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1. Metabolite Extraction
- Thaw samples on wet ice and disrupt the cells in a homogenizer with four 30 s bursts at 6,000 rpm, with 2 min cooling periods on dry ice between cycles.
- Clarify the lysates for 15 min in a pre-chilled, refrigerated microcentrifuge at maximum speed (i.e., 18,213 x g at ≤4 °C).
- Transfer the supernatant to a clean microcentrifuge tube.
- Using a micropipette, transfer a small portion of the sample to a microcentrifuge tube for the quantification of residual peptide content in step 2; store the remainder at -80 °C.
NOTE: The volume of the reserved sample varies, depending on the bicinchoninic acid (BCA) assay used in step 2.1. This sample should be stored on wet ice for immediate analysis or frozen at -80 °C.
2. Bicinchoninic Acid (BCA) Assay
- Perform a BCA assay as recommended by the kit manufacturer, using samples from step 1.4 to determine the residual peptide concentration for each sample.
3. Liquid Chromatography-Mass Spectrometry (LC-MS)
- Mix 75 µL of Staphylococcus aureus (S. aureus) extract with 75 µL of LC-MS solvent B, prepared in step 1.4.
- Vortex to mix and spin at 13,000 x g for 5 min.
- Place 100 µL of supernatant into a liquid chromatography (LC) vial and cap it. Ensure that no air bubbles are trapped in the sample.
- Load the LC vials onto the LC-MS autosampler and edit the running list in the software "Offline Worklist Editor."
- Fill out the "Sample Name" (e.g., wild-type-1), "Sample Position" (e.g., P1-A1), "Method" (e.g., Formic Acid-Negative Method), and "Data File" (e.g., wild-type-1) columns. Click the button "Save Worklist" button. Open the "Mass Spectrometry Data Acquisition Workstation" software and input the previously saved worklist. Click the "Start Worklist Run" button to start the continuous LC-MS measurement.
- Separate the samples on a column, link the column to a time of flight (TOF) spectrometer, and couple the TOF spectrometer with the LC system. Use a mobile-phase gradient as follows: 0-2 min, 85% solvent B; 3-5 min, 80% solvent B; 6-7 min, 75% solvent B; 8-9 min, 70% solvent B; 10-11.1 min, 50% solvent B; 11.1-14 min, 20% solvent B; and 14.1-24 min, 5% solvent B; end with a 10 min re-equilibration period at 85% solvent B and a flow rate of 0.4 mL min-1.
- Using an isocratic pump, infuse a reference mass solution into the run to allow for simultaneous mass axis calibration.
NOTE: This step is based on the standard TOF spectrometer manual.- Use the mixture of acetic acid D4 and hexakis (1H,1H,3H-tetrafluoropropoxy) phosphazine as the reference mass solution to perform the real-time calibration. Use the isocratic pump with a flow rate of 2.5 mL min-1 for the infusion.
4. Batch Correction of Ion Counts
- Designate any sample to serve as a reference sample for batch correction (e.g., wild-type, replicate 1).
- Calculate the sum of the ion counts for all metabolites within the reference sample. Repeat this calculation for all samples.
- Divide the total ion count of each sample by the total ion count of the reference sample to generate a ratio.
- Divide the ion count for each metabolite within a sample by the sample/reference ratio to obtain a batch-corrected ion count for each metabolite.
5. Peptide Normalization
- Divide the batch-corrected ion count values for each sample obtained in step 4 by the peptide concentration determined with the BCA assay in step 2 to yield a normalized value for each metabolite.
NOTE: The normalized, batch-batch corrected ion counts for each metabolite obtained in step 5.1 can be directly compared between strains and subjected to statistical analysis (e.g., a Mann-Whitney U-test). Alternatively, a metabolite known to be unchanged either by the treatment or genetic background may be used as a normalizer to detect changes due to metabolite decomposition. The inclusion of a known amount of L-norvaline or glutaric acid in the extraction buffer can be used to correct for loss during sample processing.