April 01, 2014
S100A8 as potential salivary biomarker of oral squamous cell carcinoma using
nanoLC-MS/MS
Yu-Jen Jou1,2
Su-Hua Huang5
Chun-Hung Hua3
Ming-Hsui Tsai3
Chia-Der Lin3
Jung-Yie Kao2,¶
Chih-Ho Lai4
Cheng-Wen Lin1,5*
1
Department of Medical Laboratory Science and Biotechnology, China Medical
University, Taichung, Taiwan
2 Department of biochemistry, College of life sciences, National Chung Hsing
University, Taichung, Taiwan
3
Department of Otolaryngology, China Medical University Hospital, Taichung,
Taiwan
4
Department of Microbiology, School of Medicine, China Medical University,
Taichung, Taiwan
5
Department of Biotechnology, College of Health Science, Asia University, Wufeng,
Taichung, Taiwan
Running title: Salivary S100A8 as an oral cancer biomarker
¶
Co-Corresponding author
*Corresponding author: Cheng-Wen Lin, PhD, Professor. Department of Medical
Laboratory Science and Biotechnology, China Medical University, No. 91,
Hsueh-Shih Road, Taichung 404, Taiwan
TEL : 886-4-22053366, ext. 7210
Fax: 886-4-22057414
Email: cwlin@mail.cmu.edu.tw
1
Abstract
Background: Oral squamous cell carcinoma (OSCC) shows low five-year survival;
early treatment greatly reduces mortality and morbidity. Saliva is a non-invasively
sample, with good potential to discover biomarkers for early detection.
Methods: NanoLC-MS/MS served to analyze saliva proteome from control subjects
(n=35) and OSCC patients T1 (n=29), T2 (n=36), T3 (n=14) and T4 (n=21) stages.
Identified biomarkers were verified by Western blot and ELISA assays.
Results: NanoLC-MS/MS analysis of salivary proteins between 10-15 kDa identified
S100A8, hemoglobin delta and gamma-G globin in T3 and T4 stage OSCC as well as
S100A7 in T1 and T2 stage OSCC. Western blot and ELISA indicated positive
correlation between salivary S100A8 increment and tumor size stage. High level of
S100A8 appeared in 3.4, 13.9, 92.9, and 100% of saliva OSCC patients with T1, T2,
T3, and T4 stages, respectively. Significant increase of salivary S100A7 was observed
in 20.7% and 11.1% of those with T1 and T2, respectively. AUROC curve indicated
high sensitivity, specificity and accuracy of S100A8-based ELISA as a detector.
Conclusions: NanoLC-MS/MS, Western blot and ELISA manifested salivary S100A8
as a specific and sensitive marker for detection of OSCC patients. Salivary S100A8
protein could be applicable in developing OSCC diagnostics.
Keywords: oral squamous cell carcinoma, saliva, LC-MS/MS, S100A8, biomarker
2
Abbreviations: OSCC, oral squamous cell carcinoma; ELISA, Enzyme-Liked
Immunosorbent Assay; NanoLC-MS/MS, Automated nano liquid chromatography
tandem mass spectrometry; AUROC, area under Receiver-Operating Characteristic.
3
Introduction
Oral squamous cell carcinoma (OSCC) is predominant in oral cancer, with more
than 300,000 cases annually worldwide that account for 3% of malignancies in men
and 2% in women [1-3]. Tobacco, alcohol, and betel quid are common risk factors for
causing large areas of mucosal change, synergistically triggering oral carcinogenesis,
even developing a secondary upper aerodigestive tract cancer [4-9]. Histological and
clinical data indicate multi-step changes: leukoplakia, erythroplakia, hyperkeratosis,
dysplasia, even carcinoma [10]. Despite advances in surgery, radiotherapy, and
chemotherapy, overall average five-year survival rate for patients has not improved
significantly (still approximately 50%) over the past 30 years, far lower than that of
laryngeal or nasopharyngeal carcinoma [11, 12]. Appropriate treatment for those with
pre-malignant oral lesions proves more effective, significantly raising the survival rate
to 80-90% [12]. Consequently, early diagnosis of oral cancer, distinguishing between
malignant or premalignant lesions, is crucial to reduce the mortality and morbidity.
Biopsy of suspicious lesions offers the gold specimens for discovery of molecular
biomarkers, but non-uniform appearance of cancerous and precancerous lesions
allows the difficulty in choosing the location of biopsy, affecting the accuracy of
potential OSCC markers [13, 14]. Developing credible, accurate, cost-effective, and
noninvasive techniques for early detection is essential.
4
S100 A1-14 and B, a group of small acidic proteins, contain EF-hand
calcium-binding motifs [15], modulating multiple biological properties in distinct
cell- and tissue-types via binding with Ca2+, Zn2+, and Cu2+. S100 proteins involve in
calcium homeostasis and cytoskeletal dynamics, as well as regulate cell proliferation
and transcriptional factor activity [16]. S100A4 protein regulates myosin dynamics by
inhibiting protein kinase C (PKC)-mediated phosphorylation on C-terminus of myosin
heavy chain [17, 18]. Secreted S100A4 is a candidate maker predicting metastatic and
prognostic potential in breast cancer [19]. S100A7 is up-regulated in inflammatory
epidermis, correlating with epithelial malignancies: e.g., breast, skin, esophagus, head
and neck [20]. S100A8 and S100A9 can be synthesized and secreted by granulocytes,
monocytes, and macrophages, identified as cytokine-like and transcriptional
factor-like molecules affecting expression of tumor necrosis factor-α, interleukin-1,
and matrix metalloproteinases [21-24]. Up-regulation of S100A8 and S100A9 is
found in gastric, colorectal, breast, lung, pancreatic, and prostate cancer, correlating
with inflammation cell proliferation and metastatic processes in tumor development
[25-27]. S100B also inhibits PKC-mediated phosphorylation on p53, reducing tumor
suppressor activity by suppressing p53-dependent transcription activation [28, 29].
Altered expression of S100 proteins is thereby associated with cancer development;
secreted form of S100 proteins could act as potential cancer markers.
5
Tissue microarray indicated significant up-expression of S100A8 in severe oral
dysplasias and OSCC tissue [30, 31]. Proteomic analysis of normal and OSCC tissues
suggested S100A7 as a positive marker for OSCC carcinogenesis and early tumor
progression that confirmed by immunofluorescence and quantitative RT-PCR analyses
[32]. Sharp decrease of S100A4 mRNA was evident in OSCC tissues [33]. Earlier we
identified S100A8, transferrin, and zinc finger protein 497 as salivary biomarkers,
using two-dimensional gel electrophoresis (2DE) and mass spectrometry (MS) [30].
Salivary samples from such patients showed elevated S100A8, necessary for further
probe or correlation with oral cancer tumor grade. This study rated potential of S100
proteins as salivary markers via proteomic analysis of low molecular weight salivary
proteins, using NanoLC-MS/MS. Protein profile indicated change of S100A7 and
S100A8 as unique markers in saliva of oral cancer patients. Western blot and direct
binding ELISA further examined levels of S100A7 and S100A8 in their saliva while
evaluating potency of these markers.
Materials and methods
Human subjects and saliva collection
In all, 35 subjects without and 100 with OSCC were enrolled for study from February
6
2007 to March 2014; OSCC patients were diagnosed via biopsy at China Medical
University Hospital in Taichung. Subjects gave informed consent prior to saliva
collection approved by the Institutional Review Board of China Medical University
Hospital (permission number DMR96-IRB- 80). Exclusion criteria for OSCC patients
and control individuals were followed, as in our prior studies [30, 34]. Table 1 lists
personal information (age, gender, and clinical features) of subjects. Tumor size (T)
and nodal metastasis (N) staging for OSCC patients, as verified by pathological
examination, was reviewed according to the universal TNM staging system of the
International Union against Cancer (UICC) [35]. Collection protocol of salivary
samples from patients and controls were performed as in prior studies [30, 34]: 5 ml
collected in 15-ml centrifuge tube, mixed 5 ul CompleteTM Protease inhibitor Cocktail
(Cat. No. 1697498, Roche), then centrifuged at 12,000 rpm (~13,400 x g) for 10 min
at 4℃. Resulting supernatants were stored at -80℃.
SDS-PAGE and Western blot
Preparation of each 500 μl sample for Western blot was performed according to
protocol of the 2-D clean up kit (Amersham). Salivary proteins were precipitated by a
combination of precipitant and co-precipitant, then rehydrated in 100 ul of rehydration
buffer (8M urea, 4% CHAPS, 0.002% bromophenol blue). Afterward, 5μg of salivary
proteins from each sample was mixed with sample buffer, heated at 100℃ for 8 min,
7
then loaded onto 12% SDS-PAGE gels stained with Coomassie blue after running. For
Western blot, dissolved proteins in gels were transferred to nitrocellulose membranes.
Resultant nitrocellulose membrane was blocked with 5% skim milk in Tris buffered
saline (TBS) buffer containing 0.1% Tween 20 (TBST) at 4℃ for 2 hours, reacted
with primary monoclonal antibodies anti-S100A7 and -S100A8 (Abnova) overnight,
then incubated with HRP-conjugated anti-mouse IgG antibodies (Invitrogen). Also,
human sIgA as control was detected in salivary samples. Immune-reactive bands of
interest were detected by ECLTM Western Blotting Detection Reagents (Amersham).
In-gel digestion of low molecular weight salivary proteins
Since S100 were low molecular weight proteins, salivary protein bands between
approximate 10-15 kDa solved by 12% SDS-PAGE were excised, washed twice with
buffer (25mM ammonia bicarbonate (ABC) in 50% acetonitrile (ACN)) for 5 min and
100% ACN for 5 min, then dried by speed vacuum concentrator. Proteins embedded
in gels were reduced by reduction buffer (10 mM dithiothreitol (DTT), 25mM ABC)
at 56℃ for 15 min, alkylated by solution (55 mM iodoacetamide (IAA), 25mM ABC)
at room temperature for 20 min in the dark, and digested with fresh trypsin solution (2
ng/μl trypsin, 25 mM ABC) at 37℃ overnight. Peptides were extracted by sonication
with 50-100% ACN and 0.1% formic acid (FA); released peptides in supernatants
were dried by speed vacuum concentrator.
8
NanoLC-MS/MS analysis of digested salivary proteins
Eluted peptides were subjected to NanoLC-MS/MS analysis, an integrated
system (QSTAR XL) comprising LC Packings NanoLC system with autosampler and
QSTAR XL Q-Tof mass spectrometer (AB Sciex) fitted with nano-LC sprayer. Eluted
samples were desalted on a LC-Packings PepMap™ C18 μ-Precolumn™ Cartidge (5
μm, 30 μm I.D. x 5mm; Dionex, Sunnyvale, CA), separated on an LC-Packings
PepMap C18 column (3μm, 15 cm x 75 μm i.D.,) at 200 nl/min, using 45 min gradient
of 5-60% ACN in 0.1% FA and analyzed by connecting inline to a mass spectrometer.
NanoESI-MS and CID MS/MS sequencing of peptides were fully automated,
synchronized with nanoLC runs under AnalystQS software. To identify proteins, 1 s
survey scans were set at mass range m/z 400-1600 and 6 s MS/MS spectral
acquisitions of multiple-charged precursor ions were detected at intensity above
predefined threshold. Acquired individual MS/MS spectra within a single LC run
were combined as a single Mascot-searchable peak list file. Peak list files served to
query Swiss-Prot database via Mascot program with these parameters: peptide mass
tolerance of 150 ppm, MS/MS ion mass tolerance of 0.15 Da, and allowing up to one
missed cleavage. Minimum score above 20 was randomly set as acceptance threshold.
Relative quantitative analysis of salivary markers by ELISA
9
Each sample containing 5 μg/ml of protein was quadruplicate pro-coated in
96-well plates at 4℃ overnight, unbound substrates washed out with TBST thrice.
After blocking with 5% skim milk in TBST, relative amount of salivary markers was
detected by monoclonal antibodies anti-S100A7 and anti-S100A8. Anti-mouse IgG
conjugated HRP was added into each well for 2-h incubation at room temperature,
immune-reactive complexes measured with ABTS/H2O substrates. Optical absorbance
was recorded at 405 nm by ELISA plate reader (ELX 808, BioTek, Winooski, USA).
Immunohistochemical staining of S100A7 and S100A8 in OSCC tissues
Tissue sections from control, T1, and T3 cases underwent immunohistochemical
staining. Sections were deparaffined and rehydrated. After blocking with 1% normal
goat serum, diluted monoclonal antibodies anti-S100A7 or anti-S100A8 were added
to tissue sections and incubated for 1 hour, followed by HRP-conjugated anti-mouse
IgG antibodies and then 3,3’-diaminobenzidine (Sigma-Aldrich) as substrate.
Results
Clinical parameters of OSCC patients and control subjects
A total of 35 controls and 100 OSCC cases were recruited, the latter grouped as
T1, T2, T3 and T4, based on tumor size stage of UICC TNM staging system (Table 1).
10
Males formed a majority in all groups; mean age of OSCC cases was over 50 years,
with controls (50.2 years) slightly younger. Among OSCC cases, tongue and buccal
sites showed most frequent oral cancer lesions; nearly half were N0 stage without
tumor cells from regional lymph nodes, 13% N1 and 29% N2 stage. Histologic
examination of lesions showed moderately differentiated OSCC in 59% and well
differentiated OSCC in 30% of cases. Table 1 lists other clinical parameters of
patients and controls.
NanoLC-MS/MS analysis of low molecular weight salivary proteins
To examine potential of salivary S100 as OSCC markers, samples from control
subjects and patients were analyzed by 12% SDS-PAGE, protein bands between 10
and 15 kDa excised and identified by NanoLC-MS/MS analysis system (Fig. 1). Table
2 details proteins from in-gel trypsin digestion of low molecular weight salivary
proteins in samples of each group by LC-MS/MS. Figures 1B-D show peptide mass
fingerprinting and peptide sequencing of S100A7, S100A8 and S100A9. Comparison
of protein profile among groups indicated S100A7 as a unique marker of T1 and T2
stages and S100A8 as a potential marker of T3 and T4 stage in OSCC patients (Table
2). S100A9 appeared in salivary samples from control and all patient groups. Western
blot analysis indicated significant rise of S100A8 in salivary samples from T3 and T4
11
groups, S100A7 elevated in T1 but falling dramatically in T3 and T4 stages (Fig. 2).
Relative levels of S100A7 and S100A8 in saliva from OSCC cases and controls
To correlate between S100A7/A8 and OSCC stage, relative levels of S100A7
and S100A8 in saliva of OSCC patients were measured via binding ELISA, compared
to those in saliva of controls (Fig. 3, Supplemental Figs. 1 and 2). Binding ELISA
indicated the significant change for S100A7 and S100A8 in tumor size stage (Fig. 3),
but no meaningful differences in lymph node status and age (Supplemental Figs. 1
and 2). The mean of OD405 nm indicated S100A8 significantly higher in saliva of
patients with T2-4 stages compared to T1 and control subjects (Fig. 3B). By contrast,
salivary S100A7 peaked at T1 stage and reached its nadir in T3 and T4 stages (Fig.
3A). When cutoff OD index of 0.3 was recommended, positive cases of S100A8 were
1 (2.9%) in controls, 1 (3.4%) in T1, 5 (13.9%) in T2, 13 (92.9%) in T3, and 21
(100%) in T4 (Table 3). Meanwhile, S100A7-positive cases tallied 6 (20.7%) in T1, 4
(11.1%) in T2, but negative in other groups. Combination of the positivity from either
one or both markers significantly improved the sensitivity in T1 and T2 stages.
ROC curve analysis of S100A8 as the potential salivary marker of OSCC
To rate sensitivity and specificity of S100A8 for OSCC detection, area under
receiver-operating characteristic curves (AUROC) discriminates between OSCC-free
12
subjects and OSCC cases at each stage, using ELISA (Fig. 4). AUROC of S100A7 for
predicting OSCC was 0.71 for T1 (95% CI: 0.88-1.04), and 0.68 for T2 (95% CI:
0.89-1.01) (Fig. 4A), that of salivary S100A8 0.99 for T3 (95% CI: 0.58-1.00), and
0.98 for T4 (95% CI: 0.63-1.06), respectively (Fig. 4B). This indicated S100A8-based
ELISA as highly accurate in detecting OSCC at T3 and T4 stages.
Overexpression of S100A8 in OSCC tissues
Immunohistochemical staining further validated levels of S100A7 and S100A8
in oral tissues from OSCC and control groups (Fig. 5). Up-regulation of S100A7 was
observed in OSCC tissues from T1, but not control and T3 groups. By contrast, strong
positive reaction for S100A8 was observed in tissues from patients, not controls.
Results indicated S100A8 moderately expressing in T1 and overexpressing in T3
stage tissue, in agreement with Western blot and ELISA analyses of saliva from
OSCC patients and controls.
Discussion
This study was the first report that NanoLC-MS/MS utilized to identify salivary
protein filing of OSCC patients and controls. NanoLC-MS/MS served as powerful
tools to discover potential serum, urine, and tissue biomarkers for colon, lung, breast,
13
colorectal, and gastric cancers [36-39]. In the present study, nanoLC-MS/MS analysis
of salivary proteins between 10-15 kDa identified S100A8 as a potential salivary
biomarker for oral cancer (Fig. 1 and Table 2), in accordance with our prior report:
up-regulation of S100A8 in OSCC patients’ saliva identified by two-dimensional gel
electrophoresis (2DE) and matrix-assisted laser desorption/ionization time-of-flight
mass spectrometry (MALDI-TOF MS) analyses [30]. Western blot showed salivary
S100A8 significantly elevated in patients compared to controls (Fig. 2). Further
ELISA evaluation indicated mean amount of S100A8 in saliva of OSCC patients as
higher than in healthy controls (Fig. 3), while AUROC of S100A8-based ELISA for
diagnosis of OSCC revealed that salivary S100A8 serves as OSCC biomarker (Fig. 4).
Results indicated high sensitivity, specificity, and accuracy of S100A8 in detecting
OSCC. In healthy control group, one case (2.9%) with high level of salivary S100A8
was followed up and diagnosed as OSCC one year post sampling. Therefore, S100A8
proves valuable as the target of OSCC diagnostics.
Comparison of salivary protein profiling between OSCC and control indicated
increment of S100A8 and hemoglobin delta in T3 and T4 as well as that of S100A7 in
T1 and T2 groups. S100A8, a calcium-binding and pro-inflammatory protein, was
overexpressed in prostate, breast, lung, gastric, pancreatic and colorectal cancers [40],
hence suggested as one critical tissue marker for aggressive breast cancer phenotype
14
[41], discriminatory ovarian cyst fluid marker for ovarian tumors [42], and serological
marker for colorectal and gastric cancers [43, 44]. In this study, quantity of S100A8 in
salivary samples was validated by direct binding ELISA (Fig. 3). NanoLC-MS/MS
and ELISA analyses revealed positive correlation between tumor size stage of OSCC
and salivary S100A8 protein level. Comparison of salivary S100A8 with the reported
candidate marker transferrin [30] indicated S100A8 had higher specificity but lower
sensitivity in detecting T1 and T2 stage OSCC than transferrin. Salivary transferrin
elevated in patients with parotid mixed tumor, alcoholic liver cirrhosis, nasal
polyposis and bronchial asthma [30]. The combination of salivary S100A8 and
transferrin could rule out the false positive and confirm the diagnosis of OSCC.
Meanwhile, AUROC curve indicated specificity, sensitivity, and accuracy of salivary
S100A8 in detecting oral cancer were similar to those of reported candidate OSCC
markers [13, 14]. Immunohistochemical staining also demonstrated S100A8
moderately up-regulated in OSCC tissues from T1 and overexpressed in T3 stage (Fig.
5). Immunohistochemical staining implied increment of salivary S100A8 derived
from secreted proteins of OSCC tissues in OSCC patients.
Previous reports demonstrated S100A7 overexpressing in tissues of oral lesions
[45] as well as regulating invasion and growth of breast and prostate cancers [46, 47].
Our study showed low increment of salivary S100A7 in OSCC cases at T1 stage as
15
well as low expression of it in T1 stage tissues (Figs. 3 and 5). Also, hemoglobin delta
and gamma globin were extracted from T3- and/or T4-stage patients’ saliva (Table 2).
Embryonic and fetal hemoglobin were expressed in human glioblastoma multiform
cells [48]. The role of S100A7, hemoglobin delta, and gamma globin in OSCC
diagnosis merits further examination. Salivary biomarkers have great potential for
detection and surveillance of OSCC progression or recurrence. This study explored
profiling of low-weight salivary proteins between 10-15 kDa, using nanoLC-MS/MS;
elucidating whole saliva proteome can identify potential biomarkers for detection or
progression of OSCC as well as pathophysiology.
In sum, nanoLC-MS/MS analysis of saliva from OSCC patients and controls
identified S100A8 as a potential salivary biomarker for the diagnosis of human oral
cancer in humans. Salivary protein level of S100A8 in OSCC patients and controls
was confirmed by Western blot and ELISA. In addition, salivary S100A8 levels
strongly correlated with tumor size stage. AUROC curve exhibited high specificity,
sensitivity, and accuracy of S100A8-based ELISA for detecting oral cancer.
Acknowledgments
This work was supported by the National Science Council of Taiwan (NSC1012320-B-039-036-MY3) and China Medical University (CMU101-ASIA-05 and
16
CMU101-S-24).
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Table and Figure captions
Table 1. Clinicopathological features of controls oral cancer patients
Table 2. NanoLC-MS/MS identification of low molecular weight salivary proteins
in samples from controls and OSCC patients at T1, T2, T3 and T4 stages.
Table 3. Number of positive ELLISA reaction of S100A7 and S100A8 in salivary
samples from controls and OSCC patients with T1, T2, T3 and T4 stages
Figure 1. Peptide mass fingerprinting and peptide sequencing of salivary protein
using nanoLC-MS/MS. Salivary samples from controls and OSCC patients
were dissolved in 12% SDS PAGE (A), protein bands between 10-15 kDa
subjected to nanoLC-MS/MS for identification. Representative LC-MS/MS
spectra of S100A7 (B), S100A8 (C), and S100A9 (D) are shown with the
calculated molecular weight (m/z values) along x-axis and relative intensity
along y-axis. Amino acid sequence (Upper panel) was determined from mass
differences in y- and b-fragment ions series.
Fig. 2. Western blot analysis of S100A7 and S100A8 in saliva from controls and
OSCC patients. Salivary samples from each group were analyzed by 12%
SDS-PAGE, then electrophoretically transferred onto nitrocellulose membrane
probed with monoclonal antibodies to S100A7, S100A8 and sIgA, and
developed with HRP-conjugated secondary antibody and chemiluminescent
26
HRP substrates. Lane 1: control group; Lane 2: T1 stage; Lane 3: T2 stage;
Lane 4: T3 stage; Lane 5: T4 stage.
Fig. 3. Direct binding ELISA to salivary S100A7 and S100A8 proteins. 2.5 μg of
salivary protein from each individual of control, T1, T2, T3, and T4 groups
were pre-coated onto microtiter plates, then incubated with 2000-fold dilution
of monoclonal antibodies anti-S100A7 or anti-S100A8 at room temperature for
1 h. Immune complexes were analyzed by ELISA procedures.
Fig. 4. Receiver-operating characteristic curves of salivary biomarkers for
predicting tumor size stages. Salivary samples were analyzed via direct
binding ELISA of S100A7 (A) and S100A8 (B).
Fig. 5. Immunohistochemical analysis of S100A7 and S100A8 expression in
OSCC. Tissue sections from healthy controls and OSCC patients with T1 or T3
stage, analyzed by immunohistochemical staining with monoclonal antibodies
anti-S100A7 and anti-S100A8. Immunoreactivity complex was developed by
HRP-conjugated anti-mouse IgG antibodies and 3,3’-diaminobenzidine as
substrate, figures photographed under ×100 magnification.
27
Table 1
Controls
(35)
OSCC Patients (100)
T1 (29)
T2 (36)
T3 (14)
T4 (21)
27
26
8
3
50.5 ± 13.6 51.2 ± 12.7
36
0
52.9 ± 11.0
13
1
56.9 ± 10.3
21
0
52.1 ± 11.1
Male
50.2 ± 14.5
50.8 ± 13.1
52.9 ± 11.0
56.9 ± 10.7
52.1 ± 11.1
Female
49.6 ± 10.3
54.7 ± 10.8
Gender
Male
Female
Mean age (y)
57.0
Cancer sites
Oral cavity
Mouth
Oropharynx
Hypopharynx
Laryngeal
27
1
1
0
0
30
5
0
1
0
11
0
0
2
1
16
0
0
4
1
Node stage
N0
N1
N2
25
1
3
17
8
11
8
2
4
8
2
11
Histology*
WD
MD
PD
Keratinizing
10
16
2
1
7
25
4
0
4
9
1
0
9
9
3
0
* WD, Well-differentiated; MD, Moderately differentiated; PD, Poorly-differentiated
28
Table 2
Control
accession
no.
protein name
mol.
weight*
Score
T1
T3
T4
Peptides Score Peptides Score Peptides Score Peptides Score Peptides
matched
matched
matched
matched
matched
cystatin SA-III=potential precursor
14181
87
5
of acquired enamel pellicle
beta-globin
15870
80
3
104
prolactin-inducible protein precursor 16562
72
7
133
protein S100-A9
13234
71
12
145
S100-A7 (psoriasin)
11450
165
hemoglobin delta
15897
gamma-G globin
16959
S100-A8 (oncodevelopmental
13086
protein)
*mol. weight refers to molecular weight given in database, as inferred from gene sequence.
gi|235948
gi|183817
gi|4505821
gi|4506773
gi|190668
gi|229172
gi|31725
gi|957232
T2
3
5
8
8
162
6
112
249
76
6
9
3
65
50
7
9
123
58
57
5
3
2
93
103
171
134
4
7
5
5
197
7
110
5
29
Table 3
OSCC Patients (100)
S100 family*
Controls
(35)
T1 (29)
T2 (36)
T3 (14)
T4 (21)
S100A7
S100A8
S100A7/S100A8＃
0
1†(2.9%)
1(2.9%)
6(20.7%)
1 (3.4%)
7(24.1%)
4(11.1%)
5 (13.9%)
9(25%)
0
13 (92.9%)
13(92.9%)
0
21 (100.0%)
21(100.0%)
*Cut-off value set at OD 405nm of 0.3.
†Case diagnosed as OSCC patient 1 year post sampling.
＃
A positive from either one or both markers.
30