Quorum Sensing

rate of 0.1 ml/min. Change solvent composition after 20 min

first to 50% eluant B and second to 0.5% eluant B.

3. Set sample temperature to 4C to keep samples stable for the
   analysis.


4. Use the following settings for each chromatographic run: apply
   a linear gradient from 0 to 90% eluant B within 5 min and with
   a flow rate of 0.1 ml/min. Keep the solvent composition stable
   for 2.5 min before equilibrating the column with 0.5% eluant
   B. Inject 5.0μl of sample. Perform UV detection at 195 nm
   with a scan rate of 10 Hz.


5. Acquire mass spectra in positive ionization mode within a mass
   range of 50–1000 m/z (seeNote 12). Measure HSL/HS
   standards from lower to higher concentrations first and then
   analyze samples in randomized order.


6. In order to ensure mass accuracy calibrate acquired spectra
   according to reference mass signals, e.g., from solvent
   impurities.


7. Create extracted ion chromatograms for protonated masses of
   HSL/HS molecules and integrate peaks to calculate peak areas
   (Fig.2).


8. Plot peak areas of HSL/HS standards and make a linear equa-
   tion. The coefficient of determination should be bigger than
   0.95.


9. Calculate quantities of samples according to linear equation of
   HSL/HS standards.

4 Notes

1. Put the solution for 5 min into an ultrasonic bath to ensure
   complete dissolution.


2. Oasis®HLB is a hydrophilic-lipophilic balanced reversed-phase
   sorbent found and supplied by Waters GmbH, Milford, USA.
   It was tested to be the best sorbent for extracting acyl-HSL/
   HS from cell culture supernatants.


3. The reaction at 200C is forming high pressure according to
   boiling retardation of the solution. Therefore, usage of a tightly
   closed stainless steel digestion vessel is indispensable. Keep the
   vessel closed until it is cooled down to room temperature
   before continuing.


4. Use all solvents always at the same temperature (best room
   temperature) to prevent volumetric differences. To ensure cor-
   rect volumetric percentages of organic solvents in mixtures,
   measure volumes by use of graduate flasks. Shake or stir organic
   solvent mixtures carefully to ensure complete mixing.

Detection of Bacterial Quorum Sensing Molecules with ELISA and UHPLC-MS 69