Supplementary material and methods
Protein sample preparation. For proteomic analysis growth arrested and proliferating
cells were obtained plating 4x106/10cm and 1.3x106/15cm dishes and maintained in
culture for 3 days or 18 hours, respectively. Cells were washed twice with 1X PBS,
scraped in ice-cold PBS and centrifuged at 100 x g. Cells were lysed in ice-cold
hypotonic buffer (10mM HEPES-KOH pH 7.9, MgCl2 1.5mM, KCl 10mM, NP40 0.05%
v/v, DTT 0.5mM, NaF 2mM, PMSF 0.5mM, Protease inhibitor cocktail 1:200) under
slow agitation for 30 min at +4°C. After centrifugation at 100 x g, supernatants were
collected as cytosolic protein fraction. Protein amounts were determined by Bradford
assay (BioRad) using BSA as standard. Protein samples for fluorescence dye labeling
were processed using a 2D Clean-Up kit (GE Healthcare) and resuspended in labeling
buffer (7 M urea, 2 M thiourea, 4% w/v CHAPS, 30 mM Tris).
Fluorescence dye labeling. Samples from growth arrested and proliferating MMH cells
were labeled with either Cy3 or Cy5 dyes while internal standard samples were labeled
with Cy2 dye. Fifty μg of proteins for each sample were labeled using 400pmol of
cyanine dyes in 1μL of anhydrous N,N dimethylformamide (GE Healthcare) for 30 min
on ice in the dark; the reaction was then quenched with 10mM lysine and additionally
incubated for 10 min. A dye-swapping protocol was used to minimize labeling-dependent
artifacts for labeling the four pair of samples variously with Cy3 or Cy5 (see
supplementary material for the experimental design). The quenched Cy3 and Cy5-labeled
samples were then combined and mixed with an aliquot of Cy2-labeled standard and an
equal volume of 2X sample buffer (7M urea, 2M thiourea, 4% w/v CHAPS, 2% w/v
DTT, 2% v/v IPG buffer 3-10 NL).
Gel electrophoresis. Isoelectric focusing (IEF) was performed using 18-cm immobilized
nonlinear pH gradient strips (NL IPG pH 3.0 to 10.0; GE Healthcare) on a IPGphor II
electrophoresis unit (GE Healthcare). Proteins were loaded by in-gel rehydratation for 9
hours using low voltage (30V), then run using a program in which the voltage was set for
1 hours at 100 V, 2 hours at 200 V, 30 min at 300 V, 3 h voltage gradient 300-3500V, 10
min at 5000V and 5 hours at 8000 V. IPG gel strips were reduced in 1% DTT for 10 min
at room temperature and free SH groups were subsequently blocked using 4%
iodoacetamide (both solutions were prepared in 50 mM Tris-HCl, pH 8.8, 6 M urea, 30%
glycerol, 2% SDS, 2% bromophenol blue). Strips were transferred to 1 mm thick 12%
polyacrylamide gels (20 x 20 cm), and the second-dimension gels were run at 50 mA for
6 h. All electrophoresis procedures were performed in the dark.
Image acquisition and spot quantification. The labeled protein spots in the gel were
visualized using the Typhoon 9400 imager (GE Healthcare). The gels were scanned using
a 488 nm laser and an emission filter of 520 nm band pass (BP) 40, a 532 nm laser and an
emission filter of 580 nm BP30, a 633 nm laser and a 670 nm BP30 emission filter to
acquire the Cy2, Cy3, and Cy5 images, respectively. One of the four gels was poststained using Sypro Ruby (BioRad) accordingly to the manufacturer’s instructions,
visualized using the Typhoon 9400 scanner and then used as preparative gel for
downstream protein identification. In order to compare protein spots across the four gels,
image analyses were conducted into two steps using DeCyder v6.5 2D Differential
Analysis Software (GE Healthcare). Briefly, processed data were filtered to reveal
statistically relevant fold changes in protein abundance, using the average volume ratios
of 1.2 and above or -1.2 and below fold differences in expression and one-way ANOVA
analysis with a p value of ≤0.05. On a total of 2500 spots detected in the master gel, 82
spots satisfied these requirements and were highlighted as spots of interest.
In-gel tryptic digestion and MALDI-TOF MS analysis
Spots were excised from the gels with an automatic spot picker (Investigator ProPic;
Genomic Solutions Inc.), subjected to in-gel tryptic digestion, concentrated with
ZipTip®μC18 pipette tips (Millipore) and coeluited directly onto a MALDI target.
MALDI-MS and MALDI-MS/MS were performed on an Applied Biosystems 4700
Proteomics Analyzer with TOF/TOF ion optics. The spectra were acquired in the positive
reflector mode by 40 subspectral accumulations (each consisting of 50 laser shots) in a
800–4000 mass range, focus mass 2,100 Da, using a 355 nm Nb:YAG laser with a 20 kV
acceleration voltage.
The interpretation of both the MS and MS/MS data was carried out with the GPS
Explorer software (Version 3.6, Applied Biosystems) which acts as an interface between
the Oracle database containing raw spectra and a local copy of the MASCOT search
engine (Version 2.1, Matrix Science, Ltd.). A combined MS peptide fingerprint and
MS/MS peptide sequencing search was performed against the Swiss Prot database
without taxon restriction using the MASCOT search algorithm. These searches specified
trypsin as the digestion enzyme, carbamidomethylation of cysteine as fixed modification,
partial oxidation of methionine and phosphorylation of serine, threonine, and tyrosine as
variable modifications, and allowed for one missed trypsin cleavage. The monoisotopic
precursor ion tolerance was set to 30 ppm and the MS/MS ion tolerance to 0.3 Da.
MS/MS peptide spectra with a minimum ion score confidence interval ≥ 95% were
accepted; this was equivalent to a median ion score cut off of approximately 35 in the
data set. Protein identifications were accepted with a statistically significant MASCOT
protein search score ≥ 65 that corresponded to an error probability of p<0.05 in our data
set.