- Iodoacetamide (IAA) solution: 50 mM IAA in 50 mM (NH 4 )
HCO 3 (seeNote 5). - 80% Acetonitrile.
- Trypsin buffer: 20 mM Tris–HCl pH 8.5, 0.1 mM CaCl 2.
- Trypsin solution: 10μg/ml sequencing grade trypsin dissolved
in trypsin buffer (seeNote 6). - 10% Formic acid in H 2 O.
2.5 Peptide
Extraction
- Wetting solution: 80% acetonitrile.
- Washing solution: 5% acetonitrile, 0.1% formic acid.
- Elution solution: 60% acetonitrile, 0.1% formic acid.
2.6 Prepare the
Samples for MS
Analysis
- Buffer A: 3% acetonitrile, 0.1% formic acid.
3 Methods
Perform all procedures at room temperature, unless stated
otherwise.3.1 Culture
Conditions
- StreakB. cenocepaciaH111 wild-type and mutant strains on LB
agar plates with respective antibiotics and incubate the plates at
37 C overnight. - Pick a single colony of each strain to inoculate 3 ml LB pre-
culture with antibiotics. Incubate the pre-cultures with vigor-
ous shaking at 37C overnight. - Inoculate 500 ml LB medium (seeNote 7) in 3 l Erlenmeyer
flasks with 3 ml overnight pre-culture. As a control, grow the
H111-rpfFBcmutant in LB medium supplemented with 10μM
BDSF. Grow the culture with agitation at 37C until early
stationary phase (i.e., an OD 600 of about 2.0).
3.2 Protein
Extraction
3.2.1 Extracellular
Protein Preparation
- Filter the individual 500 ml cultures with 0.45μm sterile filters
(seeNote 8). - Precipitate the soluble proteins in the filtered liquid with 12%
(wt/vol) TCA. To this end, add the 500 ml filtered liquid into a
bottle containing 60 g TCA. Incubate the mixture with agita-
tion at 4C overnight [24]. - Centrifugethe mixture at 12,500gfor 1 h at4C(seeNote9).
- Wash the pellet with 250 ml 100% ice-cold acetone (see
Note 10) and centrifuge again with the same condition to
harvest the precipitated proteins. - Decant the acetone and air-dry the pellet in the fume hood (see
Note 11).
196 Yilei Liu et al.