidative stresses were achieved by the addition of H2O2 (final
TR:TREATMENT_SUMMARY             	concentration 0.2 mM)
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	Metabolite sample preparation Analytical grade standards were supplied by Sigma
SP:SAMPLEPREP_SUMMARY            	Aldrich. Quenching was achieved by adaption of cold methanol protocol (56), via
SP:SAMPLEPREP_SUMMARY            	submersion of entire filter membrane. Metabolite extraction was performed on the
SP:SAMPLEPREP_SUMMARY            	resultant cell pellets using the boiling ethanol technique (57). Briefly, each
SP:SAMPLEPREP_SUMMARY            	tube was taken from the −80°C and 5 ml 75% (v/v) boiling ethanol was added
SP:SAMPLEPREP_SUMMARY            	(pre-heated). Each tube was immediately vortexed and placed in a water bath at
SP:SAMPLEPREP_SUMMARY            	80°C. After 5 min each tube was cooled on ice for 3 min, followed by
SP:SAMPLEPREP_SUMMARY            	centrifugation (5000xg, 5 minutes, -20°C, precooled). Extracts were then stored
SP:SAMPLEPREP_SUMMARY            	at -80°C until further use. Immediately prior to mass spectrometry
SP:SAMPLEPREP_SUMMARY            	experimentation all extracts were concentrated by speed vacuum at 35° C for ≈
SP:SAMPLEPREP_SUMMARY            	3 hours. Following resuspension in 500µL of LC/MS grade water samples were
SP:SAMPLEPREP_SUMMARY            	lyophilised overnight. Lyophilised samples were then resuspended in 200µL of
SP:SAMPLEPREP_SUMMARY            	0.1M formic acid, vortexed and loaded into vials.
SP:PROCESSING_STORAGE_CONDITIONS 	-80℃
SP:EXTRACTION_METHOD             	Boiling ethanol
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_TYPE           	Reversed phase
CH:INSTRUMENT_NAME               	ACQUITY SYNAPT G2-Si Mass Spectrometer
CH:COLUMN_NAME                   	1.7 µm C18 BEH column
CH:SOLVENT_A                     	95% Acetronitrile/5% water, 0.1% formic acid
CH:SOLVENT_B                     	100% water; 0.1% formic acid
CH:FLOW_GRADIENT                 	10-minute gradient from 0% to 50% acetonitrile (0.1% formic acid)
CH:FLOW_RATE                     	500nL/min
CH:COLUMN_TEMPERATURE            	35
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Waters Synapt G2 Si QTOF
MS:INSTRUMENT_TYPE               	QTOF
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	POSITIVE
MS:MS_COMMENTS                   	Data is labeled for negative or positive ionization mode
MS:MS_RESULTS_FILE               	ST002478_AN004047_Results.txt	UNITS:m/z values	Has m/z:Yes	Has RT:Yes	RT units:Minutes
#END