that membrane proteins are not underrepresented. After cutting the
individual lanes into at least ten gel slices, the proteins in these slices
are then subjected to in-gel tryptic digestion. Next, the extracted
peptides are separated by Reverse-Phase chromatography (RP-
HPLC) and analyzed by a mass spectrometer interfaced with a
nanoelectrospray source [21]. Fragment ion mass spectra are
searched against theB. cenocepaciastrain H111 protein database
and further analyzed for differential expression using an in-house
pipeline developed by Ahrens and coworkers [22]orcommercial
software solutions.2 Materials
2.1 Bacterial Strains
and Culturing
1.B. cenocepaciawild-type strain H111 [23].
2.B. cenocepaciamutant strain H111-rpfFBc[12].- 10μM BDSF (seeNote 1).
- LB liquid medium: 10 g/l Bacto Tryptone, 5 g/l Bacto yeast
extract, 4 g/l NaCl in distilled water. Adjust pH 7.4 and
autoclave. - LB agar: LB liquid medium plus 12–15 g/l agar. Autoclave.
2.2 Protein
Extraction
- Trichloroacetic acid (TCA) (seeNote 2).
- Acetone, HPLC grade.
- Tris–SDS buffer: 50 mM Tris/HCl pH 7.5 with 1% (wt/vol)
sodium dodecyl sulfate (SDS). - Bradford protein assay.
- Tris–PI buffer: 50 mM Tris/HCl pH 7.5 supplemented with
protease inhibitor. - Sonicator: sonicate samples on ice.
- French Press Cell: pre-chill the French Press Cell body before
loading the sample in order to process the samples in cold. - 100 mM Tris–HCl, pH 7.5 with 2% SDS.
2.3 SDS-PAGE 1. Colloidal blue staining buffer: 100 g/l ammonium sulfate,
20 g/l phosphoric acid (85%), 25% (vol/vol) methanol,
0.625 g/l Coomassie Brilliant Blue G250 in distilled water.
2.4 In-Gel Digestion 1. Destaining solution: 50/50 (vol/vol) methanol/50 mM
(NH 4 )HCO 3 ,(seeNote 3).
- Acetonitrile.
- Dithiothreitol (DTT) solution: 10 mM DTT in 50 mM (NH 4 )
HCO 3 (seeNote 4).
GeLC-MS/MS Proteomics to Identify Quorum Sensing Targets 195