#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