#METABOLOMICS WORKBENCH jana_carpenter_20230907_104020 DATATRACK_ID:4295 STUDY_ID:ST002854 ANALYSIS_ID:AN004676 PROJECT_ID:PR001787
VERSION             	1
CREATED_ON             	September 8, 2023, 5:59 pm
#PROJECT
PR:PROJECT_TITLE                 	HILIC-IM-MS for Simultaneous Lipid and Metabolite Profiling of Microorganisms
PR:PROJECT_TYPE                  	LC-MS quantitative analysis
PR:PROJECT_SUMMARY               	Progress in the ion mobility mass spectrometry (IM-MS) field has significantly
PR:PROJECT_SUMMARY               	increased our ability to make small molecule and lipid identifications, making
PR:PROJECT_SUMMARY               	it an attractive approach for untargeted multi-omics experiments. The
PR:PROJECT_SUMMARY               	dimensionality of collision cross section (CCS) coupled with tandem mass
PR:PROJECT_SUMMARY               	spectrometry (MS/MS) for feature annotation has become a useful tool for high
PR:PROJECT_SUMMARY               	confidence structural elucidation in complex mixtures in the absence of
PR:PROJECT_SUMMARY               	authentic standards. A comprehensive method for feature identification of small
PR:PROJECT_SUMMARY               	organisms has remained limited to exploring genetic markers and protein
PR:PROJECT_SUMMARY               	signatures, however these methods for identification only scratch the surface of
PR:PROJECT_SUMMARY               	effective methods for bacterial classification. Multi-omic methods that include
PR:PROJECT_SUMMARY               	the metabolome and lipidome have grown in popularity due to the increased
PR:PROJECT_SUMMARY               	capacity for organism specific information. We have achieved species-level
PR:PROJECT_SUMMARY               	identification of Enterococcus faecium, Staphylococcus aureus, Acinetobacter
PR:PROJECT_SUMMARY               	baumannii, and Pseudomonas aeruginosa using a modern single-phase extraction
PR:PROJECT_SUMMARY               	method with hydrophilic interaction liquid chromatography (HILIC) coupled to
PR:PROJECT_SUMMARY               	traveling wave ion mobility mass spectrometry (TWIMS). To test the robustness of
PR:PROJECT_SUMMARY               	this optimized workflow, we included internal standards as a metric for
PR:PROJECT_SUMMARY               	efficiency of the extraction, and well known calibrants for validation for our
PR:PROJECT_SUMMARY               	CCS calibration method. We observed significant differences in metabolite
PR:PROJECT_SUMMARY               	profiles at the strain level using multi-variate statistics, primarily including
PR:PROJECT_SUMMARY               	quorum sensing metabolites in Gram-negative strains, and energy production
PR:PROJECT_SUMMARY               	metabolites in the Gram-positive strains. Lipid profiles showed staggering
PR:PROJECT_SUMMARY               	differences in acyl tail compositions that effectively categorized the microbes,
PR:PROJECT_SUMMARY               	including several classes of phospholipids and glycolipids. We have demonstrated
PR:PROJECT_SUMMARY               	a powerful workflow using multi-dimensional techniques for bacterial speciation
PR:PROJECT_SUMMARY               	in a single injection.
PR:INSTITUTE                     	Univerisity of Georgia
PR:DEPARTMENT                    	Chemistry
PR:LABORATORY                    	Dr. Kelly M. Hines
PR:LAST_NAME                     	Carpenter
PR:FIRST_NAME                    	Jana
PR:ADDRESS                       	302 E Campus Rd., Athens, Georgia, 30602, USA
PR:EMAIL                         	kelly.hines@uga.edu
PR:PHONE                         	706-542-1966
#STUDY
ST:STUDY_TITLE                   	HILIC-IM-MS for Simultaneous Lipid and Metabolite Profiling of Microorganisms
ST:STUDY_SUMMARY                 	Progress in the ion mobility mass spectrometry (IM-MS) field has significantly
ST:STUDY_SUMMARY                 	increased our ability to make small molecule and lipid identifications, making
ST:STUDY_SUMMARY                 	it an attractive approach for untargeted multi-omics experiments. The
ST:STUDY_SUMMARY                 	dimensionality of collision cross section (CCS) coupled with tandem mass
ST:STUDY_SUMMARY                 	spectrometry (MS/MS) for feature annotation has become a useful tool for high
ST:STUDY_SUMMARY                 	confidence structural elucidation in complex mixtures in the absence of
ST:STUDY_SUMMARY                 	authentic standards. A comprehensive method for feature identification of small
ST:STUDY_SUMMARY                 	organisms has remained limited to exploring genetic markers and protein
ST:STUDY_SUMMARY                 	signatures, however these methods for identification only scratch the surface of
ST:STUDY_SUMMARY                 	effective methods for bacterial classification. Multi-omic methods that include
ST:STUDY_SUMMARY                 	the metabolome and lipidome have grown in popularity due to the increased
ST:STUDY_SUMMARY                 	capacity for organism specific information. We have achieved species-level
ST:STUDY_SUMMARY                 	identification of Enterococcus faecium, Staphylococcus aureus, Acinetobacter
ST:STUDY_SUMMARY                 	baumannii, and Pseudomonas aeruginosa using a modern single-phase extraction
ST:STUDY_SUMMARY                 	method with hydrophilic interaction liquid chromatography (HILIC) coupled to
ST:STUDY_SUMMARY                 	traveling wave ion mobility mass spectrometry (TWIMS). To test the robustness of
ST:STUDY_SUMMARY                 	this optimized workflow, we included internal standards as a metric for
ST:STUDY_SUMMARY                 	efficiency of the extraction, and well known calibrants for validation for our
ST:STUDY_SUMMARY                 	CCS calibration method. We observed significant differences in metabolite
ST:STUDY_SUMMARY                 	profiles at the strain level using multi-variate statistics, primarily including
ST:STUDY_SUMMARY                 	quorum sensing metabolites in Gram-negative strains, and energy production
ST:STUDY_SUMMARY                 	metabolites in the Gram-positive strains. Lipid profiles showed staggering
ST:STUDY_SUMMARY                 	differences in acyl tail compositions that effectively categorized the microbes,
ST:STUDY_SUMMARY                 	including several classes of phospholipids and glycolipids. We have demonstrated
ST:STUDY_SUMMARY                 	a powerful workflow using multi-dimensional techniques for bacterial speciation
ST:STUDY_SUMMARY                 	in a single injection.
ST:INSTITUTE                     	Univerisity of Georgia
ST:LAST_NAME                     	Carpenter
ST:FIRST_NAME                    	Jana
ST:ADDRESS                       	302 E Campus Rd., Athens, Georgia, 30602, USA
ST:EMAIL                         	kelly.hines@uga.edu
ST:PHONE                         	706-542-1966
#SUBJECT
SU:SUBJECT_TYPE                  	Bacteria
SU:SUBJECT_SPECIES               	Staphylococcus aureus/ Acinetobacter baumannii/ Enterococcus faecium/
SU:SUBJECT_SPECIES               	Pseudomonas aeruginosa
#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           	AB-A-1	P_AB-A-1	Species:Acinetobacter baumannii	Strain=NR 52187; Factor=Positive Mode; RAW_FILE_NAME=P_AB-A-1.mzML
SUBJECT_SAMPLE_FACTORS           	AB-A-2	P_AB-A-2	Species:Acinetobacter baumannii	Strain=NR 52187; Factor=Positive Mode; RAW_FILE_NAME=P_AB-A-2.mzML
SUBJECT_SAMPLE_FACTORS           	AB-A-3	P_AB-A-3	Species:Acinetobacter baumannii	Strain=NR 52187; Factor=Positive Mode; RAW_FILE_NAME=P_AB-A-3.mzML
SUBJECT_SAMPLE_FACTORS           	AB-A-4	P_AB-A-4	Species:Acinetobacter baumannii	Strain=NR 52187; Factor=Positive Mode; RAW_FILE_NAME=P_AB-A-4.mzML
SUBJECT_SAMPLE_FACTORS           	AB-A-5	P_AB-A-5	Species:Acinetobacter baumannii	Strain=NR 52187; Factor=Positive Mode; RAW_FILE_NAME=P_AB-A-5.mzML
SUBJECT_SAMPLE_FACTORS           	AB-B-1	P_AB-B-1	Species:Acinetobacter baumannii	Strain=NR 52189; Factor=Positive Mode; RAW_FILE_NAME=P_AB-B-1.mzML
SUBJECT_SAMPLE_FACTORS           	AB-B-2	P_AB-B-2	Species:Acinetobacter baumannii	Strain=NR 52189; Factor=Positive Mode; RAW_FILE_NAME=P_AB-B-2.mzML
SUBJECT_SAMPLE_FACTORS           	AB-B-3	P_AB-B-3	Species:Acinetobacter baumannii	Strain=NR 52189; Factor=Positive Mode; RAW_FILE_NAME=P_AB-B-3.mzML
SUBJECT_SAMPLE_FACTORS           	AB-B-4	P_AB-B-4	Species:Acinetobacter baumannii	Strain=NR 52189; Factor=Positive Mode; RAW_FILE_NAME=P_AB-B-4.mzML
SUBJECT_SAMPLE_FACTORS           	AB-B-5	P_AB-B-5	Species:Acinetobacter baumannii	Strain=NR 52189; Factor=Positive Mode; RAW_FILE_NAME=P_AB-B-5.mzML
SUBJECT_SAMPLE_FACTORS           	AB-C-1	P_AB-C-1	Species:Acinetobacter baumannii	Strain=NR 52190; Factor=Positive Mode; RAW_FILE_NAME=P_AB-C-1.mzML
SUBJECT_SAMPLE_FACTORS           	AB-C-2	P_AB-C-2	Species:Acinetobacter baumannii	Strain=NR 52190; Factor=Positive Mode; RAW_FILE_NAME=P_AB-C-2.mzML
SUBJECT_SAMPLE_FACTORS           	AB-C-3	P_AB-C-3	Species:Acinetobacter baumannii	Strain=NR 52190; Factor=Positive Mode; RAW_FILE_NAME=P_AB-C-3.mzML
SUBJECT_SAMPLE_FACTORS           	AB-C-4	P_AB-C-4	Species:Acinetobacter baumannii	Strain=NR 52190; Factor=Positive Mode; RAW_FILE_NAME=P_AB-C-4.mzML
SUBJECT_SAMPLE_FACTORS           	AB-C-5	P_AB-C-5	Species:Acinetobacter baumannii	Strain=NR 52190; Factor=Positive Mode; RAW_FILE_NAME=P_AB-C-5.mzML
SUBJECT_SAMPLE_FACTORS           	EF-A-1	P_EF-A-1	Species:Enterococcus faecium	Strain=ATCC 700221; Factor=Positive Mode; RAW_FILE_NAME=P_EF-A-1.mzML
SUBJECT_SAMPLE_FACTORS           	EF-A-2	P_EF-A-2	Species:Enterococcus faecium	Strain=ATCC 700221; Factor=Positive Mode; RAW_FILE_NAME=P_EF-A-2.mzML
SUBJECT_SAMPLE_FACTORS           	EF-A-3	P_EF-A-3	Species:Enterococcus faecium	Strain=ATCC 700221; Factor=Positive Mode; RAW_FILE_NAME=P_EF-A-3.mzML
SUBJECT_SAMPLE_FACTORS           	EF-A-4	P_EF-A-4	Species:Enterococcus faecium	Strain=ATCC 700221; Factor=Positive Mode; RAW_FILE_NAME=P_EF-A-4.mzML
SUBJECT_SAMPLE_FACTORS           	EF-A-5	P_EF-A-5	Species:Enterococcus faecium	Strain=ATCC 700221; Factor=Positive Mode; RAW_FILE_NAME=P_EF-A-5.mzML
SUBJECT_SAMPLE_FACTORS           	EF-B-1	P_EF-B-1	Species:Enterococcus faecium	Strain=HM 959; Factor=Positive Mode; RAW_FILE_NAME=P_EF-B-1.mzML
SUBJECT_SAMPLE_FACTORS           	EF-B-2	P_EF-B-2	Species:Enterococcus faecium	Strain=HM 959; Factor=Positive Mode; RAW_FILE_NAME=P_EF-B-2.mzML
SUBJECT_SAMPLE_FACTORS           	EF-B-3	P_EF-B-3	Species:Enterococcus faecium	Strain=HM 959; Factor=Positive Mode; RAW_FILE_NAME=P_EF-B-3.mzML
SUBJECT_SAMPLE_FACTORS           	EF-B-4	P_EF-B-4	Species:Enterococcus faecium	Strain=HM 959; Factor=Positive Mode; RAW_FILE_NAME=P_EF-B-4.mzML
SUBJECT_SAMPLE_FACTORS           	EF-B-5	P_EF-B-5	Species:Enterococcus faecium	Strain=HM 959; Factor=Positive Mode; RAW_FILE_NAME=P_EF-B-5.mzML
SUBJECT_SAMPLE_FACTORS           	EF-C-1	P_EF-C-1	Species:Enterococcus faecium	Strain=HM 952; Factor=Positive Mode; RAW_FILE_NAME=P_EF-C-1.mzML
SUBJECT_SAMPLE_FACTORS           	EF-C-2	P_EF-C-2	Species:Enterococcus faecium	Strain=HM 952; Factor=Positive Mode; RAW_FILE_NAME=P_EF-C-2.mzML
SUBJECT_SAMPLE_FACTORS           	EF-C-3	P_EF-C-3	Species:Enterococcus faecium	Strain=HM 952; Factor=Positive Mode; RAW_FILE_NAME=P_EF-C-3.mzML
SUBJECT_SAMPLE_FACTORS           	EF-C-4	P_EF-C-4	Species:Enterococcus faecium	Strain=HM 952; Factor=Positive Mode; RAW_FILE_NAME=P_EF-C-4.mzML
SUBJECT_SAMPLE_FACTORS           	EF-C-5	P_EF-C-5	Species:Enterococcus faecium	Strain=HM 952; Factor=Positive Mode; RAW_FILE_NAME=P_EF-C-5.mzML
SUBJECT_SAMPLE_FACTORS           	PA-A-1	P_PA-A-1	Species:Pseudomonas aeruginosa	Strain=NR 51588; Factor=Positive Mode; RAW_FILE_NAME=P_PA-A-1.mzML
SUBJECT_SAMPLE_FACTORS           	PA-A-2	P_PA-A-2	Species:Pseudomonas aeruginosa	Strain=NR 51588; Factor=Positive Mode; RAW_FILE_NAME=P_PA-A-2.mzML
SUBJECT_SAMPLE_FACTORS           	PA-A-3	P_PA-A-3	Species:Pseudomonas aeruginosa	Strain=NR 51588; Factor=Positive Mode; RAW_FILE_NAME=P_PA-A-3.mzML
SUBJECT_SAMPLE_FACTORS           	PA-A-4	P_PA-A-4	Species:Pseudomonas aeruginosa	Strain=NR 51588; Factor=Positive Mode; RAW_FILE_NAME=P_PA-A-4.mzML
SUBJECT_SAMPLE_FACTORS           	PA-A-5	P_PA-A-5	Species:Pseudomonas aeruginosa	Strain=NR 51588; Factor=Positive Mode; RAW_FILE_NAME=P_PA-A-5.mzML
SUBJECT_SAMPLE_FACTORS           	PA-B-1	P_PA-B-1	Species:Pseudomonas aeruginosa	Strain=NR 51589; Factor=Positive Mode; RAW_FILE_NAME=P_PA-B-1.mzML
SUBJECT_SAMPLE_FACTORS           	PA-B-2	P_PA-B-2	Species:Pseudomonas aeruginosa	Strain=NR 51589; Factor=Positive Mode; RAW_FILE_NAME=P_PA-B-2.mzML
SUBJECT_SAMPLE_FACTORS           	PA-B-3	P_PA-B-3	Species:Pseudomonas aeruginosa	Strain=NR 51589; Factor=Positive Mode; RAW_FILE_NAME=P_PA-B-3.mzML
SUBJECT_SAMPLE_FACTORS           	PA-B-4	P_PA-B-4	Species:Pseudomonas aeruginosa	Strain=NR 51589; Factor=Positive Mode; RAW_FILE_NAME=P_PA-B-4.mzML
SUBJECT_SAMPLE_FACTORS           	PA-B-5	P_PA-B-5	Species:Pseudomonas aeruginosa	Strain=NR 51589; Factor=Positive Mode; RAW_FILE_NAME=P_PA-B-5.mzML
SUBJECT_SAMPLE_FACTORS           	PA-C-1	P_PA-C-1	Species:Pseudomonas aeruginosa	Strain=NR 51517; Factor=Positive Mode; RAW_FILE_NAME=P_PA-C-1.mzML
SUBJECT_SAMPLE_FACTORS           	PA-C-2	P_PA-C-2	Species:Pseudomonas aeruginosa	Strain=NR 51517; Factor=Positive Mode; RAW_FILE_NAME=P_PA-C-2.mzML
SUBJECT_SAMPLE_FACTORS           	PA-C-3	P_PA-C-3	Species:Pseudomonas aeruginosa	Strain=NR 51517; Factor=Positive Mode; RAW_FILE_NAME=P_PA-C-3.mzML
SUBJECT_SAMPLE_FACTORS           	PA-C-4	P_PA-C-4	Species:Pseudomonas aeruginosa	Strain=NR 51517; Factor=Positive Mode; RAW_FILE_NAME=P_PA-C-4.mzML
SUBJECT_SAMPLE_FACTORS           	PA-C-5	P_PA-C-5	Species:Pseudomonas aeruginosa	Strain=NR 51517; Factor=Positive Mode; RAW_FILE_NAME=P_PA-C-5.mzML
SUBJECT_SAMPLE_FACTORS           	SA-A-1	P_SA-A-1	Species:Staphylococcus aureus	Strain=NR 46543; Factor=Positive Mode; RAW_FILE_NAME=P_SA-A-1.mzML
SUBJECT_SAMPLE_FACTORS           	SA-A-2	P_SA-A-2	Species:Staphylococcus aureus	Strain=NR 46543; Factor=Positive Mode; RAW_FILE_NAME=P_SA-A-2.mzML
SUBJECT_SAMPLE_FACTORS           	SA-A-3	P_SA-A-3	Species:Staphylococcus aureus	Strain=NR 46543; Factor=Positive Mode; RAW_FILE_NAME=P_SA-A-3.mzML
SUBJECT_SAMPLE_FACTORS           	SA-A-4	P_SA-A-4	Species:Staphylococcus aureus	Strain=NR 46543; Factor=Positive Mode; RAW_FILE_NAME=P_SA-A-4.mzML
SUBJECT_SAMPLE_FACTORS           	SA-A-5	P_SA-A-5	Species:Staphylococcus aureus	Strain=NR 46543; Factor=Positive Mode; RAW_FILE_NAME=P_SA-A-5.mzML
SUBJECT_SAMPLE_FACTORS           	SA-B-1	P_SA-B-1	Species:Staphylococcus aureus	Strain=ATCC 29213; Factor=Positive Mode; RAW_FILE_NAME=P_SA-B-1.mzML
SUBJECT_SAMPLE_FACTORS           	SA-B-2	P_SA-B-2	Species:Staphylococcus aureus	Strain=ATCC 29213; Factor=Positive Mode; RAW_FILE_NAME=P_SA-B-2.mzML
SUBJECT_SAMPLE_FACTORS           	SA-B-3	P_SA-B-3	Species:Staphylococcus aureus	Strain=ATCC 29213; Factor=Positive Mode; RAW_FILE_NAME=P_SA-B-3.mzML
SUBJECT_SAMPLE_FACTORS           	SA-B-4	P_SA-B-4	Species:Staphylococcus aureus	Strain=ATCC 29213; Factor=Positive Mode; RAW_FILE_NAME=P_SA-B-4.mzML
SUBJECT_SAMPLE_FACTORS           	SA-B-5	P_SA-B-5	Species:Staphylococcus aureus	Strain=ATCC 29213; Factor=Positive Mode; RAW_FILE_NAME=P_SA-B-5.mzML
SUBJECT_SAMPLE_FACTORS           	SA-C-1	P_SA-C-1	Species:Staphylococcus aureus	Strain=ATCC 12600; Factor=Positive Mode; RAW_FILE_NAME=P_SA-C-1.mzML
SUBJECT_SAMPLE_FACTORS           	SA-C-2	P_SA-C-2	Species:Staphylococcus aureus	Strain=ATCC 12600; Factor=Positive Mode; RAW_FILE_NAME=P_SA-C-2.mzML
SUBJECT_SAMPLE_FACTORS           	SA-C-3	P_SA-C-3	Species:Staphylococcus aureus	Strain=ATCC 12600; Factor=Positive Mode; RAW_FILE_NAME=P_SA-C-3.mzML
SUBJECT_SAMPLE_FACTORS           	SA-C-4	P_SA-C-4	Species:Staphylococcus aureus	Strain=ATCC 12600; Factor=Positive Mode; RAW_FILE_NAME=P_SA-C-4.mzML
SUBJECT_SAMPLE_FACTORS           	SA-C-5	P_SA-C-5	Species:Staphylococcus aureus	Strain=ATCC 12600; Factor=Positive Mode; RAW_FILE_NAME=P_SA-C-5.mzML
SUBJECT_SAMPLE_FACTORS           	AB-A-1	N_AB-A-1	Species:Acinetobacter baumannii	Strain=NR 52187; Factor=Negative Mode; RAW_FILE_NAME=N_AB-A-1.mzML
SUBJECT_SAMPLE_FACTORS           	AB-A-2	N_AB-A-2	Species:Acinetobacter baumannii	Strain=NR 52187; Factor=Negative Mode; RAW_FILE_NAME=N_AB-A-2.mzML
SUBJECT_SAMPLE_FACTORS           	AB-A-3	N_AB-A-3	Species:Acinetobacter baumannii	Strain=NR 52187; Factor=Negative Mode; RAW_FILE_NAME=N_AB-A-3.mzML
SUBJECT_SAMPLE_FACTORS           	AB-A-4	N_AB-A-4	Species:Acinetobacter baumannii	Strain=NR 52187; Factor=Negative Mode; RAW_FILE_NAME=N_AB-A-4.mzML
SUBJECT_SAMPLE_FACTORS           	AB-A-5	N_AB-A-5	Species:Acinetobacter baumannii	Strain=NR 52187; Factor=Negative Mode; RAW_FILE_NAME=N_AB-A-5.mzML
SUBJECT_SAMPLE_FACTORS           	AB-B-1	N_AB-B-1	Species:Acinetobacter baumannii	Strain=NR 52189; Factor=Negative Mode; RAW_FILE_NAME=N_AB-B-1.mzML
SUBJECT_SAMPLE_FACTORS           	AB-B-2	N_AB-B-2	Species:Acinetobacter baumannii	Strain=NR 52189; Factor=Negative Mode; RAW_FILE_NAME=N_AB-B-2.mzML
SUBJECT_SAMPLE_FACTORS           	AB-B-3	N_AB-B-3	Species:Acinetobacter baumannii	Strain=NR 52189; Factor=Negative Mode; RAW_FILE_NAME=N_AB-B-3.mzML
SUBJECT_SAMPLE_FACTORS           	AB-B-4	N_AB-B-4	Species:Acinetobacter baumannii	Strain=NR 52189; Factor=Negative Mode; RAW_FILE_NAME=N_AB-B-4.mzML
SUBJECT_SAMPLE_FACTORS           	AB-B-5	N_AB-B-5	Species:Acinetobacter baumannii	Strain=NR 52189; Factor=Negative Mode; RAW_FILE_NAME=N_AB-B-5.mzML
SUBJECT_SAMPLE_FACTORS           	AB-C-1	N_AB-C-1	Species:Acinetobacter baumannii	Strain=NR 52190; Factor=Negative Mode; RAW_FILE_NAME=N_AB-C-1.mzML
SUBJECT_SAMPLE_FACTORS           	AB-C-2	N_AB-C-2	Species:Acinetobacter baumannii	Strain=NR 52190; Factor=Negative Mode; RAW_FILE_NAME=N_AB-C-2.mzML
SUBJECT_SAMPLE_FACTORS           	AB-C-3	N_AB-C-3	Species:Acinetobacter baumannii	Strain=NR 52190; Factor=Negative Mode; RAW_FILE_NAME=N_AB-C-3.mzML
SUBJECT_SAMPLE_FACTORS           	AB-C-4	N_AB-C-4	Species:Acinetobacter baumannii	Strain=NR 52190; Factor=Negative Mode; RAW_FILE_NAME=N_AB-C-4.mzML
SUBJECT_SAMPLE_FACTORS           	AB-C-5	N_AB-C-5	Species:Acinetobacter baumannii	Strain=NR 52190; Factor=Negative Mode; RAW_FILE_NAME=N_AB-C-5.mzML
SUBJECT_SAMPLE_FACTORS           	EF-A-1	N_EF-A-1	Species:Enterococcus faecium	Strain=ATCC 700221; Factor=Negative Mode; RAW_FILE_NAME=N_EF-A-1.mzML
SUBJECT_SAMPLE_FACTORS           	EF-A-2	N_EF-A-2	Species:Enterococcus faecium	Strain=ATCC 700221; Factor=Negative Mode; RAW_FILE_NAME=N_EF-A-2.mzML
SUBJECT_SAMPLE_FACTORS           	EF-A-3	N_EF-A-3	Species:Enterococcus faecium	Strain=ATCC 700221; Factor=Negative Mode; RAW_FILE_NAME=N_EF-A-3.mzML
SUBJECT_SAMPLE_FACTORS           	EF-A-4	N_EF-A-4	Species:Enterococcus faecium	Strain=ATCC 700221; Factor=Negative Mode; RAW_FILE_NAME=N_EF-A-4.mzML
SUBJECT_SAMPLE_FACTORS           	EF-A-5	N_EF-A-5	Species:Enterococcus faecium	Strain=ATCC 700221; Factor=Negative Mode; RAW_FILE_NAME=N_EF-A-5.mzML
SUBJECT_SAMPLE_FACTORS           	EF-B-1	N_EF-B-1	Species:Enterococcus faecium	Strain=HM 959; Factor=Negative Mode; RAW_FILE_NAME=N_EF-B-1.mzML
SUBJECT_SAMPLE_FACTORS           	EF-B-2	N_EF-B-2	Species:Enterococcus faecium	Strain=HM 959; Factor=Negative Mode; RAW_FILE_NAME=N_EF-B-2.mzML
SUBJECT_SAMPLE_FACTORS           	EF-B-3	N_EF-B-3	Species:Enterococcus faecium	Strain=HM 959; Factor=Negative Mode; RAW_FILE_NAME=N_EF-B-3.mzML
SUBJECT_SAMPLE_FACTORS           	EF-B-4	N_EF-B-4	Species:Enterococcus faecium	Strain=HM 959; Factor=Negative Mode; RAW_FILE_NAME=N_EF-B-4.mzML
SUBJECT_SAMPLE_FACTORS           	EF-B-5	N_EF-B-5	Species:Enterococcus faecium	Strain=HM 959; Factor=Negative Mode; RAW_FILE_NAME=N_EF-B-5.mzML
SUBJECT_SAMPLE_FACTORS           	EF-C-1	N_EF-C-1	Species:Enterococcus faecium	Strain=HM 952; Factor=Negative Mode; RAW_FILE_NAME=N_EF-C-1.mzML
SUBJECT_SAMPLE_FACTORS           	EF-C-2	N_EF-C-2	Species:Enterococcus faecium	Strain=HM 952; Factor=Negative Mode; RAW_FILE_NAME=N_EF-C-2.mzML
SUBJECT_SAMPLE_FACTORS           	EF-C-3	N_EF-C-3	Species:Enterococcus faecium	Strain=HM 952; Factor=Negative Mode; RAW_FILE_NAME=N_EF-C-3.mzML
SUBJECT_SAMPLE_FACTORS           	EF-C-4	N_EF-C-4	Species:Enterococcus faecium	Strain=HM 952; Factor=Negative Mode; RAW_FILE_NAME=N_EF-C-4.mzML
SUBJECT_SAMPLE_FACTORS           	EF-C-5	N_EF-C-5	Species:Enterococcus faecium	Strain=HM 952; Factor=Negative Mode; RAW_FILE_NAME=N_EF-C-5.mzML
SUBJECT_SAMPLE_FACTORS           	PA-A-1	N_PA-A-1	Species:Pseudomonas aeruginosa	Strain=NR 51588; Factor=Negative Mode; RAW_FILE_NAME=N_PA-A-1.mzML
SUBJECT_SAMPLE_FACTORS           	PA-A-2	N_PA-A-2	Species:Pseudomonas aeruginosa	Strain=NR 51588; Factor=Negative Mode; RAW_FILE_NAME=N_PA-A-2.mzML
SUBJECT_SAMPLE_FACTORS           	PA-A-3	N_PA-A-3	Species:Pseudomonas aeruginosa	Strain=NR 51588; Factor=Negative Mode; RAW_FILE_NAME=N_PA-A-3.mzML
SUBJECT_SAMPLE_FACTORS           	PA-A-4	N_PA-A-4	Species:Pseudomonas aeruginosa	Strain=NR 51588; Factor=Negative Mode; RAW_FILE_NAME=N_PA-A-4.mzML
SUBJECT_SAMPLE_FACTORS           	PA-A-5	N_PA-A-5	Species:Pseudomonas aeruginosa	Strain=NR 51588; Factor=Negative Mode; RAW_FILE_NAME=N_PA-A-5.mzML
SUBJECT_SAMPLE_FACTORS           	PA-B-1	N_PA-B-1	Species:Pseudomonas aeruginosa	Strain=NR 51589; Factor=Negative Mode; RAW_FILE_NAME=N_PA-B-1.mzML
SUBJECT_SAMPLE_FACTORS           	PA-B-2	N_PA-B-2	Species:Pseudomonas aeruginosa	Strain=NR 51589; Factor=Negative Mode; RAW_FILE_NAME=N_PA-B-2.mzML
SUBJECT_SAMPLE_FACTORS           	PA-B-3	N_PA-B-3	Species:Pseudomonas aeruginosa	Strain=NR 51589; Factor=Negative Mode; RAW_FILE_NAME=N_PA-B-3.mzML
SUBJECT_SAMPLE_FACTORS           	PA-B-4	N_PA-B-4	Species:Pseudomonas aeruginosa	Strain=NR 51589; Factor=Negative Mode; RAW_FILE_NAME=N_PA-B-4.mzML
SUBJECT_SAMPLE_FACTORS           	PA-B-5	N_PA-B-5	Species:Pseudomonas aeruginosa	Strain=NR 51589; Factor=Negative Mode; RAW_FILE_NAME=N_PA-B-5.mzML
SUBJECT_SAMPLE_FACTORS           	PA-C-1	N_PA-C-1	Species:Pseudomonas aeruginosa	Strain=NR 51517; Factor=Negative Mode; RAW_FILE_NAME=N_PA-C-1.mzML
SUBJECT_SAMPLE_FACTORS           	PA-C-2	N_PA-C-2	Species:Pseudomonas aeruginosa	Strain=NR 51517; Factor=Negative Mode; RAW_FILE_NAME=N_PA-C-2.mzML
SUBJECT_SAMPLE_FACTORS           	PA-C-3	N_PA-C-3	Species:Pseudomonas aeruginosa	Strain=NR 51517; Factor=Negative Mode; RAW_FILE_NAME=N_PA-C-3.mzML
SUBJECT_SAMPLE_FACTORS           	PA-C-4	N_PA-C-4	Species:Pseudomonas aeruginosa	Strain=NR 51517; Factor=Negative Mode; RAW_FILE_NAME=N_PA-C-4.mzML
SUBJECT_SAMPLE_FACTORS           	PA-C-5	N_PA-C-5	Species:Pseudomonas aeruginosa	Strain=NR 51517; Factor=Negative Mode; RAW_FILE_NAME=N_PA-C-5.mzML
SUBJECT_SAMPLE_FACTORS           	SA-A-1	N_SA-A-1	Species:Staphylococcus aureus	Strain=NR 46543; Factor=Negative Mode; RAW_FILE_NAME=N_SA-A-1.mzML
SUBJECT_SAMPLE_FACTORS           	SA-A-2	N_SA-A-2	Species:Staphylococcus aureus	Strain=NR 46543; Factor=Negative Mode; RAW_FILE_NAME=N_SA-A-2.mzML
SUBJECT_SAMPLE_FACTORS           	SA-A-3	N_SA-A-3	Species:Staphylococcus aureus	Strain=NR 46543; Factor=Negative Mode; RAW_FILE_NAME=N_SA-A-3.mzML
SUBJECT_SAMPLE_FACTORS           	SA-A-4	N_SA-A-4	Species:Staphylococcus aureus	Strain=NR 46543; Factor=Negative Mode; RAW_FILE_NAME=N_SA-A-4.mzML
SUBJECT_SAMPLE_FACTORS           	SA-A-5	N_SA-A-5	Species:Staphylococcus aureus	Strain=NR 46543; Factor=Negative Mode; RAW_FILE_NAME=N_SA-A-5.mzML
SUBJECT_SAMPLE_FACTORS           	SA-B-1	N_SA-B-1	Species:Staphylococcus aureus	Strain=ATCC 29213; Factor=Negative Mode; RAW_FILE_NAME=N_SA-B-1.mzML
SUBJECT_SAMPLE_FACTORS           	SA-B-2	N_SA-B-2	Species:Staphylococcus aureus	Strain=ATCC 29213; Factor=Negative Mode; RAW_FILE_NAME=N_SA-B-2.mzML
SUBJECT_SAMPLE_FACTORS           	SA-B-3	N_SA-B-3	Species:Staphylococcus aureus	Strain=ATCC 29213; Factor=Negative Mode; RAW_FILE_NAME=N_SA-B-3.mzML
SUBJECT_SAMPLE_FACTORS           	SA-B-4	N_SA-B-4	Species:Staphylococcus aureus	Strain=ATCC 29213; Factor=Negative Mode; RAW_FILE_NAME=N_SA-B-4.mzML
SUBJECT_SAMPLE_FACTORS           	SA-B-5	N_SA-B-5	Species:Staphylococcus aureus	Strain=ATCC 29213; Factor=Negative Mode; RAW_FILE_NAME=N_SA-B-5.mzML
SUBJECT_SAMPLE_FACTORS           	SA-C-1	N_SA-C-1	Species:Staphylococcus aureus	Strain=ATCC 12600; Factor=Negative Mode; RAW_FILE_NAME=N_SA-C-1.mzML
SUBJECT_SAMPLE_FACTORS           	SA-C-2	N_SA-C-2	Species:Staphylococcus aureus	Strain=ATCC 12600; Factor=Negative Mode; RAW_FILE_NAME=N_SA-C-2.mzML
SUBJECT_SAMPLE_FACTORS           	SA-C-3	N_SA-C-3	Species:Staphylococcus aureus	Strain=ATCC 12600; Factor=Negative Mode; RAW_FILE_NAME=N_SA-C-3.mzML
SUBJECT_SAMPLE_FACTORS           	SA-C-4	N_SA-C-4	Species:Staphylococcus aureus	Strain=ATCC 12600; Factor=Negative Mode; RAW_FILE_NAME=N_SA-C-4.mzML
SUBJECT_SAMPLE_FACTORS           	SA-C-5	N_SA-C-5	Species:Staphylococcus aureus	Strain=ATCC 12600; Factor=Negative Mode; RAW_FILE_NAME=N_SA-C-5.mzML
#COLLECTION
CO:COLLECTION_SUMMARY            	All work with microorganisms was performed under Biosafety Level 2 (BSL-2)
CO:COLLECTION_SUMMARY            	conditions. Bacteria were streaked onto agar plates from stocks and incubated
CO:COLLECTION_SUMMARY            	overnight at 37°C. Single colonies were collected from the agar plates and
CO:COLLECTION_SUMMARY            	suspended in sterile deionized (DI) water to a turbidity of 2.0-2.05 McFarlands
CO:COLLECTION_SUMMARY            	(equivalent to ca. 6.0 x 108 CFU/mL). Five biological replicates were prepared
CO:COLLECTION_SUMMARY            	for each strain. Tryptic Soy Broth was inoculated at a 1:10 dilution (5 mL total
CO:COLLECTION_SUMMARY            	volume) and incubated overnight at 37°C with shaking (180 rpm). The cultures
CO:COLLECTION_SUMMARY            	were then centrifuged at 2700 rpm for 10 min at 4 °C, after which the broth was
CO:COLLECTION_SUMMARY            	discarded. The pelleted bacteria were washed and resuspended in 2 mL of sterile
CO:COLLECTION_SUMMARY            	water.
CO:SAMPLE_TYPE                   	Bacterial cells
#TREATMENT
TR:TREATMENT_SUMMARY             	No treatment
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	Prior to extraction, the suspended bacteria were normalized by turbidity to
SP:SAMPLEPREP_SUMMARY            	obtain equivalent amounts of bacteria. The suspensions were then aliquoted at
SP:SAMPLEPREP_SUMMARY            	0.5 mL into 8 mL glass culture tubes (for biphasic extraction) or 2 mL
SP:SAMPLEPREP_SUMMARY            	polypropylene microcentrifuge tubes (for single-phase extraction) and pelleted
SP:SAMPLEPREP_SUMMARY            	by centrifugation. Before extraction solvents were added, stable isotope labeled
SP:SAMPLEPREP_SUMMARY            	internal standards of lipids and metabolites were added for recovery and
SP:SAMPLEPREP_SUMMARY            	quantitation purposes. The metabolite internal standards (Cambridge Isotope
SP:SAMPLEPREP_SUMMARY            	Laboratories) included 13C5-hypoxanthine (final concentration, 1 µg/mL),
SP:SAMPLEPREP_SUMMARY            	13C6-sucrose (5 µg/mL), and 13C5-L-glutamine (10 µg/mL). The lipid internal
SP:SAMPLEPREP_SUMMARY            	standards (Avanti Polar Lipids) included phosphatidylethanolamine (PE)
SP:SAMPLEPREP_SUMMARY            	15:0/d7-18:1 (final concentration, 37.5 ng/mL), diacylglycerol (DG) 15:0/d7-18:1
SP:SAMPLEPREP_SUMMARY            	(100 ng/mL), and phosphatidylglycerol (PG) 15:0/d7-18:1 (12.5 ng/mL). For the
SP:SAMPLEPREP_SUMMARY            	biphasic Bligh and Dyer (B&D) extraction, the pelleted bacteria were
SP:SAMPLEPREP_SUMMARY            	reconstituted with 0.5 mL of HPLC grade H2O and sonicated for 30 min at 4 °C. A
SP:SAMPLEPREP_SUMMARY            	chilled solution of 1:2 CHCl3/MeOH (2 mL) was added to the sample and vortexed
SP:SAMPLEPREP_SUMMARY            	for 5 min, followed by the addition of 0.5 mL CHCl3 and 0.5 mL H2O to induce
SP:SAMPLEPREP_SUMMARY            	phase separation. After an additional 1 min of vortexing, the samples were
SP:SAMPLEPREP_SUMMARY            	centrifuged for 10 min at 3500 rpm and 4 °C. The lower organic layer and the
SP:SAMPLEPREP_SUMMARY            	upper aqueous layer of the biphasic solution were collected into separate glass
SP:SAMPLEPREP_SUMMARY            	tubes and dried under vacuum. Both dried extracts were reconstituted in 200 µL
SP:SAMPLEPREP_SUMMARY            	of 2:2:1 ACN/MeOH/H2O and stored at -80°C or directly diluted for LC-IM-MS
SP:SAMPLEPREP_SUMMARY            	analysis. A single-phase extraction solvent system based on butanol,
SP:SAMPLEPREP_SUMMARY            	acetonitrile and water (BAW) was evaluated for the recovery of both lipids and
SP:SAMPLEPREP_SUMMARY            	metabolites. We tested three compositions of the BAW extraction solution: 30%
SP:SAMPLEPREP_SUMMARY            	butanol/70% acetonitrile (30% Bu), 45% butanol/55% acetonitrile (45% Bu), and
SP:SAMPLEPREP_SUMMARY            	60% butanol/40% acetonitrile (60% Bu), with H2O constant at 20% for all three
SP:SAMPLEPREP_SUMMARY            	compositions. For the extraction, 1 mL of chilled, pre-mixed extraction solution
SP:SAMPLEPREP_SUMMARY            	was added to pelleted bacteria. The samples were vortexed and sonicated in an
SP:SAMPLEPREP_SUMMARY            	ice bath in alternating 5 min intervals for a total of 30 min. The samples were
SP:SAMPLEPREP_SUMMARY            	then chilled at 4 °C for 10 min, and then centrifuged at 3500 rpm and 4 °C for
SP:SAMPLEPREP_SUMMARY            	10 min. The supernatants were collected into fresh 2 mL microcentrifuge tubes
SP:SAMPLEPREP_SUMMARY            	and dried under vacuum. The dried single-phase extracts were reconstituted in
SP:SAMPLEPREP_SUMMARY            	200 µL of 2:2:1 ACN/MeOH/H2O and stored at -80 °C freezer or diluted for
SP:SAMPLEPREP_SUMMARY            	LC-IM-MS analysis.
SP:PROCESSING_STORAGE_CONDITIONS 	On ice
SP:EXTRACT_STORAGE               	-80℃
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_TYPE           	HILIC
CH:INSTRUMENT_NAME               	Waters Acquity
CH:COLUMN_NAME                   	Waters ACQUITY UPLC BEH Amide (100 x 2.1mm,1.7um)
CH:SOLVENT_A                     	100% water; 10 mM ammonium formate; 0.125% formic acid
CH:SOLVENT_B                     	95% ACN/5% water; 10 mM ammonium formate; 0.125% formic acid
CH:FLOW_GRADIENT                 	0-2 min at 100% B, 2-7.7 min from 100% to 70% B, 7.7-9.5 min from 70% to 40% B,
CH:FLOW_GRADIENT                 	9.5-10.25 min from 40% to 30% B, 10.25-12.75 min from 30% to 100% B, and
CH:FLOW_GRADIENT                 	12.75-17 min to re-equilibrate to 100% B
CH:FLOW_RATE                     	0.4 mL/min
CH:COLUMN_TEMPERATURE            	45
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Waters Synapt G2 XS QTOF
MS:INSTRUMENT_TYPE               	QTOF
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	NEGATIVE
MS:MS_COMMENTS                   	The UPLC was connected to the electrospray ionization source of the traveling
MS:MS_COMMENTS                   	wave ion mobility-mass spectrometer (Waters Synapt XS) and samples were injected
MS:MS_COMMENTS                   	at 5 uL. Prior to acquisition of sample data, data was acquired for a mixture of
MS:MS_COMMENTS                   	CCS calibrants using direct infusion. Randomized sample queues were analyzed in
MS:MS_COMMENTS                   	both positive and negative ionization modes. A pooled mixture of all samples was
MS:MS_COMMENTS                   	used as a quality control (QC). Data was collected across the entire 17 min
MS:MS_COMMENTS                   	chromatographic method using data-independent MS/MS acquisition. Leucine
MS:MS_COMMENTS                   	enkephalin was monitored for post-acquisition lockmass correction. Capillary -2
MS:MS_COMMENTS                   	kV; Sampling Cone 30 V; Sampling Cone 25 V; Source Offset 40 V; Source Temp 150
MS:MS_COMMENTS                   	ºC; Desolvation Temp 400 ºC; Cone gas flow 50 L/h; Desolvation gas flow 650
MS:MS_COMMENTS                   	L/h; Nebulizer gas flow 7 Bar. Mass Range 50-1200 m/z.
MS:MS_RESULTS_FILE               	ST002854_AN004676_Results.txt	UNITS:intensity	Has m/z:Yes	Has RT:Yes	RT units:Minutes
#END