Supplementary Data 1.
Tissue sample processing for global proteome profiling
The cancer-normal pair of the gastric tissues were separatively pulverized by a cryopulverizer
unit (CP02 Cryoprep, Covaris, Woburn, MA) and each of the purverlized tissues were
subsequently subjected to lysis in a buffer (4% SDS, 0.1 M Tris-HCl (pH 7.6), and PhosSTOP
phosphatase inhibitor cocktail (Roche, Mannheim, Germany) by sonication (S220 focusedultrasonicator, Covaris, Woburn, MA). The resultant proteome samples were subjected to BCA
assay (BCA Protein Assay Kit, Pierce) for protein quantification.
A slightly modified filter-
aided sample preparation (FASP) method [r3] was used for tryptic digesion as previously
described in details [r4]. The resultant peptide samples were again subjected to BCA assay for
peptide quantification.
Basis pH reverse-phase fractionation
The pooled iTRAQ labeled peptides were separated using Agilent 1260 Infinity HPLC system
(Agilent, Palp Alto, CA) equipped with an fraction collector (G1364C, Agilent, Palo Alto, CA).
An analytical column (4.6 mm  250 mm, Xbridge C18, 5μm) and a guard column (4.6 mm 
20 mm, Xbridge C18, 5μm) were used for the basic pH reverse-phase separation at the flow
rate of 0.5 mL/min. Solvent A (10mM TEAB in water, pH 7.5) and solvent B (10mM TEAB
in 90% ACN, pH 7.5) were used to generate an 130 min gradient: 0% sol B for 10 min, 0-5%
in 10 min, 5-35 % in 60 min, 35-70 % over 15 min, 70 % for 10 min, 70-0 % over 10min, and
holding at 0% over 15 min. A total of 96 fractions were collected in every 1 min from 15 min
to 110 min. The 96 fractions were pooled into 24 non-contiguously concatenated peptide
fractions as previously described [r5]. The 24 fractions were vacuum dried and stored at –80°C
until LC-MS/MS experiments.
Capillary RPLC-MS/MS analyses
10 g of labeled peptides from each of 24 fractions were individually analyzed by Q Exactive
orbitrap hybrid mass spectrometer (Thermo Scientific, Bremen, Germany) which was coupled
to the dual-online LC system through an home-built dual-column nano-electrospray source as
previously been described [r4]. The dual-online LC system is equipped with two reversephase analytical columns (75 μm i.d. × 360 μm o.d., 100cm) along with two SPE column (150
μm i.d. × 360 μm o.d., 3cm), that were packed with C18-bonded particles (Jupiter, 3 μm
diameter, 300 Å pore size, Phenomenex, Torrance, CA). A linear gradient was applied over 180
min with solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in ACN) at
flow rate of 300 nL/min (1-40 % sol B over 160 min, 40-80 % over 5 min, 80 % for 10 min
and holding at 1 % for 5 min).
The eluting peptides were ionized at the spray voltage of 2.4 kV and the desolvation capillary
temperature of 250 ℃, respectively. MS precursor scans (400 –
acquired with the resolution of 70,000 (at 400 Th), followed by data-dependent acquisition of
MS/MS data for ten most abundant ions, using higher energy collisional dissociation (HCD) at
normalized collision energy (NCE) of 30. MS/MS scans were acquired with fixed first mass of
100 Th with resolution of 17,500 and AGC target value of 1.0106, respectively. The maximum
ion injection time of MS scan and MS/MS scan were set to 20 and 60 ms, respectively.