Supplementary Material
Capillary HPLC column preparation
Briefly, a slurry (1 mg stationary phase ml-1 isopropanol was prepared and transferred
to a high pressure bomb, attached to a cylinder of oxygen-free nitrogen (OFN). A frit
was prepared at the end of a piece of 300 µM fused silica (Composite Metal, UK) by
dipping the fused silica into a reaction mixture of Kasil (PQ Corporation)/Formamide
(Sigma Aldrich) (3:1 v/v) followed by heating to 100 ºC over a15 min period. The
silica was maintained at this temperature for 4 hours and at the end of this period the
unfritted end was passed into the bomb via an entrance sealed with a ferrule and into
the stationary phase solution. Packing was achieved by application of pressure (40
bar) from the OFN cylinder.
Extraction of Proteins and preparation for protein expression analysis
Cells of each culture extracted separately. First cells were harvested and centrifuged
at 7000g (Beckman, J2-21/E) for 5 min and washed in wash buffer containing 50mM
Tris-HCL pH8.0 and recentrifuged. The pellet was resuspended in 3ml’s of ice cold
chilled extraction buffer (20mM Tris-HCL pH 8.0, 1mM Na-EDTA, 100mM NaCl,
2mM MgCl, 5mM DTT, and protease inhibitors Leupeptin 1.2ul/ml, Pepstatin 1ul/ml,
PMSF 10ul/ml) and broken through a passage by the French Press (SLM-AMINCO)
at 10,000 psi. The broken cell suspension was directly precipitated with chilled TCAacetone (10% TCA-acetone, 5mM DTT) in a ratio of 7:1 TCA-acetone to sample and
kept for 2 hours at -20C. Precipitated proteins with other cell fractions were
centrifuged at 8000g for 10 min. The pellets were further washed with several washes
of ice cold acetone to remove pigments until the wash acetone became light yellow
and clear in appearance. The pellets were then dried under air or through a stream of
nitrogen until the pellet appeared to be light blue and powdery and was kept frozen at
-80C until further use.
The pellets was dissolved in 3ml of rehydration buffer (Mechin et al., 2003)
consisting of 5 M urea, 2 M Thiourea, 2% CHAPS, 2% SB3-10, 20 mM DTT, 5 mM
TCEP, carrier ampholytes; 4-7; 0.5%, 3-10; 0.25% but omitting the reducing agents
and ampholytes at this stage. Sonication was performed under ice in a sonicator in
order.to completely dissolve the pellets. To remove cell debris and obtain a clear
supernatant centrifugation was done at 10,000g for 5 min and the resultant
supernatant was ultra centrifuged at 80,000g for 1 hour at 10C. The clear blue
supernatant was recovered and used for protein estimation using 2D quaint kit (GE
Healthcare). Proteins were aliquoted and kept at -80C until use. For rehydration,
sample volumes were adjusted to 450ul with volumes of for 24cm strips using
rehydration buffer and adding required amounts of DTT and TCEP from stock
solutions and IPG buffers.
2D gel Electrophoresis
70ug of total protein was used for IPG strips (24 cm, GE healthcare) pH 3-10 and
100ug for IPG strips 4-7. First dimension IEF was run with a horizontal
electrophoresis apparatus (Ettan IPGphor, Amersham Pharmacia Biotech) at 20C. IPG
strips were in gel rehydrated, under low voltage 15V for 14 hours. IEF was then
allowed to run initially at a low voltage to remove any interference with
contaminating salts at 200V for 1 hour. The rest was performed
according to
manufacture’s instructions. The strips were equilibrated twice, first with DTT and
secondly with idoacetamide in equlibriation buffer to improve protein transfer in the
2nd dimension(ref). The 2nd dimension gel electrophoresis was carried out on 12.5%
polyacrylamide gels (2262760.1 cm3) using Ettan Daltsix
electrophoresis system
(Amersham Pharmacia Biotech). Gels were run at 25C, using Multi TempIII
(Amersham Pharmacia Biotech) at a maximum of 100mA for approx 5 hours until the
bromophenol blue dye font reached the bottom of the gel. Co-migrating broad range
standards (BioRad) were used for estimation of molecular weight. Gels were silver
stained for protein detection purposes.
2-DE protein expression analysis
Silver stained gels were scanned using a scanner (Amersham Pharmacia Biotech). Gel
alignment, matching and statistical analysis was performed using the 2-DE software
(Progenesis same spot). Basically gels were first aligned, two individual groups in
three replicates were created and assigned as control and treated. Spots were selected
in each group based on expression changes as shown by fold change. Only spots that
showed no or marginal change were omitted. Background subtraction and
normalization was performed on each gels by selecting “lowest to boundary method”,
which divides each spot volume by the total volume of spots present in each gel.
Usually this eliminates errors associated with pippeting and staining. Spots
statistically different, were reviewed and selected by the level of significance (p) with
a t-test {t-test (p)} bellow 0.05. These spots were then analyzed for expression
changes and spots that showed an approximate two fold or greater expressions were
visually identified on the gel and tripsin digested for tentative identification by massspectrometry.
Protein digestion and identification
The 2D Gel spots of interest were excised and destained before being dried in an
Eppendorf speed Vac (Eppendorf, UK). The gel pieces were then re-swollen in a
solution of trypsin (50ng/µl in 50mM ammonium bicarbonate) for 45 minutes on ice.
After this time excess trypsin was removed and the gel piece covered in ammonium
bicarbonate and incubated overnight at 37ºC. Next the peptides were extracted from
the gel piece. Firstly the present solution was removed and kept, next 50µl of 5%
formic acid in 70|% acetonitrile was added to the gel piece which was then sonicated
for 5 minutes before removal of the liquid and its addition to the previously removed
liquid. This process was repeated three times and then the total peptide mixture was
reduced to dryness in the speed vac. Prior to analysis the peptide mixture was desalted
using ziptips (Millipore, UK) following the manufacturers instructions and reducing
the eluent to dryness and resolubilising in 0.1% formic acid in water for analysis.
Protein identification using liquid chromatography-electrospray ionizationtandem mass spectrometry (LC-ESI-MS/MS)
Extracted peptides were analyzed by nano reverse phase liquid chromatography
(Ultimate Pump, LC-Packing, Dionex, Netherlands) using an electrospray ion trap
MS (LCQ Deca XP, ThermoElectron, Hemel Hempstead, UK). LC-ESI-MS/MS
separations were performed using a 10cm x 75mm I.D. pulled-tip capillary column,
that was slurry packed in-house with 3µm, 100 Å pore size C18 silica bonded
stationary phase (PepMap, Dionex, Camberley, UK). The autosampler was fitted with
a 5µl injection loop and was refrigerated at 4ºC during analysis. After injecting 7.5µl
of the peptides, the sample was eluted onto the analytical column, over which a 5min
wash with 98% buffer A (0.1% formic acid in water v/v) was applied and peptides
were then eluted using a stepwise gradient of 0% solvent B (0.1% formic acid in
acetonitrile v/v) to 65% solvent B in 100min and then to 100% B in 10min with a
constant flow rate of 0.2 µl/min. The electrospray MS was operated in a datadependent mode in which each full MS scan (m/z 475-2000) was followed by three
MS/MS scans, in which the three most abundant peptide molecular ions were
dynamically selected for collision-induced dissociation (CID) using a normalized
collision energy of 35%. The temperature of the heated capillary and electrospray
voltage was 160ºC and 2.0kV, respectively. The data was searched against the NR
database using Sequest in Bioworks 3.2 (ThermoElectron, Hemel Hempstead, UK)
and results were filtered within Bioworks for high stringency cross correlation (Xcorr
1+, 2+, 3+ = 1.8, 2.2, 2.5).
Rationale for the AHL concentration used in the protein expression experiment
For the analysis of protein expression, C8-AHL was added to a concentration of 10
μM. This concentration is high compared with the highest steady-state concentration
observed in cultures of Gloeothece (Fig. 3). However, there are reports of pHdependent degradation of AHLs in culture medium (Yates et al., 2002). For example,
at pH 7.2 (the pH of the medium in which the Gloeothece cultures were maintained)
70% of C4-AHL (the chain length closest to C8-AHL and for which data is available)
is converted to the inactive form. Using this as a model, it is expected that the
concentration of the active form of C8-AHL that would remain in the experimental
cultures from the addition of C8-AHL to an apparent concentration of 10 µM, would
be 3.0 µM.
Figure S1. Quantitation of C8-AHL by HPLC-SRM-MS/MS. Standard curve for
C8-AHL showing linearity and precision of the HPLC-SRM-MS/MS assay developed
monitoring the transition of the protonated molecule (m/z 228) to product ion m/z 102.
Error bars indicate the standard deviation from six separate analyses for each
concentration.
250
R2 = 0.9913
SRM peak area
200
150
100
50
0
0
0.5
1
1.5
pmoles C8-HSL
2
2.5
3