Summary of Study ST002854

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001787. The data can be accessed directly via it's Project DOI: 10.21228/M8QM78 This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

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Study IDST002854
Study TitleHILIC-IM-MS for Simultaneous Lipid and Metabolite Profiling of Microorganisms
Study SummaryProgress in the ion mobility mass spectrometry (IM-MS) field has significantly increased our ability to make small molecule and lipid identifications, making it an attractive approach for untargeted multi-omics experiments. The dimensionality of collision cross section (CCS) coupled with tandem mass spectrometry (MS/MS) for feature annotation has become a useful tool for high confidence structural elucidation in complex mixtures in the absence of authentic standards. A comprehensive method for feature identification of small organisms has remained limited to exploring genetic markers and protein signatures, however these methods for identification only scratch the surface of effective methods for bacterial classification. Multi-omic methods that include the metabolome and lipidome have grown in popularity due to the increased capacity for organism specific information. We have achieved species-level identification of Enterococcus faecium, Staphylococcus aureus, Acinetobacter baumannii, and Pseudomonas aeruginosa using a modern single-phase extraction method with hydrophilic interaction liquid chromatography (HILIC) coupled to traveling wave ion mobility mass spectrometry (TWIMS). To test the robustness of this optimized workflow, we included internal standards as a metric for efficiency of the extraction, and well known calibrants for validation for our CCS calibration method. We observed significant differences in metabolite profiles at the strain level using multi-variate statistics, primarily including quorum sensing metabolites in Gram-negative strains, and energy production metabolites in the Gram-positive strains. Lipid profiles showed staggering differences in acyl tail compositions that effectively categorized the microbes, including several classes of phospholipids and glycolipids. We have demonstrated a powerful workflow using multi-dimensional techniques for bacterial speciation in a single injection.
Institute
University of Georgia
Last NameCarpenter
First NameJana
Address302 E Campus Rd., Athens, Georgia, 30602, USA
Emailkelly.hines@uga.edu
Phone706-542-1966
Submit Date2023-09-07
Raw Data AvailableYes
Raw Data File Type(s)mzML
Analysis Type DetailLC-MS
Release Date2023-09-27
Release Version1
Jana Carpenter Jana Carpenter
https://dx.doi.org/10.21228/M8QM78
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR001787
Project DOI:doi: 10.21228/M8QM78
Project Title:HILIC-IM-MS for Simultaneous Lipid and Metabolite Profiling of Microorganisms
Project Type:LC-MS quantitative analysis
Project Summary:Progress in the ion mobility mass spectrometry (IM-MS) field has significantly increased our ability to make small molecule and lipid identifications, making it an attractive approach for untargeted multi-omics experiments. The dimensionality of collision cross section (CCS) coupled with tandem mass spectrometry (MS/MS) for feature annotation has become a useful tool for high confidence structural elucidation in complex mixtures in the absence of authentic standards. A comprehensive method for feature identification of small organisms has remained limited to exploring genetic markers and protein signatures, however these methods for identification only scratch the surface of effective methods for bacterial classification. Multi-omic methods that include the metabolome and lipidome have grown in popularity due to the increased capacity for organism specific information. We have achieved species-level identification of Enterococcus faecium, Staphylococcus aureus, Acinetobacter baumannii, and Pseudomonas aeruginosa using a modern single-phase extraction method with hydrophilic interaction liquid chromatography (HILIC) coupled to traveling wave ion mobility mass spectrometry (TWIMS). To test the robustness of this optimized workflow, we included internal standards as a metric for efficiency of the extraction, and well known calibrants for validation for our CCS calibration method. We observed significant differences in metabolite profiles at the strain level using multi-variate statistics, primarily including quorum sensing metabolites in Gram-negative strains, and energy production metabolites in the Gram-positive strains. Lipid profiles showed staggering differences in acyl tail compositions that effectively categorized the microbes, including several classes of phospholipids and glycolipids. We have demonstrated a powerful workflow using multi-dimensional techniques for bacterial speciation in a single injection.
Institute:Univerisity of Georgia
Department:Chemistry
Laboratory:Dr. Kelly M. Hines
Last Name:Carpenter
First Name:Jana
Address:302 E Campus Rd., Athens, Georgia, 30602, USA
Email:kelly.hines@uga.edu
Phone:706-542-1966

Subject:

Subject ID:SU002966
Subject Type:Bacteria
Subject Species:Staphylococcus aureus; Acinetobacter baumannii; Enterococcus faecium; Pseudomonas aeruginosa
Taxonomy ID:-

Factors:

Subject type: Bacteria; Subject species: Staphylococcus aureus; Acinetobacter baumannii; Enterococcus faecium; Pseudomonas aeruginosa (Factor headings shown in green)

mb_sample_id local_sample_id Species
SA308664N_AB-B-1Acinetobacter baumannii
SA308665N_AB-B-2Acinetobacter baumannii
SA308666N_AB-A-5Acinetobacter baumannii
SA308667N_AB-A-3Acinetobacter baumannii
SA308668N_AB-A-2Acinetobacter baumannii
SA308669N_AB-B-3Acinetobacter baumannii
SA308670N_AB-A-4Acinetobacter baumannii
SA308671N_AB-B-5Acinetobacter baumannii
SA308672N_AB-C-4Acinetobacter baumannii
SA308673N_AB-C-5Acinetobacter baumannii
SA308674N_AB-C-3Acinetobacter baumannii
SA308675N_AB-C-2Acinetobacter baumannii
SA308676P_AB-A-1Acinetobacter baumannii
SA308677N_AB-C-1Acinetobacter baumannii
SA308678N_AB-B-4Acinetobacter baumannii
SA308679N_AB-A-1Acinetobacter baumannii
SA308680P_AB-C-1Acinetobacter baumannii
SA308681P_AB-B-5Acinetobacter baumannii
SA308682P_AB-C-2Acinetobacter baumannii
SA308683P_AB-C-3Acinetobacter baumannii
SA308684P_AB-C-4Acinetobacter baumannii
SA308685P_AB-B-3Acinetobacter baumannii
SA308686P_AB-B-2Acinetobacter baumannii
SA308687P_AB-A-3Acinetobacter baumannii
SA308688P_AB-A-2Acinetobacter baumannii
SA308689P_AB-A-4Acinetobacter baumannii
SA308690P_AB-A-5Acinetobacter baumannii
SA308691P_AB-B-1Acinetobacter baumannii
SA308692P_AB-C-5Acinetobacter baumannii
SA308693P_AB-B-4Acinetobacter baumannii
SA308694P_EF-B-4Enterococcus faecium
SA308695P_EF-B-5Enterococcus faecium
SA308696P_EF-B-3Enterococcus faecium
SA308697P_EF-B-2Enterococcus faecium
SA308698P_EF-A-5Enterococcus faecium
SA308699P_EF-B-1Enterococcus faecium
SA308700P_EF-A-3Enterococcus faecium
SA308701P_EF-C-1Enterococcus faecium
SA308702P_EF-C-5Enterococcus faecium
SA308703P_EF-A-1Enterococcus faecium
SA308704P_EF-C-4Enterococcus faecium
SA308705P_EF-C-3Enterococcus faecium
SA308706P_EF-C-2Enterococcus faecium
SA308707N_EF-A-2Enterococcus faecium
SA308708N_EF-A-3Enterococcus faecium
SA308709N_EF-C-2Enterococcus faecium
SA308710N_EF-C-1Enterococcus faecium
SA308711N_EF-C-3Enterococcus faecium
SA308712N_EF-C-4Enterococcus faecium
SA308713P_EF-A-4Enterococcus faecium
SA308714N_EF-C-5Enterococcus faecium
SA308715N_EF-B-5Enterococcus faecium
SA308716N_EF-B-4Enterococcus faecium
SA308717N_EF-A-5Enterococcus faecium
SA308718N_EF-A-4Enterococcus faecium
SA308719N_EF-B-1Enterococcus faecium
SA308720N_EF-B-2Enterococcus faecium
SA308721N_EF-B-3Enterococcus faecium
SA308722P_EF-A-2Enterococcus faecium
SA308723N_EF-A-1Enterococcus faecium
SA308724N_PA-A-2Pseudomonas aeruginosa
SA308725N_PA-A-3Pseudomonas aeruginosa
SA308726N_PA-A-4Pseudomonas aeruginosa
SA308727N_PA-A-5Pseudomonas aeruginosa
SA308728P_PA-A-4Pseudomonas aeruginosa
SA308729P_PA-A-5Pseudomonas aeruginosa
SA308730P_PA-B-4Pseudomonas aeruginosa
SA308731P_PA-B-3Pseudomonas aeruginosa
SA308732P_PA-B-2Pseudomonas aeruginosa
SA308733P_PA-B-1Pseudomonas aeruginosa
SA308734N_PA-B-1Pseudomonas aeruginosa
SA308735N_PA-B-2Pseudomonas aeruginosa
SA308736N_PA-C-3Pseudomonas aeruginosa
SA308737N_PA-C-4Pseudomonas aeruginosa
SA308738N_PA-C-5Pseudomonas aeruginosa
SA308739P_PA-A-3Pseudomonas aeruginosa
SA308740N_PA-C-2Pseudomonas aeruginosa
SA308741N_PA-C-1Pseudomonas aeruginosa
SA308742N_PA-B-3Pseudomonas aeruginosa
SA308743N_PA-B-4Pseudomonas aeruginosa
SA308744N_PA-B-5Pseudomonas aeruginosa
SA308745P_PA-B-5Pseudomonas aeruginosa
SA308746N_PA-A-1Pseudomonas aeruginosa
SA308747P_PA-A-2Pseudomonas aeruginosa
SA308748P_PA-C-1Pseudomonas aeruginosa
SA308749P_PA-A-1Pseudomonas aeruginosa
SA308750P_PA-C-5Pseudomonas aeruginosa
SA308751P_PA-C-2Pseudomonas aeruginosa
SA308752P_PA-C-4Pseudomonas aeruginosa
SA308753P_PA-C-3Pseudomonas aeruginosa
SA308754N_SA-B-2Staphylococcus aureus
SA308755N_SA-A-5Staphylococcus aureus
SA308756N_SA-B-1Staphylococcus aureus
SA308757N_SA-A-3Staphylococcus aureus
SA308758N_SA-B-3Staphylococcus aureus
SA308759N_SA-A-4Staphylococcus aureus
SA308760N_SA-C-2Staphylococcus aureus
SA308761N_SA-C-4Staphylococcus aureus
SA308762N_SA-C-5Staphylococcus aureus
SA308763N_SA-C-3Staphylococcus aureus
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Collection:

Collection ID:CO002959
Collection Summary:All work with microorganisms was performed under Biosafety Level 2 (BSL-2) conditions. Bacteria were streaked onto agar plates from stocks and incubated overnight at 37°C. Single colonies were collected from the agar plates and suspended in sterile deionized (DI) water to a turbidity of 2.0-2.05 McFarlands (equivalent to ca. 6.0 x 108 CFU/mL). Five biological replicates were prepared for each strain. Tryptic Soy Broth was inoculated at a 1:10 dilution (5 mL total volume) and incubated overnight at 37°C with shaking (180 rpm). The cultures were then centrifuged at 2700 rpm for 10 min at 4 °C, after which the broth was discarded. The pelleted bacteria were washed and resuspended in 2 mL of sterile water.
Sample Type:Bacterial cells

Treatment:

Treatment ID:TR002975
Treatment Summary:No treatment

Sample Preparation:

Sampleprep ID:SP002972
Sampleprep Summary:Prior to extraction, the suspended bacteria were normalized by turbidity to obtain equivalent amounts of bacteria. The suspensions were then aliquoted at 0.5 mL into 8 mL glass culture tubes (for biphasic extraction) or 2 mL polypropylene microcentrifuge tubes (for single-phase extraction) and pelleted by centrifugation. Before extraction solvents were added, stable isotope labeled internal standards of lipids and metabolites were added for recovery and quantitation purposes. The metabolite internal standards (Cambridge Isotope Laboratories) included 13C5-hypoxanthine (final concentration, 1 µg/mL), 13C6-sucrose (5 µg/mL), and 13C5-L-glutamine (10 µg/mL). The lipid internal standards (Avanti Polar Lipids) included phosphatidylethanolamine (PE) 15:0/d7-18:1 (final concentration, 37.5 ng/mL), diacylglycerol (DG) 15:0/d7-18:1 (100 ng/mL), and phosphatidylglycerol (PG) 15:0/d7-18:1 (12.5 ng/mL). For the biphasic Bligh and Dyer (B&D) extraction, the pelleted bacteria were reconstituted with 0.5 mL of HPLC grade H2O and sonicated for 30 min at 4 °C. A chilled solution of 1:2 CHCl3/MeOH (2 mL) was added to the sample and vortexed for 5 min, followed by the addition of 0.5 mL CHCl3 and 0.5 mL H2O to induce phase separation. After an additional 1 min of vortexing, the samples were centrifuged for 10 min at 3500 rpm and 4 °C. The lower organic layer and the upper aqueous layer of the biphasic solution were collected into separate glass tubes and dried under vacuum. Both dried extracts were reconstituted in 200 µL of 2:2:1 ACN/MeOH/H2O and stored at -80°C or directly diluted for LC-IM-MS analysis. A single-phase extraction solvent system based on butanol, acetonitrile and water (BAW) was evaluated for the recovery of both lipids and metabolites. We tested three compositions of the BAW extraction solution: 30% butanol/70% acetonitrile (30% Bu), 45% butanol/55% acetonitrile (45% Bu), and 60% butanol/40% acetonitrile (60% Bu), with H2O constant at 20% for all three compositions. For the extraction, 1 mL of chilled, pre-mixed extraction solution was added to pelleted bacteria. The samples were vortexed and sonicated in an ice bath in alternating 5 min intervals for a total of 30 min. The samples were then chilled at 4 °C for 10 min, and then centrifuged at 3500 rpm and 4 °C for 10 min. The supernatants were collected into fresh 2 mL microcentrifuge tubes and dried under vacuum. The dried single-phase extracts were reconstituted in 200 µL of 2:2:1 ACN/MeOH/H2O and stored at -80 °C freezer or diluted for LC-IM-MS analysis.
Processing Storage Conditions:On ice
Extract Storage:-80℃

Combined analysis:

Analysis ID AN004675 AN004676
Analysis type MS MS
Chromatography type HILIC HILIC
Chromatography system Waters Acquity Waters Acquity
Column Waters ACQUITY UPLC BEH Amide (100 x 2.1mm,1.7um) Waters ACQUITY UPLC BEH Amide (100 x 2.1mm,1.7um)
MS Type ESI ESI
MS instrument type QTOF QTOF
MS instrument name Waters Synapt G2 XS Waters Synapt G2 XS
Ion Mode POSITIVE NEGATIVE
Units internsity intensity

Chromatography:

Chromatography ID:CH003518
Instrument Name:Waters Acquity
Column Name:Waters ACQUITY UPLC BEH Amide (100 x 2.1mm,1.7um)
Column Temperature:45
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, 9.5-10.25 min from 40% to 30% B, 10.25-12.75 min from 30% to 100% B, and 12.75-17 min to re-equilibrate to 100% B
Flow Rate:0.4 mL/min
Solvent A:100% water; 10 mM ammonium formate; 0.125% formic acid
Solvent B:95% ACN/5% water; 10 mM ammonium formate; 0.125% formic acid
Chromatography Type:HILIC

MS:

MS ID:MS004422
Analysis ID:AN004675
Instrument Name:Waters Synapt G2 XS
Instrument Type:QTOF
MS Type:ESI
MS Comments:The UPLC was connected to the electrospray ionization source of the traveling wave ion mobility-mass spectrometer (Waters Synapt XS) and samples were injected at 5 uL. Prior to acquisition of sample data, data was acquired for a mixture of CCS calibrants using direct infusion. Randomized sample queues were analyzed in both positive and negative ionization modes. A pooled mixture of all samples was used as a quality control (QC). Data was collected across the entire 17 min chromatographic method using data-independent MS/MS acquisition. Leucine enkephalin was monitored for post-acquisition lockmass correction. Capillary +3 kV; Sampling Cone 30 V; Sampling Cone 25 V; Source Offset 40 V; Source Temp 150 ºC; Desolvation Temp 400 ºC; Cone gas flow 50 L/h; Desolvation gas flow 650 L/h; Nebulizer gas flow 7 Bar. Mass Range 50-1200 m/z.
Ion Mode:POSITIVE
  
MS ID:MS004423
Analysis ID:AN004676
Instrument Name:Waters Synapt G2 XS
Instrument Type:QTOF
MS Type:ESI
MS Comments:The UPLC was connected to the electrospray ionization source of the traveling wave ion mobility-mass spectrometer (Waters Synapt XS) and samples were injected at 5 uL. Prior to acquisition of sample data, data was acquired for a mixture of CCS calibrants using direct infusion. Randomized sample queues were analyzed in both positive and negative ionization modes. A pooled mixture of all samples was used as a quality control (QC). Data was collected across the entire 17 min chromatographic method using data-independent MS/MS acquisition. Leucine enkephalin was monitored for post-acquisition lockmass correction. Capillary -2 kV; Sampling Cone 30 V; Sampling Cone 25 V; Source Offset 40 V; Source Temp 150 ºC; Desolvation Temp 400 ºC; Cone gas flow 50 L/h; Desolvation gas flow 650 L/h; Nebulizer gas flow 7 Bar. Mass Range 50-1200 m/z.
Ion Mode:NEGATIVE
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