Supplementary information 1
Materials and Methods
Protein extraction
Frozen tissue samples were solubilized in lysis buffer (30mM Tris-HCl pH 8.5, 7M
urea,
2M
thiourea,
4%
(w/v)
3-[(3-cholamidopropyl)
dimethylammonio]-1-propanesulfonate (CHAPS)) containing protease inhibitor
mixture (Complete; Roche, Mannheim, Germany) using a Polytron homogenizer
(Kinematica, Littau-Luzern, Switzerland) followed by centrifugation (100,000x g) for 1
hr at 4 degrees centigrade.
CyDye labeling
Protein extracts were labeled using fluorescent cyanine dyes developed for 2D-DIGE
technology (GE Healthcare UK Ltd, Buckinghamshire, England) following the
manufacturer’s recommended protocols. A 50 μg protein extract was labeled with 400
pmol fluorescent dye (Cy2, Cy3 or Cy5). Each non-cancer sample was labeled with
Cy3, and each cancer sample was labeled with Cy5. Internal standard, created by
pooling aliquots of all samples and labeled with Cy2, was used to normalize protein
abundance measurements across multiple gels in each experiment (1). An equal volume
of 2 x sample buffer (7M urea, 2M thiourea, 4% CHAPS, 2% dithiothreitol (DTT), 2%
PharmalyteTM, broad range pH3-10) was added to each of the labeled protein samples,
and these samples were mixed prior to IEF on agarose IEF gel.
Agarose 2D-DIGE and 2-DE
Agarose gels were prepared as described previously (2). Mixed labeled protein extract
(150 μg) was applied to the agarose IEF gel and first-dimensional IEF was conducted
at 12000 Vhr at 4 degrees centigrade, followed by fixation in 10% trichloroacetic acid
and 5% sulfosalicylic acid for 45 min at room temperature. After washing with
deionized water for 45 min, the agarose gel was then transferred to a 6-10% gradient of
polyacrylamide gel, and second-dimensional SDS-PAGE was performed. The
second-dimensional gel was scanned using a fluorescence image scanner, Typhoon
9400TM (GE Healthcare) and analyzed using image analyzing software DeCyderTM
(GE Healthcare). Non-labeled 500 μg of whole cell lysates (containing equal volumes
of 2x sample buffer) were separated by conventional agarose 2-DE. 2-DE gel was first
incubated in 30% methanol and 10% acetic acid, and then stained with PhastGel Blue R
(GE Healthcare).
Enzymatic in-gel digestion of proteins
Protein spots were cut from conventional agarose 2-DE gel, and in-gel tryptic digestion
of proteins was performed. Briefly, gels were cut into small pieces, destained in 50%
acetonitrile/50mM NH4HCO3, and washed with deionized water. Gel pieces were
dehydrated in 100% acetonitrile for 15 min, and then dried in a SpeedVac Evaporator
(Wakenyaku, Kyoto, Japan) for 45 min. Gel pieces were rehydrated in 10-30 μl of
25mM Tris-Cl (pH 9)/20% acetonitrile containing 25 ng/μl trypsin (Trypsin sequence
grade, Roche, Mannheim, Germany) for 45 min. After removal of unabsorbed solution,
gel pieces were incubated in 10-20 μl of 50mM Tris-Cl (pH9)/ 20% acetonitrile for 20
hr at 37 degrees centigrade. The solution containing digested fragments of proteins was
transferred to a new tube, and the peptide fragments remaining in the gel were also
extracted in 5% formic acid/50% acetonitrile for 20 min at room temperature.
Identification of proteins
Digested peptides equivalent to the maximum of 10-20 pmol of a protein in a 2-DE spot
were injected into an Aquapore RP-300 column (Perkin-Elmer, Shelton, CT), a 2.1-mm
diameter and 30-mm length C8 reversed phase, and attached to Nanospace SI-2
(Shiseido Fine Chemicals, Tokyo, Japan), a high-performance liquid chromatography
system. The flow rate of the mobile phase was 200 μl/min using buffer A (0.05%
HCOOH) and buffer B (90% acetonitrile and 0.05% HCOOH). Following an initial
wash with buffer A for 5 min, peptides were eluted with a linear gradient from 0-60%
buffer B over an interval of either 3 or 30 min. The purified peptides were sprayed from
high-performance liquid chromatography via a metal needle-attached AP12
(electrospray ionization adapter) to LCQ-Deca (ThermoQuest, San Jose, CA), ion trap
mass spectrometry. Data-dependent measurements of mass and tandem mass spectra of
the peptides were performed according to the manufacturer’s operating specifications.
SEQUEST (ThermoQuest) was used to identify proteins from mass and tandem mass
data. When the SEQUEST score for the best candidate protein was <100, we inspected
raw mass and tandem mass data of peptides to judge their qualities.
Quantification of mRNA
Total RNA was extracted from tumor and non-tumor tissues with RNeasy Plus Mini Kit
(Qiagen, Tokyo, Japan). RNA quality was monitored by both the ratio of absorbance at
260/280nm and 2% agarose gel electrophoresis. cDNA was synthesized from total
RNA with the first-strand cDNA synthesis kit for reverse transcription-PCR (Roche).
Real-time quantification of CHC, Ku86, vinculin, FTCD, rhodanese mRNA was
performed using a LightCycler instrument (Roche Diagnostics GmbH, Mannheim,
Germany) with primer sets and hybridization probes obtained from Nihon Gene
Research Labs Inc. (Sendai, Japan). β-actin mRNA was also quantified with primer
sets and hybridization probes obtained from Roche. Primer sets (forward, reverse) and
hybridization probes for the real-time PCR procedure are presented as follows;
CHC (NM_004859) : 5'-AGCTACAGAGACACAACC-3' (forward primer),
5'-AGACGATGTTTCAGTACG-3'
(reverse
5'-CATGCTGTACCCAAAGCCAGGCTGTG-FL-3'
primer),
(FL-probe),
5'-LCR640-CTGTCCATACGGTGGTGCGGTATAACC-3' (LCR640-probe), Ku86
(NM_021141):
5'-AGCCTCTGGAAGTTCTGTCA-3',
5'-TGTTAGAACTCCCAGCATCAC-3',
5'-CCCATACATCCACGACCTATATCATGTCCAAT-FL-3',
5'-LCR640-AATCGTCCACATCACCACCTTCTTCAAATACA-3',
(NM_003373)
:
vinculin
:
5'-5'-CAGTGCACAGATAAACGGA-3',
5'-TGCTCAGACTCCTCATCAC-3',
5'-CCTCTTACAGGTATGTGAGCGAATCCCAA-FL-3',
5'-LCR640-CATAAGCACCCAGCTCAAAATCCTGTCC-3', FTCD (NM_006657) :
5'-AGCCAGGACGTCTGAAGA-3',
5'-TGAAGAGGTTCTCCTTCTCG-3',
5'-TGTGCAGTGCCGTGACCTCAAAGTCC-FL-3',
5'-LCR640-GAAGATTGGTGGACACCTGAGCCAGGTT-3',
(NM_003312)
:
rhodanese
5'-TGGTGGATTCAAGGTCTCAA-3',
5'-TCTTGGTCTGGAACAGAGCA-3',
5'-ATGCAGTAGGACTGGACTCGGGCCAT-FL-3',
5'-LCR640-TCCGTGGTGCCGTCAACATGCCTT-3', respectively.
Supplementary information References
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Yan JX, Devenish AT, Wait R, Stone T, Lewis S, Fowler S. Fluorescence two-dimensional
difference gel electrophoresis and mass spectrometry based proteomic analysis of Escherichia coli.
Proteomics 2002;2:1682-1698.
2.
Oh-Ishi M, Satoh M, Maeda T. Preparative two-dimensional gel electrophoresis with agarose
gels in the first dimension for high molecular mass proteins. Electrophoresis 2000;21:1653-1669.