J O U RN A L OF P ROT EO M IC S 8 3 ( 2 01 3 ) 1 9 7 –2 13

Available online at www.sciencedirect.com

www.elsevier.com/locate/jprot

Comparative proteomics, network analysis and

post-translational modification identification reveal
differential profiles of plasma Con A-bound glycoprotein
biomarkers in gastric cancer

Yih-Huei Uena, b, c, d, 1 , Kai-Yuan Linc, e, 1 , Ding-Ping Sunb, 1 , Chen-Chung Liaof, 1 ,

Ming-Song Hsiehg, h, i , Yung-Kai Huangj , Yen-Wei Chenk , Pei-Hsuan Huangf ,
Wei-Jung Chenl , Chih-Chun Taim , Kuan-Wei Leei , You-Chia Cheni , Ching-Yu Lini,⁎
a
Superintendent's Office, Chi-Mei Hospital Chiali, Tainan 722, Taiwan
b
Division of General Surgery, Department of Surgery, Chi-Mei Medical Center, Tainan, 710, Taiwan
c
Department of Medical Research, Chi-Mei Medical Center, Tainan 710, Taiwan
d
Institute of Biomedical Engineering, Southern Taiwan University of Science and Technology, Tainan 710, Taiwan
e
Department of Biotechnology, Chia Nan University of Pharmacy and Science, Tainan 717, Taiwan
f
Proteomics Research Center, National Yang-Ming University, Taipei, Taiwan
g
Department of Infection Control, Microbiological Laboratory, Taipei Medical University-Shuang-Ho Hospital, Taipei Medical University,
New Taipei City, Taiwan
h
Department of Medical Laboratory Science and Biotechnology, Central Taiwan University, Taichung, Taiwan
i
School of Medical Laboratory Science and Biotechnology, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan
j
School of Oral Hygiene, College of Oral Medicine, Taipei Medical University, Taiwan
k
Department of Life Science and Institute of Bioinformatics and Structural Biology, College of Life Science, National Tsing Hua University,
Hsin Chu 30013, Taiwan
l
Institute of Biotechnology, National Ilan University, Ilan 26047, Taiwan
m
Department of Laboratory Medicine, Taipei Medical University-Shuang-Ho Hospital, Taipei Medical University, New Taipei City, Taiwan

AR TIC LE I N FO ABS TR ACT

Article history: In the study, we used Con A affinity chromatography, 1-D gel electrophoresis, and
Received 11 May 2012 nano-LC–MS/MS to screen biomarker candidates in plasma samples obtained from 30 patients
Accepted 10 March 2013 with gastric cancer and 30 healthy volunteers. First, we pooled plasma samples matched by age
Available online 26 March 2013 and sex. We identified 17 differentially expressed Con A-bound glycoproteins, including 10
upregulated proteins and 7 downregulated proteins; these differences were significant (Student's
Keywords: t-test, p-value < 0.05). Furthermore, 2 of the upregulated proteins displayed expression levels
Gastric cancer that were increased by 2-fold or more in gastric cancer samples when compared with normal
Glycoproteins control samples. These proteins included leucine-rich alpha-2-glycoprotein (LRG1) and inter-
Concanavalin A alpha-trypsin inhibitor heavy chain H3 (ITIH3), and the expression levels were validated by
Mass spectrometry Western blot analysis. Pathway and network analysis of the differentially expressed proteins by
Post-translational modification Ingenuity Pathway Analysis revealed vital canonical pathways involving acute phase response
Human plasma signaling, the complement system, LXR/RXR activation, hematopoiesis from pluripotent

⁎ Corresponding author at: School of Medical Laboratory Science and Biotechnology, College of Medical Science and Technology, Taipei Medical
University, No. 250, Wuxing Street, Taipei 11031, Taiwan. Tel.: +886 2 2736 1661x3326; fax: +886 2 27324510.
E-mail address: cylin@tmu.edu.tw (C.-Y. Lin).
1
Equal contribution.

1874-3919/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jprot.2013.03.007
198 J O U RN A L OF P ROTE O M IC S 8 3 ( 2 01 3 ) 1 9 7 –21 3

stem cells, and primary immunodeficiency signaling. Our results suggest that Con A-bound LRG1
and ITIH3 may not be practically applicable as a robust biomarker for the early detection of
gastric cancer. Additionally, three novel PTMs in ITIH3 were identified and include hexose-
N-acetyl-hexosamine at asparagine-41, trimethylation at aspartic acid-290, and flavin adenine
dinucleotide at histidine-335.

Biological significance
Our study was to describe a combinatorial approach of Con A affinity chromatography, 1-D
SDS-PAGE, and nano-LC/MS/MS that provides a label-free, comparative glycoproteomic
quantification strategy for the investigation of glycoprotein profiles in plasma from gastric
cancer patients versus healthy volunteers and to identify glycoprotein biomarkers for the
early clinical detection of gastric cancer. Three novel PTMs, HexHexNAc, trimethylation
and FAD, in Con A-bound ITIH3 were identified and built in molecular modeling. The
aspartic acid-290 trimethylation site was located in a metal ion-dependent adhesion site (MIDAS
motif; 290-DXSXS…T…D-313) that may influence important function for binding protein ligands.
© 2013 Elsevier B.V. All rights reserved.

1. Introduction be as low as 50% RSD, but an unlimited number of samples can be

Gastric cancer is one of the most frequent cancers worldwide
and shows a wide geographical variation with low incidence
areas in the Americas and high incidence areas in Africa, Europe,
and Asia [1]. Gastric cancer is the fourth most common cancer
and the third leading cause of cancer deaths worldwide [2,3]. In
the year 2000, approximately 880,000 people were diagnosed
with gastric cancer, and approximately 650,000 people died of
the disease [1]. In 2008, it was the seventh most prevalent cancer
and the sixth leading cause of cancer-related deaths in Taiwan
[4]. Epidemiological studies have indicated that the risks for
gastric cancer include family history, age, gender, consump-
tion of salt-preserved foods and dietary nitrites, gastrectomy,
Helicobacter pylori (H. pylori) infection, smoking, and alcohol
use [5]. The patients with gastric cancer have a relatively poor
prognosis; the global 5-year survival rate is approximately
20%. However, the rate in Taiwan is less than 40% [4,6]. The
available serum tumor markers carcinoembryonic antigen
(CEA), cancer antigen 19-9 (CA 19-9), and cancer antigen 72-4 (CA
72-4) used in the clinic do not demonstrate sufficient sensitivity
for the early detection of gastric cancer [7]. Therefore, there is an
urgent need to discover better biomarkers for gastric cancer.
An important criterion for biomarker candidate discovery is
the quantitative comparison between disease samples and
control samples. Quantification methods include a labeling
approach that requires incorporation of the stable isotope
labels 12/13C, 14/15N, and/or 16/18O into proteins or peptides prior to
MS analysis or the use of a label-free method. Labeling methods
include methanol (substituted with hydrogen or deuterium)
esterification reactions [8], tryptic digestion in the presence
of H2(16/18)O [9], 12/13C-acrylamide-cysteine labeling [10], 13CNBS
(2-nitrobenzenesulfenyl) labeling [11], isotope-coded protein
labeling (ICPL) [12], ICAT [13], stable isotope labeling with amino
acids in cell culture (SILAC) [14] and iTRAQ [15]. In general,
labeling methods demonstrate high precision for quantification
(typically 10% RSD), but restrictions due to isotope labels and
experimental operation limit the number of samples that can be
tested for biomarker discovery [16]. In contrast, the quantitative
precision of label-free techniques based on spectral counting can
analyzed to independently acquire MS data [17]. However, the
reproducibility of label-free quantification based on spectral
counting by 1-D SDS-PAGE coupled to nano-LC–MS/MS ranges
from 23.4% to 26.1% RSD [18]. Quantification based on peak
intensities (extracted ion chromatograms, XIC) is analyzed
separately in LC–MS/MS runs and can usually provide quantita-
tive accuracy below 30% [19]. The advantages of label-free
quantification include its low cost, high proteome coverage,
high analytical depth, and high dynamic range. Both label-free
and isotopic labeling quantification require sufficient sample
sizes to overcome individual variability in clinical samples [17]. A
combinatorially pooled sample, 1-D SDS-PAGE and label-free
nano-LC–MS/MS may reduce the complexity of the sample by
mixing different proteomes.
Over the past decade, clinical proteomics has successfully
combined the development of advanced technology and bioin-
formatics to determine the protein/peptide biomarkers responsi-
ble for disease through altered levels of expression and/or PTMs
and different protein variants [20]. Biomarkers can be divided into
three types based on the analytes and assay methods. First,
altered expression of the total protein is measured by competitive
ELISA. Second, single, specific PTMs of proteins are detected with
mAb by competitive and sandwich ELISA assays. Third, multiple
PTMs of proteins are detected with different specific mAbs by the
sandwich assay [21]. Anderson [22] has analyzed all of the
protein-based assays cleared or approved by the US Food and
Drug Administration (FDA) (Jan 1993 to Jan 2009), and these
assays have revealed 109 unique protein analytes in the plasma
and/or serum, 62 additional tests, and 96 laboratory-developed
tests (LDTs), yielding a total of 205 unique proteins. The 62
additional tests consisted of peptides (7 tests), protein PTMs (18
tests), protein complexes (3 tests), autoantibodies (20 tests), and
blood cell proteins (14 tests). The 18 protein PTM biomarkers
included fucosylated glycoforms of AFP (AFP-L3%), glycated
albumin, bone-specific alkaline phosphatase (BAP), CA 19-9,
type I collagen cross-linked deoxypyridinoline, type I collagen
cross-linked N-terminal telopeptide, type I collagen cross-
linked pyridinium, type I collagen C-terminal telopeptide,
D-dimer, des-gamma-carboxy prothrombin (DCP), DR-70,
 J O U RN A L OF P ROT EO M IC S 8 3 ( 2 01 3 ) 1 9 7 –2 13
fibrin monomers, fibrin(ogen) split/degradation products
(FSP/FDP), fibrinopeptide A, glycated low-density lipopro-
tein (LDL), plasmin, prothrombin fragment 1.2 (F1.2), and
carbohydrate-deficient transferrin.
PTMs are the chemical modification of proteins after mRNA
translation, and they play pivotal roles in cellular physiology and
disease progression [23–25]. Currently, more than 300 different
types of PTMs have been identified [26]. These PTMs include the
addition of chemical groups (e.g., phosphate, citrulline, biotin,
or acetate) or more complex molecules (e.g., carbohydrates,
nucleotide, or lipids), the covalent linkage of small proteins
(ubiquitin or ubiquitin-like proteins), the modification of
side-chain residues of specific amino acids (acetylation,
deimination, deamidation, farnesylation, glycosylation, hydrox-
ylation, methylation, nitration, oxidation, palmitoylation, phos-
phorylation, sulfation, SUMOylation, and ubiquitination) and
proteolysis [27]. Protein PTMs have led to the alteration of gene
and protein expression levels to activate specific signaling
pathways, enhance cell proliferation, and dysregulate apoptosis
during cancer progression [28–30]. The identification of each type
of PTM may be developed as a unique cancer-specific biomarker,
and the PTM peptide sequence has also been considered a
neo-epitope. PTM analysis of the plasma proteome has thus
become an important area for biomarker discovery [27].
Glycosylated proteins exist as multiple glycoforms that can be
captured by hydrazide resin and lectin affinity chromatography,
among other methods [31]. The tumor markers CEA, CA 19-9, and
CA 72-4 are glycoproteins used for the detection of gastric cancer
[7]. Most FDA-approved cancer biomarkers are glycoproteins or
glycans, and none demonstrate the specificity required for
diagnosis when used alone [32]. Therefore, protein-based multi-
plex assays, such as the in vitro diagnostic multivariate index
assay (IVDMIA) and the FDA-approved OVA1™ test (Sep 2009)
[33], will be critical in achieving the high sensitivity and specificity
required for early cancer diagnosis. The panel of 5 biomarkers in
the OVA1™ test contains of CA125, transthyretin (prealbumin),
apolipoprotein A1 (APOA1), beta-2 microglobulin (B2M), and
transferrin (TF). The OVA1 score for serum ranges from 0 to 10
and is determined by an algorithm analysis that combines 5
immunoassays into a single numerical result. Different
cut-off values are used for premenopausal women (5.0) and
postmenopausal women (4.4). The sensitivity of OVA1™ is 89% in
premenopausal women and 98% in postmenopausal women [34].
Table 1 – The demographic and clinical characteristics of the individual subjects contributing to the pooled plasma for
gastric cancer patients and normal control samples.
Characteristic Discovery set
(N = 60)
Cancer, n (%)
(n = 30)
Age (years), mean ± SD 65.33 ± 9.03
Gender
Male 16 (53.33%)
Female 14 (46.67%)
Stage of gastric cancer
I 10 (33.33%)
II 5 (16.67%)
III 10 (33.33%)
IV 5 (16.67%)
199
Therefore, we need an approach to determine potentially novel
glycosylation biomarkers.
In this study, our aim was to describe a combinatorial
approach of Con A affinity chromatography, 1-D SDS-PAGE,
and nano-LC/MS/MS that provides a label-free, comparative
glycoproteomic quantification strategy for the investigation of
glycoprotein profiles in plasma from gastric cancer patients
versus healthy volunteers and to identify glycoprotein bio-
markers for the early clinical detection of gastric cancer. We
also used bioinformatic tools to search for protein ontology,
category classifications, molecular modeling, PTM peptides, PTM
sites, and pathway analyses. Furthermore, Western blot and Con
A analyses were performed to confirm the differential expres-
sion levels of glycoprotein biomarkers. The potential biological
roles of these altered expression glycoproteins and novel PTM
sites in gastric cancer plasma are discussed.
2. Materials and methods
2.1. Patient samples
Plasma samples from 90 patients with gastric cancer and 90
age- and sex-matched healthy volunteers (normal controls)
were obtained from the Tumor and Serum Bank of Chimei
Medical Center and the Department of Laboratory Medicine,
Shuang-Ho Hospital in Taiwan (Table 1). This study was
approved by the hospital ethics committee, and all patients
and volunteers provided informed consent for the study. The
gastric cancer plasma samples were from patients with stage I
tumors (27 samples), stage II tumors (21 samples), stage III
tumors (21 samples), and stage IV tumors (21 samples). For
discovery, pooled plasma samples were obtained from 30
gastric cancer patients and 30 normal controls (the discovery
set), and the protein panels were further validated against
individual plasma samples from 60 gastric cancer patients
and 60 normal controls (the validation set) by Western blot
analysis. The validation set of plasma from gastric cancer
patients and normal controls was separated into three groups
containing plasma from 120 samples [stage I/II tumors (33
samples), stage III/IV tumors (27 samples), and normal
controls (60 samples)]. For the validation experiment of the
discovery set, 30 normal controls (63.3 ± 8.50 years old) and 30
Validation set
(N = 120)
Control, n (%) Cancer, n (%) Control, n (%)
(n = 30) (n = 60) (n = 60)
63.3 ± 8.50 59.92 ± 13.10 58.30 ± 12.80
16 (53.33%) 36 (60.0%) 36 (60.0%)
14 (46.67%) 24 (40.0%) 24 (40.0%)
17 (28.33%)
16 (26.67%)
11 (18.33%)
16 (26.67%)
200 J O U RN A L OF P ROTE O M IC S 8 3 ( 2 01 3 ) 1 9 7 –21 3
gastric cancer patients (65.33 ± 9.03 years old) were selected.
Sixty normal controls (58.30 ± 12.80 years old) and 60 gastric
cancer patients (59.92 ± 13.10 years old) participated in the
validation experiment of the validation set.
2.2. Con A affinity chromatography
For glycoprotein enrichment, we used agarose-bound Con A
according to the manufacturer's protocol (Vector Laboratories,
Burlingame, California, USA). Briefly, 300 μL of agarose-bound
Con A was added to a 1.5 ml centrifuge tube. The agarose-bound
Con A was washed with 3 volumes of binding buffer (25 mM Tris,
1 mM MnCl2, 1 mM CaCl2, and 500 mM NaCl, pH 7.6). Then,
100 μL of pooled plasma was added and mixed with agarose-
bound Con A at 4 °C overnight. After centrifugation, the
agarose-bound Con A was washed with 8 volumes of binding
buffer to remove nonspecifically bound proteins. Finally, the
captured glycoproteins were eluted 6 times with 200 μL of elution
buffer (500 mM alpha-methyl mannoside in 20 mM Tris, pH 7.6).
The eluted fractions were pooled and concentrated with a
Microcon YM-10 centrifugal filter unit (Millipore Corporation,
Billerica, Massachusetts, USA), and the Con A-captured glycopro-
teins were subjected to quantification by the Bradford assay
(Bio-Rad Laboratories, Hercules, California, USA). The features of
the plasma-derived Con A-bound glycoproteins were confirmed
using N-deglycosylation strategies, including PNGase F (New
England BioLabs, Ipswich, MA, USA) treatment, re-Con A affinity
chromatography and analysis by LC–MS/MS. PNGase F treatment
of the plasma-derived Con A-bound glycoproteins was performed
according to the manufacturer's protocol. Briefly, a 10 μL reaction
including 50 μg of plasma-derived Con A-bound glycoproteins
and 1× glycoprotein denaturing buffer was denatured by heating
at 100 °C for 10 min. A 20 μL reaction containing denatured
glycoproteins, 1× G7 reaction buffer, 1% NP-40 and 1000 U of
PNGase F was incubated at 37 °C for 1 h. A parallel reaction
without PNGase F served as a control.
2.3. Depletion of high abundance proteins from plasma
Albumin removal using the Albumin and IgG Depletion SpinTrap
column (GE Healthcare Life Sciences, Piscataway, New Jersey,
USA) was performed according to the manufacturer's protocol.
Briefly, 30 μL of pooled plasma was diluted with binding buffer
(20 mM sodium phosphate and 150 mM NaCl, pH 7.4) to a final
volume of 100 μL. Then, 100 μL of the sample was applied to the
column, and the column was incubated at 4 °C for 1 h. The
column was eluted with 5 volumes of binding buffer to collect the
depleted samples. The depleted samples were pooled and
concentrated with a Microcon YM-10 centrifugal filter unit
(Millipore Corporation, Billerica, Massachusetts, USA), and pro-
tein quantification was determined with the Bradford assay
(Bio-Rad Laboratories, Hercules, California, USA).
2.4. Periodic acid-Schiff (PAS) staining for glycoproteins
The glycoprotein sugar moieties in polyacrylamide gels were
stained using a Pierce Glycoprotein Staining Kit (Thermo
Scientific, Rockford, Illinois, USA) according to the manufacturer's
protocol. Briefly, 50 μg of proteins in the gel was fixed with 100 ml
of 50% methanol for 30 min. The gel was washed twice with
100 ml of 3% acetic acid for 10 min. The gel was then washed
with 25 ml of oxidizing solution for 15 min and washed three
times with 100 ml of 3% acetic acid for 5 min. The gel was next
soaked with 25 ml of glycoprotein staining reagent for 15 min
and with 25 ml of reducing solution for 5 min. Finally, the gel was
washed with 3% acetic acid followed by deionized water. The
N-deglycosylation reaction of plasma-derived Con A-bound
glycoproteins used PNGase F (New England BioLabs, Ipswich,
MA, USA) treatment and was verified by PAS staining. A duplicate
gel was run and stained with CBB G-250 as a loading control.
2.5. SDS-PAGE and in-gel digestion
Plasma protein samples were fractionated by 12% SDS-PAGE
(Hoefer®, Inc., Holliston, Massachusetts, USA) according to a
previously described method [35]. The gels were stained with CBB
staining solution (Bio-Rad Laboratories, Hercules, California,
USA), and 50 μg of each protein sample was loaded onto the gel
in triplicate. The gel lanes were cut into 10 fractions based on
molecular weight, and the gel slices were destained repeatedly in
a solution of 25 mM NH4HCO3 and 50% (v/v) CAN (1:1) until no
protein bands were visible. After drying in a Speed-Vac (Thermo
Electron, Waltham, Massachusetts, USA), the slices were
incubated with 2% β-mercaptoethanol and 25 mM NH4HCO3 for
20 min at room temperature in the dark to reduce the disulfide
bonds in the proteins. For cysteine alkylation, an equal volume of
10% 4-vinylpyridine in 25 mM NH4HCO3 and 50% CAN was added
for 20 min. After soaking the slices in 1 ml of 25 mM NH4HCO3 for
10 min, the slices were dried in a Speed-Vac for 20 min. Then,
100 ng of modified trypsin (Promega, Mannheim, Germany) in
25 mM NH4HCO3 was added, and tryptic digestion was performed
at 37 °C overnight. The tryptic digest was removed from the gel,
dried in a Speed-Vac and stored at −20 °C until further analysis.
The tryptic peptides were resuspended in 0.1% formic acid
immediately before use [18,36].
2.6. Mass spectrometric analysis
Triplicate tryptic peptide mixtures were injected into a nano-flow
HPLC system (Agilent Technologies 1200 series, Waldbronn,
Germany) coupled to an LTQ-Orbitrap Discovery™ hybrid mass
spectrometer with a nanoelectrospray ionization source (Thermo
Electron, Waltham, Massachusetts, USA). The tryptic peptides
were separated on an Agilent C18 column (100 × 0.075 mm,
3.5 μm in diameter) with mobile phases of 0.1% formic acid in
water (solvent A) and 0.1% formic acid in CAN (solvent B) at a flow
rate of 0.5 μL/min using a 30 min linear gradient of 5% to 35%
solvent B followed by 95% solvent B for 10 min. Following each
full scan (m/z range of 200–2000), a data-dependent acquired MS/
MS scan for a series of precursor ions was selected on the basis of
the conventional MS spectra (Survey Scan) triggered at high
resolution (M/DM, 60,000 full width half maximum). The former
acquired the spectrum (CID spectrum or MS/MS spectrum) for the
fragment ions generated by CID, whereas the latter examined the
accurate mass and the charge state of the selected precursor ion.
2.7. Database searching
A MS/MS dataset generated by nano-LC/MS/MS was analyzed
by Xcalibur 2.0 SR1 software (Thermo Electron, Waltham,
 J O U RN A L OF P ROT EO M IC S 8 3 ( 2 01 3 ) 1 9 7 –2 13 201

Massachusetts USA) to generate peak lists. The SEQUEST
algorithm (TURBO) was used to identify peptide sequences
against the Universal Protein Resource Knowledgebase, a
human protein database (UniProt; http://www.uniprot.org/) that
contains ion scans obtained from MS/MS data. Information on
category classifications in the protein annotation, including
cellular components, molecular functions, and biological pro-
cesses, was recorded. The database searches permitted fixed
modification of the cysteine residue (carboxyamidomethylation,
57 Da), variable modification of the methionine residue (oxida-
tion, 16 Da), peptide mass tolerance of 1.5 Da, and fragment mass
tolerance of 1 Da. These peptides were filtered by Xcorr versus
charge state with a p-value threshold < 0.05 to determine
significance. Xcorr was used as a match with 1.9 for singly
charged ions, 2.2 for doubly charged ions and 3.75 for triply
charged ions. Additional filtering was performed for proteins
scoring above ten and those having at least two hits (two
different peptides or one peptide detected by at least two
different fractions or scan times) for protein identification.
The individual molecules participating in the protein–protein
interactions were searched using Ingenuity Pathway Analysis
software (IPA; Ingenuity Systems, USA). Additionally, the mass
spectra of candidate biomarkers [Con A-bound leucine-rich
alpha-2-glycoprotein (LRG1) and inter-alpha-trypsin inhib-
itor heavy chain H3 (ITIH3)] were searched to allow a wide
range of modifications using our in-house PTM finder
program that subjects the protein sequence to alignment
to identify PTM peptide sequences, PTM sites, and quanti-
fication [36]. MS spectral counts were normalized on the
sum of the spectral counts per biological sample in
quantitative analyses [18]. Furthermore, all modified MS1
spectra were manually identified in this study, and the
fragmented ions (from the nano-LC/MS/MS) were labeled as
b, y, y-NH3, and b-H2O ions.

Table 2 – Differentially expressed Con A-bound glycoprotein biomarkers in plasma identified by 1-D SDS-PAGE and LC–MS/
MS analysis in gastric cancer and normal samples.
Protein Symbol Protein name Normalized average p-Value Fold Glycosylation N-Deglycosylation
ID spectral counts change a
(mean ± RSD)

Cancer Normal

P02750 LRG1 Leucine-rich 12.97 ± 4.00 ± 0.004 ↑2.24 N-linked/ N.D.

alpha-2-glycoprotein 18.73% 19.41% O-linked
Q06033 ITIH3 Inter-alpha-trypsin 21.54 ± 7.03 ± 0.022 ↑2.06 N-linked N.D.
inhibitor heavy chain H3 32.05% 9.36%
P02790 HPX Hemopexin 41.96 ± 20.78 ± 0.026 ↑1.02 N-linked/ +
7.97% 48.51% O-linked
P05155 SERPING1 Plasma protease 25.43 ± 15.14 ± 0.027 ↑0.68 N-linked/ +
C1 inhibitor 13.49% 26.18% O-linked
P04217 A1BG Alpha-1B-glycoprotein 94.32 ± 63.68 ± 0.022 ↑0.48 N-linked +
11.23% 15.80%
P01876 IGHA1 Ig alpha-1 chain C region 143.84 ± 102.52 ± 0.010 ↑0.40 N-linked/ +
10.55% 3.85% O-linked
P00751 CFB Complement factor B 38.42 ± 27.81 ± 0.004 ↑0.38 N-linked +
2.47% 10.37%
P02751 FN1 Fibronectin 80.09 ± 59.85 ± 0.002 ↑0.34 N-linked/ +
5.33% 4.12% O-linked
P0CG06 IGLC3 Ig lambda-3 chain 5.75 ± 4.37 ± 0.039 ↑0.32 N.A. c N.D.
C regions 10.60% 11.52%
P19827 ITIH1 Inter-alpha-trypsin 72.44 ± 55.75 ± 0.010 ↑0.30 N-linked/ +
inhibitor heavy chain H1 5.76% 8.44% O-linked
P01859 IGHG2 Ig gamma-2 chain C region 11.57 ± 25.50 ± 0.016 ↓1.20 N-linked +
52.07% 1.87%
P01871 IGHM Ig mu chain C region 32.69 ± 54.07 ± 0.024 ↓0.65 N-linked +
18.26% 15.79%
P10909 CLU Clusterin 17.58 ± 27.37 ± 0.038 ↓0.56 N-linked N.D.
22.32% 14.32%
P05090 APOD Apolipoprotein D 15.80 ± 20.89 ± 0.024 ↓0.32 N-linked +
10.60% 8.80%
P00747 PLG Plasminogen N.D. b 5.41 ± 0.002 – N-linked/ N.D.
25.38% O-linked
P01031 C5 Complement C5 N.D. 4.13 ± 0.028 – N-linked N.D.
51.18%
P02746 C1QB Complement C1q N.D. 5.38 ± 0.000 – N.A. N.D.
subcomponent subunit B 9.56%
a
Fold change represent increment (↑) and reduction (↓) fold change compared with gastric cancer versus normal samples.
b
N.D., not detectable.
c
N.A., not available.
202 J O U RN A L OF P ROTE O M IC S 8 3 ( 2 01 3 ) 1 9 7 –21 3
2.8. Molecular modeling
Molecular modeling, figure building, and sequence align-
ment of ITIH3 were performed using SWISS-MODEL (http://
swissmodel.expasy.org/) [37–39], PyMOL (http://www.pymol.org/)
and multiple sequence alignment (MSA) (http://workbench.sdsc.
edu) software, respectively [36].
2.9. Ingenuity Pathway Analysis
Functional pathway and network analyses of 17 differen-
tially expressed Con A-bound glycoproteins in the plasma of
gastric cancer samples versus normal controls were
assessed through the use of IPA analysis. No expression
value cut-off was selected, and we investigated both
upregulated and downregulated proteins. The modulated
proteins are grouped by known relationships into the
“Functional Analysis” and “Canonical Pathway Analysis”
of a network, with significance calculated using Fisher's
exact test (p-value < 0.05) to determine the probability of
each biological function and/or disease. The canonical
pathway may be explained by chance alone. For the IPA
network analysis, hypothetical protein interaction clusters
between the molecules were analyzed. IPA computes a
p score to rank networks according to the differentially
expressed proteins. The score is calculated as p score = − log
10 (p value), which indicates the probability of matching the
input proteins in a protein–protein interaction from the
Ingenuity Knowledgebase by random chance. A score of 3
or higher indicates at least a 99.9% confidence level for
excluding random chance [40].
2.10. Western blot analyses and Con A blotting
The protein samples (the albumin- and IgG-depleted and
the plasma-derived Con A-captured glycoprotein samples)
were separated by electrophoresis on a 10% SDS polyacryl-
amide gel (Hoefer®, Inc., Holliston, Massachusetts, USA)
and transferred to a PVDF membrane (GE Healthcare Life
Sciences, Piscataway, New Jersey, USA). Briefly, a total of
50 μg of protein was loaded in each well. The proteins were
resolved in Tris–glycine buffer (0.1% SDS (w/v), 25 mM Tris,
192 mM glycine, pH 8.3). After electrophoresis, the separat-
ed proteins were transferred to PVDF membranes in
transfer buffer (20% methanol, 25 mM Tris base, 192 mM
glycine, pH 8.0). The blots were blocked with 1 × Carbo-Free
™ Blocking Solution (Vector Laboratories, Burlingame,
California, USA) in deionized water with 0.05% Tween-20
and then incubated with the following antibodies: ITIH3
(dilution 1:200; Santa Cruz Biotechnology, Inc., USA), LRG1
(mAb 2E3, dilution 1:500; Abnova, Taiwan), and biotinylated
Con A (dilution 1:1000; Vector Laboratories, Burlingame,
California, USA). After washing, the blots were subsequently
incubated with HRP-conjugated monoclonal IgG antibody
(dilution 1:10,000; Millipore Corporation, Billerica, Massachusetts,
USA) as the secondary antibody. The reactive protein bands were
detected using the Immobilon™ Western Chemiluminescent
HRP Substrate (Millipore Corporation, Billerica, Massachusetts,
USA). The band intensity was analyzed and estimated using
an ImageQuant 400™ Imager (GE Healthcare Life Sciences,
Piscataway, New Jersey, USA) and ImageJ software (National
Institutes of Health, Bethesda, Maryland, USA). Western blot
analysis of each protein was performed in duplicate, and a
duplicate gel was run and stained with CBB as a loading control.
2.11. Statistical analyses
Student's t tests were performed to determine the significance of
peptide mass spectra and blot densitometry differences. The
spectral count data are presented as the mean ± RSD. The RSD is
a measure of precision and is usually called the CV. It is often
calculated as a percentage. Fold change is defined as (mean of
cancer normalized spectral counts − mean of normal normalized
spectral counts) / (mean of normal normalized spectral counts).
The threshold for an upregulated or downregulated protein was a
1.0-fold change in expression. Proteins that were upregulated by
2-fold or more were selected for validation by Western blot
analysis. An ANOVA and Scheffe's post hoc test were used to test
the LRG1 and ITIH3 protein levels in Con A-captured plasma
among normal controls and early- and late-stage gastric cancer
samples. SAS version 9.3 (SAS Institute Inc., Cary, North Carolina,
USA) was used for all statistical analyses. The ROC curve was
generated to evaluate the diagnostic performance of Con
A-bound LRG1 and ITIH3 using the Stata 9.0 software package
(StataCrop LP, College Station, TX, USA). For all statistical tests,
the level of significance was established at 0.05.
3. Results
3.1. Detection and enrichment of Con A-captured plasma
Approximately 88.2% (15/17) and 41.2% (7/17) of the identified
differentially expressed proteins were N-linked and O-linked
glycoproteins, respectively, as annotated by the UniProt
database (Table 2). We utilized an N-deglycosylation strategy to
confirm the characteristics of plasma-derived Con A-bound
glycoproteins. Of the identified glycoproteins, 41.2% (7/17) were
not detectable by LC–MS/MS (Table 2). The PAS staining approach
was used to identify glycoproteins present in the plasma. With
this technique, periodic acid oxidizes the 1,2-glycol functional
groups present in glucose or polysaccharides, resulting in
aldehydes that react with Schiff regent to turn a purple-magenta
or pink color. After SDS-PAGE, proteins in the molecular weight
(MW) range of 35 to 250 kDa were more highly stained with PAS
in the Con A-captured plasma samples than in the albumin- and
IgG-depleted samples as shown in Supplementary Fig. 1A (see
Supporting Information). This result strongly indicated that the
Con A-captured plasma samples contain more glycoproteins
than the albumin- and IgG-depleted plasma samples. However,
proteins with a MW less than 35 kDa may be less efficiently
stained by PAS. To study the differential profiles of glycoprotein
content in gastric cancer versus normal control plasma samples,
the albumin- and IgG-depleted samples and the Con A-captured
plasma samples were separated by SDS-PAGE, transferred to
PVDF membranes and visualized with biotin-labeled Con A
lectins (Supplementary Fig. 1B). The Con A blotting results
showed differential glycoprotein profiles in both samples
depending on the broad glycan specificity and preferential
 J O U RN A L OF P ROT EO M IC S 8 3 ( 2 01 3 ) 1 9 7 –2 13
binding to oligomannosidic/glucosidic, hybrid, and biantennary
N-glycans. A few Con A-bound proteins were detected in the
MW range of 55 to 110 kDa in the albumin- and IgG-depleted
plasma samples. Most Con A-bound proteins were present as
strong protein bands in the MW range of 35 to 250 kDa in the
Con A-captured plasma samples. The comparative results of
the PSA staining and Con A blotting were highly consistent for
the Con A-captured plasma samples. The detection range of the
Con A blotting is smaller than that of the PSA staining as shown
in the albumin- and IgG-depleted plasma samples. The gel band
patterns of plasma-derived Con A-bound glycoproteins after
the N-deglycosylation reaction were obviously changed, and
most glycoproteins were not stained by the PAS reaction
(Supplementary Fig. 1C–D).
3.2. Identification of the differentially expressed Con A-bound
glycoprotein biomarkers by nano-LC–MS/MS
The enrichment of Con A-captured plasma samples from a
single pair of each of the 30 pooled plasma samples (gastric
cancer versus normal control samples) was analyzed in triplicate
by 1-D SDS-PAGE coupled to nano-LC–MS/MS. A list of 82
proteins and peptides was identified at a false discovery rate
(FDR) of 0.3% (Supplementary Table 1). Seventeen of the
differentially expressed proteins varied significantly (Student's
t-test, p-value < 0.05), as shown in Table 2. There were 10
upregulated proteins and 7 downregulated proteins. Relative to
the control plasma pools, expression of 2 of the identified
proteins was increased or decreased by more than 2-fold in the
gastric cancer plasma pools, and expression of 12 proteins was
increased or decreased by 0.30- to 1.20-fold. Three proteins were
undetectable in the gastric cancer plasma pools. The vast
majority of proteins are tumor-associated proteins (88.2%),
including LRG1, ITIH3, hemopexin (HPX), plasma protease C1
inhibitor (SERPING1), alpha-1B-glycoprotein (A1BG), comple-
ment factor B (CFB), fibronectin (FN1), inter-alpha-trypsin
inhibitor heavy chain H1 (ITIH1), Ig gamma-2 chain c region
(IGHG2), Ig mu chain c region (IGHM), clusterin (CLU), apolipo-
protein D (APOD), plasminogen (PLG), complement C5 (C5), and
complement C1q subcomponent subunit B (C1QB). These pro-
teins have been found to be differentially expressed in cancers
such as ovarian [41,42], lung [43–45], liver [46], pancreatic [47,48],
bladder [49], biliary tract [50], breast [51–55], nasopharyngeal [56],
renal [57], papillary thyroid [58], colon [59], prostate [60,61], and
cutaneous malignant melanoma [62,63]. The summary of
the category classification of the differentially expressed Con
A-bound glycoproteins according to cellular component, molec-
ular function, biological process, and their predicted functional
partners is shown in Supplementary Table 2. This category
classification provided useful information regarding protein
ontology in response to gastric cancer.
3.3. Validation of differential expression by Western blot
To validate the LC–MS/MS data, we analyzed the levels of Con
A-bound LRG1 and ITIH3 by Western blot analysis in the
discovery and validation sets (Fig. 1 and Supplementary Fig. 2).
These are potential tumor-specific proteins. Plasma-derived Con
A-bound LRG1 and ITIH3 were identified as being upregulated by
2-fold or more in the gastric cancer samples compared with
203
normal controls according to peptide identification and/or
quantification values by LC–MS/MS. Therefore, we further
investigated the Con A-bound LRG1 and ITIH3 expression levels
in human plasma to determine whether these proteins might be
valuable biomarker candidates for gastric cancer diagnosis. We
used the albumin- and IgG-depleted samples and the Con A-
captured plasma pools for the initial validation, as the presence
of high-abundance plasma proteins may interfere with the
detection of low-abundance proteins by Western blot analysis.
In a previous study, the mAb against LRG1 recognized one
protein band at ~47 kDa in human plasma [41]; however, we
determined that the major molecular weight of LRG1 in plasma
was ~55 kDa. Likewise, the predicted molecular weight of ITIH3
is approximately 80 or 125 kDa [64], but ITIH3 was detected as a
major band at ~125 kDa in this study. In the discovery set, the
densitometry of the Western blot data showed 1.20- (average
densitometry value: 14,248 versus 11,866; p = 0.58) and 1.40-fold
(average densitometry value: 33,835 versus 24,249; p = 0.001)
overexpression of LRG1, respectively, in the albumin- and
IgG-depleted samples and the Con A-captured plasma pools of
gastric cancer compared with normal controls. Similarly, ITIH3
expression levels were 1.35- (average densitometry value: 28,315
versus 21,003; p = 0.005) and 1.56-fold (average densitometry
value: 21,881 versus 14,022; p = 0.013) higher in gastric cancer
samples than in normal controls. Equal loadings of LRG1 and
ITIH3 amounts in these experiments were also examined
(Fig. 1A–C). The validation set was further examined by
subjecting individual Con A-captured plasma to immunoblotting
with the LRG1 and ITIH3 antibodies. The LRG1 expression levels
in early- (stage I/II) and late-stage (stage III/IV) gastric cancers
were increased 1.30- (average densitometry value: 87,759 versus
67,715; p > 0.05) and 1.59-fold (average densitometry value:
107,411 versus 67,715; p < 0.05), respectively, compared with
normal controls as shown in Fig. 1D. Additionally, the levels of
ITIH3 in early- and late-stage gastric cancer samples were higher
than normal controls by 1.80- (average densitometry value:
101,740 versus 56,345; p < 0.05) and 1.73-fold (average densitom-
etry value: 97,723 versus 56,345; p < 0.05), respectively, as shown
in Fig. 1D. The box plot generated by ANOVA statistical analysis
revealed that the difference in LRG1 (p = 0.0004) and ITIH3
(p < 0.0001) expression levels was significant among normal
controls and early- and late-stage gastric cancer patients
(Fig. 1E). These experiments were performed in duplicate.
The difference in the levels of Con A-bound LRG1 and ITIH3
between early- and late-stage gastric cancer patients was not
statistically significant. The average densitometry signals of
LRG1 and ITIH3 expression for individual Con A-captured
plasma were calculated, and an asterisk (*) indicates a
p-value < 0.05 (Fig. 1). A ROC curve was plotted based on
these results to calculate the sensitivity and specificity of Con
A-bound LRG1 and ITIH3 in gastric cancer detection. As shown
in Fig. 1F, the AUC value of the ROC curve was found to be 0.65
(95% CI: 0.55 - 0.75) at an average densitometry cut-off of 75,000,
giving Con A-bound LRG1 a sensitivity and specificity of 56.7%
and 66.7%, respectively. Furthermore, at an average densitom-
etry cut-off of 75,000, the AUC value of the ROC curve for Con
A-bound ITIH3 was found to be 0.75 (95% CI: 0.66–0.84), yielding
a sensitivity and specificity of 73.3% and 76.7%, respectively
(Fig. 1F). The sensitivity and specificity were estimated at 95%
confidence for gastric cancer detection.
204 J O U RN A L OF P ROTE O M IC S 8 3 ( 2 01 3 ) 1 9 7 –21 3

A LRG1 B C
*
kDa 1 2 3 4 40000
kDa 1 2 3 4
70
55 30000 250

Intensity
20000 130
Albumin-and IgG-depleted plasma 100
Normal control average: 11,866 10000
Gastric cancer average: 14,248 70
Con A-captured plasma 55
0
Normal control average: 24,249 1 2 3 4
Gastric cancer average: 33,835

35

ITIH3 *
35000 25
kDa 1 2 3 4 *
30000

25000
130

Intensity
20000 15
100
15000
Albumin-and IgG-depleted plasma
Normal control average: 21,003 10000
Gastric cancer average: 28,315 5000
Con A-captured plasma
Normal control average: 14,022 0
Gastric cancer average: 21,881
1 2 3 4

D E

F
 J O U RN A L OF P ROT EO M IC S 8 3 ( 2 01 3 ) 1 9 7 –2 13 205

3.4. Networks and functional pathway analysis
Supplementary Table 3 lists the top 5 canonical pathways that
involve the 17 differentially expressed Con A-bound glycopro-
tein biomarkers in plasma from patients with gastric cancer.
The analysis shows the canonical pathways of acute phase
response signaling, the complement system, LXR/RXR activa-
tion, hematopoiesis from pluripotent stem cells, and primary
immunodeficiency signaling. The interactive relationship be-
tween the proteins in the top canonical pathway is shown in
Fig. 2.
3.5. A novel type of PTM identified in ITIH3
LRG1 and ITIH3 are known glycoproteins. There are 4N- and 1
O-linked glycosylation sites in LRG1 at positions asparagine-79,
asparagine-186, asparagine-269, asparagine-325 [65,66], and
threonine-37, respectively, as indicated by the UniProt database.
No novel PTMs to LRG1 were found in this study. The three
types of PTMs found on ITIH3 were N-linked glycosylation
(asparagine-91 and asparagine-580) [65], 1-(chondroitin
4-sulfate)-ester modification (aspartic acid-651) [67], and phos-
phorylation (threonine-705 and threonine-727) [Human Protein
Reference database, http://www.hprd.org/], and they are shown
in Supplementary Fig. 3. Three novel types of PTMs on ITIH3
were identified by manual examination of the modified spectra
using our in-house PTM finder program. The identified peptides
and the corresponding modified peptides are listed in Table 3.
The peptide 33-RSLPEGVAHNGIEVYSTK-49 was used to identify
ITIH3 and was found to be glycated at asparagine-41, and
identification of the peptide moiety of the glycopeptide was
based on the presence of b- and y-series ions derived from
the peptide. The glycation residue is a hexose-N-acetyl-
hexosamine (HexHexNAc), and this residue was confirmed by
an unmodified y7 ion followed by a modified b9 ion, corre-
sponding to a mass increase of 365.1322 Da (Fig. 3A). In addition
to glycation, we were able to identify a series of other novel
modifications by manual examination. Two other modifica-
tions in ITIH3 were found and include trimethylation at aspartic
acid-290 and flavin adenine dinucleotide (FAD) covalently bound
at histidine-335. Trimethylation of aspartic acid-290 in the
modified peptide 284-NVAFVITDISGSMAGR-298 was shown in
Fig. 3B. The spacing of the b and y ions was used to decipher the
sequence of the modified peptide. The novel trimethylation site
was discovered by detection of the unmodified b6 ion.
Additionally, the b7 ion was shifted by the 43.0548 Da, the
mass of a trimethyl group. We also observed a novel FAD
modification in ITIH3. The modified peptide was identified
from the unmodified y16 ion and modified b2 and b3 ions with a
mass shift of 783.1415 Da. The sequence of the peptide in the
spectrum showed a peptide fragmentation pattern that demon-
strated the b and y ion series. These data show that the modified
peptide 334-EFHLVQATPENLQEARTFVK-352 contained a FAD
covalently bound at histidine- 335, which was confirmed by
the b and y ion series as shown in Fig. 3C and Table 3.
3.6. Molecular modeling
When searched against the UniProt database, alignment of
the human plasma ITIH3 sequence showed similarity to the
known sequences of ITIH3 from other species, including
Rattus norvegicus (Q63416), Mus musculus (Q61704), Mesocricetus
auratus (P97280), Pongo abelii (Q5RB37), Bos taurus (P56652), and
Sus scrofa (F1SH94). In the alignment shown in Supplementary
Fig. 3, two domains, the vault protein inter-alpha-trypsin
(VIT) domain and a von Willebrand factor (vWF) type A
(VWFA) domain, are conserved among all ITIH3 members,
and the three novel modifications documented in the present
study were identified inside these domains. The glycation
residue (HexHexNAc) was found in the VIT domain (amino
acid sequence 29 to 158), and the other two modifications (the
trimethyl group and the FAD) were observed in the VWFA
domain (amino acid sequence 284 to 467). Taking these
identified modification sites into consideration, we performed
molecular modeling of human plasma ITIH3 in the SWISS-
MODEL Repository. Two templates, bacterial micronemal pro-
tein 2 (PDB ID: 2xggB; modeled residue range 280 to 428; 22.8%
identity; E value 4.6E-13) and human anthrax toxin receptor 2
(PDB ID: 1shuX; modeled residue range 282 to 468; 19.1%
identity; E value 4E-18), were better matched. We selected
human anthrax toxin receptor 2 (the VWFA domain of human
capillary morphogenesis protein 2) as a template to model the
three-dimensional (3-D) structure of the VWFA domain in ITIH3
using PyMol. The PTM sites are shown on Fig. 4; the aspartic
acid-290 trimethylation site was shown in red and the
histidine-335 FAD site is shown in blue. However, the SWISS-
MODEL did not predict the asparagine-41, most likely due to its
low identity or reduced conservation. The conserved amino
acid residues of a metal ion-dependent adhesion site (MIDAS
motif; 290-DXSXS…T…D-313) are labeled (Fig. 4).

Fig. 1 – Validation of Con A-bound LRG1 and ITIH3 expression levels by Western blot analysis. A representative Western blot of the
albumin- and IgG-depleted samples and the Con A-captured plasma pools for the discovery set (A–C). The average densitometry
values were calculated from the validation set and used to construct box plots from the duplicate data (D–E). A total of 50 μg of
plasma proteins was resolved by SDS-PAGE and detected using anti-LRG1 and ITIH3 antibodies and Western blot analysis
(A and D). In the discovery set: lanes 1–2, albumin- and IgG-depleted plasma protein; lanes 3–4, Con A-captured plasma protein;
lanes 1 and 3, plasma pools from 30 normal controls; lanes 2 and 4, plasma pools from 30 gastric cancer patients. A duplicate gel
was stained with CBB as a loading control (C). In the validation set: lane 1, individual plasma sample selected from a set of 60 controls;
lane 2, individual plasma sample selected from 33 early-stage (stage I/II) gastric cancers; lane 3, individual plasma sample selected
from 27 late-stage (stage III/IV) gastric cancers. The average densitometry reading of the protein expression level for each
glycoprotein biomarker was calculated. Significant differences in expression levels (indicated with an asterisk *, p-value < 0.05)
were found between normal controls and gastric cancers. The ROC curve was generated using the average densitometry readings
of Con A-bound LRG1 and ITIH3 for all individual plasma samples (F).
206 J O U RN A L OF P ROTE O M IC S 8 3 ( 2 01 3 ) 1 9 7 –21 3

Fig. 2 – The top 1 canonical pathway was assigned by the Ingenuity Pathway Analysis for differentially expressed Con A-bound
glycoproteins in plasma with gastric cancer. The red and green colors in each shape indicate the up- or down-regulation of
pathway-associated protein expression when gastric cancer samples were compared with normal control samples. The proteins
in a gray color are not identified as differentially expressed in the list but are associated with the possible computationally
generated networks based on the Ingenuity database. The node (protein) and edge (protein relationship) symbols are described in
the bottom part of the figure. Each line and arrow between proteins represents known functional and physical interactions, with
solid lines indicating direct relationships (the two molecules have physical contact) and dashed lines representing indirect
or hypothetical interactions (does not require physical contact). The node shapes denote enzymes ( ), peptidases ( ), translation
regulator ( ), transmembrane receptor ( ), cytokines (□), growth factor (□), transporter ( ), Complex / Group (Ⓞ) and other
(○).

tissue polypeptide antigen (TPA; cytokeratins 8, 18, and 19),

4. Discussion
The most common serum-based tumor markers for gastric
cancer in clinical use include CEA, CA 19-9, CA 72-4,
cytokeratins [CYFRA 21-1 (soluble cytokeratin 19 fragment),
tissue polypeptide-specific antigen (TPS; cytokeratin 18)], and
the free beta-subunit of HCG. The early detection rate of
tumor markers in gastric cancer diagnosis is approximately
15 to 35% [68]; the majority of these markers are glycopro-
teins. Therefore, there is an urgent need for early detection
 J O U RN A L OF P ROT EO M IC S 8 3 ( 2 01 3 ) 1 9 7 –2 13
biomarkers to screen for gastric cancer and to enhance the
survival rate. Previous reports on the glycoprotein biomarkers
discovered in gastric cancer plasma and/or serum include
mucin-1 (MUC1), mucin-5AC (MUC5AC), IgG, haptoglobin (HP),
TF, alpha-1-acid glycoprotein (AGP), A1BG, vitamin D binding
protein (GC), alpha-1-antitrypsin (SERPINA1), antithrombin
(SERPINC1), angiotensin (AGT), albumin (ALB), CFB, kininogen-1
(KNG1), serpin peptidase inhibitor, Clade A (SERPINA3), IGHM,
LRG1, C4A variant protein (C4A), Zn-alpha-2-glycoprotein
(AZGP1), CLU, alpha-2-HS-glycoprotein (AHSG), ITIH2, comple-
ment factor H (CFH), ceruloplasmin (CP), interalpha-trypsin
inhibitor HCRP, and SERPING1 [69–72]. Nine of these proteins
have been previously identified as Con A-bound glycoproteins
and include ITIH2, CFH, CP, ITIH4, SERPING1, C4A, A1BG,
SERPINA3, and HP. These glycoproteins were enriched from
depleted serum using the Pierce Con A Glycoprotein Kit and
were identified by LC-MS/MS as 12 protein bands labeled in gel
[72]. Here, we used the Con A-captured plasma samples as
starting materials; these samples were purified directly from
plasma by Con A affinity chromatography and identified by 1-D
SDS-PAGE and nano-LC–MS/MS. In the present study, a total of
82 proteins were identified, and 17 proteins were differentially
expressed at a significant level. In this study, the 17
differentially expressed proteins identified in triplicate from
the 10 gel bands from 1-D SDS-PAGE coupled to LC-MS/MS
exhibited an average RSD of 16.32% (ranges from 1.87% to
52.07%) for the spectral counts (Table 2). Piersma et al. suggested
an average RSD (ranges from 23.4% to 26.1%) for the spectral
counts for proteins detected in triplicate experiments from 10
gel bands from 1-D SDS-PAGE coupled to LC–MS/MS [18]. Gautier
et al. indicated a RSD (median of 7.19%, maximum of 145.40%) in
label-free quantification after 3 LC-MS/MS analyses of the 12 gel
bands from 1-D SDS-PAGE shotgun analysis [73]. These studies
used protein sample fractionation by SDS-PAGE followed by LC–
MS/MS analysis and yielded similar results. These Con A-bound
proteins revealed reactivity with the α-D-mannose/ α-D-
glucosyl-specific Con A lectin and reduced the complexity of
the plasma sample. Of the identified Con A-bound proteins,
78.0% (64/82) were N-linked glycoproteins as annotated by the
UniProt database (data not shown). This result was confirmed
by the data from protein samples comparing CBB staining, PAS
staining, and Con A blotting (Fig. 1 and Supplementary Fig. 1).
After N-deglycosylation, 58.8% (10/17) of glycoproteins were still
detected (Table 2). PNGase F (N-glycosidase F) is an amidase that
cleaves the bond between the innermost GlcNAc and all
asparagine-linked high mannose, hybrid, and complex oligo-
saccharides unless the core contains an α(1 → 3)-fucose from
the N-linked glycoprotein [74]. It is likely that the incubation time
with PNGase F must be increased as steric constraints in N-glycan
may prevent enzyme activity or additional exoglycosidases may
be needed to remove other N- and O-glycans. Albumin and IgG,
the most abundant proteins in human serum, must be removed
by immunodepletion in the study of proteomics. Koutroukides
et al. indicated that depleted serum had been co-depleted of 147
non-target serum proteins using an immunoaffinity kit [75].
This depleted serum is known to provide new insights into
clinical proteomics. The use of lectin-captured plasma and/or
serum could prevent the loss of important information.
Two proteins with a potential role in carcinogenesis were
identified, Con A-bound LRG1 and ITIH3 (Table 2). The isoforms
207
and physiological function of LRG1 are unknown. Increment
levels of LRG1 in the serum and/or plasma have been reported
in various diseases, including gastric cancer [76], ovarian cancer
[77], biliary tract cancer [50], pancreatic cancer [47], lung cancer
[78], uterine leiomyomas [79], microbial infections [80], rheu-
matoid arthritis [81], heart failure [82], and graft-versus-host
disease [83]. LRG1 has also been detected as a urinary biomarker
in lung cancer [84] and bladder cancer [85]. The expression level
of LRG1 in the serum was increased in gastric cancer samples
compared with normal controls, and the levels increase in
relation to the increase in cancer progression stage (stage I,
1.2-fold; stage II, 1.6-fold; stage III, 1.7-fold; stage IV, 1.9-fold)
[76]. Upregulated expression of LRG1 was identified in other
cancer versus normal controls by proteomic tools, including a
1.20 to 1.50-fold increase in ovarian cancer (2-D-DIGE) [77], a
4.49-fold increase in biliary tract cancer (2-DE) [50], a 6.0-fold
increase in lung cancer (2-D-DIGE) [78], and a 2.05 to 2.84-fold
increase in pancreatic cancer (2-D-DIGE) [47]. Similar to our study,
the trend of upregulated expression of plasma Con A-bound LRG1
in gastric cancer samples versus normal controls was consistent
in the study by Kim et al. (Fig. 1) [76]. LRG1 contains 8 repetitive
consensus sequences known as the leucine-rich repeat (LRR)
domain, and its primary function may be to provide a curved
solenoid structural framework that is particularly suitable for
protein–protein interactions [86]. LRG1 binds cytochrome c,
which may play a role in cell survival [80]. Shirai et al. found
that LRG1 was a secretory type 1 acute-phase protein, and its
expression was induced by IL-6, IL-1β, and TNFα [87].
ITIH3 is one of the five heavy chains (ITIH1-5) belonging to
the family of inter-alpha-trypsin inhibitors (ITIs). The ITI family
is built from one light chain, bikunin, and a variable set of heavy
chains. ITIs act as carriers of hyaluronan in the serum or as
binding proteins between hyaluronan and other matrix pro-
teins to stabilize the extracellular matrix (ECM) and prevent
tumor metastasis; they are also involved in plasma protease
inhibition [88]. Paris et al. have shown that overexpression of
the ITI family chains causes inhibition of tumor growth and
metastatic spreading [89]. ITIH3 contains the two domains
conserved in all known ITIHs, the VIT and VWFA domains
[89,90]. The function of the VIT domain in ITIH3 is unknown.
The VWFA domain has the capacity to bind integrins, collagens,
proteoglycans, and heparin and mediates adhesion via the
metal ion-dependent adhesion site [91,92]. ITIH3 has also been
shown to be upregulated in the serum and/or plasma of lung
cancer and gastric cancer samples versus normal controls
[44,90]. Chong et al. found that the plasma ITIH3 expression
level in gastric cancer samples was 1.9-fold higher when
compared with normal controls by Western blot analysis [90].
Furthermore, the expression level of Con A-bound ITIH3 in
gastric cancer samples versus normal controls in our study was
similar to that observed in a study by Chong et al. (Fig. 1) [90].
However, the sensitivity (96.0% versus 73.7%) and specificity
(66.0% versus 76.7%) of ITIH3 versus Con A-bound ITIH3 in
gastric cancer detection calculated from the ROC curve were
inconsistent [90]. Our preliminary experiment for PTM peptide
quantification compared gastric cancer samples with normal
controls. The relative modified peptide ratios of the three novel
modifications of ITIH3 were identified and included HexHexNAc
(↓ 1.90-fold, p = 0.0071), trimethylation (↑ 1.78-fold, p = 0.00001),
and FAD (↓ 2.9-fold, p = 0.02). There was a newly found
208 J O U RN A L OF P ROTE O M IC S 8 3 ( 2 01 3 ) 1 9 7 –21 3

Table 3 – The identification of novel types of post-translational modifications in ITIH3 using mass spectrometry.
Protein a PTM b Site Modified peptides c Position d Obs. Calc. dM (Obs.– dM
m/z e m/z f calc.) g (ppm) h

ITIH3 Hexose-N-acetyl-hexosamine Asparagine-41 RSLPEGVA*NGIEVYSTK 33–49 1093.058 1093.046 0.013 11.497

(HexHexNAc)
Trimethylation Aspartic NVAFVI*DISGSMAGR 284–298 532.611 532.615 -0.004 -7.923
acid-290
Flavin adenine dinucleotide Histidine-335 E*HLVQATPENLQEARTFVK 334–352 998.445 998.438 0.007 6.573
(FAD)
a
Name of the modified protein.
b
Type of modification.
c
Site of modified peptide is marked with an asterisk (*) and boldface.
d
Amino acid positions of the first and the last residues in accession number.
e
Obs: observed m/z of the modified peptides.
f
Cal: calculated m/z of the modified peptides.
g
dM (Obs.–calc.): mass accuracy.
h
Modified peptide mass accuracy (ppm).

HexHexNAc residue at asparagine-41, and two glycation residues carcinogenesis (Supplementary Fig. 3). In the molecular modeling
were located within the VIT domain. The ratio of N-linked of ITIH3, no suitable template structure of the VIT domain
glycosylation modification was decreased in the process of was selected because of the reduced conservation between

Fig. 3 – (A) A representative MS/MS spectrum of the peptide sequence of 33-RSLPEGVAHNGIEVYSTK-49 and the modified peptide
bearing the asparagine glycation. The glycation residue is a HexHexNAc. (B) The MS/MS spectrum of 284-NVAFVITDISGSMAGR-298
and the modified peptide bearing the aspartic acid trimethylation. (C) The MS/MS spectrum of the peptide 334-EFHLVQATPENLQEARTF
VK-352 with a FAD covalently bound at a histidine residue.
 J O U RN A L OF P ROT EO M IC S 8 3 ( 2 01 3 ) 1 9 7 –2 13 209

Fig. 4 – Novel PTM patterns of Con A-bound ITIH3 VWFA domain and the 3-D structure developed with PyMol. The highlighted amino
acid residues include D290 (aspartic acid- 290; red), S292 (serine- 292; red), S294 (serine- 294; red), T303 (threonine- 303; green),
D313 (aspartic acid-313; green) and H335 (histidine-335; blue). The conserved amino acids of the MIDAS motif are 290-DXSXS…T…D-313.
The PTM sites include trimethylation at D290 and FAD at H335. The A and B view with a different orientation.

species. In the alignment shown in Supplementary Fig. 3, 4
of the 8 amino acid residues at modification sites (N-linked
glycosylation at asparagine- 91, trimethylation at aspartic
acid- 290, 1-(chondroitin 4-sulfate)-ester modification at
aspartic acid-651, and phosphorylation at threonine-727) are
conserved in all ITIH3 members. These highly conserved
regions included asparagine-91 in 74-AKEVSFDVELPKTAFIT
NFTLTIDGV-99, aspartic acid-290 in 287-FVIDISGSM-295, aspartic
acid-651 in 644-PYYYVDGDPHFIIQ-657, and threonine-727 in
725
-EVTTE-729. The heavy chains of ITI are linked to bikunin
through chondroitin 4-sulfate esterification to the alpha-carboxyl
of the C-terminal at aspartic acid-651 after propeptide cleavage
[93]. The VWFA domain adopts a classic alpha/beta secondary
structure (Rossmann fold; order beta-alpha-beta-alpha-beta) and
contains a MIDAS motif for binding protein ligands [94]. Bajt and
Loftus demonstrated that mutation of the DXSXS sequence
abolished ligand binding [95]. It is interesting that aspartic
acid-290 in 290-DISGSMAGRKLEQTKEALLRILED-313 was modified
by trimethylation in the present study. Many previous studies
have indicated that the trimethylation of histones is a tumor-
associated PTM [96–99]. Zhang et al. found that significant
alterations in the trimethylation of histone H3 at lysine 27
(H3K27me3) in gastric cancer tissues were involved in the
pathogenesis of gastric cancer [98]. Li et al. indicated that the
transcription factor CBX7 could initiate trimethylation of histone
H3 at lysine 9 (H3K9me3) at the p16 promoter to cause epigenetic
silencing of the p16 gene in gastric cancer [99]. Huq et al. found
trimethylation at lysine-347 in retinoic acid receptor-alpha
(RARalpha) protein; this trimethylation enhanced the molecular
interaction of RARalpha with its modulators by site-specific
hydrophobicity [100]. Whether trimethylation of the VWFA
domain in ITIH3 enhances its binding capacity with ITI, ITIH1,
ITIH2, TGFB, and HNF4A requires further study (Supplementary
Table 2). FAD is an essential coenzyme in cellular oxidation-
reduction reactions. Werner et al. hypothesized that riboflavin
deficiency leads to hepatocarcinoma cell stress by rapidly
inducing cell cycle arrest [101]. An animal model by Pinto et al.
supported the hypothesis that riboflavin deprivation causes a
disturbance in the formation of FAD and covalently bound flavins
in hepatomas [102]. Koturbash et al. indicated that FAD may be a
significant regulator of the cellular epigenome [103]. The relative
modified peptide ratio of FAD in ITIH3 may be affected by
metabolic changes in the carcinogenesis process. Our MS/MS
spectra findings provide the first evidence of trimethylation and
FAD modification in the VWFA domain (Fig. 3). In this study, the
PTM analysis has revealed a Con A-bound biomarker in addition
to glycosylation in gastric cancer samples versus normal controls.
Some protein PTMs associated with gastric cancer carcinogenesis
have been studied and include glycosylation of mucin [70] and
IgG [71], phosphorylation of H. pylori CagA [104], citrullination of
peptidylarginine deiminase type 4 (PAD4) [105], methylglyoxal
modification of heat-shock protein 27 [106], polyubiquitylation of
tripartite motif 31 (TRIM31) [107], trimethylation of histone H3
[98,99], and others.
Consistent with the crucial role of the inflammatory
response in carcinogenesis [108], the upregulation of plas-
ma Con A-bound LRG1 and ITIH3 participated in the
inflammatory response, and both are mediated by interleukin-
210 J O U RN A L OF P ROTE O M IC S 8 3 ( 2 01 3 ) 1 9 7 –21 3
6 (IL-6) signaling [72,87]. Ikeguchi et al. reported that high serum
IL-6 levels may be associated with gastric cancer progression
[109]. Unfortunately, IL-6 was undetectable, which indicates
that IL-6 may not react with Con A lectin in this study. In Fig. 2
and Supplementary Table 3, the expression levels of proteins
participating in acute phase response signaling are shown,
including SERPING1, HPX, ITIH3, FN1, and CFB. These data are
consistent with the study by Bones et al., apart from their
additional findings of C5 and PLG [72].
The ROC curve of Con A-bound LRG1 and ITIH3 in early- and
late-stage gastric cancer was estimated (data not shown). The
cut-off for Con A-bound LRG1 and ITIH3 in early- or late-stage
gastric cancer was set at 75,000. Con A-bound LRG1 sensitivity
and specificity in detecting early-stage gastric cancer were 51.5%
and 66.7%, respectively; the AUC value of the ROC curve was 0.57
(95% CI: 0.44–0.71). Con A-bound LRG1 sensitivity and specificity
in detecting late-stage gastric cancer were 63.0% and 66.7%,
respectively; the AUC value of the ROC curve was 0.73 (95% CI:
0.61–0.85). Con A-bound ITIH3 sensitivity and specificity in
detecting early-stage gastric cancer were 57.6% and 76.7%,
respectively; the AUC value of the ROC curve was 0.67 (95% CI:
0.56–0.81). Con A-bound ITIH3 sensitivity and specificity in
detecting late-stage gastric cancer were 92.6% and 76.7%,
respectively; the AUC value of the ROC curve was 0.82 (95% CI:
0.73–0.91). Such small differences (<2-fold) in the protein levels of
Con A-bound LRG1 and ITIH3 may not be of practical use as a
clinical biomarker for the early detection of gastric cancer (Fig. 1).
In conclusion, we have performed a comparative glyco-
proteomic analysis to discover and investigate specific, differ-
entially expressed Con A-bound biomarkers in plasma from
gastric cancer samples and normal controls. Plasma Con
A-bound LRG1 and ITIH3 were validated as biomarkers by
Western blot analysis against a discovery set by pooled plasma
and a validation set of individual plasma. We further identified
novel types of PTMs by using an in-house program to analyze
the mass spectra of both candidate biomarkers. The use of
molecular modeling and multiple sequence alignments will
assist in the discussion of the biological consequences of novel
PTMs of Con A-bound ITIH3. IPA also assisted in the pathway
analysis of 17 differentially expressed proteins. It supports the
important biological roles of acute phase response signals in
gastric cancer and reveals differential physiological responses
related to gastric cancer carcinogenesis. This strategy may serve
as the basis for a glycoproteomic platform for additional cancer
diagnostic and therapeutic applications.
Supplementary data to this article can be found online at
http://dx.doi.org/10.1016/j.jprot.2013.03.007.
Competing interests
The authors declare that there are no competing interests.
Acknowledgments
We thank Professor Yuan-Chii Lee for the discussions
regarding the IPA analysis. This study was supported by grant
99CM-TMU-12 from the Foundation of Chi Mei Medical Center
and grant NSC101-2622-B-038-001-CC3 from the National
Science Council in Taiwan.
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