#METABOLOMICS WORKBENCH danielpardo88_20240930_065556 DATATRACK_ID:5250 STUDY_ID:ST003511 ANALYSIS_ID:AN005765 PROJECT_ID:PR002156
VERSION             	1
CREATED_ON             	October 3, 2024, 10:01 pm
#PROJECT
PR:PROJECT_TITLE                 	Lipidomic analysis of Malassezia globosa at different growth stages and the
PR:PROJECT_TITLE                 	dynamics of uptake and secreted lipids with growth media
PR:PROJECT_TYPE                  	Lipidomics
PR:PROJECT_SUMMARY               	Malassezia is one of the most abundant genera found on human skin; specifically,
PR:PROJECT_SUMMARY               	M. globosa is one of the yeast species dominant in this organ as it has been
PR:PROJECT_SUMMARY               	associated with several skin diseases. Malassezia cannot synthesize fatty acids.
PR:PROJECT_SUMMARY               	In response, the yeast cell intakes external fatty acids from the host or the
PR:PROJECT_SUMMARY               	growth media for survival. Several studies have focused on investigating the
PR:PROJECT_SUMMARY               	identity of lipids and enzymes in M. globosa to understand its lipid metabolism
PR:PROJECT_SUMMARY               	and the biology of the yeast cell-host interaction. In this work, we performed a
PR:PROJECT_SUMMARY               	supernatant lipidomic analysis on the mDixon media and the supernatant and on
PR:PROJECT_SUMMARY               	the M. globosa at early and late stationary phase (72h and 90h, respectively) to
PR:PROJECT_SUMMARY               	determine the lipid dynamics (lipids consumed vs. lipids secreted) between the
PR:PROJECT_SUMMARY               	growth media and the two stages of growth. We were able to identify 87 lipids
PR:PROJECT_SUMMARY               	within 17 classes of lipids; during the analysis, the increment of several
PR:PROJECT_SUMMARY               	lipids increased throughout time concerning the growth media, suggesting a
PR:PROJECT_SUMMARY               	secretion pattern from the cell to the media; some lipids found in this group
PR:PROJECT_SUMMARY               	were conjugated Sterols (ST) such as Glycochenodeoxycholic acid (GCDCA),
PR:PROJECT_SUMMARY               	Glycerophospholipids (GP), specifically phosphocholine's (PCs), Cardiolipins
PR:PROJECT_SUMMARY               	(CL), in particular those with chains of (47 to 54 carbons) and Sphingolipids
PR:PROJECT_SUMMARY               	(SP) such as Cer-PI which might have some role in pathogenicity. Likewise, the
PR:PROJECT_SUMMARY               	increment of some lipids decreased, but some only reduced at the late stationary
PR:PROJECT_SUMMARY               	phase (90h) only when the nutrients available was minimal. Finally, we observed
PR:PROJECT_SUMMARY               	a third pattern in which the amount of some lipids decreased throughout time
PR:PROJECT_SUMMARY               	(starting in the early stationary phase and finishing in the late stationary
PR:PROJECT_SUMMARY               	phase), hinting at a distinctive consumption pattern. The principal lipids
PR:PROJECT_SUMMARY               	consumed wereSterols (ST) bile acids, cholic acid, and its derivates, some
PR:PROJECT_SUMMARY               	phosphocholines (PCs), Fatty acyls (FA), and cardiolipins (CL). The consumption
PR:PROJECT_SUMMARY               	of these lipids was associated with different metabolic roles of the lipids in
PR:PROJECT_SUMMARY               	the cell as it lacks production of these lipids in M. globosa.
PR:INSTITUTE                     	Universidad de los Andes
PR:LAST_NAME                     	Cala
PR:FIRST_NAME                    	Monica
PR:ADDRESS                       	CALLE 46 N 3 35 Bogota-Colombia
PR:EMAIL                         	mp.cala10@uniandes.edu.co
PR:PHONE                         	+573164316037
#STUDY
ST:STUDY_TITLE                   	Lipidomic analysis of Malassezia globosa at different growth stages and the
ST:STUDY_TITLE                   	dynamics of uptake and secreted lipids with growth media
ST:STUDY_SUMMARY                 	Malassezia is one of the most abundant genera found on human skin; specifically,
ST:STUDY_SUMMARY                 	M. globosa is one of the yeast species dominant in this organ as it has been
ST:STUDY_SUMMARY                 	associated with several skin diseases. Malassezia cannot synthesize fatty acids.
ST:STUDY_SUMMARY                 	In response, the yeast cell intakes external fatty acids from the host or the
ST:STUDY_SUMMARY                 	growth media for survival. Several studies have focused on investigating the
ST:STUDY_SUMMARY                 	identity of lipids and enzymes in M. globosa to understand its lipid metabolism
ST:STUDY_SUMMARY                 	and the biology of the yeast cell-host interaction. In this work, we performed a
ST:STUDY_SUMMARY                 	supernatant lipidomic analysis on the mDixon media and the supernatant and on
ST:STUDY_SUMMARY                 	the M. globosa at early and late stationary phase (72h and 90h, respectively) to
ST:STUDY_SUMMARY                 	determine the lipid dynamics (lipids consumed vs. lipids secreted) between the
ST:STUDY_SUMMARY                 	growth media and the two stages of growth. We were able to identify 87 lipids
ST:STUDY_SUMMARY                 	within 17 classes of lipids; during the analysis, the increment of several
ST:STUDY_SUMMARY                 	lipids increased throughout time concerning the growth media, suggesting a
ST:STUDY_SUMMARY                 	secretion pattern from the cell to the media; some lipids found in this group
ST:STUDY_SUMMARY                 	were conjugated Sterols (ST) such as Glycochenodeoxycholic acid (GCDCA),
ST:STUDY_SUMMARY                 	Glycerophospholipids (GP), specifically phosphocholine's (PCs), Cardiolipins
ST:STUDY_SUMMARY                 	(CL), in particular those with chains of (47 to 54 carbons) and Sphingolipids
ST:STUDY_SUMMARY                 	(SP) such as Cer-PI which might have some role in pathogenicity. Likewise, the
ST:STUDY_SUMMARY                 	increment of some lipids decreased, but some only reduced at the late stationary
ST:STUDY_SUMMARY                 	phase (90h) only when the nutrients available was minimal. Finally, we observed
ST:STUDY_SUMMARY                 	a third pattern in which the amount of some lipids decreased throughout time
ST:STUDY_SUMMARY                 	(starting in the early stationary phase and finishing in the late stationary
ST:STUDY_SUMMARY                 	phase), hinting at a distinctive consumption pattern. The principal lipids
ST:STUDY_SUMMARY                 	consumed wereSterols (ST) bile acids, cholic acid, and its derivates, some
ST:STUDY_SUMMARY                 	phosphocholines (PCs), Fatty acyls (FA), and cardiolipins (CL). The consumption
ST:STUDY_SUMMARY                 	of these lipids was associated with different metabolic roles of the lipids in
ST:STUDY_SUMMARY                 	the cell as it lacks production of these lipids in M. globosa.
ST:INSTITUTE                     	Universidad de los Andes, Colombia
ST:LAST_NAME                     	Cala
ST:FIRST_NAME                    	Mónica
ST:ADDRESS                       	CALLE 46 N 3 35
ST:EMAIL                         	mp.cala10@uniandes.edu.co
ST:PHONE                         	+573164316037
ST:NUM_GROUPS                    	6
#SUBJECT
SU:SUBJECT_TYPE                  	Fungi
SU:SUBJECT_SPECIES               	Malassezia globosa
SU:TAXONOMY_ID                   	76773
#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           	-	Sg1_1	Sample source:mDixon(media) | Sample_type:mDixon | Time of collection:na	RAW_FILE_NAME=GL(-)-G1_001.mzXML
SUBJECT_SAMPLE_FACTORS           	-	Sg1_2	Sample source:mDixon(media) | Sample_type:mDixon | Time of collection:na	RAW_FILE_NAME=GL(-)-G1_002.mzXML
SUBJECT_SAMPLE_FACTORS           	-	Sg1_3	Sample source:mDixon(media) | Sample_type:mDixon | Time of collection:na	RAW_FILE_NAME=GL(-)-G1_003.mzXML
SUBJECT_SAMPLE_FACTORS           	-	Sg2_4	Sample source:Yeast | Sample_type:Mglobosa | Time of collection:72h	RAW_FILE_NAME=GL(-)-G2_004.mzXML
SUBJECT_SAMPLE_FACTORS           	-	Sg2_5	Sample source:Yeast | Sample_type:Mglobosa | Time of collection:72h	RAW_FILE_NAME=GL(-)-G2_005.mzXML
SUBJECT_SAMPLE_FACTORS           	-	Sg2_6	Sample source:Yeast | Sample_type:Mglobosa | Time of collection:72h	RAW_FILE_NAME=GL(-)-G2_006.mzXML
SUBJECT_SAMPLE_FACTORS           	-	Sg3_7	Sample source:Yeast | Sample_type:Mglobosa | Time of collection:90h	RAW_FILE_NAME=GL(-)-G3_007.mzXML
SUBJECT_SAMPLE_FACTORS           	-	Sg3_8	Sample source:Yeast | Sample_type:Mglobosa | Time of collection:90h	RAW_FILE_NAME=GL(-)-G3_008.mzXML
SUBJECT_SAMPLE_FACTORS           	-	Sg3_9	Sample source:Yeast | Sample_type:Mglobosa | Time of collection:90h	RAW_FILE_NAME=GL(-)-G3_009.mzXML
SUBJECT_SAMPLE_FACTORS           	-	Sg4_10	Sample source:Yeast | Sample_type:Supernatant | Time of collection:72h	RAW_FILE_NAME=GL(-)-G4_010.mzXML
SUBJECT_SAMPLE_FACTORS           	-	Sg4_11	Sample source:Yeast | Sample_type:Supernatant | Time of collection:72h	RAW_FILE_NAME=GL(-)-G4_011.mzXML
SUBJECT_SAMPLE_FACTORS           	-	Sg4_12	Sample source:Yeast | Sample_type:Supernatant | Time of collection:72h	RAW_FILE_NAME=GL(-)-G4_012.mzXML
SUBJECT_SAMPLE_FACTORS           	-	Sg4_13	Sample source:Yeast | Sample_type:Supernatant | Time of collection:72h	RAW_FILE_NAME=GL(-)-G4_013.mzXML
SUBJECT_SAMPLE_FACTORS           	-	Sg4_14	Sample source:Yeast | Sample_type:Supernatant | Time of collection:72h	RAW_FILE_NAME=GL(-)-G4_014.mzXML
SUBJECT_SAMPLE_FACTORS           	-	Sg4_15	Sample source:Yeast | Sample_type:Supernatant | Time of collection:72h	RAW_FILE_NAME=GL(-)-G4_015.mzXML
SUBJECT_SAMPLE_FACTORS           	-	Sg5_16	Sample source:Yeast | Sample_type:Supernatant | Time of collection:90h	RAW_FILE_NAME=GL(-)-G5_016.mzXML
SUBJECT_SAMPLE_FACTORS           	-	Sg5_17	Sample source:Yeast | Sample_type:Supernatant | Time of collection:90h	RAW_FILE_NAME=GL(-)-G5_017.mzXML
SUBJECT_SAMPLE_FACTORS           	-	Sg5_18	Sample source:Yeast | Sample_type:Supernatant | Time of collection:90h	RAW_FILE_NAME=GL(-)-G5_018.mzXML
SUBJECT_SAMPLE_FACTORS           	-	Sg5_19	Sample source:Yeast | Sample_type:Supernatant | Time of collection:90h	RAW_FILE_NAME=GL(-)-G5_019.mzXML
SUBJECT_SAMPLE_FACTORS           	-	Sg5_20	Sample source:Yeast | Sample_type:Supernatant | Time of collection:90h	RAW_FILE_NAME=GL(-)-G5_020.mzXML
SUBJECT_SAMPLE_FACTORS           	-	Sg5_21	Sample source:Yeast | Sample_type:Supernatant | Time of collection:90h	RAW_FILE_NAME=GL(-)-G5_021.mzXML
SUBJECT_SAMPLE_FACTORS           	-	QC_10	Sample source:Yeast | Sample_type:Quality control | Time of collection:na	RAW_FILE_NAME=GL(-)-QC_010.mzXML
SUBJECT_SAMPLE_FACTORS           	-	QC_11	Sample source:Yeast | Sample_type:Quality control | Time of collection:na	RAW_FILE_NAME=GL(-)-QC_011.mzXML
SUBJECT_SAMPLE_FACTORS           	-	QC_12	Sample source:Yeast | Sample_type:Quality control | Time of collection:na	RAW_FILE_NAME=GL(-)-QC_012.mzXML
SUBJECT_SAMPLE_FACTORS           	-	QC_13	Sample source:Yeast | Sample_type:Quality control | Time of collection:na	RAW_FILE_NAME=GL(-)-QC_013.mzXML
SUBJECT_SAMPLE_FACTORS           	-	QC_14	Sample source:Yeast | Sample_type:Quality control | Time of collection:na	RAW_FILE_NAME=GL(-)-QC_014.mzXML
#COLLECTION
CO:COLLECTION_SUMMARY            	The reference strain Malassezia globosa CBS 7966 (Westerdijk Institute, Utrecht,
CO:COLLECTION_SUMMARY            	The Netherlands) was used for the whole study. A frozen stock was reactivated
CO:COLLECTION_SUMMARY            	and precultured at 33°C for seven days in modified Dixon (mDixon) agar [36 g
CO:COLLECTION_SUMMARY            	L-1 mycosel agar [BD, USA], 20 g L-1 Ox Bile [Sigma Aldrich, USA], 36 g L-1 malt
CO:COLLECTION_SUMMARY            	extract [Oxoid, UK], 0.02% glycerol [Sigma Aldrich, USA], 0.02% oleic acid
CO:COLLECTION_SUMMARY            	[Sigma Aldrich, USA], and 0.1% Tween 40 [Sigma Aldrich, USA]]. Then, one colony
CO:COLLECTION_SUMMARY            	was transferred to a new mDixon agar plate and was incubated for five days at
CO:COLLECTION_SUMMARY            	33°C. From this plate, yeasts were suspended in 3 mL inoculum in water plus
CO:COLLECTION_SUMMARY            	0.1% Tween 80 [Sigma Aldrich, USA] to a top standard of 2 on the McFarland scale
CO:COLLECTION_SUMMARY            	and were used to inoculate 27 mL of mDixon broth [36 g L-1 malt extract [Oxoid,
CO:COLLECTION_SUMMARY            	UK], 6 g L-1 peptone [BD, USA], 20 g L-1 Ox bile [Sigma Aldrich, USA, 0.02%
CO:COLLECTION_SUMMARY            	glycerol [Sigma Aldrich, USA], 0.02% oleic acid [Sigma Aldrich, USA], and 0.1%
CO:COLLECTION_SUMMARY            	Tween 40 [Sigma Aldrich, USA] for 96 hours at 33°C and 180 rpm. An aliquot of
CO:COLLECTION_SUMMARY            	300 mL was used to inoculate 29.7 mL of fresh mDixon broth and incubated at
CO:COLLECTION_SUMMARY            	33°C and 180 rpm for 72 h and 90 h to reach the early stationary and stationary
CO:COLLECTION_SUMMARY            	phase. (doi:10.1007/978-3-642-03616-3_2; doi:10.3389/fcimb.2020.00338).
CO:SAMPLE_TYPE                   	Yeast cells
#TREATMENT
TR:TREATMENT_SUMMARY             	No treatments were applied to the samples.
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	Samples of M. globosa were collected at different growth stages, then
SP:SAMPLEPREP_SUMMARY            	centrifuged at 4,500 rpm for 10 minutes, and the supernatants were collected.
SP:SAMPLEPREP_SUMMARY            	Subsequently, 5 mL of isopropanol was added to the supernatant, followed by
SP:SAMPLEPREP_SUMMARY            	centrifugation at 4,500 rpm for another 10 minutes. Lipid extraction was
SP:SAMPLEPREP_SUMMARY            	performed according to Bligh and Dyer lipid extraction with some modifications
SP:SAMPLEPREP_SUMMARY            	(doi:10.1038/nprot.2016.040; doi:10.1016/j.jchromb.2017.06.045;
SP:SAMPLEPREP_SUMMARY            	doi:10.1021/acs.analchem.8b02839). 2mL of a citric acid buffer [0.1 M sodium
SP:SAMPLEPREP_SUMMARY            	citrate tribasic dihydrate, 1 M sodium chloride, pH 3.6], 2 mL of MeOH, and 4 mL
SP:SAMPLEPREP_SUMMARY            	of chloroform were added to 8 mL of supernatant collected previously. The
SP:SAMPLEPREP_SUMMARY            	mixture was homogenized with vortex for 15 min and sonicated for 30 min. The
SP:SAMPLEPREP_SUMMARY            	extracted lipids' organic phase was collected and dried on a Speed Vac. Then,
SP:SAMPLEPREP_SUMMARY            	the dry extract was re-dissolved in 1 mL of ACN containing 0.1% NH3·H2O (v/v),
SP:SAMPLEPREP_SUMMARY            	followed by strong anion-exchange solid-phase extraction using Strata SAX
SP:SAMPLEPREP_SUMMARY            	SPE-cartridge (55 uM, 70 A, 100 mg, 1 mL Phenomenex) which was pre-conditioned
SP:SAMPLEPREP_SUMMARY            	with 3 mL ACN. After sampling 1 mL of the lipid extract, the cartridge was
SP:SAMPLEPREP_SUMMARY            	washed with 3 mL acetone/H2O (1/9, v/v), 3mL acetone, and eluted with 3 mL
SP:SAMPLEPREP_SUMMARY            	formic acid/acetone (1/99, v/v) followed by evaporation using a Speed Vac.
SP:SAMPLEPREP_SUMMARY            	Samples were stored at -80°C for one week and dissolved in 1 mL of MeOH for
SP:SAMPLEPREP_SUMMARY            	further analysis (doi:10.1038/nprot.2016.040; doi:10.1016/j.jchromb.2017.06.045;
SP:SAMPLEPREP_SUMMARY            	doi:10.1021/acs.analchem.8b02839).
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_SUMMARY        	Chromatographic analysis was carried out at 65°C and constant flow 0.6 mL/min
CH:CHROMATOGRAPHY_SUMMARY        	using gradient elution with phase A (60:40 ACN: type I water with 10 mM of
CH:CHROMATOGRAPHY_SUMMARY        	ammonium formate and 0.1% v/v of formic acid) and mobile phase B (90:10
CH:CHROMATOGRAPHY_SUMMARY        	Isopropanol: Acetonitrile with 10 mM of ammonium formate and 0.1% v/v of formic
CH:CHROMATOGRAPHY_SUMMARY        	acid). The elution gradient was 0 min 15% (B), 0–4 min 30% (B), 4–5 min 48%
CH:CHROMATOGRAPHY_SUMMARY        	(B), 5–22 min 82% (B), 22–23 min 95% (B), 23–25 min 95% (B), 25–26 min
CH:CHROMATOGRAPHY_SUMMARY        	15% (B), and 26–31 min 15% (B).
CH:CHROMATOGRAPHY_TYPE           	Reversed phase
CH:INSTRUMENT_NAME               	Agilent 1260 Infinity LC System
CH:COLUMN_NAME                   	Agilent InfinityLab Poroshell 120 EC-C18 (100 x 3mm,2.7um)
CH:SOLVENT_A                     	60% acetonitrile/40% water; 10 mM ammonium formate; 0.1% formic acid
CH:SOLVENT_B                     	90% isopropanol/10% acetonitrile; 10 mM ammonium formate; 0.1% formic acid
CH:FLOW_GRADIENT                 	The elution gradient was 0 min 15% (B), 0–4 min 30% (B), 4–5 min 48% (B),
CH:FLOW_GRADIENT                 	5–22 min 82% (B), 22–23 min 95% (B), 23–25 min 95% (B), 25–26 min 15%
CH:FLOW_GRADIENT                 	(B), and 26–31 min 15% (B)
CH:FLOW_RATE                     	0.6 mL/min
CH:COLUMN_TEMPERATURE            	65 °C
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Agilent 6545 QTOF
MS:INSTRUMENT_TYPE               	QTOF
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	NEGATIVE
MS:MS_COMMENTS                   	Mass spectrometry detection was performed in negative ionization mode in a full
MS:MS_COMMENTS                   	scan from 100 m/z to 1200 m/z. The mass correction was performed during the
MS:MS_COMMENTS                   	analysis with reference masses: m/z 121.0509 (C5H4N4) y m/z 922.0098
MS:MS_COMMENTS                   	(C18H18O6N3P3F24).
MS:MS_RESULTS_FILE               	ST003511_AN005765_Results.txt	UNITS: Peak area	Has m/z:Yes	Has RT:Yes	RT units:Minutes
#END