Characterization of the Human Cervical Mucous Proteome.

Abstract

INTRODUCTION: Cervical cancer is among the most common cancers in women worldwide. Discovery of biomarkers for the early detection of cervical cancer would improve current screening practices and reduce the burden of disease. OBJECTIVE: In this study, we report characterization of the human cervical mucous proteome as the first step towards protein biomarker discovery. METHODS: The protein composition was characterized using one- and two-dimensional gel electrophoresis, and liquid chromatography coupled with mass spectrometry. We chose to use this combination of traditional biochemical techniques and proteomics to allow a more comprehensive analysis. RESULTS AND CONCLUSION: A total of 107 unique proteins were identified, with plasma proteins being most abundant. These proteins represented the major functional categories of metabolism, immune response, and cellular transport. Removal of high molecular weight abundant proteins by immunoaffinity purification did not significantly increase the number of protein spots resolved. We also analyzed phosphorylated and glycosylated proteins by fluorescent post-staining procedures. The profiling of cervical mucous proteins and their post-translational modifications can be used to further our understanding of the cervical mucous proteome.

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Characterization of the Human Cervical Mucous Proteome
Gitika Panicker & Yiming Ye & Dongxia Wang &
Elizabeth R. Unger
Published online: 9 March 2010
# The Author(s) 2010. This article is published with open access at Springerlink.com
Abstract
Introduction Cervical cancer is among the most common
cancers in women worldwide. Discovery of biomarkers for
the early detection of cervical cancer would improve current
screening practices and reduce the burden of disease.
Objective In this study, we report characterization of the
human cervical mucous proteome as the first step towards
protein biomarker discovery.
Methods The protein composition was characterized using
one- and two-dimensional gel electrophoresis, and liquid
chromatography coupled with mass spectrometry. We chose
to use this combination of traditional biochemical techniques
and proteomics to allow a more comprehensive analysis.
Results and Conclusion A total of 107 unique proteins
were identified, with plasma proteins being most abundant.
These proteins represented the major functional categories
of metabolism, immune response, and cellular transport.
Removal of high molecular weight abundant proteins by
immunoaffinity purification did not significantly increase the
number of protein spots resolved. We also analyzed phos-
phorylated and glycosylated proteins by fluorescent post-
stainingprocedures.The profilingofcervicalmucousproteins
and their post-translational modifications can be used to
further our understanding of the cervical mucous proteome.
Keywords Cervical mucous.Cervical cancer biomarkers.
Mass spectrometry.2-D electrophoresis.LC/MS/MS
Introduction
Advances in proteomic technologies have greatly accelerated
the field of protein biomarker discovery. High-throughput
technologies like surface-enhanced laser desorption and
ionization-time of flight mass spectrometry (SELDI-TOF
MS) or the combination of one- or two-dimensional gel
electrophoresis (1-DE, 2-DE) methods with multidimensional
chromatographic separation and MS provide a snapshotof the
proteome. Validated protein biomarkers could be useful in
early detection of disease, monitoring disease progression or
monitoring response to treatment. Several studies have thus
focused on biomarker discovery in body fluids which would
be advantageous for eventual clinical implementation.
Cervical mucous is potentially an ideal sample to screen
for biomarkers for early detection of cervical cancer. As
cervical mucous is produced in the microenvironment
where cervical neoplasia arises, it is likely to include
proteins produced by the lesion as well as by the host in
response to the lesion.
Cervical mucous is a complex mixture of proteins
consisting of an aqueous and glycoprotein phase [1], but
is thought to have a restricted protein profile compared to
other body fluids like serum or plasma. It is recognized that
The findings and conclusions in this report are those of the author(s)
and do not necessarily represent the views of the funding agency.
Gitika Panicker, Yiming Ye, and Dongxia Wang contributed equally to
this paper.
G. Panicker:E. R. Unger (*)
Division of Viral and Rickettsial Diseases,
National Center for Zoonotic, Vector-Borne, & Enteric Diseases,
Centers for Disease Control and Prevention,
1600 Clifton Road, Mail Stop G-41, Atlanta, GA 30333, USA
e-mail: eru0@cdc.gov
Y. Ye:D. Wang
Division of Scientific Resources,
National Center for Preparedness, Detection,
& Control of Infectious Diseases,
Centers for Disease Control and Prevention,
1600 Clifton Road, Mail Stop G-36, Atlanta, GA 30333, USA
Clin Proteom (2010) 6:18–28
DOI 10.1007/s12014-010-9042-3

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the cervix is hormonally responsive and differences in
protein composition and concentration due to menopause,
menstrual cycle, and hormonal contraceptives will impact
sample to sample comparison. CVF includes, in addition to
cervical mucous, contributions from vulvar secretions,
vaginal wall transudate, exfoliated cells, endometrial and
oviductal fluids, and metabolic products of vaginal micro-
flora [2]. Thus, proteomic characterization of CVF is suited
for exploring markers of genital tract infections or pregnancy
status or overall evaluation of reproductive tract. However,
protein contributions from multiple sites could significantly
dilute the cervical contribution that would be most represen-
tative of cervical neoplasia localized to the cervix.
To date, there have been six studies of CVF [3–8], four
of these were restricted to pregnant women [3–6]. These
studies identified from 15 to 685 proteins using gel
separation/MALDI-TOF MS ([4, 8]; 15 and 59 proteins
respectively), LC-MS/MS ([5]; 39 proteins), gel separation
and LC-MS/MS ([7]; 685 proteins), and gel separation and
LC-LC-MS/MS ([3, 6]; 150 and 205 proteins respectively).
Consistently, many CVF proteins were noted to be of
plasma origin, suggesting CVF could largely be plasma
transudate. Other major functional categories were repre-
sented by immune response, metabolism, and cellular
transport. Only one study examined cervical mucous [9],
and it focused on samples from normal women obtained
during various points of the menstrual cycle. Using gel
separation/LC-MS/MS they identified 194 proteins and
again identified plasma proteins. Cervical mucous and
CVF would be expected to share similarities in their
proteome, but for reasons stated above, the cervical mucous
is expected be more representative of cervical disease and
the local host response to cervical disease.
In our study, the cervical mucous proteome was
characterized by 2-DE and gel-based liquid chromatogra-
phy–mass spectrometry (GeLC-MS/MS) techniques. Of the
current proteomic tools, no single method can resolve an
entire proteome. Combination of several pre-fractionation
methods like gel separation and LC prior to mass
spectrometry allowed for visualization of the proteins as
well as the identification of proteins that could otherwise be
missed by a single method [10]. Also, characterization of
sub-proteomes like the glycoproteome or phosphoproteome
and immunoaffinity depletion of high abundant proteins
was attempted in order to reduce complexity and facilitate
better resolution of proteins in cervical mucous.
Materials and Methods
Sample Collection and Processing Women attending urban
colposcopy clinics were enrolled as part of an ongoing study
of cervical neoplasia [11]. Cervical mucous samples were
collected at the time of colposcopy by absorption into two
Weck-Cel® sponges (Xomed Surgical Products, Jacksonville,
FL) placed, one at a time, into the cervical os. Samples were
stored at −80°C until use. We processed 40 Weck-Cel®
sponges (one or two sponges per subject depending on
availability) with no visual blood contamination from 25
subjects who were randomly selected. The median age of the
women in this subset was 27 years (range 18–57); 90% were
black; 52% currently used hormonal contraceptive (oral,
depo-provera, or both); 62% were HPV positive and 52%
had pre-invasive cervical disease. No data was available on
the menstrual cycle at the time of sample collection.
Total protein was extracted using M-PER® extraction
reagent (M-PER, 0.15 M NaCl) (Pierce Biotechnology,
Inc., Rockford, IL) as previously established [12]. All
samples were combined and the pooled sample was
aliquoted for storage at −80°C. A pooled sample was used
in order to have sufficient volume that would allow for
optimization of the 2-DE and GeLC-MS/MS methods
described below. Total protein content was measured using
the Coomasie PlusTMkit (Pierce Biotechnology Inc.) as per
the manufacturer’s protocol.
Depletion of Albumin and IgG Immunodepletion of the
pooled mucous sample was carried out using IgY-C12 spin
column (GenWay Biotech. Inc., San Diego, CA) following
manufacturer’s protocol. In brief, an aliquot of protein
extract (∼200 µg) was first mixed with 1× Tris-buffered saline
(TBS, 10 mM Tris, 150 mM NaCl, pH 7.4) at 1:1 ratio. The
mixture was added to IgY-C12 spin column and incubated at
room temperature with shaking for 20 min. The unbound
proteins were eluted and the column washed three times with
1× TBS buffer. The eluates from all the above steps were
combined and dried down using a lyophilizer.
Two-Dimensional Gel Electrophoresis For each gel, 80 µg
of protein extract was prepared with the 2-D Clean Up Kit
(GE Healthcare) to remove interfering components. All
reagents were purchased from Sigma-Aldrich (St. Louis,
MO) unless otherwise specified. The samples (with or
without depletion) were mixed in a rehydration buffer (7 M
urea, 2 M thiourea, 2% CHAPS, 50 mM DTT, 1%
Pharmalyte) and incubated for 12 h followed by focusing
on an 11-cm immobilized pH gradient (IPG) strip (pH 3-11
NL, GE Healthcare) in a PROTEAN IEF cell (Bio-Rad,
Hercules, CA). Focusing was performed under the condi-
tion: 500 V constant for 500 V h; linear gradient to 1,000 V
for 800 V h; gradient to 6,000 V for 7,000 V h; gradient to
6,000 V for 3,700 V h. After the first-dimensional
isoelectric focusing, IPG strips containing proteins were
treated for 15 min with gentle shaking in the equilibration
buffer (6 M urea, 30% glycerol, 2% SDS, 50 mM Tris, pH
8.8) containing 2.5% tributyl phosphine (TBP) for protein
Clin Proteom (2010) 6:18–2819

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reduction followed by protein alkylation with 3% iodoace-
tamide (IAA) to replace TBP in the buffer for another
15 min. The second-dimensional SDS-PAGE was run on an
8–16% linear gradient criterion Tris–HCl gel (Bio-rad) at
110 V for 2 h. The gels were stained with colloidal
Coomassie or SYPRO Ruby (Invitrogen, Carlsbad, CA)
dyes as per the manufacturer’s protocol.
One-Dimensional Gel Electrophoresis The sample (80 µg)
was treated with the 2-D Clean Up kit as described above.
The precipitate was solubilized in lysis buffer (1 M Tris–
HCl, 8 M urea, and 4% CHAPS, pH 8.5) and run on an 8–
16% linear gradient criterion Tris–HCl gel at 110 V for 1 h.
After colloidal Coomassie staining, the entire lane was
sliced into 40 pieces of equal size for further analysis.
Gel Staining The 2-DE gel for the characterization of post-
translational modifications on human cervical mucous pro-
teins was stained using three different dyes: Pro-Q Diamond
for phosphoproteins, Pro-Q Emerald 488 for glycoproteins,
and SYPRO Ruby for total proteins (Invitrogen) following
manufacturer’s protocol. In brief, the gels were fixed
overnight at room temperature in 500 ml fixation solution
(50% methanol, 10% acetic acid), followed by two 10-min
washsteps with ultrapure water withgentleagitation.Staining
was performed with 500 ml Pro-Q Diamond with gentle
agitation in the dark for 90 min. The gel was destained by
washing twice with 500 ml destain solution (20% acetonitrile,
50 mM sodium acetate, pH 4.0) with gentle agitation. The gel
imageswere acquiredonTyphoon9400(GEHealthcare)with
532 nm excitation and 560 nm long pass emission. After
image scanning, gels were washed two times in 3% glacial
acetic acid for 20 min each and then incubated in oxidizing
solution (1% periodic acid in 3% acetic acid) for 1 h. The
oxidizing solution was removed by washing three times with
3% glacial acetic acid for 20 min each. The gels were then
incubatedinfreshPro-QEmerald488stainingsolutionfor3h
in the dark. Destaining was done by washing the gel twice in
3% acetic acid at room temperature for 30 min each. Gel
images were acquired by Typhoon 9400 at 510 nm excitation
and 520 nm emission wavelengths. Finally, the gels were
visualized by colloidal Coomassie staining and the gel spots
of interest were excised for further characterization.
In-Gel Trypsin Digestion Sample in-gel digestion and
micro-purification were carried out on a ZipPlate micro-
SPE according to manufacturer’s In-Gel Digestion Protocol
(Millipore, Billerica,MA) withminor modifications. Briefly,
gel spots or bands were destained in buffer 1 (25 mM
ammonium bicarbonate and 5% acetonitrile) for 30 min and
then in buffer 2 (25 mM ammonium bicarbonate and 50%
acetonitrile) for an additional 30 min. Following reduction
with 10 mM dithiothreitol and alkylation with 55 mM IAA,
the gel pieces were incubated in 15 or 30 µL of trypsin
(165 ng) solution and incubated overnight at 37°C. The
peptides were extracted, micro-purified, vacuum-dried and
resuspended in 1% formic acid and 2% acetonitrile for mass
spectrometric analysis.
Mass Spectrometry and Database Analysis Nanocapillary
LC-MS/MS analysis was performed in a Micromass Q-Tof
Ultima mass spectrometer equipped with a nanospray ion
source and coupled with an nanoAcquity ultraperformance
liquid chromatography system (UPLC; Waters, Milford, MA).
The protein digest (2 µl) was loaded onto an in-house packed
reverse phase C18 capillary column (∼15 cm, 75 µm inner
diameter) and separated using a linear gradient of 15% to 45%
of buffer B (acetonitrile, 0.1% formic acid) in buffer A (water,
0.1% formic acid) in 60 min (2-DE gel samples) or 80 min
(1-DE gel samples) at a flow rate of 0.5 µl/min.
All mass spectra were obtained in the positive-ion mode.
An electric potential of 3.5 kV was applied to the emitter in
the ion source. The acquisition of data was performed on a
MassLynx data system (version 4.0; Waters) using a data-
dependent mode where the four most intense precursors in
a survey scan were isolated for collision-induced dissoci-
ation. Resulting MS/MS data were searched for protein
candidates with automated database searching against
NCBI nr database using MASCOT Daemon software
(Matrix Sciences, Boston, MA). The peptide mass
tolerance was set to ±50 ppm, and fragment mass tolerance
was set to ±0.3 Da. Carboxyamidomethyl cysteine and
oxidized methionine were set as variable modifications. All
peptides identified with score less than 50 and one matched
peptide during MASCOTsearches were examined by manual
inspection. Functional categories were annotated by literature
surveys in PubMed or based on classification from the
Database for Annotation, Visualization and Integrated
Discovery (DAVID) [13].
Results and Discussion
Identification of Cervical Mucous Proteins by Proteomic
Techniques Complex protein mixtures can be resolved
effectively according to their isoelectric points and molecular
weights by 2-DE. While gel-based approaches provide good
separation and direct visualization of individual proteins,
highly accurate LC-MS/MS analysis on protein digests
extracted from gel spots leads to high-confidence identifica-
tionofthese proteins.Therefore,the combination ofthese two
techniques becomes a powerful tool in characterizing a
proteome. The proteins extracted from pooled cervical
mucous samples were first separated by 2-DE method
(Fig. 1a). 183 spots were excised and subjected to in-gel
trypsin digestion followed by liquid chromatography coupled
20 Clin Proteom (2010) 6:18–28

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to mass spectrometric analysis. High-confidence protein
identification was accomplished by searching non-
redundant protein databases with tandem mass spectra of
the peptide mixtures. A total of 134 protein spots were
successfully identified that represented 79 unique proteins
(Table 1). Several spots resulted in identical protein
identification, presumably due to the formation of protein
isoforms bearing various modified residues introduced by
post-translational modification or during sample processing.
Applying different sample preparation and fractionation
methods to a single sample could increase the total number of
unique proteins identified. To get a broader proteome
coverage, we analyzed the protein composition using two
other techniques; 2-DE after immunoaffinity-based depletion
ofhighlyabundantproteins,and the combinationof1-DEand
liquid chromatography coupled mass spectrometry (GeLC-
MS/MS). Removal of highly abundant proteins in serum or
other body fluid samples can effectively improve the number
MW
150
100
75
pH 31111
11
69696969

−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
11
6262
6363
64646464
7979
4444
7474
9999 9999

6060
57 57
65 65
99
61 61
7373
83 83
100100
9898
45 45
50
71717171
5959
4646
40404040
6868
101101
102102
9393
8989
4242
393939 39
41414141
4747
6666
6767
8080
8787
8888
9292
123123
127127
131131

37
5858
7070
7272
4343
5656
484848 48
3838
103103
9797
9696
9494
9191
8585
9090
37 37 3737
110110
8282
95 95
124124
125125
126 126

33 33
3131
505050 50
53 53
3636
3434
105 105
104 104
3535
111111
32 32
84 84
112 112
122 122
132 133132 133
25
3030
29 29
51 51
54 5454
121 121
55 555555
28 28
49 49
52 52
7676
86 86
106106
129129

20
107107
7575
113113
115115
114114
108108
109109

54
1818 1818
2727
117117
120120
118 118 118118
1717
81 81
116116
128 128
15
44
11 11
1010
1616
26 26
151515 15
119119
22
55
88
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7777
78 78
6666
2020
25 25
22 222222
24 24
11
33
7777
9999
12 12
1313
2121
10
23 23
1414
130130
134134

MW pH 3
150
100

75
5050
53 53
2121
5555
7777
33
38 38
37 37
4949
2222
19 19
15 1515 15
50 50
5151
48 48
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29 292929
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373737
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4647
2525
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1616
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7373
74747474
4647
9191
78 7878 78
4545
4747
8181
80808080
7979
8888
9696
72727272
82828282

25
62 62
20
6666
7070

5353
15
61 61
11
22
60 60
10
6464
MW
150
100
75
50
37
25
20
15
10
a
b
c
Fig. 1 Gel electrophoresis of the proteins extracted from cervical mucous. a 2-DE gel image (Coomassie staining); b 2-DE gel after depletion
(SYPRO Ruby staining); and c 1-DE gel image (Coomassie staining). Only spots representing identified proteins are labeled on gel images
Clin Proteom (2010) 6:18–2821

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Table 1 Cervical mucous proteins identified through proteomic analyses
Protein nameAcc. no.a
MW
(kDa)
pIMASCOT scoreMatched
peptides
Sequence
coverage (%)
Methodb
Spot no.c
14-3-3 protein epsilon P6225829.1 4.6392310T51
14-3-3 protein sigmaP3194727.74.68 713 10T51
14-3-3 protein zeta/deltaP63104 27.7 4.73181525T50
6-phosphogluconate
dehydrogenase
Alcohol dehydrogenase
[NADP+]d
Alpha-1-acid glycoprotein
1 precursore
Alpha-1-antichymotrypsin
precursore
Alpha-1-antitrypsin precursore
P5220953.16.805125T,G46
P1455036.56.327526T122
P02763 23.44.93 1946 34T,G56
P0101147.65.332857 21T98–100
P0100946.75.3710921648T,G, D45, 57, 59, 97, 123, d19
Alpha-enolaseP0673347.1 7.01338919T,G 46–47, 80
Annexin A1P0408338.76.5718931243T,G, D 35–36, 85, 124,
d78–82, d91
Annexin A2 P07355 38.57.575731343T,G, D82, 111–112, d37,
d45–47, d73, d85
Annexin A3P1242936.45.637581242T,G, D32–33, 84, d88, d96
Annexin A5P0875835.94.94167725 T,G31
Antileukoproteinase 1 precursorP0397314.3 9.115426G
Anterior gradient protein 2
homolog precursor
Apolipoprotein A-I precursore
O95994 19.9 9.03126426 T,G118–119
P0264730.85.5614641656T,G30, 49, 106–109
Arginase-1P0508934.76.7236310T125
ATP synthase subunit beta
Beta-actine
Calregulind
P0657656.55.266424D d51
P6070941.75.299261034T,G58, 96
P2779748.14.299238T, D101, d48
Carbonic anhydrase 1 P0091528.96.59133522T, D34, 53, 54, d72
CatalaseP0404059.76.93811D d13
Cathelicidin antimicrobial
peptide precursord
Ceruloplasmin precursor
P4991319.3 9.481352 10T, G 119–120
P00450 122.1 5.4412444T,G, D69, d53
Complement C3 precursor 1637187.06.022541511T,G, D 34, 43, d5, d7
Complement C4-A precursor1618192.76.656021Dd38
Complement factor B precursorP0075185.56.675745G
Cystatin-AP0104011.05.38126230T,G21, 24
Cystatin-B P0408011.16.96 85224G
Elongation factor 1-alpha 1 P6810450.19.10 7036T87
Endoplasmin precursorP14625 92.44.7616868T, D 62, d50
F-actin-capping protein
subunit alpha-1
Fatty-acid-binding
protein, epidermal
Flavin reductased
P5290732.95.454313T32
Q0146915.26.603156 40T,G10
P3004322.17.13 35 17T 121
Fructose-bisphosphate
aldolase A
Glutamine synthetased
Glutathione-S-transferase Pe
P04075 39.48.304413D d44
P15104 42.06.43 995 19G
P09211 23.35.43 732 13T,G29
Glyceraldehyde-3-phosphate
dehydrogenase
Haptoglobin precursor
P04406 36.08.57 168625 T,G37, 48
P0073845.2 6.13 2265 14 T,G72
Heat-shock 70 kDa protein 1
Heat-shock 70 kDa protein 1 Ld
P0810769.95.48311 11 17T, D126, d19
P34931 70.35.76153710T 126
22Clin Proteom (2010) 6:18–28