PSM secretion was assayed as before (19 (link)) with modifications. Starter cultures were diluted into TSB without glucose supplemented with chloramphenicol and tetracycline to an OD600 = 0.200. Cells were incubated, shaking at 37 °C, 180 rpm for 4 h. D-xylose was added to 0.5% (w/v), and cells were cultured for one additional hour. Cells were pelleted by centrifugation at 13,200 rpm for 2 min in a microcentrifuge, and the supernatants (extracellular fraction) were collected and analyzed for PSMs by injecting 100 µL into a HPLC (Agilent 1260) connected inline to a quadrupole mass spectrometer (MS, Agilent 6120) performed exactly as before (44 (link)) using a 2.1 × 5 mm C8 column with increasing gradients of acetonitrile (ACN) + 0.1% trifluoroacetic acid (TFA) to wash the column and elute PSMs. Peptides were detected based on elution time and ion m/z. PSM secretion was quantified by summing the extracted ion chromatogram (EIC) peak area from two ionized (m/z) species per PSM.
Protocol detail
Quantitative Analysis of Phenol-Soluble Modulins
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Other organizations : National Institute of Allergy and Infectious Diseases, National Institute of General Medical Sciences, University of Maryland, College Park, Virginia–Maryland College of Veterinary Medicine
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Variable analysis
independent variables
- Addition of D-xylose to a final concentration of 0.5% (w/v)
- PSM secretion
- Starter cultures were diluted into TSB without glucose supplemented with chloramphenicol and tetracycline to an OD600 = 0.200
- Cells were incubated, shaking at 37 °C, 180 rpm for 4 h
- Cells were pelleted by centrifugation at 13,200 rpm for 2 min in a microcentrifuge
- The supernatants (extracellular fraction) were collected and analyzed for PSMs by injecting 100 µL into a HPLC (Agilent 1260) connected inline to a quadrupole mass spectrometer (MS, Agilent 6120) using a 2.1 × 5 mm C8 column with increasing gradients of acetonitrile (ACN) + 0.1% trifluoroacetic acid (TFA) to wash the column and elute PSMs
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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