- Zorbax Eclipse XDB-C18 reverse phase column (Analytical,
4.6150 mm, 5-Micron).
6. 20 C Refrigerator. - UPLC/MS system: UPLC system coupled with an accurate
mass TOF-MS equipped with a Jet Stream (JS) electrospray
ionization (ESI) source. - Diode array detector (model: G4212A).
- Software: MassHunter Workstation Data Acquisition Software
(revision B.04). - Methanol (HPLC grade).
- Mobile phase or elution buffer for UPLC analysis: 80:20
methanol:water (v/v).
3 Methods
3.1 Preparation
ofXccCultures
- Streak theXccmutant strainΔrpfCon NA agar plate supple-
mented with 50μg/ml rifampicin. Incubate the plate at 28C
for 3 days (seeNote 4). - Pick a single colony and inoculate in 10 ml of NA broth
supplemented with 50μg/ml rifampicin in a 50 ml Erlenmeyer
flask. Incubate the culture at 28C with shaking at 200 rpm
for 36 h. - Measure the optical density at 600 nm wavelength (OD 600 )of
theΔrpfCpre-culture with a spectrophotometer. Adjust the
OD 600 of the pre-culture to approximately 1.0 with NA broth. - Add 1 ml of the adjusted pre-culture to 50 ml of NA broth in a
250 ml Erlenmeyer flask with vent cap. Incubate the culture at
28 C with shaking at 200 rpm for approximately 36 h, until it
reaches an OD 600 of 2.8. This culture will be used for DSF
extraction.
3.2 DSF Signal
Extraction and Sample
Preparation for UPLC/
MS Analysis
- Centrifuge at 8000gfor 15 min at room temperature 1.0 ml
of theΔrpfCculture from Subheading3.1, point 4, into a
1.5 ml tube. - Transfer the supernatant into a 5 ml tube and adjust the super-
natant’s pH to 4.0 by adding 10μl of 6 M hydrochloric acid.
Check the pH with pH test strips (seeNotes 5and 6 ). - Add 1 ml of ethyl acetate to the adjusted supernatant and
vortex the tube vigorously for 5 min to extract DSF family
signals. Centrifuge at 8000gfor 10 min to separate the
ethyl acetate fraction from the aqueous fraction. - Transfer the ethyl acetate fractions (upper layer, about 0.9 ml)
into a clean 1.5 ml tube.
100 Lian Zhou et al.