Tropical Medicine and Health Vol. 40 No. 3, 2012, pp. 89-102
doi:10.2149/tmh.2012-16
Copyright 2012 by The Japanese Society of Tropical Medicine 89
Original article
TMHImmunoproteomics Identification of Major IgE and IgG4 Reactive 
Schistosoma japonicum Adult Worm Antigens Using Chronically 
Infected Human Plasma
Daniel Boamah1, Mihoko Kikuchi1, Nguyen Tien Huy1, Kenta Okamoto1, Honggen Chen2, Irene Ayi3, 
Daniel Adjei Boakye3, Kwabena Mante Bosompem3 and Kenji Hirayama1*
9 Received 4 July, 2012 Accepted 5 July, 2012 Published online 24 October, 2012
© 20A1b2s tJrapcat:neIsmem Suoncoieptyid oefm Tiorloopgiical Mstuediiecsi nfreom endemic areas have revealed age-dependent resistance correla-
tion with increased level of IgE and decreased level of IgG4 antibodies in responses to schistosomes’ soluble
worm antigen. However, there have been limited studies on analyses of major antigens that provoke IgE and IgG4
immune response during chronic stage of schistosomiasis. In this study, for the first time, immunoproteomics
approach has been applied to identify S. japonicum worm antigens in liquid fractions that are recognized by IgE
and IgG4 antibody using plasma from chronically infected population. ProteomeLabPF 2D fractionated 1-D and
2-D fractions of SWA antigens were screened using pooled high IgE/IgG4 reactive plasma samples by dot-blot
technique. In 1-D fractions, IgE isotype was detected by fewer antigenic fractions (43.2%). The most recognized
isotype was IgG3 (79.5%) followed by IgG1 (75.0%) and IgG4 (61.4%). Liquid chromatography MS/MS protein
sequencing of reactive 2-D fractions revealed 18 proteins that were identified, characterized and gene ontology cat-
egories determined. 2-D fractions containing proteins such as zinc finger, RanBP2-type, domain-containing protein
were strongly recognized by IgE and moderately by IgG4 whereas fractions containing proteins such as ubiquitin-
conjugating enzyme and cytosolic II 5'-nucleotidase strongly recognizing by IgG subclasses (IgG1, IgG3 and
IgG4) but not IgE. By this study, a simple and reproducible proteomic method has been established to identify
major immunoreactive S. japonicum antigens. It is anticipated that this will stimulate further research on the
immunogenicity and protective potential of proteins identified as well as discovery of novel compounds that have
therapeutic importance.
Key words: Schistosoma japonicum, IgE, IgG4, Proteome, Mass Spectrometry, Genome
ing infections has been a topic of great concern. Particu-
INTRODUCTION
larly, the role of antibodies in resistance to reinfection. In
The pathophysiology of schistosomiasis is mainly due schistosomiasis, the balance between IgE and IgG4 anti-
to the immune response against tissue trapped eggs with body isotypes is thought to play a role in resistance or sus-
consequent clinical manifestations being typical of the spe- ceptibility to infection. Immunoepidemiological studies
cies infecting, intensity of worm burden as well as the im- from endemic areas have revealed age-dependent resis-
munity of the infected host. The variety of antigens released tance correlation with specific antibody isotype responses
by dead worms or secreted by the worms or shed during the to the schistosome antigens, particularly IgE responses to
various developmental stages of the worm life cycle (cer- Schistosoma mansoni adult worm antigens (AWA). The IgE
cariae, schistosomula, adult male and female, and eggs) levels are low in children and high in adults, whereas for
provide strong sustained stimuli to the host’s humoral and IgG4 the reverse has been reported [2–4]. Furthermore,
T-lymphocyte-mediated immune responses [1]. In recent since IgE and IgG4 can exhibit parallel specificity profile, it
years, immune response regulation by the schistosomes dur- has been suggested that IgG4 subclass acts as a blocking
1  Department of Immunogenetics and Global Centre of Excellence, Institute of Tropical Medicine, Nagasaki University, 1-12-4 Sakamoto, 
Nagasaki City, Nagasaki 852-8523, Japan
2  Jiangxi Provincial Institute of Parasitic Disease, Nanchang 330046, Jiangxi, PRC, China
3  Noguchi Memorial Institute for Medical Research, College of Health Sciences, University of Ghana, Legon, Post Office Box LG 581 Legon, 
Accra, Ghana
*Corresponding authors: 
E-mail: hiraken@nagasaki-u.ac.jp
Abbreviations: BCA, Bicinchoninic acid; TCEP, Tris (2-Carboxyethyl) Phosphine Hydrochloride; n-OG, n-Octylglucoside
90 Tropical Medicine and Health Vol.40 No.3, 2012
antibody against killing of the parasites by inhibiting IgE achieved improvements in platforms and the standard pro-
antibody-dependent cellular cytotoxicity (ADCC) mediated teomics approaches rely on the second dimensional (2-D)
by monocytes, platelets or eosinophiles. Similar effect has separation of complex protein mixtures using second di-
also been suggested for IgM and IgG2 antibodies [2, 5–8]. mensional gel electrophoresis (2-DE) [17, 18]. In some
The IgG3 antibody level also correlated with susceptibility cases, the 2-DE may be combined with difference in gel
to and biomarkers in liver fibrosis [6]. The production of electrophoresis (DIGE) as a profiling platform and proteins
IgE is stimulated by interleukin-l3 (IL-13) and IL-4, and are identified by ESI-MS/MS of trypsin-derived peptides.
modulated by IL-12 and interferon-gamma (IFN-γ) while However, 2-DE has a number of shortcomings including
the production of IgG4 is also stimulated by IL-4 [4]. The limited loading capacity; inability to resolve proteins of ex-
IL-4-dependent production of IgE and IgG4 is blocked by treme pI values; limitation in resolution of hydrophobic
IFN-γ, though the level required to block IL-4-dependent proteins and inability to resolve proteins of smaller molecu-
IgE production is much lower than that needed to block lar weights. Therefore, fractionating complex protein mix-
IgG4. In the sequential events of class switching, IgG4 is tures while maintaining intact proteins by liquid chromatog-
synthesized thereafter IgE, caused by sequential involve- raphy (LC) is most desirable for downstream analyses (top-
ment of different lymphokines raising the possibility that down proteomics) [19].
development of protection against schistosomes would de- The proteomeLabPF 2D instrument introduced by
pend on population of lymphocytes producing cytokine [4, Beckman-Coulter (Beckman Coulter, Fullerton, CA, USA)
9, 10]. features a rapid semi-automated 2-D HPLC system that uses
In spite of many studies demonstrating importance of two different methods to separate proteins; ion-exchange in
antibody-mediated protection against re-infection of schis- the 1-D and non-porous reversed phase in the 2-D chroma-
tosomes both in experimental and epidemiological models, tography [20–22]. Unlike gel electrophoresis, it offers an
many of the human schistosome vaccine research based on added advantage that collected fractions are in liquid phase
antibody-mediated protection have not progressed to the and can be utilized directly for any of various analytical
phase III clinical trials. This in part might be due to the lim- procedures, such as enzymatic digests, mass spectrometer
ited understanding of protective anti-schistosome response analysis, additional fractionation, western blot, or a combi-
against specific proteins [11]. Relatively, limited target anti- nation of analytical tests. Additionally, it has been shown to
gens have been analyzed in the context of selective anti- be suitable for high-throughput large-scale analysis of intact
body isotype recognition for IgE or IgG4 especially in proteins [23–26] and high loading capacity (up to 5 mg)
S. japonicum infection [2–4, 6]. Antigens that are IgE, IgG4 than with gel electrophoresis, thus significantly increasing
or both antibodies preferred can be very useful for studying the sensitivity of protein identification. Liquid-based frac-
mechanisms associated with antibody related resistance to tionation and separation systems offer great flexibility and
schistosomiasis. can be suitable for large-scale proteomic profiling in a
Many of the antigenic substances produced by the quantitative analysis [25, 26].
schistosomes at the various life cycle stages consist of pro- This study focused on isolating, identifying, and char-
teins, glycoproteins and polysaccharides in nature [12]. So acterizing immunogenic S. japonicum proteins that are pref-
far, characterization of schistosome antigens has involved erentially detected by IgE and IgG4 antibodies using sero-
studying crude parasite extracts that had no detailed charac- logical proteomics approach. Identifying and characterizing
teristics of reactive immunoglobulins. Some studies have antigenic proteins detected by the isotypes studied would
also focused on proteins or glycoprotein components of contribute to understanding of schistosome-specific adap-
schistosomes either directly or by cloning in bacteria sys- tive immunity. This also, highlights the importance of vac-
tems [5, 13]. Although, elevated IgE level is important for cine research focusing on induction of protective isotype-
development of resistance to reinfection in schistosomia- specific antibody response to specific peptides as a single
sis, only a limited number of studies have been conducted protein from the parasite might possess undetermined anti-
to isolate and characterize IgE-specific antigens from genic determinants capable of stimulating various antibody
S. mansoni [14] with a homologous antigen identified in S. productions.
haematobium [15] and S. japonicum [16]. Therefore, the an-
tigenic source of variation in IgE antibody isotype-specific
MATERIALS AND METHODS
response to S. japonicum is limited.
The mass spectrometry (MS) based proteomics has fa- Soluble Worm Antigen Preparation
cilitated identification of large numbers of proteins from Soluble worm antigen (SWA) extract was prepared
complex biological systems. Proteomics has in recent years from frozen Chinese strain S. japonicum adult worms fol-
D. Boamah et al. 91
lowing the procedures previously described [27] with slight followed by addition of stop solution (1N H2SO4, WAKO).
modifications. Briefly, adult worms (600 mg) were homog- The OD was measured at 450 nm (iMark Microplate
enized in 3.25 ml cold Diethyl Ether (Wako Pure Chemical Absorbance Reader, Bio-Rad laboratories, Inc. Japan). The
Industries, Ltd. Osaka, Japan). The homogenate was centri- mean ODs obtained were Log-transformed after subtracting
fuged at 2,000 g, 5 min to remove lipids together with the the mean ODs of the negatives and samples within the up-
diethyl ether. Thereafter, the pellet was freeze-thawed sev- per quartile (95 percentile) (51/184) were pooled for dot-
eral times in 3.5 ml of lysis buffer (6 M Urea, 2 M Thiourea, blot reactivity against 1-D and 2-D fractionated SWA
10% Glycerol, 50 mM Tris-HCl, pH 7.8, 2% n-OG, 5 mM (Fig. 1A).
TCEP) mixed with 0.1 mM PMSF and 2 μg/ml Leupeptin.
This was dialyzed in PBS (pH 7.5) containing 8 M Urea at Buffer Exchanging and Chromatofocusing
4°C with stirring. The homogenate was centrifuged at The PD-10 desalting column containing 8.3 ml of
20,000 g for 1 hr at 4°C and then filtered through 0.22 μm Sephadex G-25 medium (85 to 260 μm particle size), (GE
filter (Millex GP Filter Unit, Millipore Ireland Ltd. Healthcare Bio-Sciences K. K. Tokyo, Japan) was applied
Tullagreen, Carrigtwohill Co Cork, Ireland). Protein con- in buffer exchange of SWA before chromatofocusing fol-
centration was determined by BCA Protein Assay Kit (Bio- lowing the manufacturer’s recommendation. Briefly, the
Rad Laboratories Inc., Tokyo, Japan) and stored at –80°C PD-10 column was equilibrated with proprietary buffer,
until used. “ProteoSep Start” buffer (Eprogen, Darien, IL, USA) by al-
lowing it to enter the packed bed completely. The flow-
Measurement of Anti-worm Antibody Levels through was discarded. This was repeated with a total of 25
An ELISA was carried out using SWA to screen ml “ProteoSep Start” buffer. The 2 mg of SWA was resus-
plasma samples obtained from individuals with liver fibro- pended in 1.25 ml of “ProteoSep Start” buffer, loaded onto
sis (n = 31 grade 0; n = 62 grade 1; n = 91 grade 2 and 3 in- the equilibrated PD-10 column and allowed to enter the
dividuals) due to schistosomiasis japonica as previously de- column completely. The flow-through was again discarded.
scribed [28, 29]. The project proposal including the reuse of Elution was performed with 3.5 ml “ProteoSep Start”
the stored samples was processed to the Institutional buffer added onto the column and the eluent collected
Review Board at NEKKEN and was approved (No. into a new 15 ml tube under gravity and applied in 1-D
12081793). Five plasma samples originally confirmed by Chromatofusing.
microscopy and ultrasound were included as positive con- Chromatofocusing was performed using the
trols. Three plasma samples were also included as negative ProteomeLab PF 2D protein separation system (Fig. 1B)
controls which were obtained from healthy Japanese indi- with 32 Karat user interface software. The pH gradient was
viduals without schistosomiasis history. Briefly, plates formed using two proprietary buffers: “ProteoSep Start”
(Nunc-Immuno Plate, Nunc, Denmark) were coated with 5 buffer ([essentially contained urea, Tris-HCl and n-OG at
μg/ml of SWA. After washing unbound antigens two times pH of 8.5) and “ProteoSep Elution” buffer (Eprogen,
(2×) with PBS containing 0.05% Tween 20 (PBST, pH 7.4), Darien, IL, USA) (essentially Urea, Polybuffer 74-HCl, n-
the plates were blocked with 5% non-fat skimmed milk in OG and iminodiacetic acid, pH 4.0). The High Performance
PBST for 60 min at room temperature (RT) followed by 2× Chromatofocusing Column (A51685 ProteoSep HPCF
washing. Plasma samples were diluted 1:20 for detection of Column, 250 mm × 2 mm, Eprogen. Darien, IL, USA) was
IgE and IgG4 and 1:800 for detection of IgG1 and IgG3 treated according to the manufacturer’s instructions.
with 1% blocking solution at followed by incubation at Briefly, the column was washed with 10 volumes of auto-
37°C for 60 min and then 3× washing. The procedure claved MilliQ water at a flow rate of 0.2 ml/min for 45 min
continued with 60 min incubation (37°C) with horseradish and then equilibrated with 30 volumes of “ProteoSep Start”
peroxidase-conjugated mouse anti-human IgG1, IgG3 buffer for 130 min at 0.2 ml/min, ambient temperature. The
(Southern Biotechnology Associates Inc., Birmingham, buffer exchanged SWA sample was introduced with a man-
AL, USA), IgG4 (MP Biomedicals. LLc, France) or biotin- ual injector into the column. Proteins bound to the strong
conjugated goat anti-human IgE (Invitrogen Corporation, anion exchanger in the HPCF column were eluted with a
Camarillo, CA, USA) in 1% blocking solution at 1:1000, continuous decreasing pH from 8.5 to 4.0. Twenty minutes
1:1000 1:400 or 1:400 respectively. For detection of IgE, after sample injection, the valve automatically switched
the plates were further treated with 1:400 horseradish from “ProteoSep Start” buffer to “ProteoSep Elution”
peroxidase-conjugated streptavidin (DakoCytomation, buffer at a flow rate of 0.2 ml/min over 95 min. The pH be-
Copenhagen, Denmark). Finally, plates were developed gan to decrease after about 45 min. Fractions were automat-
with stabilized chromogen (SB01, Invitrogen) in the dark ically collected every 0.3 pH units into a 96-well deep-plate
92 Tropical Medicine and Health Vol.40 No.3, 2012
Fig. 1. Workflow of the experiment. In order to obtain anti-worm antibody, plasma from chronically S. japonicum infected individu-
als were screened in ELISA system (A) and highly reactive samples pooled for dot-blot screening. Soluble worm antigen
(SWA) was fractionated by Chromatofocusing (B) using ProteomeLab PF 2D (Beckman Coulter, Fullerton, CA, USA) fol-
lowed by dot-blot screening (C). Reactive fractions were further fractionated by reversed phase chromatography which was
again screened by dot-blot and reactive fractions trypsinized for ESI-MS/MS protein identification (E).
(Part No. 26700J, Beckman Coulter, Fullerton, CA, USA). TFA in ACN (Solvent B). At the end of each run, equilibra-
At 170 min, the HPCF column was washed for 45 min with tion of the column was achieved with initial mobile phase
10 column volumes of a third buffer of high ionic strength (Solvent A) for 10 min followed by Solvent B for 5 min
solution (1 M NaCl) and re-stored by 10 column volumes of prior to each injection. All 2-D chromatography was con-
distilled water for 45 min. The absorbance of the column ef- ducted at a column temperature of 50°C and buffer flow rate
fluent was monitored at 280 nm with an online pH flow cell. of 0.75 ml/min with the absorption of the effluent monitored
The percentage concentration eluted over the different pH at 214 nm. From the selected 1-D fractions, 200 μl was au-
conditions was estimated using the peak area of the fraction tomatically injected into the PF 2D HPRP column and ran
monitored at 280 nm, a wavelength at which the peak area for 6 min. The column was eluted at a flow rate of 0.75 ml/
is directly proportional to the quantity of the proteins [30]. min with a 0–100% linear gradient of solvent A and solvent
The 1-D fractions obtained were screened by dot-blot assay B for 35 min. Thereafter, Solvent B was continued for 5
and selected reactive fractions directly applied to the 2-D min, followed by re-equilibration with 100% Solvent A for
reversed phase unit. 10 min. The fractions were collected at a flow rate of 0.18
min into 96-well microplate (Product code 3363, Corning
Second Dimension Reversed Phase Chromatography International K. K. Tokyo, Japan) placed in an automated
The 2-D separation (Fig. 1D) was performed using fraction collector (Gilson FC 204 Fraction Collector, M & S
Reversed Phase High Performance Column (391106 PF 2D Instruments Inc. Osaka, Japan). The 2-D fractions were
HPRP Column, Beckman Coulter, Fullerton, CA, USA) and stored at –80°C while some screened by dot-blot and some.
two solvents, 0.1% TFA in water (Solvent A) and 0.08%
D. Boamah et al. 93
Dot-blot Screening spots were included in the entire test for positive and back-
The 44 1-D fractions as well as 80 2-D fractions (de- ground control respectively. The IgG1 and IgG3 were included
rived from each 1-D fraction) obtained were screened for to aid in selection of IgG4 and IgE preferred fractions.
reactivity against circulating anti-schistosome IgE, IgG4, For the analysis of all the antigenic spots intensity, the
Ig3 and IgG1 antibody isotypes in dot-blot assays in search background intensity was subtracted and then the value ob-
of novel reactive proteins. The dot-blot was conducted tained divided by that of the positive control to generate a
using Bio-Dot SF Micro filtration apparatus (Bio Rad relative reactivity index of ‘1’ for positive control, ‘0’ for
Laboratories, Inc., CA, USA) as previously described with negative control. Using these criteria each antigenic spot
modifications [27]. Briefly, 30 μl 1-D fraction was loaded was designated ‘not-reactive’, ‘weakly reactive’, ‘moder-
onto polyvinylidene fluoride (PVDF) membrane (Amersham ately reactive’ or ‘strongly reactive’ with respect to isotype
Hybond-P PVDF Membrane, GE Healthcare Bio-Sciences recognition using the pixel analysis and manual verifica-
K. K. Tokyo, Japan) imbedded in transfer buffer (192 mM tion. These were calculated for each isotype separately so
Glycine, 25 mM Tris, pH 7.4) [31] and fixed into the Bio- that the spot intensity was standardized within the mem-
Dot SF Micro filtration apparatus. Following blocking, 30 brane. The intensity scoring rather than absolute values
μl of diluted pooled plasma (51 samples) (IgG1, 1:4000; could be compared among the fractions. This approached
IgG3, 1:4000; IgG4, 1:100; or IgE, 1:800) in TBS washing was applied since it was not expected for any fraction to
buffer (20 mM Tris, 137 mM NaC1, 0.01% Tween 20, pH have equal intensity by the four isotypes for determining re-
7.6) was applied to each respective well blotted with the activity intensity across the antibody isotypes.
fractions. Bound antibodies were incubated with respective
conjugated enzymes using horseradish peroxidase- In-solution Tryptic Digestion
conjugated mouse anti-human IgG1, IgG3 (Southern Fractions from basic, neutral and acidic regions were
Biotechnology Associates Inc.), IgG4 (MP Biomedicals, treated with trypsin prior to protein sequencing. Briefly, liq-
LLc) or biotin-conjugated goat anti-human IgE (Invitrogen) uid fractions (50 to 150 μl) were precipitated at –80°C over-
in dilutions of 1:16,000, 1:16,000 1:1,000 or 1:4,000 re- night in about 10-bed volume of pre-chilled acetone
spectively. The IgE antibody bound membrane was further (WAKO) followed by centrifugation at 20,000 g for 30 min
treated with 1:6,000 horseradish peroxidase-conjugated at 4°C. After removing most of the supernatant, the samples
streptavidin (DakoCytomation). Blocking (5% skimmed were speed-vacuumed to eliminate the remaining ACN and
milk/TBS washing buffer) and conjugate reaction of the TFA together with the acetone. Then 15 μL of denaturation
membranes were conducted in a separate container. The re- solution (8 M urea; 500 mM Tris-HCl, pH 8.5; 2.5 mM
activity was revealed by ImmunoStar Reagents (WAKO EDTA) was added and incubated for 10 min at 100°C fol-
Pure Chemicals Industries, Ltd. Osaka, Japan) for chemilu- lowed by cooling at RT. Addition of 5 μl of reduction solu-
minescence detection following the manufacturer’s proto- tion (40 mM DTT), (WAKO) in 25 mM NH4HCO3
col. Digital images were obtained by the Las-4000EPUV (WAKO), incubated 1 hr at 56°C with shaking followed.
Mini with an interface Las-4000 Image Reader (Fujifilm Alkylation reaction performed with 5 μL of 250 mM io-
Corporation. Tokyo, Japan). The acquired antigenic spots doacetamide (Tokyo Chemical Industry Co, Ltd, Tokyo,
were further transformed into pixels units for quantification Japan) in 25 mM NH4HCO3 and incubated in the dark for 45
of the recognition intensity using ATTO Lane and Spot min at 25°C with shaking. A volume of 180 μL 50 mM
Analyzer 6.0 software (ATTO Corporation. Tokyo, Japan). NH4HCO3 was added to dilute out the urea and to terminate
In screening of 2-D fractions by dot-blot assay, similar steps all the reactions prior to trypsin proteolytic digestion.
were followed. Briefly, 50 μl of each fraction in transfer Trypsin proteolysis was conducted using 50 μl (10 μg/ml)
buffer after drying to remove most of the ACN and TFA to sequence grade modified trypsin (Promega Corporation,
prevent interference of membrane blotting was loaded onto Madison, WI, USA) in 50 mM NH4HCO3 overnight at 37°C
the PVDF membrane. Test plasma were diluted for IgG1, followed by addition of 5 μl 5% LC-MS grade Formic Acid
1:25,000; IgG3, 1:15,000; IgG4, 1:400; and IgE, 1:1,000 (WAKO) in LC-MS grade ultra pure water (WAKO) to ter-
and applied to each respect 80 wells blotted with the 2-D minate the reaction. The trypsinized peptides were again
fractions. Bound antibody was again incubated with conju- speed-vacuumed and resuspended in 25 μl of 0.3% formic
gated enzymes using horseradish peroxidase-conjugated acid before filtered in Spin-X centrifuge tubes (0.22 μm
mouse anti-human IgG1, IgG3, IgG4 and biotin-conjugated Nylon, Costar Corning Inc., Corning, NY, USA) which was
goat anti-human IgE accordingly, in dilutions of 1:30,000, span at 2,300 g for 30 sec. The filtrate was transferred into
1:30,000 1:3,000 or 1:6,000 respectively. Two crude SWA MS vials (0.3 ml, TPX Snap Vial, GL Sciences Inc, Tokyo,
and BSA (Sigma A4503, Sigma-Aldrich Co. MO, USA) Japan) for loading onto the ESI-MS/MS system.
94 Tropical Medicine and Health Vol.40 No.3, 2012
Mass Spectrometric Analysis and Database Search searched in a locally established database for S. japonicum
The MS and tandem-MS (MS/MS) spectra of tryptic sequences downloaded from Chinese National Human
peptides (Fig. 1E) were obtained using the NanoFrontier Genome Centre at Shanghai (http://www.chgc.sh.cn/
nLC and NanoFrontier eLD Liquid Chromatography Mass japonicum/Resources.html) (containing 12,657 predicted
Spectrometer (Hitachi High-technologies, Tokyo, Japan). proteins) [33] using the MS/MS Ion Search provided by
The nanoLiquid Chromatography/ ElectroSpray Ionization/ MASCOT Sequence Query sever version 2.3 (http://
Linear Ion Trap/ Time of Flight (nLC-ESI/LIT/TOF) and www.matrixscience.com). With the MASCOT search, the
collision induced dissociation (CID) modes were used for following search parameters were used, enzyme: trypsin,
MS detection and peptide fragmentation as described [32]. variable modifications: carbamidomethylation, carboxyme-
In the NanoFrontier nLC, the trypsinized peptides (1–10 thyl (C) and oxidation (M), mass values: monoisotopic, pro-
μL) suspended in 0.3% formic acid were trapped on mono- tein mass: unrestricted, peptide mass tolerance: ± 0.5 Da,
lith trap column [C18-50-150 column, (0.05 mm I.D. × 150 fragment mass tolerance: ± 0.2 Da (CID data), maximum
mm L). Hitachi High-technologies] and separated on a missed cleavages: 1 and Instrument type: ESI-TRAP. For
packed nano-capillary column [NTCC-360/75-3-123, MASCOT output, significant peptides were determined by
(0.075 mm I.D. × 100 mm L, particle diameter 3 μm), the peptides score from the probability-based molecular
Nikkyo Technos Co., Ltd, Tokyo, Japan] at a flow rate of weight search (MOWSE) which identifies proteins from the
200 nL/min. The peptides in the column were eluted using a molecular weight of peptides created by the proteolytic di-
stepwise ACN gradient (mobile phase A: 2% ACN, 0.1% gestion [34]. Peptide score > 27 indicate identity or exten-
formic acid; mobile phase B: 98% ACN, 0.1% formic acid, sive homology (p<0.05). Further stringency was added by
The A:B concentration gradient was 0.0 min: (A:B = eliminating any single peptide that could be assigned to
100:0%) → 60 min (0:100%). In the NanoFrontier eLD more than one protein. To ensure a non-redundancy, the
spectrometer, the eluted peptides were ionized with a capil- protein identifications were examined manually in the data-
lary voltage of 1700 V and detected in a detector potential base for possible redundancies including multiple names
TOF range of 2050–2150 V. and homologies.
Acquired MS and MS/MS spectra were converted into
Mascot generic format (mgf) using a Data Processing soft- Characterization of Identified Proteins
ware 2008 (Hitachi High-technologies) and subsequently Identified S. japonicum proteins were characterized
Fig. 2. Elution profile and percentage concentration of SWA into various fractions during chromatofocusing. Chromatofocusing was
performed using the ProteomeLab PF 2D protein separation system. The pH gradient was formed using two proprietary buff-
ers: “ProteoSep Start” buffer at a pH 8.5 and “ProteoSep Elution” buffer at a pH 4.0. As shown, the SWA was well fractionat-
ed with fraction 31 containing most of the void proteins. Proteins identified were obtained from fractions numbers indicated
by arrows (2, 3, 18 and 3).
D. Boamah et al. 95
according to antigenic propensity, hydrophobicity, antigenic this phenomenon is observed in 2-D electrophoresis and is
determinant, domains and gene ontology (GO) types with useful for identifying proteins with different modification
respect to their antibody isotype recognition. An estimate of condition [38].
hydrophobicity was determined from the grand average of Four human circulating antibody isotypes (IgE, IgG1,
hydropathicity index (GRAVY) [35] using ProtParam from IgG3 and IgG4) recognition by the 1-D fractions were eval-
ExPASy [36]. The GRAVY index is the average hydropathy uated using dot-blot ELISA. The reactivity intensity of each
score for all the amino acids in the protein. The positive fraction was transformed into pixel unit and then expressed
GRAVY index indicates hydrophobic protein and negative, as relative reactivity intensity. The IgE was bound by 19
hydrophilic protein. The immune peptides or antigenic de- fractions (43.18%) with mean intensity of 0.22 ± 0.18 while
terminants and antigenic propensity were also estimated IgG1 had 33 (75.00%) with mean intensity, 0.24 ± 0.23. For
[37]. With reference to conserved domain sequences (CDS) IgG3 and IgG4 there were 35 (79.55%) and 27 (61.36%)
that were similar to known genes and domains, the proteins fractions detected with mean intensity of 0.28 ± 0.26 and
were further characterized into GO terms and annotations 0.36 ± 0.30, respectively. The IgE isotype was detected by
(biological processes, molecular functions and cellular fewer fractions as compared to the remaining isotypes. The
components) using UniProt-GOA server (ver. 106) (http:// most detected isotype was IgG3 followed by IgG1 and IgG4
www.ebi.ac.uk/QuickGO/). accordingly. It was observed that fractions 2 to 6 reacted
with all the isotypes with varying intensity (Fig. 3). Like-
Statistical Analysis wise, the 2-D fractions (2, 3, 18 and 31) were further
The data generated were analyzed by Microsoft Excel screened by dot-blot analysis to identify wells containing
and GraphPad Prism ver. 5. All the data were expressed as proteins that were IgE or IgG4 preferred (Fig. 3). The posi-
the mean ± SD. Differences between groups were analyzed tive dot-blot fractions of such 2-D wells were selected from
for statistical significance by one-way analysis of variance basic fractions (F2-C5, F2-C7, F2-C3; F3-E1, F3-H7), neu-
and the t-test (Unpaired t test) using the GraphPad Prism. A tral fractions (F18-B1, F18-C3) and acidic fractions (F31-
p-value of <0.05 was considered as statistically significant. C3, F31-E1, F31-H7) which were processed for LC-MS/
MS. Fig. 4A shows a representative 2-D dot-blot reactivity.
RESULTS
ESI-MS/MS Analysis
Fractions and Dot-blot Screening A total of 25 non-redundant proteins were identified
Protein fractionation by chromatofocusing is used to by two-dimensional fractionation through ESI MS/MS
enrich proteins with similar isoelectric point (IEP or pI) and analysis of 10 fractions (Table 1). A representative tandem
collected in one fraction [38]. Liquid fractionation of crude mass spectrum is presented in Fig. 4B. Proteins identified
SWA into 1- and 2-D was achieved using the ProeteomLab with peptides obtained from the MS/MS mostly had a single
PF 2D instrument. In the chromatofocusing, SWA proteins confirmed peptide matches with a minimum Mascot score
were separated and eluted into 44 fractions. Fig. 3 shows of 27. The 2-D wells (F2-C3, F2-C5 and F2-C7) originating
the fraction number against the elution profile (average pI) from 1-D F2; 1-D F3 (F3-E1 and F3-H7); 1-D F18 (F18-B1
and percentage concentrations while Fig. 3, 2-D elution and F18-C3) and 1-D F31 (F31-C3, F31-E1 and F31-H7),
profiles of fractions 2, 3, 18 and 31. As expected, the basic yielded 3, 14, 4, 4 proteins respectively. Two proteins (chro-
proteins started to elute (fractions 1 to 17). The elution con- matin licensing and DNA replication factor 1 and zinc fin-
tinued with the neutral proteins (18 to 20) followed by the ger, RanBP2-type, domain-containing) were sequenced
weak and strong acidic proteins (21 to 44). Most of the pro- from two sequential fractions (F2 and F3) and at the same
teins were eluted in fraction 31 (15.42%) with mainly acidic time one of them (chromatin licensing and DNA replication
proteins. Fractions 37 to 44 had undetectable level of per- factor 1), in other two non-sequential fractions (F18 and
centage concentration. The pIs of almost all the proteins F31). Proteins with a wide range of molecular weight were
identified in the higher pH gradient were consistent with the identified from less than 20 kDa to more than 300 kDa
expected pI (Table 1). However, lower pH gradient showed (Table 1). The pI and the molecular weight profiles there-
a weak correlation to the expected pI range even though fore indicate the benefit of protein fractionation which is an
proteins with the expected pI were also found in these frac- important aspect of protein profiling in identifying proteins
tions (F31-H7). Such unexpected phenomenon is possible with different biochemical properties. The majority of the S.
because the last few fractions can be enriched with proteins japonicum proteins were recognized by IgE, IgG3 and IgG4
that carry post-translational modifications (PTM) such as but not IgG1.
phosphorylation which can cause a shift in their pI. Usually,
96 Tropical Medicine and Health Vol.40 No.3, 2012
Fig. 3. Second dimensional elution profile of 1-D fractions 2 (A), 3 (B), 18 (C) and 31 (D). 1-D fractions were run in the 2-D re-
versed phase chromatography using the ProteomeLab PF 2D. As shown is the elution profile with respect to time and absor-
bance units (AU). The elution gradient was achieved using two solvents, 0.1% TFA in water (A) and 0.08% TFA in ACN (B).
The 2-D fractionation was run at column temperature of 50°C and a buffer flow rate of 0.75 ml/min with the absorption mon-
itored at 214 nm. The column was eluted with a 0–100% linear gradient of solvent A and B for 35 min. Insert, 2-D UV differ-
ence maps obtained by ProteoVue of the fractions and the pH range; 31 (pH 4.29–4.34), 18 (pH 6.03–6.33), 3 (pH 8.22–8.45)
and 2 (pH 8.45–8.47). Arrows indicate fractions from which proteins were sequenced. F2 P# = 14, 23, 29. F3 P# = 14, 23, 29.
F18 P# = 3/4, 13. F31 P# = 6, 16, 47. P#, peak numbers.
Characterization (GO:0051301), mitotic cell cycle (GO:0000278), RNA in-
It is worth noting that all the proteins identified have a terference (GO:0016246) and protein physiological pro-
GRAVY index within negative range (Table 2). Meaning cesses such as potassium ion transport (GO:0006813), ATP
the proteins are hydrophilic in consistent with 2-D reversed binding (GO:0005524) and protein modification process
phase elution profile (Fig. 3). Meiosis-specific nuclear (GO:0006464). Others were associated with regulation, de-
structural protein 1 (MNS1) was found to be the most hy- velopment or stress response including cell differentiation
drophilic (–1.306) and BRO1 domain-containing protein (GO:0030154), reproduction (GO:0000003) and response
BROX, less hydrophilic (–0.177) protein. With respect to to oxidative stress (GO:0006979) and so on. In terms of cel-
the number of possible epitope each protein have, ubiquitin- lular components, the proteins were found to be associated
conjugating enzyme E2 N had 8 antigenic determinants, be- with cytoplasm (GO:0005737), intracellular (GO:0005622),
ing the least yet with average antigenic propensity of nucleus (GO:0005634), plasma membrane (GO:0005886)
1.0386. and extracellular region (GO:0005576). The PDZ and LIM
The GO terms for the annotated proteins in terms of bi- domain protein 3 expressed in the cytoplasm is associated
ological processes, molecular function and cellular compo- with response to oxidative stress (GO:0006979). In addi-
nents were identified for almost all the proteins (Table S1). tion, a membrane protein, Small conductance calcium-
In terms of biological processes, some are associated with activated potassium channel (SK) protein 2 with
DNA metabolic and physiological processes including nu- Calmodulin binding domain (CaMBD) (GO:0015269), has
cleotide metabolic process (GO:0009117), cell division been found to be a secreted protein [39–42].
D. Boamah et al. 97
Table 1. Fractions and identified proteins by ESI MS/MS. S. japonicum adult worms extract was liquid fractionated using ProteomeLab PF 2D system into 1-D by chromato-
focusing and reversed phase chromatography followed which were screened by dot-blot. Immunoreactive 2-D fractions were subjected to ESI MS/MS and peptide
identification using MASCOT server with a locally established S. japonicum protein sequences downloaded from S. japonicum database at http://www.chgc.sh.cn/
japonicum/Resources.html. MP, MASCOT peptides score. *Total MASCOT Peptide Score.
Fraction Unique 
Protein ID Match to m/z z error (Da) Peptide MP Score pI/Mw
Well peptide
F2-C3 Sjc_0203170 Chromatin licensing and DNA replication factor 1 1 530.81 2 0.0092 DVIDLVKMK 56 9.74/64520
F2-C5 Sjc_0213700 Zinc finger, RanBP2-type, domain-containing 1 450.29 2 0.0517 INLSSLPR 27 9.34/57532
F2-C6 Sjc_0213700 Zinc finger, RanBP2-type, domain-containing 1 450.29 2 0.0503 INLSSLPR 30 9.34/57532
F3-E1 Sjc_0034740 Meiosis-specific, nuclear structural protein 1 1 689.85 2 –0.0309 RELEAINAYTAK 40 6.79/45874
Sjc_0203170 Chromatin licensing and DNA replication factor 1 1 530.8 2 –0.008 DVIDLVKMK 36 9.74/64520
Sjc_0058190 NACHT and WD repeat domain-containing protein 1 1 590.33 2 0.0613 MCEQLLKTR 34 5.28/228171
Sjc_0037420 Triple functional domain protein 1 606.37 2 0.0134 QFLAK 30 6.13/173854
Sjc_0083690 RNA Helicase 1 606.37 2 0.0465 MQLAK 30 9.07/86337
Sjc_0040110 Protein kinase 1 699.86 2 0.0254 ENFVLYDEIEK 29 8.86/209937
Sjc_0302250 BRO1 domain-containing protein BROX 1 564.33 2 0.0046 EKAGQAIAALR 31 8.28/47921
Sjc_0111110 Cell division control protein CDC7 2 464.79 2 0.1187 LLEPCPEK 32* 9.5/36413
716.92 2 0.0794 TTDLNISENNRR
Sjc_0063180 Centriolin 3 572.88 2 0.1375 GELEQIKAEK 51* 7.04/278991
723.43 2 0.0615 KISDQSELKLER
730.45 2 0.2049 RDYSLMRSCVR
Sjc_0046400 Conductance calcium-activated potassium channel protein 2 2 481.32 2 0.1387 FISLCNHK 31* 7.09/169207
899.53 2 0.1672 NLVTSVMGVLSDYMPR
Sjc_0009730 CREB-binding protein 2 679.52 2 0.153 MILMR 32* 8.00/113822
414.23 2 0.0905 YTVCER
Sjc_0106490 PDZ and LIM domain protein 3 3 564.31 2 0.0968 VPMHPECLK 30* 9.08/35351
506.97 2 0.2293 VPMHPECLKCCK
534.22 2 0.0493 CCKCGIGLR
Sjc_0054640 Prolyl-tRNA synthetase 3 488.93 2 0.0718 KGTQQGLRCCVR 31* 7.5/128901
699.86 2 0.0117 CKVEPHVRTGSK
621.88 2 0.0013 LALQNTVLSKR
F3-H6 Sjc_0213700 Zinc finger, RanBP2-type, domain-containing 1 450.29 2 0.0415 INLSSLPR 30 9.34/57532
F18-B1 Sjc_0045150 E3 ubiquitin-protein ligase HUWE1 1 466.78 2 0.0381 RWTNLSR 29 5.25/301151
Sjp_0042440 5'-nucleotidase, cytosolic II 1 384.8 2 0.1307 VTSVHLL 27 7.17/55038
F18-C3 Sjc_0203170 Chromatin licensing and DNA replication factor 1 1 530.83 2 0.0376 DVIDLVKMK 40 9.74/64520
Sjc_0042100 Ubiquitin-conjugating enzyme E2 N 2 487.32 2 0.0935 QNEAEALAK 28* 5.42/19728
601.4 2 0.1123 LGRICLDILK
F31-C3 Sjc_0203170 Chromatin licensing and DNA replication factor 1 1 530.8 2 0.0104 DVIDLVKMK 36 9.74/64520
F31-E1 Sjc_0001260 Putative Serine/threonine-protein kinase C05D10.2 2 683.38 2 0.0558 LCDFGLARSLK 34* 9.18/120799
590.36 2 0.1803 CQNGNKINCK
F31-H7 Sjc_0203170 Chromatin licensing and DNA replication factor 1 1 530.81 2 0.0056 DVIDLVKMK 36 9.74/64520
Sjc_0058190 NACHT and WD repeat domain-containing protein 1 1 590.33 2 0.0763 MCEQLLKTR 31 5.28/228171
98 Tropical Medicine and Health Vol.40 No.3, 2012
have also presented a global proteomics approach using 2-D
gels to identify major S. japonicum excretory and secretory
proteins as well as adult worm and egg extracts [46–50]. In
this study, for the first time, proteomics approach was ex-
tended to identifying S. japonicum proteins in ProteomeLab
FP 2D derived liquid fractions reactive to antibody isotypes
in plasma samples from S. japonicum-infected population.
ESI MS/MS was applied to sequence fractions containing
immunreactive proteins. In all, 18 proteins were identified;
characterized and GO categories determined to enhance un-
derstanding of the immunological significance of these pro-
teins.
In IgE, IgG1, IgG3 and IgG4 antibody isotype recogni-
tion of 1-D fractions, it has been shown that some fractions
from the adult S. japonicum proteome are preferentially rec-
ognized by certain isotypes. For instance, IgE isotype was
detected by fewer antigenic fractions (43.2%). The most
recognized isotype was IgG3 (79.5%) followed by IgG1
(75.0%) and IgG4 (61.4%) accordingly. The IgG3 response
was directed against a larger repertoire of antigens in the
fractions. This suggests that there were fewer dominant an-
tigens stimulating IgE response. Earlier report [51, 52]
showed where IgE reactivity to glycolipids extracted from
schistosome eggs (SEA) or adult worms (SWA) was more
than IgG4 and that proteins alone do not constitute the ma-
jor binding targets of IgE, and that this isotype is substan-
tially directed towards carbohydrate moieties portion of
glycolipids on proteins present in SEA or AWA. Weiss et al.
Fig. 4. Representative 2-D fraction dot-blot reactivity and tan- [53] showed that a carbohydrate epitope recognized by a
dem mass spectrum. Represented by A, IgE reactivity monoclonal antibody that was raised against the cercarial
intensity of F2-C3, F2-C5 and F2-C7 with crude SWA
glycocalyx was present on glycoproteins and glycolipids of
and BSA controls. B, a tryptic INLSSLPR peptide of
various schistosomes’ life cycle stages. This might explain
Zinc finger, RanBP2-type, domain-containing protein,
Sjc_0213700 (GenBank: CAX74641.1). The precursor why IgG4 recognized more proteins than IgE in the 1-D
ion was m/z 450.30(2+). Sjc_0213700 was sequenced dot-blot.
from F2-C5 and F2-C7 with strong preference for IgE. Furthermore, zinc finger, RanBP 2-type, domain-
containing protein was strongly recognized by IgE but mod-
erately by IgG3 and IgG4 and weakly by IgG1 indicating
that it might play less role in IgG subclasses directed im-
mune response. The antigens recognized strongly by IgE
DISCUSSION
are of interest as such antigens could be associated with de-
Following skin penetration by cercariae, S. japonicum velopment of resistance to schistosomiasis [2–4, 6]. The E3
adult worms migrate to the hepatic portal system, where ubiquitin-protein ligase, ubiquitin-conjugating enzyme E2
they mature and survive for many years where the female N and 5'-nucleotidase, cytosolic II were strongly recogniz-
occasionally migrating to the smallest venules to lay eggs ing by IgG subclasses (IgG1, IgG3 and IgG4) but not IgE
[43]. The adult schistosomes are constantly exposed to the suggesting these enzymes might be of IgG subclasses pre-
host immune system with antibodies produced against frac- ferred. The serine/threonine-protein kinase with a relatively
tions of the worms. These antibodies are often used as po- higher antigenic propensity of 1.0118 was sequenced from a
tential diagnostic tools. Several immunoepidemiological single 2-D well strongly reactive with all the four isotypes
studies have examined antibody isotype responses to schis- indicating strong preference for IgE, IgG1, IgG3 as well as
tosomal protein extracts in the form of isolated proteins, re- IgG4 suggesting shared antigenic determinates or multiple
combinant proteins or crude antigen [44, 45]. Many studies epitopes. This highlights the importance of vaccine research
D. Boamah et al. 99
Table 2. Biochemical properties and immunoreactivity pattern of the proteins. Dot-blot assay was performed for each second dimension fraction containing the proteins iden-
tified. The reactivity was quantified into pixels unit and graded according to reactivity intensity with respect to that of the crude parasite antigen to obtain relative
reactive intensity. Using the relative reactive intensity, each reactive spot was scored as ‘weak reactivity’ (–), ‘moderate reactivity’ (±) or ‘strong reactivity’ (+). In
addition to reactive pattern, proteins were characterized using GRAVY index (ProtParam tool), number of antigenic determinant contained in the full amino acid
length and the antigenic propensity where >1 indicates high antigenic character. In this Table antibody reactivity pattern is not repeated for proteins from the same
fraction well.
Fraction GRAVY Amino Antigenic Antigenic Antibody reactivity pattern
Protein identified
Well index acids determinants propensity IgE IgG1 IgG3 IgG4
F2-C3 Chromatin licensing/DNA replication factor 1 –0.348 578 20 1.0414 + – – +
F2-C5 Zinc finger, RanBP2-type, domain-containing –0.986 513 21 1.0108 + – – ±
F2-C7 Zinc finger, RanBP2-type, domain-containing –0.986 513 21 1.0108 ± – – ±
F3-E1 Meiosis-specific, nuclear structural protein 1 –1.306 376 11 0.9936 + ± + +
Chromatin licensing/DNA replication factor 1 –0.348 578 20 1.0414 + ± + +
NACHT/WD repeat domain-containing protein 1 –0.282 2005 81 1.0271
Triple functional domain protein –0.414 1554 68 1.0336
RNA Helicase [EC:3.6.1.-] –0.409 762 25 1.0308
Protein kinase [EC:2.7.1.-] –0.22 1921 63 1.0397
BRO1 domain-containing protein BROX –0.177 426 16 1.0459
Cell division control protein CDC7 –0.333 330 13 1.0363
Centriolin –0.847 2444 92 1.0139
Small conductance calcium-activated potassium channel protein 2, putative –0.393 1536 58 1.0294
CREB-binding protein –0.708 986 29 1.0269
PDZ and LIM domain protein 3 –0.674 317 13 1.0199
Prolyl-tRNA synthetase [EC6.1.1.15] –0.473 1135 41 1.0295
F3-H7 Zinc finger, RanBP2-type, domain-containing –0.986 513 21 1.0108 – – ± –
F18-B1 E3 ubiquitin-protein ligase HUWE1 –0.423 2720 105 1.0231 – + + +
5'-nucleotidase, cytosolic II –0.2 476 21 1.0368
F18-C3 Chromatin licensing/DNA replication factor 1 –0.348 578 20 1.0414 – + + +
Ubiquitin-conjugating enzyme E2 N –0.228 173 8 1.0386
F31-C3 Chromatin licensing/DNA replication factor 1 –0.348 578 20 1.0414 – + + +
F31-E1 Putative serine/threonine-protein kinase C05D10.2 –0.774 1061 39 1.0118 + + + +
F31-H7 Chromatin licensing/DNA replication factor 1 –0.348 578 20 1.0414 – – – –
NACHT/WD repeat domain-containing protein 1 –0.282 2005 81 1.0271
100 Tropical Medicine and Health Vol.40 No.3, 2012
focusing on induction of protective isotype-specific anti- International Cooperation Agency (JICA). This research re-
body response to specific peptides since a single protein ceived further financial support from Grants Kakenhi
from the parasite might possess undetermined antigenic de- (22406009, 23590489) and the Global Centre of Excellence
terminants capable of stimulating various antibody produc- (GCOE), Nagasaki University, Japan. Special thanks are ad-
tions. Therefore, further investigations employing peptide dressed to Noguchi Memorial Institute for Medical
mapping techniques will be essential in determining spe- Research, University of Ghana-Legon, Ghana and Department
cific antigenic determinants for the isotypes. of Immunogenetics, Institute of Tropical Medicine,
There were also proteins found with strong immuno- Nagasaki University, Japan, for their cooperation.
genic activity to IgE, IgG3 and IgG4 but not IgG1. Some of
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