#METABOLOMICS WORKBENCH KasparasP_20240820_065447 DATATRACK_ID:5129 STUDY_ID:ST003420 ANALYSIS_ID:AN005619 PROJECT_ID:PR002115
VERSION             	1
CREATED_ON             	August 20, 2024, 7:04 am
#PROJECT
PR:PROJECT_TITLE                 	TRAM–LAG1–CLN8 family proteins are acyltransferases regulating phospholipid
PR:PROJECT_TITLE                 	composition
PR:PROJECT_SUMMARY               	The diversity of cellular phospholipids, crucial for membrane homeostasis and
PR:PROJECT_SUMMARY               	function, arises from enzymatic remodeling of their fatty acyl chains. In this
PR:PROJECT_SUMMARY               	work, we reveal that poorly understood TRAM–LAG1–CLN8 domain-containing
PR:PROJECT_SUMMARY               	(TLCD) proteins are phospholipid remodeling enzymes. We demonstrate that TLCD1
PR:PROJECT_SUMMARY               	is an evolutionarily conserved lysophosphatidylethanolamine acyltransferase,
PR:PROJECT_SUMMARY               	which regulates cellular phospholipid composition and generates novel fatty acid
PR:PROJECT_SUMMARY               	and thiamine (vitamin B1) esters as its secondary products. Furthermore, we
PR:PROJECT_SUMMARY               	establish that human TLCD protein CLN8, mutations in which cause fatal
PR:PROJECT_SUMMARY               	neurodegenerative Batten disease, is a lysophosphatidylglycerol acyltransferase.
PR:PROJECT_SUMMARY               	We show that CLN8 catalyzes the essential step in the biosynthesis of
PR:PROJECT_SUMMARY               	bis(monoacylglycero)phosphate, a phospholipid critical for lysosome function.
PR:PROJECT_SUMMARY               	Our study unveils a new family of acyltransferases integral to cellular membrane
PR:PROJECT_SUMMARY               	phospholipid homeostasis and human disease.
PR:INSTITUTE                     	University of Cambridge
PR:LAST_NAME                     	Petkevicius
PR:FIRST_NAME                    	Kasparas
PR:ADDRESS                       	The Keith Peters Building, Cambridge, Cambridgeshire, CB2 0XY, United Kingdom
PR:EMAIL                         	kp416@mrc-mbu.cam.ac.uk
PR:PHONE                         	+447500233355
PR:PUBLICATIONS                  	TRAM–LAG1–CLN8 family proteins are acyltransferases regulating phospholipid
PR:PUBLICATIONS                  	composition
#STUDY
ST:STUDY_TITLE                   	Analysis of lipid composition of control, YPR114w and YJR116w yeast mutants
ST:STUDY_TITLE                   	grown under exponential phase
ST:STUDY_SUMMARY                 	Untargeted lipidomics experiments were conducted to analyze the differences in
ST:STUDY_SUMMARY                 	lipid composition between control, YPR114w and YJR116w yeast (Saccharomyces
ST:STUDY_SUMMARY                 	cerevisiae) mutants . See a reference publication (Sheokand et al) for details.
ST:INSTITUTE                     	University of Cambridge
ST:LAST_NAME                     	Petkevicius
ST:FIRST_NAME                    	Kasparas
ST:ADDRESS                       	The Keith Peters Building
ST:EMAIL                         	kp416@mrc-mbu.cam.ac.uk
ST:PHONE                         	07500233355
ST:PUBLICATIONS                  	TRAM–LAG1–CLN8 family proteins are acyltransferases regulating phospholipid
ST:PUBLICATIONS                  	composition
#SUBJECT
SU:SUBJECT_TYPE                  	Cultured cells
SU:SUBJECT_SPECIES               	Saccharomyces cerevisiae
SU:TAXONOMY_ID                   	4932
#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           	-	Yeast process blank	Genotype:Process blank | Sample source:Process blank | Replicate:Process blank	RAW_FILE_NAME(Raw file name)=Yeast_process_blank.raw
SUBJECT_SAMPLE_FACTORS           	-	Wild-type replicate 1	Genotype:Wild-type | Sample source:Saccharomyces cerevisiae | Replicate:1	RAW_FILE_NAME(Raw file name)=Yeast_Wild-Type_exponential_phase_Replicate_1.raw
SUBJECT_SAMPLE_FACTORS           	-	Wild-type replicate 2	Genotype:Wild-type | Sample source:Saccharomyces cerevisiae | Replicate:2	RAW_FILE_NAME(Raw file name)=Yeast_Wild-Type_exponential_phase_Replicate_2.raw
SUBJECT_SAMPLE_FACTORS           	-	Wild-type replicate 3	Genotype:Wild-type | Sample source:Saccharomyces cerevisiae | Replicate:3	RAW_FILE_NAME(Raw file name)=Yeast_Wild-Type_exponential_phase_Replicate_3.raw
SUBJECT_SAMPLE_FACTORS           	-	YJR116w KO replicate 1	Genotype:YJR116w KO | Sample source:Saccharomyces cerevisiae | Replicate:1	RAW_FILE_NAME(Raw file name)=YJR116w_KO_exponential_phase_Replicate_1.raw
SUBJECT_SAMPLE_FACTORS           	-	YJR116w KO replicate 2	Genotype:YJR116w KO | Sample source:Saccharomyces cerevisiae | Replicate:2	RAW_FILE_NAME(Raw file name)=YJR116w_KO_exponential_phase_Replicate_2.raw
SUBJECT_SAMPLE_FACTORS           	-	YJR116w KO replicate 3	Genotype:YJR116w KO | Sample source:Saccharomyces cerevisiae | Replicate:3	RAW_FILE_NAME(Raw file name)=YJR116w_KO_exponential_phase_Replicate_3.raw
SUBJECT_SAMPLE_FACTORS           	-	YPR114w KO replicate 1	Genotype:YPR114w KO | Sample source:Saccharomyces cerevisiae | Replicate:1	RAW_FILE_NAME(Raw file name)=YPR114w_KO_exponential_phase_Replicate_1.raw
SUBJECT_SAMPLE_FACTORS           	-	YPR114w KO replicate 2	Genotype:YPR114w KO | Sample source:Saccharomyces cerevisiae | Replicate:2	RAW_FILE_NAME(Raw file name)=YPR114w_KO_exponential_phase_Replicate_2.raw
SUBJECT_SAMPLE_FACTORS           	-	YPR114w KO replicate 3	Genotype:YPR114w KO | Sample source:Saccharomyces cerevisiae | Replicate:3	RAW_FILE_NAME(Raw file name)=YPR114w_KO_exponential_phase_Replicate_3.raw
#COLLECTION
CO:COLLECTION_SUMMARY            	Yeast cells were grown in synthetic medium (SC) containing 2% glucose, 0.17%
CO:COLLECTION_SUMMARY            	yeast nitrogen base (Cat. No. 233520, Difco, BD, Franklin Lakes, NJ), 0.5%
CO:COLLECTION_SUMMARY            	ammonium sulfate, and amino acid drop-out (60 mg/L leucine, 55 mg/L adenine, 55
CO:COLLECTION_SUMMARY            	mg/L uracil, 55 mg/L tyrosine, 20 mg/L of arginine, 10 mg/L histidine, 60 mg/L
CO:COLLECTION_SUMMARY            	isoleucine, 40 mg/L lysine, 60 mg/L phenylalanine, 50 mg/L threonine, 10 mg/L
CO:COLLECTION_SUMMARY            	methionine, 40 mg/L tryptophan). For lipidomics, cells were grown overnight to
CO:COLLECTION_SUMMARY            	exponential phase (OD600 0.4-0.6), then were washed once in water and snap
CO:COLLECTION_SUMMARY            	frozen in liquid nitrogen. For acyl-thiamine measurements, cultures were grown
CO:COLLECTION_SUMMARY            	to the exponential phase overnight, then split in two; one half was left
CO:COLLECTION_SUMMARY            	untreated while the second half was supplemented with 100 mM thiamine. All
CO:COLLECTION_SUMMARY            	cultures were then grown for 2h 30 min. Cells were washed once in water and snap
CO:COLLECTION_SUMMARY            	frozen in liquid nitrogen.
CO:SAMPLE_TYPE                   	Yeast cells
CO:STORAGE_CONDITIONS            	-80℃
#TREATMENT
TR:TREATMENT_SUMMARY             	Cells were untreated (collected under standard culturing conditions).
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	The extraction of total lipids from cellular matrices, immunoprecipitated
SP:SAMPLEPREP_SUMMARY            	mitochondria and in vitro assays was conducted employing the butanol-methanol
SP:SAMPLEPREP_SUMMARY            	(BUME) method, as described in detail (37). Note that the chloroform-methanol
SP:SAMPLEPREP_SUMMARY            	lipid extraction methods result in the partitioning of acyl-thiamines into the
SP:SAMPLEPREP_SUMMARY            	polar phase. 2 ml screw cap plastic tubes were used (3469-11, Thermo
SP:SAMPLEPREP_SUMMARY            	Scientific), and a blank extraction was always performed in parallel to account
SP:SAMPLEPREP_SUMMARY            	for the plastic-related contaminants. The extraction commenced with the
SP:SAMPLEPREP_SUMMARY            	homogenization of frozen cell pellets, mitochondrial beads or in vitro assay
SP:SAMPLEPREP_SUMMARY            	mixtures in a 0.5 ml ice-cold solution of butanol to methanol in a 3:1 ratio.
SP:SAMPLEPREP_SUMMARY            	For lipidomics samples, BUME solution was enriched with SPLASH internal standard
SP:SAMPLEPREP_SUMMARY            	mix (330707, Avanti Polar Lipids). For the extraction, a further 0.5 ml of 1%
SP:SAMPLEPREP_SUMMARY            	acetic acid and 0.5 ml of a heptane:ethyl acetate 3:1 mixture were added,
SP:SAMPLEPREP_SUMMARY            	followed by vigorous vortexing for a total of 5 minutes. The mixture was then
SP:SAMPLEPREP_SUMMARY            	centrifuged at 6000 g for 5 minutes, allowing for the separation of phases,
SP:SAMPLEPREP_SUMMARY            	after which the upper organic phase was carefully decanted into glass vials. A
SP:SAMPLEPREP_SUMMARY            	second extraction was conducted on the remaining aqueous phase, with the newly
SP:SAMPLEPREP_SUMMARY            	acquired upper phase being combined in the same glass vials as the first. Post
SP:SAMPLEPREP_SUMMARY            	extraction, the solvents were evaporated under a stream of nitrogen, and the
SP:SAMPLEPREP_SUMMARY            	resultant dry lipid extracts were preserved at −70°C pending further
SP:SAMPLEPREP_SUMMARY            	analysis.
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_SUMMARY        	Dried samples were reconstituted in 100 μL of isopropanol, acetonitrile, and
CH:CHROMATOGRAPHY_SUMMARY        	water (2:1:1 ratio) and thoroughly vortexed. Liquid chromatography was conducted
CH:CHROMATOGRAPHY_SUMMARY        	using a Shimadzu HPLC System, with 10 μL of the sample introduced onto a Waters
CH:CHROMATOGRAPHY_SUMMARY        	Acquity Premier UPLC® CSH column (1.7 μm pore size, 2.1 mm × 50 mm), which
CH:CHROMATOGRAPHY_SUMMARY        	was maintained at 55 °C. The mobile phase A comprised a 6:4 ratio of
CH:CHROMATOGRAPHY_SUMMARY        	acetonitrile to water with 10 mM ammonium formate, and mobile phase B consisted
CH:CHROMATOGRAPHY_SUMMARY        	of a 9:1 ratio of isopropanol to acetonitrile with 10 mM ammonium formate. A
CH:CHROMATOGRAPHY_SUMMARY        	flow rate of 500 μL per minute was maintained with a gradient protocol for
CH:CHROMATOGRAPHY_SUMMARY        	mobile phase B as follows: 0.00 minutes_40% mobile phase B; 1.5 minutes_40%
CH:CHROMATOGRAPHY_SUMMARY        	mobile phase B; 8.00 minutes_99% mobile phase B; 10.00 minutes_99% mobile phase
CH:CHROMATOGRAPHY_SUMMARY        	B; 10.10 minutes_40% mobile phase B; 12.00 minutes_40% mobile phase. The sample
CH:CHROMATOGRAPHY_SUMMARY        	injection needle was rinsed with a 9:1 isopropanol and acetonitrile solution
CH:CHROMATOGRAPHY_SUMMARY        	(strong wash) and isopropanol, acetonitrile, and water (2:1:1, weak wash).
CH:CHROMATOGRAPHY_TYPE           	Reversed phase
CH:INSTRUMENT_NAME               	Shimadzu 10A
CH:COLUMN_NAME                   	Waters ACQUITY UPLC CSH C18 (100 x 2.1mm,1.7um)
CH:SOLVENT_A                     	6:4 of acetonitrile to water with 10 mM ammonium formate
CH:SOLVENT_B                     	9:1 of isopropanol to acetonitrile with 10 mM ammonium formate
CH:FLOW_GRADIENT                 	Gradient protocol for mobile phase B as follows: 0.00 minutes_40% mobile phase
CH:FLOW_GRADIENT                 	B; 1.5 minutes_40% mobile phase B; 8.00 minutes_99% mobile phase B; 10.00
CH:FLOW_GRADIENT                 	minutes_99% mobile phase B; 10.10 minutes_40% mobile phase B; 12.00 minutes_40%
CH:FLOW_GRADIENT                 	mobile phase.
CH:FLOW_RATE                     	500 μL per minute
CH:COLUMN_TEMPERATURE            	55
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Thermo Q Exactive Orbitrap
MS:INSTRUMENT_TYPE               	Orbitrap
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	UNSPECIFIED
MS:MS_COMMENTS                   	Mass spectrometric detection was carried out on a ThermoFisher Scientific
MS:MS_COMMENTS                   	Q-Exactive Orbitrap equipped with a heated electrospray ionization source. The
MS:MS_COMMENTS                   	mass spectrometer was calibrated immediately before sample analysis using
MS:MS_COMMENTS                   	positive and negative ionization calibration solution (recommended by Thermo
MS:MS_COMMENTS                   	Scientific). The electrospray ionization parameters were optimized with a 50:50
MS:MS_COMMENTS                   	mix of mobile phase A and B for spray stability, setting the capillary
MS:MS_COMMENTS                   	temperature at 300 °C, source heater temperature at 420 °C, with the sheath,
MS:MS_COMMENTS                   	auxiliary, and spare gas flows at specific arbitrary units (40, 15 and 3,
MS:MS_COMMENTS                   	respectively), and source voltage at 4 kV. The mass spectrometer operated at a
MS:MS_COMMENTS                   	scan rate of 4 Hz, yielding a resolution of 35,000 at m/z 200, over a full-scan
MS:MS_COMMENTS                   	range from m/z 120 to 1800, with continuous switching between positive and
MS:MS_COMMENTS                   	negative modes.
#MS_METABOLITE_DATA
MS_METABOLITE_DATA:UNITS	nmol
MS_METABOLITE_DATA_START
Samples	Yeast process blank	Wild-type replicate 1	Wild-type replicate 2	Wild-type replicate 3	YJR116w KO replicate 1	YJR116w KO replicate 2	YJR116w KO replicate 3	YPR114w KO replicate 1	YPR114w KO replicate 2	YPR114w KO replicate 3
Factors	Genotype:Process blank | Sample source:Process blank | Replicate:Process blank	Genotype:Wild-type | Sample source:Saccharomyces cerevisiae | Replicate:1	Genotype:Wild-type | Sample source:Saccharomyces cerevisiae | Replicate:2	Genotype:Wild-type | Sample source:Saccharomyces cerevisiae | Replicate:3	Genotype:YJR116w KO | Sample source:Saccharomyces cerevisiae | Replicate:1	Genotype:YJR116w KO | Sample source:Saccharomyces cerevisiae | Replicate:2	Genotype:YJR116w KO | Sample source:Saccharomyces cerevisiae | Replicate:3	Genotype:YPR114w KO | Sample source:Saccharomyces cerevisiae | Replicate:1	Genotype:YPR114w KO | Sample source:Saccharomyces cerevisiae | Replicate:2	Genotype:YPR114w KO | Sample source:Saccharomyces cerevisiae | Replicate:3
PE_(32:1)	0	0.213276409	0.222070599	0.20444661	0.250931665	0.240778332	0.187420943	0.297076631	0.349799744	0.308327365
PE_(32:2)	0	0.538111654	0.543252812	0.525006262	0.612678723	0.577156982	0.495485451	0.437204547	0.459162065	0.411103778
PE_(34:1)	0	0.144757584	0.151616987	0.135784191	0.162962302	0.146185043	0.115896705	0.148333956	0.155132689	0.147414284
PE_(34:2)	0	0.292285491	0.278620632	0.263338387	0.344018988	0.308976599	0.268697549	0.22464156	0.211606061	0.204235539
PE_(36:1)	0	0.031090807	0.030556418	0.028334764	0.031523946	0.028605243	0.022156137	0.034917874	0.038214952	0.034698892
PE_(36:2)	0	0.103578863	0.101188183	0.092808515	0.128684416	0.169321634	0.093691	0.098126893	0.076156003	0.067123301
PC_(28:1)	0	0.098712744	0.106333834	0.092871078	0.138744216	0.122373396	0.120962497	0.094846558	0.098410761	0.087467723
PC_(30:1)	0	0.117496035	0.125758476	0.105958257	0.134306713	0.110458806	0.101342071	0.214260203	0.245950709	0.17935015
PC_(30:2)	0	0.045570283	0.049240054	0.047699065	0.070137645	0.058955199	0.052850628	0.068533207	0.073541438	0.06178775
PC_(32:1)	0	0.203062877	0.220610167	0.200993009	0.226342159	0.184110594	0.174916997	0.289978068	0.313179233	0.274730221
PC_(32:2)	0	1.170464959	1.246431758	1.204106606	1.595365724	1.320692309	1.279042226	1.205339489	1.314667576	1.187002393
PC_(34:1)	0	0.068362173	0.064846418	0.063532718	0.079023475	0.063466172	0.059496166	0.069913634	0.075685009	0.073746631
PC_(34:2)	0	0.528856665	0.605288516	0.555142484	0.790028983	0.646997488	0.570770225	0.484448021	0.491409979	0.447194154
PC_(36:1)	0	0.005630952	0.005515127	0.005505952	0.007940455	0.006158653	0.005823518	0.005672185	0.005257069	0.00514105
PC_(36:2)	0	0.029309574	0.027630282	0.027884154	0.033728701	0.025054793	0.022680225	0.051998066	0.051160199	0.048423948
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	m/z [M+H]+	Average Rt(min)
PE_(32:1)	690.50623	4.229
PE_(32:2)	688.49121	3.639
PE_(34:1)	718.54077	4.784
PE_(34:2)	716.52209	4.303
PE_(36:1)	746.5697	4.751
PE_(36:2)	744.55377	4.265
PC_(28:1)	676.49152	2.281
PC_(30:1)	704.52283	3.296
PC_(30:2)	702.5069	2.494
PC_(32:1)	732.55371	4.075
PC_(32:2)	730.5379	3.44
PC_(34:1)	760.58527	4.666
PC_(34:2)	758.56903	4.156
PC_(36:1)	788.61652	5.136
PC_(36:2)	786.60046	4.709
METABOLITES_END
#END