A proteomic view of probiotic Lactobacillus rhamnosus GG

Materials and Methods
3.2.3. 2-D GE and image analysis
Th e labeled proteins were separated by IEF.
IPG strips (24 cm, pH 3–10, nonlinear,
Bio-Rad) were rehydrated in 500 μl buffer,
which contained 7 M urea, 2 M thiourea, 4%
3-[(3-cholamidopropyl)dimethylammonio]-
1-propanesulfonate, 50 mM DTT, 2 mM tributylphosphine,
and 1% Bio-Lyte pH 3–10
(Bio-Rad), overnight at 20 °C using a Protean
IEF Cell (Bio-Rad). Samples containing
approximately 100 μg of protein in 50 mM
DTT, 4 mM tributylphosphine, and 1% Bio-
Lyte pH 3–10 were applied to the IPG strips
via cup-loading near the acidic end of the
strips. IEF was performed using a Protean
IEF Cell at 20 °C for 80,000 V. Aft er IEF, the
strips were equilibrated in a buff er containing
50 mM Tris-HCl (pH 6.8), 6 M urea, 2% SDS,
20% glycerol, and either 2% DTT (buff er A) or
2.5% iodoacetamide (buff er B), fi rst in buff er
A for 25 min and then in buff er B for 25 min.
The strips were loaded on 12% acrylamide
gels, and the gels were subjected to
electrophoresis in an Ettan DALTsix Electrophoresis
Unit (GE Healthcare). Th e gels were
imaged for the Cy dyes Cy2, Cy3, and Cy5
using an FLA-5100 laser scanner (Fujifi lm),
and the images were cropped to an identical
size by removing areas extraneous to the protein
spots with ImageQuant TL 7.0 soft ware
(GE Healthcare). Aft er scanning, the gels were
fi xed and silver-stained. Image and statistical
analyses for the cropped DIGE gels were performed
using DeCyder 2D 6.5 soft ware (GE
Healthcare).
3.3. Protein iden� fi ca� on by MS
From the silver-stained 2-D DIGE gels, protein
spots of interest were excised manually
and digested in-gel with trypsin, and the peptides
were recovered. Th e resulting peptides
were analyzed by peptide mass fingerprint-
32
ing (PMF) or by fragment ion analysis with
LC-MS/MS. For PMF, the mass spectra were
acquired using an Ultrafl ex TOF/TOF instrument
(Bruker Daltonics). LC-MS/MS analysis
for the tryptic peptides was performed using
an Ultimate 3000 nano-LC system (Dionex)
and QSTAR Elite hybrid quadrupole TOF
mass spectrometer (Applied Biosystems/MDS
Sciex) with nano-ESI ionization. The PMF
spectra were processed with FlexAnalysis version
3.0. Th e PMF and LC-MS/MS data were
searched with the local Mascot version 2.2
(Matrix Science) against the in-house database
of the published ORF set of L. rhamnosus
GG using the Biotools 3.0 (Bruker Daltonics)
and ProteinPilot 2.0.1 (Applied Biosystems)
interface, respectively.
3.4. Transcriptomic methods
3.4.1. Experimental design, RNA
methods, cDNA synthesis,
and labeling
In studies II–IV, the RNA samples from 3 or
4 independent biological replicates at each
time point or treatment were hybridized to
microarrays. Hybridizations were performed
using a balanced dye swap design. From the
bacterial cultures, 1–4 ml was mixed with 2–8
ml of RNAprotect Bacteria reagent (Qiagen)
and treated according to the manufacturer’s
instructions. The cell pellets were stored
at –70 °C for subsequent RNA extraction.
Th e cells were lysed, and the cell lysate was
homogenized. Cell debris was removed by
centrifugation, the lysate was extracted with
chloroform, and the phases were separated by
centrifugation. Th e aqueous phase was recovered
and purified with an RNeasy Mini kit
(Qiagen). During RNA purifi cation, DNA was
removed using RNase-free DNase (Qiagen).
The concentration and purity of the RNA
samples were determined. For each sample, 5