Identification of hepatocellular-carcinoma-associated antigens and autoantibodies by serological proteome analysis combined with protein microarray.
ABSTRACT To comprehensively study autoantibodies in patients with hepatocellular carcinoma (HCC), we used an approach-based serology and proteomics technologies. Total proteins extracted from HepG2 cells and HepG2.2.15 cells were separated by two-dimensional gel electrophoresis (2DE) and then transferred onto polyvinylidene difluoride (PVDF) membranes, which were subsequently incubated with sera from HCC patients or from normal controls. As a result, 13 HCC-associated antigens were identified. Antigenicity of eight proteins was further confirmed using recombinant proteins by Western blotting (WB) and protein microarray. The results of antigen microarray analysis showed strong signals of keratin 8 and lamin A/C in chronic hepatitis controls; therefore, the autoantibodies to keratin 8 and lamin A/C may not be HCC-specific. These two antigens were removed from subsequent analyses. The frequencies of positive reactions to DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, eukaryotic translation elongation factor 2 (eEF2), apoptosis-inducing factor (AIF), heterogeneous nuclear ribonucleoprotein A2 (hnRNP A2), prostatic binding protein, and triosephosphate isomerase (TIM) were significantly higher in HCC than in chronic hepatitis and normal individuals. Positive reactions to DEAD box polypeptide 3, eEF2, AIF, and prostatic binding protein were significantly more frequent in HCC than in any other cancer. The sensitivity of any individual antigen in HCC at stage I ranged from 50 to 85%. When the combinations of six antigens were analyzed, the sensitivity increased to 90%. We conclude that the detection of autoantibodies against the six antigens may have value on early diagnosis of HCC.
- SourceAvailable from: Fernando J Corrales[show abstract] [hide abstract]
ABSTRACT: Wnt/β-catenin pathway controls biochemical processes related to cell differentiation. In committed cells the alteration of this pathway has been associated with tumors as hepatocellular carcinoma or hepatoblastoma. The present study evaluated the role of Wnt/β-catenin activation during human mesenchymal stem cells differentiation into hepatocytes. The differentiation to hepatocytes was achieved by the addition of two different conditioned media. In one of them, β-catenin nuclear translocation, up-regulation of genes related to the Wnt/β-catenin pathway, such as Lrp5 and Fzd3, as well as the oncogenes c-myc and p53 were observed. While in the other protocol there was a Wnt/β-catenin inactivation. Hepatocytes with nuclear translocation of β-catenin also had abnormal cellular proliferation, and expressed membrane proteins involved in hepatocellular carcinoma, metastatic behavior and cancer stem cells. Further, these cells had also increased auto-renewal capability as shown in spheroids formation assay. Comparison of both differentiation protocols by 2D-DIGE proteomic analysis revealed differential expression of 11 proteins with altered expression in hepatocellular carcinoma. Cathepsin B and D, adenine phosphoribosyltransferase, triosephosphate isomerase, inorganic pyrophosphatase, peptidyl-prolyl cis-trans isomerase A or lactate dehydrogenase β-chain were up-regulated only with the protocol associated with Wnt signaling activation while other proteins involved in tumor suppression, such as transgelin or tropomyosin β-chain were down-regulated in this protocol. In conclusion, our results suggest that activation of the Wnt/β-catenin pathway during human mesenchymal stem cells differentiation into hepatocytes is associated with a tumoral phenotype.PLoS ONE 01/2012; 7(4):e34656. · 3.73 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Activation of plasminogen on the cell surface initiates a cascade of protease activity with important implications for several physiological and pathological events. In particular, components of the plasminogen system participate in tumor growth, invasion and metastasis. Plasminogen receptors are in fact expressed on the cell surface of most tumors, and their expression frequently correlates with cancer diagnosis, survival and prognosis. Notably, they can trigger multiple specific immune responses in cancer patients, highlighting their role as tumor-associated antigens. In this review, three of the most characterized plasminogen receptors involved in tumorigenesis, namely Annexin 2 (ANX2), Cytokeratin 8 (CK8) and alpha-Enolase (ENOA), are analyzed to ascertain an overall view of their role in the most common cancers. This analysis emphasizes the possibility of delineating new personalized therapeutic strategies to counteract tumor growth and metastasis by targeting plasminogen receptors, as well as their potential application as cancer predictors.Experimental hematology & oncology. 04/2013; 2(1):12.
- [show abstract] [hide abstract]
ABSTRACT: Lymph node metastasis (LNM) is recognised as an important factor involved in malignant tumour progression by interfering with a favourable prognosis. It is involved in a variety of cancers. Proteins are believed to play important roles in the LNM of cancers. The rapid achievements of state-of-the-art proteomic techniques have emerged as the key technologies successfully applied to identify markers for cancers at high-throughput level by providing novel targets and creating possible therapeutic interventions in cancer research. This review summarises recent progress in proteomic research in hepatocarcinoma, gastric cancer, oesophageal cancer, colorectal cancer, breast cancer, lung cancer and nasopharyngeal cancer. Actin, heat-shock proteins (HSPs), annexins, cytokeratin 10 (CK10), CK19, protein gene product 9.5 (PGP9.5) and protein disulfide isomerase (PDI) are the most common proteins in lymphatic metastases of cancers revealed by proteomic and protein functional studies. Other protein candidates specifically associated with LNMs of certain cancers are also summarised and discussed.Clinical and Translational Oncology 01/2012; 14(1):21-30. · 1.28 Impact Factor
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Identification of Hepatocellular-Carcinoma-Associated
Antigens and Autoantibodies by Serological
Proteome Analysis Combined with Protein Microarray
Lan Li, Su-hong Chen, Chao-hui Yu, You-ming Li, and Sheng-qi Wang
J. Proteome Res., 2008, 7 (2), 611-620 • DOI: 10.1021/pr070525r • Publication Date (Web): 28 December 2007
Downloaded from http://pubs.acs.org on February 5, 2009
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Identification of Hepatocellular-Carcinoma-Associated Antigens and
Autoantibodies by Serological Proteome Analysis Combined with
Lan Li,†,‡,§Su-hong Chen,†,§Chao-hui Yu,‡You-ming Li,*,‡and Sheng-qi Wang*,†
Beijing Institute of Radiation Medicine, Number 27 Taiping Road, Beijing 100850, People’s Republic of China,
and Department of Gastroenterology, The First Affiliated Hospital, Medical College, Zhejiang University,
Number 79 Qingchun Road, Hangzhou 310003, People’s Republic of China
Received August 11, 2007; Accepted October 31, 2007
To comprehensively study autoantibodies in patients with hepatocellular carcinoma (HCC), we used
an approach-based serology and proteomics technologies. Total proteins extracted from HepG2
cells and HepG2.2.15 cells were separated by two-dimensional gel electrophoresis (2DE) and then
transferred onto polyvinylidene difluoride (PVDF) membranes, which were subsequently incubated
with sera from HCC patients or from normal controls. As a result, 13 HCC-associated antigens were
identified. Antigenicity of eight proteins was further confirmed using recombinant proteins by
Western blotting (WB) and protein microarray. The results of antigen microarray analysis showed
strong signals of keratin 8 and lamin A/C in chronic hepatitis controls; therefore, the autoantibodies
to keratin 8 and lamin A/C may not be HCC-specific. These two antigens were removed from
subsequent analyses. The frequencies of positive reactions to DEAD (Asp-Glu-Ala-Asp) box
polypeptide 3, eukaryotic translation elongation factor 2 (eEF2), apoptosis-inducing factor (AIF),
heterogeneous nuclear ribonucleoprotein A2 (hnRNP A2), prostatic binding protein, and triose-
phosphate isomerase (TIM) were significantly higher in HCC than in chronic hepatitis and normal
individuals. Positive reactions to DEAD box polypeptide 3, eEF2, AIF, and prostatic binding protein
were significantly more frequent in HCC than in any other cancer. The sensitivity of any individual
antigen in HCC at stage I ranged from 50 to 85%. When the combinations of six antigens were
analyzed, the sensitivity increased to 90%. We conclude that the detection of autoantibodies against
the six antigens may have value on early diagnosis of HCC.
Keywords: Autoantibody • autoimmunity • hepatocellular carcinoma • serological proteome analysis
Hepatocellular carcinoma (HCC) is one of the most frequent
and lethal malignancies worldwide, and the 5 year survival rate
after hepatectomy is 30–50%.1Given the high incidence and
mortality of HCC, it is important to develop biomarkers for
assessing onset or prediction of therapy outcome as well as to
identify targets for the development of novel therapies. Al-
though there are multiple promising diagnostic biomarkers of
HCC in the development phase, there are at the present time
no definitive antibody-based serologic markers for its early
diagnosis in clinic.2,3Several experimental studies have led to
the identification of autoantigens via recognition by autoan-
tibodies present in HCC sera.4Antigens that have been shown
to induce a humoral response in HCC include p535and diverse
other nuclear proteins.6Autoantibodies to cyclin B17and to a
novel cytoplasmic protein with RNA-binding motifs8have also
been reported. However, efforts to consistently predict HCC
based on autoimmunity to antigens have not resulted in
serologic markers with definitive specificity and sensitivity.
Serological proteome analysis (SERPA) is a powerful tool for
the identification of a large group of candidate cancer bio-
markers recognized as autoantigens by the sera of cancer
patients.9This approach permits the transfer and immobiliza-
tion of proteins to a semirigid support, allowing for the
subsequent immunodetection of relevant antigens among
thousands of individual proteins separated by two-dimensional
gel electrophoresis (2DE). To survey autoantibodies associated
with HCC, we used this SERPA approach and identified 13
proteins that induced a humoral response in HCC patients but
not in healthy individuals and subsequently investigated the
prevalence of the autoantibodies in a large number of HCC
patients and negative controls.
* To whom correspondence should be addressed: Beijing Institute of
Radiation Medicine, Number 27 Taiping Road, Beijing 100850, People’s
Republic of China. Telephone: +86-10-68210077X932211. Fax: +86-10-
68210077X932211. E-mail: firstname.lastname@example.org (S.-q.W.); The First Affiliated
Hospital, Medical College, Zhejiang University, Number 79 Qingchun Road,
Hangzhou 310003, People’s Republic of China. Telephone: +86-571-
87236603. Fax: +86-571-87236611. E-mail: email@example.com (Y.-m.L.).
†Beijing Institute of Radiation Medicine.
§These authors contributed equally to this work.
10.1021/pr070525r CCC: $40.75
2008 American Chemical Society
Journal of Proteome Research 2008, 7, 611–620 611
Published on Web 12/28/2007
Materials and Methods
Serum Samples and Cell Lines. Serum samples for SERPA
were obtained from 28 HCC patients during routine blood tests
prior to any treatment, consisting of 18 hepatitis B virus (HBV)-
related HCC patients and 10 virus-negative HCC patients. Sera
from 18 healthy volunteers were used as controls. Healthy
volunteers without a history of cancer or autoimmune disease
were asymptomatic and had normal physical exams and
normal routine blood tests of liver function.
Serum samples for blind validation by Western blotting
(WB)/microarray were collected from 146 HCC patients at the
time of initial cancer diagnose. Among 146 HCC patients, 91
patients were positive for HBV, 30 patients were positive for
hepatitis C virus (HCV), and 25 patients were negative for
hepatitis virus. Sera from 63 patients chronically infected with
HBV or HCV without HCC, from 66 patients with other cancers,
including 17 with lung cancer, 16 with esophageal cancer, 15
with breast cancer, and 18 with gastric cancer, and from 53
healthy volunteers were used as controls. Aliquots of sera were
immediately frozen at -80 °C until used and were never
refrozen. Clinical characteristics of the research subjects are
shown in Table 1.
Patients were only included in the study if they had provided
written consent to participate in the study after receiving oral
and written information regarding its course and purpose.
Approval for the study was received from the Ethics Committee
of the host institution.
The human hepatoma cell lines HepG2 were cultured in
Dulbecco’s modified Eagle medium (Invitrogen, Carlsbad, CA)
supplemented with 10% fetal calf serum (FCS; Invitrogen,
Carlsbad, CA), 100 units/mL penicillin, and 100 units/mL
streptomycin. HepG2.2.15 cells were kindly provided by Beijing
Medical University, who received them from Mount Sinai
Medical Center in New York. The HepG2.2.15 cells were
cultured in minimum essential medium (Invitrogen, Carlsbad,
CA) supplemented with 10% fetal bovine serum (Invitrogen,
Carlsbad, CA), 380 µg/mL antibiotic G-418 sulfate (Promega,
Madison, WI), 2 mM L-glutamine (Sigma, St. Louis, MO), and
200 units/L amikaxin sulfate (QiLu medicine company, Shan-
Two-Dimensional Gel Electrophoresis (2DE). All reagents
for 2DE were from Amersham Biosciences, except mentioned
specially. Cultured cells were solubilized in lysis buffer contain-
ing 40 mM Tris, 7 M urea, 2 M thiourea, 4% 3-[(3-cholami-
dopropyl)dimethylamonio]-1-propanesulfonate (CHAPS), 40
mM dithiothreitol (DTT), 2% immobilized pH gradient (IPG)
buffer (pH 3–10), protease inhibitor mix, and nuclease mix. The
protein concentration was measured with Bradford’s method.
The IPG strips (pH 3–10, 7 cm longer) were rehydrated
overnight in 7 M urea, 2 M thiourea, 2% CHAPS, 18 mM DTT,
0.5% IPG buffer, and trace bromophenol blue. The sample (120
µg) was applied to gels by rehydration loading. Isoelectric
focusing (IEF) was initiated at 100 V for 1 h, 500 V for 1 h, 1000
V for 1 h, and then gradually increased to 5000 V for 5–6 h.
Focus was carried out for 20 000 V h. After IEF, IPG strips were
equilibrated for 2 × 15 min. Equilibration buffers contained
75 mM Tris-HCl (pH 8.8), 6 M urea, 29.3% glycerol, 2% sodium
dodecyl sulfate (SDS), and trace bromophenol blue, with 1%
DTT for the first step and 2.5% iodoacetamide for the second
step. For the second-dimension electrophoresis, 12.5% SDS-
polyacrylamide gels were used. Proteins were transferred to
polyvinylidene difluoride (PVDF) membranes (Schleicher and
Schuell Biosciences, Keene, NH) or visualized by Colloidal
Coomassie staining of the gels.
Two-Dimensional Immunoblot Analysis. After transfer,
membranes were incubated in Tris-buffered saline (TBS)
containing 5% nonfat dry milk and 0.1% Tween 20 for 3 h at
room temperature. The membranes were incubated overnight
at 4 °C with a mixture of serum samples diluted at 1:100 with
blocking buffer. After washing 6 times for 15 min each in TBS
with 0.1% Tween 20, membranes were incubated with horse-
radish peroxidase-conjugated mouse anti-human IgG (South-
ern Biotechnology Associates, Inc., Birmingham, AL) for 1 h at
room temperature. Immunodetection was performed with
enhanced chemiluminescence reagent (Santa Cruz Biotech-
nology, Inc., Santa Cruz, CA), followed by autoradiography on
Kodak film. Two-dimensional electrophoresis gels and immu-
nobloting maps were scanned using ImageScanner II (Amer-
sham Biosciences, Buckinghamshire, U.K.) and analyzed by
ImageMaster 2D Platinum 5.00 software (Amersham Bio-
sciences Buckinghamshire, U.K.). The proteomic profile of
proteins from the hepatoma cell line was used as a reference
map for spot analysis. Spots on immunoblotting maps were
matched to the reference map, and those observed from HCC
sera but not from normal sera were excised for protein
In-Gel Digestion and Mass Spectrometry (MS) Identifica-
tion. In-gel digestion of proteins from 2D gels was performed
as described by Steiner et al.10Spots were excised and destained
with 25 mM ammonium bicarbonate/50% acetonitrile and
dried in a vacuum concentrator (Savant, Holbook, NY). The
dried gel pieces were rehydrated with 5 µL of 10 mg/L trypsin
(Roche Molecular Biochemicals, Mannheim, Germany) in 25
mM ammonium bicarbonate and digested at 37 °C for 18–20
h. Tryptic peptides were first extracted using 5% trifluoroacetic
acid (TFA) at 40 °C for 1 h and then 2.5% TFA/50% acetonitrile
at 30 °C for 1 h. The extracted solutions were mixed in an
Eppendorf tube and dried in a vacuum concentrator. The
peptides mixture was solubilized with 0.5% TFA for MS analysis.
MS was performed on a matrix-assisted laser desorption/
Table 1. Clinical Characteristics of the Research Subjects
subjectsn age (mean)
used for SERPA
used for WB and microarray
aAssessment based on TNM classification by the International Union Against Cancer.
Li et al.
612 Journal of Proteome Research • Vol. 7, No. 2, 2008
ionization-time-of-flight (MALDI-TOF) mass spectrometer
(Bruker Daltonics, Bremen, Germany), with saturated R-cyano-
4-hydroxycinnamic acid (CHCA) solution in 0.1% TFA/50%
acetonitrile as the matrix. Mass spectra were externally cali-
brated with autodigest peaks of trypsin (MH+: 906.505, 1020.504,
1153.574, 2163.057, and 2273.160 Da). Database searching
(NCBI or Swiss-Prot) was performed using the Internet-
available program Mascot (Matrix Science, London, U.K.).
Search parameters were set up as follows: the taxonomy was
selected as Homo sapiens; the mass tolerance was 0.3 Da; the
missed cleavage sites were allowed up to 1; and the variable
modifications were selected as carbamidomethyl (cysteine) and
oxidation (methionine). The criteria for protein identifications
included the extent of sequence coverage, the number of
peptides matched, and the probability-based Mowse score, and
also, the molecular weight and isoelectric point of identified
proteins should match the estimated values obtained from
Preparation of Recombinant Proteins. According to the
reported cDNA sequence of the protein identified, we prepared
corresponding oligonucleotide primers to amplify DNA frag-
ments by reverse transcription and polymerase chain reaction.
The nucleotide sequences of the primers included the restric-
tion enzyme recognition sequence for subcloning. The resulting
cDNA fragments were subcloned separately into the plasmid
expression vector pGEX-4T-1, pET-28a(+), and pET-43.1a(+).
Using these constructs, recombinant proteins were produced
in Escherichia coli BL21 (DE3) as fusion proteins with corre-
sponding tags. After induction with isopropyl-?-D-thiogalac-
topyranoside, the recombinant proteins were separately puri-
fied on Glutathione Sepharose columns (Amersham Biosciences,
Buckinghamshire, U.K.) or Ni-NTA columns (Qiagen, Hilden,
Germany) according to the instructions of the manufacturer.
Next, serum samples either from HCC patients or from normal
controls were used for WB with recombinant proteins and the
corresponding tags as negative controls.
Probing and Scanning of Antigen Microarrays. Antigens
were diluted to 0.5 µg/µL in phosphate-buffered saline (PBS)
with 0.02% SDS and 1% glycerol and robotically attached in
ordered arrays on aldehyde-activated glass slides, using a
computer-controlled microchip spotting instrument (Cartisan
Pixsys 3000). Samples were transferred from 384 microtiter
plates to glass slides using stainless-steel microspotting pins
(400 µm in diameter). Each pin was estimated to transfer ∼2
nL of sample to the slide. The size of the glass slide was 25.4 ×
76.2 mm, including 10 matrixes (7.5 × 7.5 mm). Each antigen
was printed in two replicates within a matrix. Human IgG
(Sigma, St. Louis, MO) was used as a positive control, and
purified glutathione-S-transferase (GST) protein and Nus pro-
tein were used as negative controls in microarrays. Protein
microarrays were blocked in PBS containing 25% FCS and
0.05% Tween 20 for 3 h at 37 °C and probed with 1:10 dilutions
of HCC serum or negative control serum for 30 min, followed
by washing 5 times for 10 s each in PBS with 0.1% Tween 20
and incubation with a 1:400 dilution of Cy3-conjugated mouse
antihuman IgG secondary antibody (Sigma, St. Louis, MO).
Arrays were scanned using the GenePix 4000 scanner (Axon,
Foster City, CA), and the signal intensities of the spots and
background values were determined using GenePix Pro version
5.1 (Axon, Foster City, CA).
The technical steps of the WB/microassay in the blinded
validation phase were performed by individuals who did not
know the diagnoses of patients and who had not been directly
involved in the initial SERPA.
Statistical Analysis. ?2tests were used to determine the
difference of positive reactions to HCC-associated antigens
between different classes and the correlation between antigens
and the clinicopathological parameters. A value of p < 0.05 was
considered statistically significant.
Proteomic Analysis of Autoantigens in Patients with
HCC. We separated total proteins extracted from the two cell
lines (HepG2 and HepG2.2.15) by 2DE and transferred them
onto PVDF membranes. Initially, mixed serum samples from
eight HBV-related HCC patients were incubated with HepG2
proteins. Normal sera were used as controls. Then, mixed
serum samples from 10 HBV-related HCC patients and 10 virus-
negative HCC patients were separately reacted with HepG2.2.15
proteins. We detected six antigenic protein spots of HepG2 cells
that reacted to HCC sera but did not reacted to normal controls.
In the case of HepG2.2.15, we detected 10 antigenic protein
spots that reacted to HCC sera but not to normal controls.
Among the 10 proteins, 5 reacted to sera from all HCC patients
with or without HBV infection, 2 reacted only to sera without
HBV infection, and 3 reacted only to sera with HBV infection,
reflecting a difference between the prevalence of autoantibod-
ies in HBV-related HCC and virus-negative HCC.
Identification of the Autoantigens. We tried to identify the
proteins detected by SERPA. The proteins of interest were
excised from stained Colloidal Coomassie Brilliant G-250 gels
and submitted to digest with trypsin. The peptide mixtures were
analyzed by MALDI-TOF MS. The resulting peptide mass maps
were used for protein database searches using Mascot software.
Six protein spots from the HepG2 cell were identified as four
distinct proteins, including keratin 8, lamin A/C, DEAD (Asp-
Glu-Ala-Asp) box polypeptide 3, and eukaryotic translation
elongation factor 2 (eEF2). Of the 10 protein spots from the
HepG2.2.15 cell, 2 reactive proteins with autoantibodies in
virus-negative HCC sera were identified as triosephosphate
isomerase (TIM) and prostatic binding protein, 3 reactive
proteins with autoantibodies in HBV-related HCC sera were
identified as pyruvate kinase, phosphoglycerate kinase 1, and
phosphoglycerate mutase isozyme B, and the rest, which
reacted to both of the HCC sera, were identified as programmed
cell death 8 (apoptosis-inducing factor, AIF), heterogeneous
nuclear ribonucleoprotein (hnRNP) A2, cyclophilin A, and
aspartate aminotransferase 1. Locations of the identified
protein spots on 2D gels are shown in Figures 1 and 2. The
results of identification are summarized in Table 2.
Analysis of the Prevalence of Autoantibodies against the
Identified Antigens by WB. Antigenicity of the identified
protein was confirmed by WB. Four identified antigens from
the HepG2 cell and three from the HepG2.2.15 cell were
randomly selected for the expression of recombinant proteins.
The protein TIM was purchased from Sigma-Aldrich Company.
Keratin 8 cDNA was subcloned into pGEX-4T-1 vector, produc-
ing a fusion protein with a GST tag. cDNA from lamin A/C,
eEF2, and prostatic binding protein was subcloned into
pET28a(+) vector, respectively, and recombinant proteins were
expressed as fusion proteins with His tags. cDNA from DEAD
box polypeptide 3, hnRNP A2, and AIF was separately sub-
cloned into pET43.1a(+) vector for protein expression as fusion
proteins with Nus and His tags. The purified fusion proteins
were separated on 10% SDS-PAGE and stained with Coomassie
HCC-Associated Antigens and Autoantibodies by SERPA
Journal of Proteome Research • Vol. 7, No. 2, 2008
Blue. The purity of the recombinant proteins was over 90%,
which was evaluated with the software Bandscan.
Using the eight proteins as antigens, sera from 28 HCC
patients and 10 normal controls were screened individually by
Figure 1. Two-dimensional electrophoresis map of proteins from the HepG2 cell line and the corresponding immunoblotting maps.
The proteins from the hepatoma cell line HepG2 were separated by IEF (pH 3–10) and then 12.5% SDS-PAGE and subsequently stained
with Colloidal Coomassie Brilliant Blue (a) or transferred on PVDF membranes for WB experiments using mixed sera from eight patients
with HCC (b) or from eight healthy subjects (c) as a first antibody. The position of the six identified reactive protein spots are labeled
on the 2D patterns.
Figure 2. Two-dimensional electrophoresis map of proteins from the HepG2.2.15 cell line and the corresponding immunoblotting maps.
The proteins from the hepatoma cell line HepG2.2.15 were separated by IEF (pH 3–10) and then 12.5% SDS-PAGE and subsequently
stained with Colloidal Coomassie Brilliant Blue (a) or transferred on PVDF membranes for WB experiments using mixed sera from 10
HBV-related HCC patients (b), from 10 virus-negative HCC patients (c), or from 10 healthy subjects (d) as a first antibody. The position
of the 10 identified reactive protein spots are labeled on the 2D patterns.
Li et al.
614Journal of Proteome Research • Vol. 7, No. 2, 2008