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.
| Study ID | ST002854 |
| Study Title | HILIC-IM-MS for Simultaneous Lipid and Metabolite Profiling of Microorganisms |
| Study 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 | University of Georgia |
| Last Name | Carpenter |
| First Name | Jana |
| Address | 302 E Campus Rd., Athens, Georgia, 30602, USA |
| kelly.hines@uga.edu | |
| Phone | 706-542-1966 |
| Submit Date | 2023-09-07 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | mzML |
| Analysis Type Detail | LC-MS |
| Release Date | 2023-09-27 |
| Release Version | 1 |
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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℃ |
