ComparativeProteomicAnalysisofProteinsInvolvedin ...downloads.hindawi.com/archive/2010/726968.pdf · carcinomas of the bladder, prostate, or upper tracts. Very few studies have been

Hindawi Publishing CorporationInternational Journal of ProteomicsVolume 2010, Article ID 726968, 14 pagesdoi:10.1155/2010/726968

Research Article

Comparative Proteomic Analysis of Proteins Involved inthe Tumorigenic Process of Seminal Vesicle Carcinoma inTransgenic Mice

Wei-Chao Chang,1 Chuan-Kai Chou,2 Chih-Chiang Tsou,3 Sheng-Hsiang Li,4

Chein-Hung Chen,1 Yu-Xing Zhuo,2 Wen-Lian Hsu,3 and Chung-Hsuan Chen1, 5, 6

1 Genomics Research Center, Academia Sinica, Taipei, Taiwan2 National Applied Research Laboratories, National Laboratory Animal Center, Taipei, Taiwan3 Institute of Information Science, Academia Sinica, Taipei, Taiwan4 Department of Medical Research, Mackay Memorial Hospital, Tamshui, Taiwan5 Chemistry Department, National Taiwan University, Taipei, Taiwan6 Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei, Taiwan

Correspondence should be addressed to Chung-Hsuan Chen, [email protected]

Received 18 December 2009; Accepted 18 February 2010

Academic Editor: Alexander I. Archakov

Copyright © 2010 Wei-Chao Chang et al. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.

We studied the seminal vesicle secretion (SVS) of transgenic mice by using one-dimensional gel electrophoresis combined withLTQ-FT ICR MS analysis to explore protein expression profiles. Using unique peptide numbers as a cut-off criterion, 79 proteinswere identified with high confidence in the SVS proteome. Label-free quantitative analysis was performed by using the IDEAL Qsoftware program. Furthermore, western blot assays were performed to validate the expression of seminal vesicle proteins.Sulfhydryl oxidase 1, glia-derived nexin, SVS1, SVS3, and SVS6 showed overexpression in SVS during cancer development. Withhigh sequence similarity to human semenogelin, SVS2 is the most abundance protein in SVS and is dramatically decreased duringthe tumorigenic process. Our results indicate that these protein candidates could serve as potential targets for monitoring seminalvesicle carcinoma. Moreover, this information can provide clues for investigating seminal vesicle secretion-containing seminalplasma for related human diseases.

1. Introduction

Primary seminal vesicle carcinoma is an extremely rareneoplasm; only a few cases have been reported [1, 2].Seminal vesicle carcinoma is usually associated with diffusecarcinomas of the bladder, prostate, or upper tracts. Veryfew studies have been published on seminal vesicle invasion[3], and the tumor biology of seminal vesicle carcinomais not well understood. The diagnoses of seminal vesiclecarcinoma are generally based on a combination of morpho-logic, immunohistochemical, and radiological examinations.However, it is difficult to make a definitive diagnosis usinglimited biopsy material.

The seminal vesicle makes up part of the male accessorysexual glands. After puberty, the glands produce a fluid called

seminal vesicle secretion (SVS), which accumulates in thelumen of the seminal vesicles. SVS contains both proteinand nonprotein components and composes the majority ofseminal plasma. The extirpation of seminal vesicles fromadult rodents greatly reduces fertility, indicating that SVSplays an important role in sperm activity and modification[4]. Some SVS proteins have been analyzed to characterizetheir functions and physiological activities [5–9], but theconstituents of the SVS proteome have not been well studied.

Proteomic studies are broadly applied to the diagnostic,prognostic, and therapeutic fields. In terms of diseasediagnosis and prognosis, body fluid analysis proves to bemore attractive than tissue analysis because it providesseveral advantages including low invasiveness, minimumcost, and easy sample collection and processing [10]. The

2 International Journal of Proteomics

linear ion trap-Fourier transform ion cyclotron resonancemass spectrometer (LTQ-FT ICR MS) has been exploitedas a tool for identifying and characterizing isolated proteins[11]. Combining LTQ-FT ICR MS with liquid chromatog-raphy, large-scale and high-quality proteomic analyses ofseveral body fluids are achievable, including tears, urine,and seminal plasma [12–14]. By virtue of its capacity forexhaustive investigation, proteomic research has arousedhigh expectations for the discovery of biomarkers of variousdiseases [15, 16].

In this work, we used the transgenic mouse TAg as ananimal model to study seminal vesicle carcinoma. Transgenicmouse TAg (C57BL/6-TgN (TRAMP) 8247Ng) expresses anSV40 large T antigen that is driven by the probasin promoter[17]. In addition to being models for prostate cancer, TAgmice also develop other tumors, including neuroendocrinetumors in the prostate and neoplasms in the seminal vesicles.We analyzed the SVS proteome by using one-dimensionalPAGE and LC LTQ-FT ICR MS. Accurate masses of trypticpeptides were measured by FT ICR MS, and tandem mass(MS/MS) experiments were performed in LTQ to acquireadequate data for proteomic identification. The resultingmzXML format data and Mascot search results were appliedto perform label-free quantitative analysis. Moreover, weused western blot assays to validate the quantitative results.

2. Materials and Methods

2.1. Animals and Histopathology. TAg mice were purchasedfrom the Jackson Laboratory (Maine, USA), and wild typeC57BL/6JNarl normal control mice were obtained fromthe National Laboratory Animal Center (Taipei, Taiwan).They were housed in a specifically pathogen-free facilityand handled in accordance with the guidelines of Guide forthe Care and Use of Laboratory Animals. All of the micewere given free access to a standard murine chow diet andreverse osmosis water and were maintained on a 14:10hours light-dark cycle at 21–23◦C. In this study, TAg mice(n = 21; n = 7 for each subgroup) of various ages (32–40weeks) and normal control mice (n= 6) were euthanized andthe seminal vesicles and prostate glands were processed forhistopathologic examination.

2.2. Sample Collection and Preparation. The seminal vesicleswere collected by careful dissection to free them from theadjacent coagulating glands, and SVS was squeezed directlyinto 5 mL of 8 M urea solution. Small amounts of coagulatedpellets were removed through centrifugation at 15,000 g for10 minutes. The protein concentration of the supernatantwas determined using the Bio-Rad Protein Assay (Bio-RadLaboratories, Calif, USA) by the measurement of A595. Each20 μg of SVS proteins was applied to 13% PAGE, and thegel was subsequently visualized through coomassie blue-staining. Gel lanes were divided into 10 sections and allslices were cut into small gel pieces (<1 mm3), followed byin-gel digestion. Additionally, five of the observed majorbands were cut for independent assays. Briefly, the procedureof in-gel digestion included the following steps in order:

(a) destaining with 50% acetonitrile (ACN) and 25 mMammonium bicarbonate (ABC); (b) reduction using freshlyprepared 10 mM dithiothreitol for 45 minutes at 58◦C;(c) alkylation using freshly prepared 55 mM iodoacetamidefor 45 min at room temperature in the dark; (d) enzymedigestion with 4 ng/μL sequencing grade trypsin in 25 mMABC solution at 37◦C for 16–18 hours; (e) extraction oftryptic peptides using a 60% ACN/1% trifluoroacetic acidsolution. After drying to remove the solvent, the re-dissolvedtryptic peptides were subjected to LC LTQ-FT ICR MSanalysis.

2.3. LC LTQ-FT ICR MS Analysis. MS/MS experiments wereperformed with an LTQ-FT ICR MS (Thermo Electron,Calif, USA) equipped with a nanoelectrospray ion source(New Objective, Mass, USA), an Agilent 1100 Series binaryHPLC pump (Agilent Technologies, Calif, USA) and aFamos autosampler (LC Packings, Calif, USA). Trypticpeptide mixtures were injected at a 10 μL/min flow rateinto a self packed precolumn in line with a reverse phaseC18 nanocolumn (75 μm I.D. × 200 mm) that used MagicC18AQ resin (particle size, 5 μm; pore size, 200 A; MichromBioresources, Calif, USA). The analytic program was setat a linear gradient from 10% to 50% ACN with a 60minutes running cycle and a split flow rate of 300 nL/min.The full-scan survey MS experiment (m/z 320–2,000) wasexecuted in FT ICR MS with a mass resolution of 100,000at m/z 400. The top ten most abundant multiply chargedions, if they were above a minimum threshold of 1,000counts, were sequentially isolated for MS/MS by LTQ.Singly charged ions were rejected for MS/MS sequenc-ing.

2.4. Mascot Search. The raw files of spectra were convertedto mgf files with Mascot Daemon (data import filter: massrange, 600–5400; grouping tolerance, 1.4) and merged intoa single file for searching by the MASCOT (version 2.1,Matrix Science Ltd., London, UK) software platform basedon the IPI mouse database (v 3.36). The following MASCOTparameter settings were used; the peptide tolerance was15 ppm with 2+ and 3+ peptide charges and the MS/MStolerance was 0.6 Da. Two missed cleavages by trypsinwere allowed, carbamidomethyl (C) was used as a fixedmodification and oxidation (M) and deamidated (NQ) wereused as variable modifications. The significance threshold forthe identification was set to P < .01.

2.5. Label-Free Quantitative Analysis. A software programIDEAL-Q (ID-based Elution time Alignment by Linearregression Quantification) was developed in-house toanalyze LC-MS/MS data for label-free quantitative analysis[18]. The program was used to process the LC-MS dataand the search results obtained from the Mascot searchengine to extract the quantification information. The wholequantitative analysis consisted of the following tasks. (I) Datapreparation and construction of the protein list. The raw datafiles generated from the mass instrument were convertedinto the mzXML data format by the ReAdW program

International Journal of Proteomics 3

(http://tools.proteomecenter.org/wiki/index.php?title=Soft-ware:ReAdW). The data of each fractional LC-MS/MSruns coming from the same sample were merged and thensearched by the Mascot search engine to establish a proteinlist, which contained identified proteins and their relatedpeptide information. The mzXML files coupled with thepeptide and protein identification results were input to theIDEAL-Q program. (II) Extracting quantitative informationfrom each LC-MS run. For quantitative analysis of a peptidein an LC-MS run, we extracted the LC-MS data within therange of ±1.5 minutes of its elution time and ±3.5 Da of theprecursor m/z value. The peak clusters located within theselected elution time and the precursor m/z value from theextracted data underwent a peptide validation process. Forpeptide validation, the following three criteria were applied:signal-to-noise ratio (S/N), charge state (CS), and isotopepattern (IP). The S/N criterion checks whether the precursorpeak has a valid S/N ratio (>2). The CS criterion eliminatedthe peak clusters with an incorrect charge state by examiningwhether the distance between adjacent peaks is equal to 1/z(tolerance ±1/10 1/z). Finally, the IP criterion examined thecorrelation between isotopic distribution of the observedpeak intensities and the theoretical isotopic distributionof the peptide. The correlation was then evaluated by aChi-square goodness of fit test (<0.218). The purpose ofpeptide validation was to filter out false peptide signals; onlythe peptide passing the validation criteria were processedto subsequent quantification. We used the extracted ionchromatogram (XIC) to determine peptide abundance inan LC-MS run. (III) Peptide abundance and peptide ratioprocessing. First we determined the abundances of validpeptides in each LC-MS run. Then we calculated the peptideabundance in a fraction by averaging the peptide abundancesof all repeated runs. We summed the peptide abundances inall fractions to represent the peptide quantity in the sample.Following, the peptide ratio between samples could becalculated. (IV) Protein abundance processing. We selectednondegenerate unique peptides and performed Dixon’stest to eliminate outliers of peptide ratios for each protein.We then used the top three highly abundant peptides ofone protein to represent the quantity of this protein by aweighted average.

2.6. Western Blot. Polyclonal antibodies against SVS1, SVS2,SVS3 [19], SVS5, SVS7 [20], sulfhydryl oxidase 1, glia-derived nexin, carcinoembryonic antigen-related cell adhe-sion molecule 10 (CEACAM 10) [7], Lysozyme C-type M,secreted seminal-vesicle Ly-6 protein 1 (SSLP-1) [21], serineprotease inhibitor kazal-like protein (SPINKL) [22], and ser-ine protease inhibitor Kazal-type 3 (SPINK3) [9] were raisedin New Zealand White rabbits. Antibody against humanalbumin, which also crossreacted with mouse albumin, waspurchased from Calbiochem (Darmstadt, Germany). AfterPAGE separation, proteins were transferred to a PVDFmembrane using a semidry blotter (ATTA, Tokyo, Japan).The electrophoresis program was set with a constant current(1.5 mA/cm2) for 1 hour. The membrane was blocked with5% (w/v) skim milk in phosphate-buffered saline (PBS) at

room temperature for O/N reaction, and then incubatedwith primary antibody (1 : 5000) in PBS with 2% (w/v) skimmilk for 1 hour. After gentle agitation in four changes ofPBST (PBS with 0.05% Tween 20) for 15 minutes each, themembrane was immuno-reacted with secondary antiserum(horseradish peroxidase-conjugated goat antirabbit IgG,Amersham Pharmacia) diluted to 1 : 10000 in PBS with 2%(w/v) skim milk for 1 hour. Immuno-reactive bands wererevealed using an enhanced ECL substrate according to themanufacturer’s instructions (Pierce, Illinois, USA).

3. Results

3.1. Histopathology and Tumor Categories. According to thehistopathologic results, the neoplasm development of theaccessory sex gland was categorized into three stages: (I)hyperplasia of the seminal vesicle (Hp), (II) adenoma ofthe seminal vesicle (Ad), and (III) adenocarcinoma of theseminal vesicle with prostate cancer (Ac) (Figure 1). TheHp stage showed a typical hyperplasia area. These areas,which were characterized by small size and uniformityof cells and nuclei, appeared to have increased glandularenfolding and lack of mitoses. The Ad stage showed thereplacement of most of the normal glandular parenchymawith tumor masses encompassing two main components,epithelial cells and stromal cells. The cuboidal to columnarepithelial cells were arranged in multiple papillary fronds andglandular structures, which were separated and supportedby abundant, immature, and fibrovascular stroma. However,mitotic figures were infrequent. At the Ac stage, the seminalvesicular tumor showed an appearance similar to the Adstage, except that sometimes hemorrhage occurred in theseminal vesicles. Prostatic adenocarcinoma of the Ac stageappeared as intraluminar cribriform to papillary epithelialproliferations that completely or almost completely filled thelumen of several adjacent alveoli. The epithelium that linedthe tumors was composed of bland-appearing, cuboidal totall columnar epithelial cells with maintenance of nuclearpolarity and sometimes with basophilic cytoplasm.

3.2. Protein Pattern and Proteomic Analysis. Three SVS sam-ples of each group were pooled for the proteomic measure-ments. SVS proteins were separated by PAGE and their pro-file was presented by coomassie blue staining (Figure 2). Theprotein profile was dominated by a small number of highlyexpressed proteins. The protein distribution of normal SVSwas different from those coming from the tumorous SVS,which displayed more complex protein contents. Based onprotein pattern comparison, five major bands were chosenas reference indicators. Bands A and E showed upregulatedexpression, while band B showed downregulated expressionin the tumorous SVS. MS/MS was used to analyze the majorproteins in bands A to E, which were identified as albumin,seminal vesicle secretory protein 2 (SVS2), SVS4, SVS5, andhemoglobin beta, respectively. The serum proteins, albumin,and hemoglobin beta, are obviously increased in SVS in theearly stages of tumorigenesis.


(a) (b)

(c) (d)

Figure 1: Histopathologic section and tumor categories. Tissue slices from seminal vesicles of normal and various tumor stage mice werestained with hematoxylin-eosin. (a) Normal seminal vesicle, (b) Hp stage seminal vesicle, (c) Ad stage seminal vesicle, and (d) Ac stageprostate with carcinoma. Photographs display the images with original magnification ×10. Scale bar, 75 μm.

The proteomic analyses of four groups of SVS, whichwere normal, Hp, Ad, and Ac, were performed by repeatedexperimental runs. As a consequence of these analyses,excluding single peptide or single unique peptide matchedproteins and keratins that were frequent contaminants inproteomic analysis, 179 proteins met the search criterionand were identified in the proteome by combining fourkinds of samples. In order to further improve confidence inthe identified proteins, the reproducibility of the matchedunique peptides found in every SVS sample was consideredas an index to sift through the protein list. The determinationof unique characteristics for a peptide was based on theproteotypic peptide sequence that could only match to oneprotein through the IPI mouse database. The number ofunique peptides for one protein was summed with the resultsof proteomic analyses of the four groups of SVS; therefore,not all peptide signals could be found in the four groups ofSVS. Generally, a smaller number of unique peptides coupledwith a lower detection rate. We defined the undetectable rateas the average percentage that could not find the matchedunique peptides at the designated unique peptide number inthe four groups of SVS. With the unique peptide numbers 2,3, 4, and 5, the undetectable rate was 27.7%, 28.0%, 27.6%,

and 12.5%, respectively. When the unique peptide numberwas more than 5, the undetectable rate decreased to zero(Figure 3). Thus, we chose the unique peptide number 5as a cut-off criterion to finalize the SVS proteome. Basedon this condition, 79 proteins were included in the SVSproteome (Table 1). Among this proteome, 47 proteins weresecreted or serum proteins that were the most abundant inthis proteome and 16 proteins belonged to seminal vesicleproteins that have been identified or characterized at theprevious studies. In addition, a small number of intracellularproteins, including cytoplasm protein, lysosome protein,endoplasmic reticulum protein, and membrane protein werealso found in this proteome.

3.3. Label-Free Quantitative Analysis. To assess the relativeprotein expression levels at normal and various cancer stages,quantitative proteomic analysis was pursued by using a label-free quantitative approach, which adopted the average peak-area of the three most intense peptides for the identifiedproteins to represent the absolute protein abundance [23].The in-house developed program IDEAL-Q [18] was usedto analyze LC-MS data and the corresponding MASCOT


15

20

25

35

50

70

100

140

240kDa N Hp Ad Ac Major protein

(A) Serum albumin

(B) Seminal vesicle protein 2

(C) Seminal vesicle protein 4(D) Seminal vesicle protein 5

(E) Hemoglobin β

Figure 2: Protein pattern of SVS. SVS proteins were separated by 13% PAGE and visualized by coomassie blue staining. N, normal; Hp, Adand Ac, various tumor stages. Molecular weights of protein markers are shown on the left. Designated bands A–E were identified by MS/MS,and the names of the major proteins are shown on the right.

2 3 4 5 6 > 60

5

10

15

20

25

30

Number of unique peptide

Un

dete

ctab

lera

te(%

)

Figure 3: The relationship of unique peptide number and theundetectable rate. The undetectable rate was defined as thepercentage of proteins without identifiable peak-areas in the sameunique peptide number group.

search results to extract the quantification information.The results obtained from this quantitative calculation areillustrated in Table 1. The stoichiometric distribution ofprotein abundance was from 3.26 × 104 (Dickkopf-likeprotein 1) to 2.77 × 109 (SVS2), which meant that thedynamic range for protein detection reached five orders ofmagnitude by using the described method. To estimate theapplied quantity of SVS proteins for proteomic analysis, thetotal protein abundances were summed and were 3.31 ×109, 9.74 × 108, 1.05 × 109, and 9.85 × 108 for normal, Hp,Ad, and Ac, respectively. The results showed that the quantity

of SVS proteins between various cancer stages was within a10% difference, but the quantity of normal SVS was threetimes more than other samples, which appeared to conflictwith the gel patterns (Figure 2). We further compared thedifference of expression levels of SVS2 and SVS4 (or SVS5)of normal SVS, which displayed almost equal intensity onPAGE gel by dye staining. SVS2 showed a protein abundanceof 2.77 × 109, which was about 200-fold higher than SVS4at 1.29 × 107 (or 150-fold higher than SVS5 at 1.76 × 107).This result implied that quantification errors could occurwith the comparison of various molecular weight proteins.To acquire the protein content distribution for realizingthe effect of using this quantitative analysis method, theabundance of every protein was converted to the percentageof content in every SVS sample. The results illustrated thatSVS2, SVS4, and SVS5 were 83.7%, 0.4% and 0.5% in normalSVS proteins, respectively (Table 1). In the SVS of the Hpstage, the low molecular weight proteins SVS4, SVS5, andhemoglobin beta were 1.4%, 1.2%, and 2.0%, respectively,which were significantly less than SVS2 at 33.0%. Comparedwith the gel pattern, the levels of low molecular weightproteins tended to be underestimated, which lead to errors inmeasuring protein quantity or determining the percentage ofprotein content.

The relative expression levels of tumorous versus nor-mal conditions were achieved by calculating the ratios ofabsolute SVS protein abundances (Table 1). Among sem-inal vesicle proteins, SVS1, SVS3, and glia-derived nexinshowed upregulated expression with ratios greater than 2.With respect to downregulated expression, lysozyme C and


Ta

ble

1:La

bel-

free

quan

tita

tive

anal

ysis

ofm

ouse

SVS

prot

eom

e.

Acc

essi

onn

um

ber

Un

iqu

ePe

ptid

e(A

)pr

otei

nab

ouda

nce

(B)

aver

age

CV

ofpe

ptid

es(%

)(C

)re

lati

veex

pres

sion

rati

o(D

)p

erce

nta

geof

con

ten

t(E

)W

este

rnbl

otas

say

Pro

tein

Mas

s/pI

Hp

Ad

Ac

NH

pA

dA

cN

Hp

/NA

d/N

Ac/

NH

pA

dA

cN

(1)

Sem

inal

vesi

cle

prot

ein

IPI0

0408

931

Car

cin

oem

bryo

nic

anti

gen

-rel

ated

cell

adh

esio

nm

olec

ule

1029

455/

6.84

242.

43E

+07

2.16

E+

072.

68E

+07

4.29

E+

0712

.44.

34.

210

.00.

570.

500.

632.

502.

062.

721.

30•

IPI0

0115

065

Glia

-der

ived

nex

in44

179/

9.85

444.

12E

+06

5.10

E+

064.

68E

+06

2.34

E+

0611

.77.

712

.517

.61.

762.

182.

000.

420.

490.

470.

07•

IPI0

0469

211

Sulf

hydr

ylox

idas

e1,

isof

orm

363

296/

8.17

535.

00E

+06

7.24

E+

068.

69E

+06

4.98

E+

067.

23.

63.

814

.31.

001.

451.

740.

510.

690.

880.

15•

IPI0

0347

009

Secr

eted

sem

inal

-ve

sicl

eLy

-6pr

otei

n1

1114

7/8.

727

1.49

E+

062.

16E

+06

1.25

E+

061.

35E

+06

31.1

6.3

11.5

22.6

1.10

1.61

0.93

0.15

0.21

0.13

0.04

•

IPI0

0133

448

Sem

inal

vesi

cle

anti

gen

1826

6/5.

5726

2.89

E+

074.

27E

+07

2.10

E+

077.

16E

+07

12.0

8.1

5.6

19.7

0.40

0.60

0.29

2.97

4.08

2.13

2.17

IPI0

0227

548

Sem

inal

vesi

cle

secr

etor

ypr

otei

n1

9347

0/8.

9862

9.58

E+

061.

08E

+07

1.40

E+

073.

18E

+06

9.1

4.2

5.7

15.8

3.02

3.41

4.42

0.98

1.03

1.43

0.10

•

IPI0

0331

164

Sem

inal

vesi

cle

secr

etor

ypr

otei

n2

4082

4/9.

8920

33.

21E

+08

3.25

E+

082.

63E

+08

2.77

E+

096.

73.

64.

310

.90.

120.

120.

0932

.95

31.0

626

.67

83.7

4•

IPI0

0227

543

Sem

inal

vesi

cle

secr

etor

ypr

otei

n3

beta

2998

3/9.

3742

3.32

E+

073.

67E

+07

4.44

E+

071.

64E

+07

9.6

7.9

3.9

13.1

2.03

2.24

2.71

3.41

3.51

4.51

0.49

•

IPI0

0314

341

Sem

inal

vesi

cle

secr

etor

ypr

otei

n3a

2994

8/9.

397

1.16

E+

061.

60E

+06

2.19

E+

067.

24E

+05

28.7

18.1

13.0

12.9

1.60

2.21

3.03

0.12

0.15

0.22

0.02

•

IPI0

0134

286

Sem

inal

vesi

cle

secr

etor

ypr

otei

n4

1250

7/8.

0418

1.39

E+

071.

20E

+07

1.73

E+

071.

29E

+07

7.4

7.4

3.9

12.5

1.08

0.93

1.34

1.43

1.15

1.76

0.39

IPI0

0135

709

Sem

inal

vesi

cle

secr

etor

ypr

otei

n5

1301

1/9.

5222

1.20

E+

071.

05E

+07

6.42

E+

061.

76E

+07

8.7

8.5

4.8

14.2

0.68

0.60

0.36

1.23

1.01

0.65

0.53

•

IPI0

0133

431

Sem

inal

vesi

cle

secr

etor

ypr

otei

n6

1147

5/5.

9315

1.95

E+

061.

90E

+06

1.48

E+

061.

41E

+06

13.4

8.7

5.8

12.0

1.38

1.35

1.05

0.20

0.18

0.15

0.04

IPI0

0877

305

Sem

inal

vesi

cle

secr

etor

ypr

otei

n7

1106

0/8.

9321

1.03

E+

071.

04E

+07

6.59

E+

061.

47E

+07

6.2

9.5

6.9

14.2

0.70

0.70

0.45

1.06

0.99

0.67

0.45

•

IPI0

0229

083

Seri

ne

prot

ease

inh

ibit

orka

zal-

like

prot

ein

1053

5/7.

556

1.40

E+

061.

39E

+06

8.81

E+

051.

73E

+06

13.2

11.9

12.7

14.5

0.81

0.81

0.51

0.14

0.13

0.09

0.05

•

IPI0

0132

416

Seri

ne

prot

ease

inh

ibit

orK

azal

-typ

e3

8482

/8.2

67

5.58

E+

067.

01E

+06

4.54

E+

069.

53E

+06

4.5

6.3

7.0

7.7

0.59

0.73

0.48

0.57

0.67

0.46

0.29

•

IPI0

0308

892

sim

ilar

toly

sozy

me

C,

typ

eM

1703

9/9.

7017

4.15

E+

061.

32E

+06

7.05

E+

055.

29E

+06

17.3

32.9

21.1

12.1

0.78

0.25

0.13

0.43

0.13

0.07

0.16

•


Ta

ble

1:C

onti

nu

ed.

Acc

essi

onn

um

ber

Un

iqu

ePe

ptid

e(A

)pr

otei

nab

ouda

nce

(B)

aver

age

CV

ofpe

ptid

es(%

)(C

)re

lati

veex

pres

sion

rati

o(D

)p

erce

nta

geof

con

ten

t(E

)W

este

rnbl

otas

say

Pro

tein

Mas

s/pI

Hp

Ad

Ac

NH

pA

dA

cN

Hp

/NA

d/N

Ac/

NH

pA

dA

cN

(2)

Seru

mpr

otei

nan

dSe

cret

edpr

otei

n

IPI0

0131

695

Alb

um

in68

648/

5.75

109

8.12

E+

076.

21E

+07

3.52

E+

072.

17E

+07

12.4

4.6

4.6

12.1

3.74

2.86

1.62

8.34

5.94

3.57

0.66

•

IPI0

0129

755

Alp

ha-

1-an

titr

ypsi

n1-

245

946/

5.32

87.

59E

+05

7.53

E+

053.

16E

+05

1.31

E+

066.

66.

35.

527

.50.

580.

570.

240.

080.

070.

030.

04

IPI0

0123

924

Alp

ha-

1-an

titr

ypsi

n1-

445

969/

5.24

248.

49E

+06

1.10

E+

076.

45E

+06

7.10

E+

0614

.96.

77.

116

.41.

201.

540.

910.

871.

050.

650.

21

IPI0

0123

927

Alp

ha-

1-an

titr

ypsi

n1-

545

862/

5.44

98.

01E

+05

2.14

E+

062.

17E

+06

3.01

E+

0625

.54.

727

.110

.60.

270.

710.

720.

080.

200.

220.

09

IPI0

0117

857

Alp

ha-

1-an

titr

ypsi

n1-

645

794/

5.25

101.

45E

+06

1.18

E+

065.

62E

+05

3.36

E+

057.

97.

55.

843

.64.

323.

511.

670.

150.

110.

060.

01

IPI0

0128

249

Alp

ha-

2-H

S-gl

ycop

rote

in37

302/

6.04

97.

34E

+05

7.96

E+

057.

52E

+05

1.20

E+

066.

016

.79.

013

.90.

610.

660.

630.

080.

080.

080.

04

IPI0

0624

663

Alp

ha-

2-m

acro

glob

ulin

1671

77/6

.38

113.

69E

+07

4.63

E+

075.

60E

+07

3.66

E+

0715

.27.

16.

215

.81.

011.

271.

533.

794.

435.

691.

11

IPI0

0169

625

An

tiac

idph

osph

atas

eva

riab

lelig

ht

chai

n18

1695

5/8.

505

ND

ND

ND

6.34

E+

04N

DN

DN

D0.

0N

DN

DN

DN

DN

DN

D0.

00

IPI0

0136

642

An

tith

rom

bin

-III

5197

1/6.

1013

3.27

E+

055.

48E

+05

1.13

E+

062.

61E

+05

32.0

9.2

3.0

12.3

1.26

2.10

4.35

0.03

0.05

0.12

0.01

IPI0

0877

236

Apo

lipop

rote

inA

-I30

569/

5.64

373.

99E

+06

5.29

E+

069.

58E

+06

1.05

E+

0615

.410

.29.

520

.43.

805.

049.

130.

410.

510.

970.

03

IPI0

0377

351

Apo

lipop

rote

inA

-IV

4500

1/5.

4123

2.86

E+

072.

82E

+07

3.54

E+

072.

59E

+07

16.7

17.6

19.8

20.7

1.10

1.09

1.36

2.94

2.69

3.59

0.78

IPI0

0322

463

Bet

a-2-

glyc

opro

tein

138

593/

8.59

95.

68E

+05

2.74

E+

053.

49E

+05

3.87

E+

0429

.55.

68.

236

.414

.67

7.08

9.01

0.06

0.03

0.04

0.00

IPI0

0331

286

Bet

a-2-

mic

rogl

obu

lin13

770/

8.55

158.

61E

+06

1.19

E+

079.

06E

+06

9.84

E+

067.

56.

46.

010

.00.

881.

210.

920.

881.

130.

920.

30

IPI0

0320

420

Clu

ster

in51

623/

5.46

471.

89E

+07

1.06

E+

072.

07E

+07

4.31

E+

0623

.811

.010

.328

.04.

382.

454.

811.

941.

012.

110.

13

IPI0

0323

624

Com

plem

ent

C3,

isof

orm

Lon

g(F

ragm

ent)

1865

38/6

.29

531.

41E

+07

1.62

E+

071.

66E

+07

9.51

E+

0618

.49.

19.

325

.81.

491.

701.

751.

451.

551.

690.

29

IPI0

0130

010

Com

plem

ent

com

pon

ent

fact

orH

1411

79/6

.85

221.

17E

+07

1.43

E+

074.

89E

+06

9.29

E+

0614

.819

.15.

015

.11.

261.

540.

531.

201.

370.

500.

28

IPI0

0114

065

Com

plem

ent

fact

orB

(Fra

gmen

t)84

951/

7.18

91.

54E

+06

1.56

E+

061.

78E

+06

6.84

E+

0514

.46.

44.

814

.22.

252.

272.

600.

160.

150.

180.

02

IPI0

0123

744

Cys

tati

n-C

1552

1/9.

1810

1 .03

E+

061.

10E

+06

8.86

E+

054.

57E

+05

8.7

9.9

6.1

33.9

2.26

2.41

1.94

0.11

0.11

0.09

0.01

IPI0

0331

487

Dic

kkop

f-lik

epr

otei

n1

2662

3/7.

986

2.76

E+

052.

86E

+05

1.36

E+

053.

26E

+04

14.3

8.1

23.7

8.7

8.46

8.77

4.16

0.03

0.03

0.01

0.00

IPI0

0115

522

Fibr

inog

en,a

lph

a61

288/

7.16

132.

69E

+06

3.24

E+

066.

91E

+06

2.55

E+

067.

212

.99.

212

.31.

061.

272.

710.

280.

310.

700.

08


Ta

ble

1:C

onti

nu

ed.

Acc

essi

onn

um

ber

Un

iqu

ePe

ptid

e(A

)pr

otei

nab

ouda

nce

(B)

aver

age

CV

ofpe

ptid

es(%

)(C

)re

lati

veex

pres

sion

rati

o(D

)p

erce

nta

geof

con

ten

t(E

)W

este

rnbl

otas

say

Pro

tein

Mas

s/pI

Hp

Ad

Ac

NH

pA

dA

cN

Hp

/NA

d/N

Ac/

NH

pA

dA

cN

IPI0

0279

079

Fibr

inog

en,b

eta

5471

8/6.

689

8.79

E+

067.

32E

+06

6.24

E+

063.

98E

+05

22.6

18.5

22.8

13.0

22.0

918

.40

15.6

70.

900.

700.

630.

01

IPI0

0122

312

Fibr

inog

en,g

amm

a50

317/

5.38

166.

52E

+05

5.58

E+

053.

92E

+05

2.45

E+

0514

.25.

09.

46.

92.

672.

281.

600.

070.

050.

040.

01

IPI0

0113

539

Fibr

onec

tin

2723

19/5

.39

114.

53E

+05

8.32

E+

055.

61E

+05

2.85

E+

068.

01.

112

.710

.60.

160.

290.

200.

050.

080.

060.

09

IPI0

0117

167

Gel

solin

,iso

form

185

888/

5.83

51.

58E

+06

1.26

E+

061.

40E

+06

4.13

E+

0533

.622

.822

.417

.93.

843.

053.

390.

160.

120.

140.

01

IPI0

0124

640

Gra

nu

lin64

972/

6.70

171.

93E

+06

2.19

E+

062.

57E

+06

1.14

E+

0610

.53.

615

.618

.61.

701.

922.

250.

200.

210.

260.

03

IPI0

0409

148

Hap

togl

obin

3872

7/5.

887

3.24

E+

053.

78E

+05

3.99

E+

052.

44E

+05

2.8

8.7

10.7

13.5

1.32

1.54

1.63

0.03

0.04

0.04

0.01

IPI0

0469

114

Hem

oglo

bin

,alp

ha

1507

6/7.

967

2.99

E+

073.

87E

+07

9.02

E+

071.

39E

+07

7.4

6.9

6.1

24.0

2.15

2.79

6.49

3.07

3.70

9.15

0.42

IPI0

0110

658

Hem

oglo

bin

,bet

a15

193/

8.96

51.

94E

+07

4.57

E+

076.

68E

+07

1.01

E+

072.

815

.07.

932

.41.

924.

516.

602.

004.

366.

780.

31

IPI0

0128

484

Hem

opex

in51

308/

7.92

272.

18E

+06

8.42

E+

069.

28E

+06

7.59

E+

0519

.511

.53.

07.

82.

8711

.10

12.2

30.

220.

800.

940.

02

IPI0

0322

304

His

tidi

ne-

rich

glyc

opro

tein

HR

G60

401/

7.26

111.

50E

+07

3.43

E+

071.

65E

+07

4.43

E+

076.

44.

45.

216

.70.

340.

770.

371.

543.

281.

671.

34

IPI0

0114

958

Isof

orm

HM

Wof

Kin

inog

en-1

7305

6/6.

0511

2.56

E+

072.

97E

+07

1.68

E+

079.

15E

+06

14.1

16.4

9.7

19.8

2.79

3.25

1.83

2.63

2.84

1.70

0.28

IPI0

0317

340

Lac

totr

ansf

erri

n77

788/

8.86

352.

63E

+07

2.06

E+

071.

52E

+07

1.81

E+

0726

.421

.318

.618

.61.

461.

140.

842.

701.

971.

540.

55

IPI0

0114

403

Met

allo

prot

ein

ase

inh

ibit

or1

2261

3/9.

145

2.37

E+

062.

26E

+06

2.60

E+

063.

27E

+06

13.4

4.4

6.6

26.0

0.72

0.69

0.79

0.24

0.22

0.26

0.10

IPI0

0123

223

Mu

rin

oglo

bulin

-116

5193

/6.0

019

4.28

E+

066.

65E

+06

7.31

E+

064.

64E

+06

5.2

10.5

3.9

18.9

0.92

1.43

1.57

0.44

0.64

0.74

0.14

IPI0

0115

941

Neu

trop

hil

gela

tin

ase-

asso

ciat

edlip

ocal

in22

861/

8.96

162.

22E

+06

4.08

E+

063.

67E

+06

1.35

E+

064.

85.

04.

814

.61.

653.

032.

720.

230.

390.

370.

04

IPI0

0309

704

Nu

cleo

bin

din

-250

273/

5.05

246.

56E

+05

2.82

E+

061.

38E

+06

4.88

E+

059.

614

.16.

816

.21.

345.

782.

830.

070.

270.

140.

01

IPI0

0322

936

Pla

smin

ogen

9072

3/6.

2116

2.89

E+

053.

11E

+05

2.34

E+

052.

07E

+05

6.2

4.5

4.3

14.3

1.39

1.50

1.13

0.03

0.03

0.02

0.01

IPI0

0135

156

Pro

tein

FAM

3B26

135/

8.97

117.

40E

+05

5.28

E+

055.

86E

+05

3.63

E+

0519

.914

.07.

013

.82.

041.

461.

620.

080.

050.

060.

01

IPI0

0311

104

Sem

aph

orin

-3C

8520

6/8.

865

3.16

E+

051.

72E

+05

3.22

E+

056.

56E

+04

41.7

27.1

32.4

50.4

4.82

2.63

4.91

0.03

0.02

0.03

0.00

IPI0

0131

830

Seri

ne

prot

ease

inh

ibit

orA

3K46

850/

5.05

191.

09E

+06

1.12

E+

065.

17E

+05

6.73

E+

0413

.58.

24.

718

.016

.28

16.6

37.

690.

110.

110.

050.

00

IPI0

0139

788

Sero

tran

sfer

rin

7667

4/6.

9490

1.46

E+

071.

28E

+07

1.07

E+

074.

87E

+06

13.6

7.6

13.7

16.3

2.99

2.62

2.20

1.50

1.22

1.09

0.15

IPI0

0309

214

Seru

mam

yloi

dP-

com

pon

ent

2623

0/5.

985

5.96

E+

055.

52E

+05

3.30

E+

055.

09E

+05

47.5

36.3

24.6

6.4

1.17

1.08

0.65

0.06

0.05

0.03

0.02

IPI0

0321

190

Sulf

ated

glyc

opro

tein

161

381/

5.07

85.

36E

+07

5.04

E+

075.

34E

+07

3.39

E+

0728

.018

.05.

335

.81.

581.

491.

585.

504.

825.

421.

02

IPI0

0136

556

Tran

scob

alam

in-2

4755

5/5.

908

9.37

E+

051.

50E

+06

1.85

E+

061.

85E

+06

7.1

4.4

12.7

13.0

0.51

0.81

1.00

0.10

0.14

0.19

0.06

International Journal of Proteomics 9T

abl

e1:

Con

tin

ued

.

Acc

essi

onn

um

ber

Un

iqu

ePe

ptid

e(A

)pr

otei

nab

ouda

nce

(B)

aver

age

CV

ofpe

ptid

es(%

)(C

)re

lati

veex

pres

sion

rati

o(D

)p

erce

nta

geof

con

ten

t(E

)W

este

rnbl

otas

say

Pro

tein

Mas

s/pI

Hp

Ad

Ac

NH

pA

dA

cN

Hp

/NA

d/N

Ac/

NH

pA

dA

cN

IPI0

0127

560

Tran

sthy

reti

n15

766/

5.77

89.

05E

+05

1.29

E+

061.

54E

+06

2.16

E+

0612

.714

.910

.79.

60.

420.

600.

710.

090.

120.

160.

07

IPI0

0129

102

Uro

kin

ase-

typ

epl

asm

inog

enac

tiva

tor

4823

6/8.

5321

3.09

E+

072.

50E

+07

1.70

E+

071.

74E

+07

11.7

15.7

24.2

20.7

1.78

1.44

0.97

3.18

2.39

1.72

0.53

IPI0

0126

184

Vit

amin

D-b

indi

ng

prot

ein

5356

5/5.

3919

1.22

E+

061.

58E

+06

1.37

E+

069.

77E

+05

16.0

20.5

8.9

14.7

1.25

1.62

1.40

0.13

0.15

0.14

0.03

(3)

Cyt

opla

smpr

otei

n

IPI0

0110

850

Act

in,c

ytop

lasm

ic1

4171

0/5.

2911

4.63

E+

055.

79E

+05

3.36

E+

052.

72E

+05

13.0

18.6

5.1

17.1

1.70

2.13

1.23

0.05

0.06

0.03

0.01

IPI0

0223

757

Ald

ose

redu

ctas

e35

709/

6.71

81.

21E

+06

1.41

E+

061.

94E

+06

6.78

E+

0520

.811

.66.

98.

81.

782.

082.

860.

120.

130.

200.

02

IPI0

0462

072

Alp

ha-

enol

ase

4711

1/6.

377

1.81

E+

051.

57E

+05

1.89

E+

051.

44E

+05

47.1

12.2

16.5

6.1

1.26

1.09

1.31

0.02

0.01

0.02

0.00

IPI0

0230

320

Car

bon

ican

hydr

ase

128

303/

6.44

57.

77E

+05

1.14

E+

062.

63E

+05

ND

50.4

1.7

60.3

ND

ND

ND

ND

0.08

0.11

0.03

ND

IPI0

0275

232

Mat

rix

met

allo

prot

ein

ase

730

009/

6.79

69.

13E

+05

1.40

E+

061.

36E

+06

2.44

E+

055.

712

.27.

211

.13.

745.

755.

550.

090.

130.

140.

01

IPI0

0121

788

Pero

xire

doxi

n-1

2216

2/8.

269

1.90

E+

051.

92E

+05

2.86

E+

051.

76E

+05

14.7

8.2

7.0

19.9

1.08

1.09

1.62

0.02

0.02

0.03

0.01

(4)

Lyso

som

epr

otei

n

IPI0

0111

013

Cat

hep

sin

D44

925/

6.71

62.

14E

+05

1.69

E+

053.

55E

+05

2.82

E+

0512

.710

.715

.812

.70.

760.

601.

260.

020.

020.

040.

01

IPI0

0128

154

Cat

hep

sin

L137

523/

6.37

77.

14E

+05

1.80

E+

061.

38E

+06

5.97

E+

058.

67.

815

.817

.71.

203.

022.

310.

070.

170.

140.

02

IPI0

0311

305

Deo

xyri

bon

ucl

ease

-2-

beta

4076

8/9.

4916

6.69

E+

056.

34E

+05

4.55

E+

059.

34E

+05

9.6

11.4

25.8

20.8

0.72

0.68

0.49

0.07

0.06

0.05

0.03

IPI0

0381

303

Lyso

som

alal

pha-

man

nos

idas

e11

4532

/8.3

024

4.99

E+

064.

56E

+06

5.26

E+

066.

23E

+06

11.4

11.2

7.0

19.7

0.80

0.73

0.84

0.51

0.44

0.53

0.19

(5)

Endo

plas

mic

Ret

icul

umpr

otei

n

IPI0

0319

992

78kD

agl

uco

se-r

egu

late

dpr

otei

n72

377/

5.07

134.

17E

+05

4.91

E+

052.

11E

+05

1.42

E+

0528

.525

.129

.89.

82.

933.

451.

480.

040.

050.

020.

00

IPI0

0187

353

ER

O1-

like

prot

ein

beta

5348

4/8.

3913

1.35

E+

051.

55E

+05

8.01

E+

043.

52E

+05

11.6

10.4

26.3

8.0

0.38

0.44

0.23

0.01

0.01

0.01

0.01

IPI0

0138

342

Liv

erca

rbox

yles

tera

seN

6113

3/5.

108

5.41

E+

058.

27E

+05

5.24

E+

051.

83E

+06

6.6

14.4

15.1

12.2

0.30

0.45

0.29

0.06

0.08

0.05

0.06

IPI0

0135

686

Pept

idyl

prol

ylis

omer

ase

B23

699/

9.56

147.

22E

+06

9.49

E+

064.

73E

+06

4.16

E+

0610

.25.

97.

213

.01.

742.

281.

140.

740.

910.

480.

13

(6)

Mem

bran

epr

otei

n

IPI0

0553

419

Des

mop

laki

n33

2706

/6.4

310

7.42

E+

066.

91E

+06

5.89

E+

065.

74E

+06

18.3

16.4

9.6

13.2

1.29

1.20

1.03

0.76

0.66

0.60

0.17

IPI0

0881

007

En

doth

elia

lch

lori

dech

ann

el99

794/

6.72

51.

87E

+05

2.15

E+

051.

12E

+05

4.45

E+

0416

.610

.127

.912

.44.

214.

832.

520.

020.

020.

010.

00


seminal vesicle antigen were decreased two times lower thantheir normal value. SVS2, with a 10-fold decrease, wasthe most differentially expressed protein. Similar differenceswere also observed in the gel patterns. In addition, mostserum proteins (35/46, 76%) had an upregulated expressionin the tumorous SVS. This phenomenon could relate to thephysiological change of seminal vesicles that increases thevascular permeability for serum proteins to enter the lumenof the seminal vesicle.

To evaluate the reproducibility of MS analysis, coefficientof variation (CV) values (%) of the top three highly abundantpeptides in repeated runs were calculated and the average CVvalues are listed in Table 1. In this work, the total averageCV values for normal, Hp, Ad, and Ac samples were 16.4%,15.1%, 10.8%, and 11.5%, respectively.

3.4. Western Blot Validation. The effects of tumorigenesis onthe expression of seminal vesicle secretory proteins were con-firmed by western blot analyses, which were also performedto validate the quantification results of the MS analyses(Table 1). Because no protein has been demonstrated tohave constitutive or consistent expression in SVS, we usedthe same loading quantity as the quantitative control inthis study. Based on MS quantification, more than 75%of serum proteins showed upregulated expression in thetumorous SVS. Using western blot analysis, for example, thehigher levels of albumin were clearly demonstrated in thetumorous SVS, which was consistent with the MS results(Figure 4). Many SVS proteins were also analyzed by theproduced antibodies to verify their expression. The signalintensities of western blot were quantitatively measured bythe software ImageJ (http://rsb.info.nih.gov/ij/) [24], and therelative expression ratios compared with the normal sampleswere calculated (Figure 4(c)). Sulfhydryl oxidase 1, glia-derived nexin, SVS1 and SVS3 showed upregulated expres-sion, but SVS2, CEACAM 10, lysozyme C-type M, SVS5,SSLP-1, SVS7, SPINKL, and SPINK3 showed downregulatedexpression. Regardless of upregulation or downregulation,most SVS proteins changed their secretion depending onthe carcinogenesis progress. The protein expression levelsrevealed a few discrepancies in comparison with the westernblot and MS quantification methods. For example, SSLP-1showed little change during tumorigenic process by using MSanalysis; however, it had a clear decreasing pattern by westernblot. On the whole, the western blot results were in goodagreement with the MS quantification.

4. Discussion

4.1. Tumorigenesis Affects the Composition of the SVS Pro-teome. SVS, one of the major contributors to seminal fluid,is important for semen coagulation and plays an importantrole in promoting sperm motility and suppressing immuneactivity in the female reproductive tract. Only 16 specificseminal vesicle secretory proteins have been identified withhigh confidence in the SVS proteome, which is a relativelysimple system compared to other body fluids. Almost all SVSproteins make changes in their levels during the tumorigenic

process. Compared with increasing protein expression, thesuppression effect produced by tumorigenesis on proteinexpression is more remarkable. The expression of SVS2,SVS5, and lysozyme C-type M decreased 5- to 10-fold atthe Ac stage by MS analysis or western blot assay. Althoughthe functions of most SVS proteins in the reproductivesystem are not fully understood, previous studies havedemonstrated that SVS proteins involved in formation of thecopulatory plug have the abilities to enhance sperm motilityand act as decapacitation factors to modulate the fertilizingability of spermatozoa. They also have activity of serineprotease inhibitors and antimicrobial activity, which maybe involved in fertility (Table 2). According to the currentstudy, the physiological functions of SVS proteins did notshow a definite correlation with the effects of tumorigenesis.For example, SVS1, SVS2, and SVS3 are the componentsof copulatory plug formation; however, SVS1 and SVS3showed upregulated expression, but SVS2 showed downreg-ulated expression. As serine protease inhibitors, glia-derivednexin and SVS6 showed upregulated expression, but SVS5,SPINKL, and SPINK3 showed downregulated expression.Thus, the changes of seminal vesicle proteins during thetumorigenic process need to be explored individually. Basedon our studies, almost all seminal vesicle secretory proteinswere aberrantly expressed by tumor cells. Only a few seminalvesicle proteins have been demonstrated to correlate withthe occurrence of carcinoma (Table 2). The expression ofsulfhydryl oxidase 1, glia-derived nexin, and SVS6 increasedin seminal vesicle carcinoma and other carcinomas. Theresults suggest that the expression of these proteins could beunder similar regulation in various carcinoma systems.

SVS2, the most abundant protein in SVS, decreasedduring seminal vesicle carcinoma. SVS2 shows 55% sequencesimilarity to human semenogelin, which is the major struc-tural component of gelatinous coagulum. Semenogelin isable to protect sperm from protein tyrosine phosphorylationand to prevent induction of the acrosome reaction [38].A member of the gene family that encodes this semen-coagulating protein is commonly found in mammalianspecies [6, 39–41]. As a decapacitation factor, SVS2 can bindsperm to affect fertility in the female reproductive tract.Decreased levels of SVS2 at the tumorigenic stages coulddiminish its inhibition of sperm mobility in a concentration-dependent manner [33]. In addition to varying biologicalactivities, the change of the SVS2 protein level could providea useful target to monitor the progress of seminal vesiclecarcinoma.

4.2. Vascular Permeability Contributes to the Elevated Levels ofSerum Proteins in SVS. Many serum proteins showed upreg-ulation in the tumorous SVS (Table 1). The most strikinglyexpressed proteins, albumin and hemoglobin β, could beobserved on the coomassie blue-stained gel (Figure 2). Theexpression of albumin was also confirmed by western blotassay (Figure 4). Basal levels of serum proteins could beconsidered to come from contamination by surgical leaks.However, elevated levels of serum proteins may originatefrom other sources. First, the mutant secretory cells of


Upregulated expression

Downregulated expression Relative expression ratio

N Hp Ad Ac

N Hp Ad Ac

N Hp Ad Ac

N Hp Ad Ac

N Hp Ad Ac

N Hp Ad Ac

N Hp Ad Ac

N Hp Ad Ac

N Hp Ad Ac

N Hp Ad Ac

N Hp Ad Ac

N Hp Ad Ac

N Hp Ad Ac

(1) Albumin

(2) Sulfhydryl oxidase

(3) Glia-derived nexin

(4) SVS 1

(5) SVS 3

(1) SVS 2

(2) CEACAM 10

(3) Lysozyme C, type M

(4) SVS 5

(5) SSLP-1

(6) SVS 7

(7) SPINKL

(8) SPINK 3

Hp/N10.69

Ad/N9.72

Ac/N4.45

Hp/N0.87

Ad/N1.41

Ac/N1.82

Hp/N1.9

Ad/N2.29

Ac/N1.66

Hp/N1.26

Ad/N1.26

Ac/N1.08

Hp/N1.03

Ad/N1.11

Ac/N1.22

Hp/N0.21

Ad/N0.19

Ac/N0.16

Hp/N1.08

Ad/N0.86

Ac/N0.9

Hp/N0.65

Ad/N0.48

Ac/N0.45

Hp/N0.3

Ad/N0.34

Ac/N0.21

Hp/N0.76

Ad/N0.66

Ac/N0.29

Hp/N0.81

Ad/N0.59

Ac/N0.68

Hp/N0.7

Ad/N0.64

Ac/N0.55

Hp/N0.46

Ad/N0.44

Ac/N0.32

(a)

(b) (c)

Figure 4: Western blot analysis. The expression of albumin, sulfhydryl oxidase 1, glia-derived nexin, SVS1, SVS3, SVS2, CEACAM 10,Lysozyme C-type M, SVS5, SSLP-1, SVS7, SPINKL, and SPINK3 were examined using polyclonal antibodies. (a) proteins with upregulatedexpression; (b) proteins with downregulated expression; (c) relative expression ratios compared to normal SVS.

the seminal vesicles may produce and secrete them intothe lumen, meaning that they are endogenous proteins.Second, angiogenesis or vascular permeability results in themovement of proteins from the blood into the seminalvesicle lumen. To evaluate this phenomenon, we usedmicroarray gene chips to examine the mRNA expression inthe seminal vesicles from the four stages. The results showedthat the expression of albumin, hemoglobin, apolipoprotein,and α1-antitrypsin remained at background levels (datanot shown). This suggested that these serum proteins areexcluded from being newly synthesized under tumorigenicconditions in seminal vesicles, and that angiogenesis orvascular permeability is responsible for the increase in theirlevels. Vascular permeability is a tightly regulated process that

is often an essential response accompanying angiogenesis,tumor metastasis, or inflammation [42]. Thus, there is areasonable explanation for late (Ac) stage SVS to have a pinkor red appearance.

4.3. Label-Free Quantitative Analysis. Combining one-dimensional PAGE fractionation techniques with LC MS/MSanalysis is a popular approach for proteomic research[43, 44]. Due to simplicity and flexibility, the label-freequantitative strategy is an attractive alternative for thequantification of LC MS/MS-based proteomics. In this study,our experimental data were analyzed by using the self-developed program IDEAL-Q, which was designed to reduce


Table 2: Summary of seminal vesicle secretory proteins identified in mouse SVS.

Expression

Protein Function Reference Mass Western Cancer-related studies

Upregulated expression

Sulfhydryl oxidase 1Catalyze disulfide bondformation

[25] ⇑ ⇑ [26, 27]

Glia-derived nexin Serine protease inhibitor [8] ⇑ ⇑ [28, 29]

Seminal vesicle secretory protein 1Amine oxidaseactivity/Copulatory plugformation

[5] ⇑ ⇑ —

Seminal vesicle secretory protein 3aCopulatory plugformation

[19] ⇑ ⇑ —

Seminal vesicle secretory protein 3βCopulatory plugformation

[30] ⇑ ⇑ —

Seminal vesicle secretory protein 6Serine proteinaseinhibitor

[31] ⇑ — [32]

Downregulated expression

Seminal vesicle secretory protein 2Semen-coagulatingprotein/Spermdecapacitation factor

[6, 33] ⇓ ⇓ [34]

Carcinoembryonic antigen-related celladhesion molecule 10

Sperm binding andenhancement of spermmotility

[7] ⇓ ⇓ —

Lysozyme C, type M Primary bacteriolysis — ⇓ ⇓ —

Seminal vesicle secretory protein 5Serine proteinaseinhibitor

[35] ⇓ ⇓ —

Secreted seminal-vesicle Ly-6 protein 1Potential cellularadhesion and signaling

[21] ⇔ ⇓ —

Seminal vesicle secretory protein 7Sperm capacitationfactor

[20] ⇓ ⇓ —

Serine protease inhibitor kazal-likeprotein

Serine proteaseinhibitor/Spermdecapacitation factor

[22] ⇓ ⇓ —

Serine protease inhibitor Kazal-type 3Serine proteaseinhibitor/Spermdecapacitation factor

[9] ⇓ ⇓ —

Seminal vesicle antigenSperm decapacitationfactor

[36] ⇓ — —

Seminal vesicle secretory protein 4Anti-inflammatory andimmune-modulatingagent

[37] ⇔ — —

the errors in peak detection and drift of retention time, andto validate the selected peptides by the criteria of signal-to-noise ratio, charge state, and isotope pattern. The averageCV values were in the range of 10–17% for the top threemost abundant peptides, which demonstrated that accuratepeak detection and reproducibility could be acquired byusing the analytical program IDEAL-Q. However, errorswill occur in the analysis of low molecular weight proteinsby using the top three most abundant peptides for quan-titative analysis, as found in a previous study [38]. Theenzymatic reaction produces various peptides with a widerange of ionization efficiency during MS analysis. Smallerproteins will have fewer enzymatic peptides to choose fromthe highest ionization efficiency region to represent their

quantity. Therefore, underestimation is inevitable for thequantification of low molecular weight proteins. For therelative quantification of specific protein abundance changesunder various conditions, such kinds of quantification errormay not affect the comparison of protein ratios. However,to achieve the accurate absolute quantification, modifyingthe original results with parameters regarding molecular sizeneeds to be further pursued.

5. Conclusion

To obtain SVS samples from humans is quite difficult;therefore, SVS samples from a mouse model system were


chosen as an alternative research target, although the volumeand composition of SVS differs significantly between variousspecies of mammals. Based on proteomic analysis, weidentified 79 proteins, including 16 seminal vesicle secretoryproteins, in the SVS proteome. Label-free quantitative anal-ysis was performed to estimate the quantity of the identifiedproteins. Moreover, most seminal vesicle secretory proteinswere subjected western blot assay to further validate theirexpression. Our data showed that both approaches were ingood agreement with each other. We confirmed that manySVS proteins had differential expression profiles during thetumorigenic process, especially SVS2, with a sequence similarto human semenogelin, which was dramatically decreasedduring the primary stage of seminal vesicle carcinoma. Theinformation obtained from the transgenic mice could behelpful in understanding tumor biology and could be furtherapplied as a reference to study related human diseases.

Acknowledgments

This work was supported by the Genomics Research Center.We express our thanks to Y. C. Chien for the cDNA microar-ray experiments and P. Y. Wang for the ICP MS experiments.Help on manuscript preparation and submission from Ku-Ju Cherry Lin is also acknowledged. W.-C. Chang and C.-K.Chou contributed equally to this work.

References

[1] T. Ralf and E. Peter, “Primary adenocarcinoma of the seminalvesicles,” Journal of Urology, vol. 168, no. 5, pp. 1891–1896,2002.

[2] L. Egevad, R. Ehrnstrom, U. Hakansson, and M. Grabe,“Primary seminal vesicle carcinoma detected at transurethralresection of prostate,” Urology, vol. 69, no. 4, pp. 778.e11–778.e13, 2007.

[3] A. Billis, D. A. Teixeira, R. F. Stelini, M. M. Quintal, M.S. Guimaraaes, and U. Ferreira, “Seminal vesicle invasion inradical prostatectomies: which is the most common route ofinvasion?” International Urology and Nephrology, vol. 39, no.4, pp. 1097–1102, 2007.

[4] B. Peitz and P. Olds-Clarke, “Effects of seminal vesicle removalon fertility and uterine sperm motility in the house mouse,”Biology of Reproduction, vol. 35, no. 3, pp. 608–617, 1986.

[5] A. Lundwall, J. Malm, A. Clauss, C. Valtonen-Andre, andA. Y. Olsson, “Molecular cloning of complementary DNAencoding mouse seminal vesicle-secreted protein SVS I anddemonstration of homology with copper amine oxidases,”Biology of Reproduction, vol. 69, no. 6, pp. 1923–1930, 2003.

[6] A. Lundwall, “The cloning of a rapidly evolving seminal-vesicle-transcribed gene encoding the major clot-formingprotein of mouse semen,” European Journal of Biochemistry,vol. 235, no. 1–2, pp. 424–430, 1996.

[7] S.-H. Li, R. K. Lee, Y.-L. Hsiao, and Y.-H. Chen, “Demon-stration of a glycoprotein derived from the Ceacam10 gene inmouse seminal vesicle secretions,” Biology of Reproduction, vol.73, no. 3, pp. 546–553, 2005.

[8] J.-D. Vassalli, J. Huarte, D. Bosco, et al., “Protease-nexin I as anandrogen-dependent secretory product of the murine seminalvesicle,” EMBO Journal, vol. 12, no. 5, pp. 1871–1878, 1993.

[9] L.-Y. Chen, Y.-H. Lin, M.-L. Lai, and Y.-H. Chen, “Devel-opmental profile of a caltrin-like protease inhibitor, P12, inmouse seminal vesicle and characterization of its binding siteson sperm surface,” Biology of Reproduction, vol. 59, no. 6, pp.1498–1505, 1998.

[10] T. D. Veenstra, T. P. Conrads, B. L. Hood, A. M. Avellino, R.G. Ellenbogen, and R. S. Morrison, “Biomarkers: mining thebiofluid proteome,” Molecular and Cellular Proteomics, vol. 4,no. 4, pp. 409–418, 2005.

[11] W. Haas, B. K. Faherty, S. A. Gerber, et al., “Optimizationand use of peptide mass measurement accuracy in shotgunproteomics,” Molecular and Cellular Proteomics, vol. 5, no. 7,pp. 1326–1337, 2006.

[12] G. A. de Souza, L. M. F. Godoy, and M. Mann, “Identificationof 491 proteins in the tear fluid proteome reveals a largenumber of proteases and protease inhibitors,” Genome Biology,vol. 7, no. 8, article R72, 2006.

[13] J. Adachi, C. Kumar, Y. Zhang, J. V. Olsen, and M. Mann, “Thehuman urinary proteome contains more than 1500 proteins,including a large proportion of membrane proteins,” GenomeBiology, vol. 7, no. 9, article R80, 2006.

[14] B. Pilch and M. Mann, “Large-scale and high-confidence pro-teomic analysis of human seminal plasma,” Genome Biology,vol. 7, no. 5, article R40, 2006.

[15] R. Etzioni, N. Urban, S. Ramsey, et al., “The case for earlydetection,” Nature Reviews Cancer, vol. 3, no. 4, pp. 243–252,2003.

[16] M. A. Reymond and W. Schlegel, “Proteomics in cancer,”Advances in Clinical Chemistry, vol. 44, pp. 103–142, 2007.

[17] N. M. Greenberg, F. DeMayo, M. J. Finegold, et al., “Prostatecancer in a transgenic mouse,” Proceedings of the NationalAcademy of Sciences of the United States of America, vol. 92, no.8, pp. 3439–3443, 1995.

[18] C.-C. Tsou, C.-F. Tsai, Y.-H. Tsui, et al., “IDEAL-Q, anautomated tool for label-free quantitation analysis usingan efficient peptide alignment approach and spectral datavalidation,” Molecular and Cellular Proteomics, vol. 9, no. 1, pp.131–144, 2010.

[19] H.-J. Lin, C.-W. Luo, and Y.-H. Chen, “Localization ofthe transglutaminase cross-linking site in SVS III, a novelglycoprotein secreted from mouse seminal vesicle,” Journal ofBiological Chemistry, vol. 277, no. 5, pp. 3632–3639, 2002.

[20] C.-W. Luo, H.-J. Lin, and Y.-H. Chen, “A novel heat-labilephospholipid-binding protein, SVS VII, in mouse seminalvesicle as a sperm motility enhancer,” Journal of BiologicalChemistry, vol. 276, no. 10, pp. 6913–6921, 2001.

[21] S.-H. Li, R. K.-K. Lee, M.-H. Lin, et al., “SSLP-1, a secretedLy-6 protein purified from mouse seminal vesicle fluid,”Reproduction, vol. 132, no. 3, pp. 493–500, 2006.

[22] M.-H. Lin, R. K.-K. Lee, Y.-M. Hwu, et al., “SPINKL,a Kazal-type serine protease inhibitor-like protein purifiedfrom mouse seminal vesicle fluid, is able to inhibit spermcapacitation,” Reproduction, vol. 136, no. 5, pp. 559–571, 2008.

[23] J. C. Silva, M. V. Gorenstein, G.-Z. Li, J. P. C. Vissers, and S. J.Geromanos, “Absolute quantification of proteins by LCMSE:a virtue of parallel MS acquisition,” Molecular and CellularProteomics, vol. 5, no. 1, pp. 144–156, 2006.

[24] M. D. Abramoff, P. J. Magalhaes, and S. J. Ram, “Imageprocessing with imageJ,” Biophotonics International, vol. 11,no. 7, pp. 36–42, 2004.

[25] S. Matsuba, Y. Suga, K. Ishidoh, et al., “Sulfhydryl oxidase(SOX) from mouse epidermis: molecular cloning, nucleotide


sequence, and expression of recombinant protein in thecultured cells,” Journal of Dermatological Science, vol. 30, no.1, pp. 50–62, 2002.

[26] C. Morel, P. Adami, J.-F. Musard, D. Duval, J. Radom, and M.Jouvenot, “Involvement of sulfhydryl oxidase QSOX1 in theprotection of cells against oxidative stress-induced apoptosis,”Experimental Cell Research, vol. 313, no. 19, pp. 3971–3982,2007.

[27] B. L. Moss, L. Taubner, Y. K. Sample, D. A. Kazmin, V. Copie,and J. R. Starkey, “Tumor shedding of laminin binding proteinmodulates angiostatin production in vitro and interferes withplasmin-derived inhibition of angiogenesis in aortic ringcultures,” International Journal of Cancer, vol. 118, no. 10, pp.2421–2432, 2006.

[28] Y. Li and Y.-Y. Lu, “Applying a highly specific and reproduciblecDNA RDA method to clone garlic up-regulated genes inhuman gastric cancer cells,” World Journal of Gastroenterology,vol. 8, no. 2, pp. 213–216, 2002.

[29] S. Thelin-Jarnum, C. Lassen, I. Panagopoulos, N. Mandahl,and P. Aman, “Identification of genes differentially expressedin TLS-CHOP carrying myxoid liposarcomas,” InternationalJournal of Cancer, vol. 83, no. 1, pp. 30–33, 1999.

[30] J. Kawai, A. Shinagawa, K. Shibata, et al., “Functional annota-tion of a full-length mouse cDNA collection,” Nature, vol. 409,no. 6821, pp. 685–690, 2001.

[31] C. Guilbaud, A. M. Simon, G. Veyssiere, and C. Jean, “Cloningand sequence analysis of a cDNA encoding an androgen-dependent mouse seminal vesicle secretory protein,” Journalof Molecular Endocrinology, vol. 10, no. 3, pp. 279–288, 1993.

[32] H.-J. Kim, I. Sohng, C.-H. Hwang, and J.-Y. Park,“Cytokeratin-20 and seminal vesicle secretory protein VIas possible marker proteins in urinary bladder preneoplasticlesions induced by N-butyl-N-(4-hydroxybutyl) nitrosamine,”International Journal of Urology, vol. 13, no. 2, pp. 142–147,2006.

[33] N. Kawano and M. Yoshida, “Semen-coagulating protein,SVS2, in mouse seminal plasma controls sperm fertility,”Biology of Reproduction, vol. 76, no. 3, pp. 353–361, 2007.

[34] Y. Zhang, Z. Wang, H. Liu, F. J. Giles, and S. H. Lim, “Patternof gene expression and immune responses to Semenogelin 1in chronic hematologic malignancies,” Journal of Immunother-apy, vol. 26, no. 6, pp. 461–467, 2003.

[35] A. Clauss, H. Lilja, and A. Lundwall, “The evolution of agenetic locus encoding small serine proteinase inhibitors,”Biochemical and Biophysical Research Communications, vol.333, no. 2, pp. 383–389, 2005.

[36] L.-C. Yu, J.-L. Chen, W.-B. Tsai, and Y.-H. Chen, “Primarystructure and characterization of an androgen-stimulatedautoantigen purified from mouse seminal-vesicle secretion,”Biochemical Journal, vol. 296, no. 3, pp. 571–576, 1993.

[37] Y. H. Chen, B. T. Pentecost, J. A. McLachlan, and C.T. Teng, “The androgen-dependent mouse seminal vesiclesecretory protein IV: characterization and complementarydeoxyribonucleic acid cloning,” Molecular Endocrinology, vol.1, no. 10, pp. 707–716, 1987.

[38] E. De Lamirande, K. Yoshida, T. M. Yoshiike, T. Iwamoto,and C. Gagnon, “Semenogelin, the main protein of semencoagulum, inhibits human sperm capacitation by interferingwith the superoxide anion generated during this process,”Journal of Andrology, vol. 22, no. 4, pp. 672–679, 2001.

[39] S. Dorus, P. D. Evans, G. J. Wyckoff, S. S. Choi, and B. T.Lahn, “Rate of molecular evolution of the seminal protein

gene SEMG2 correlates with levels of female promiscuity,”Nature Genetics, vol. 36, no. 12, pp. 1326–1329, 2004.

[40] S. E. Harris, M. A. Harris, C. M. Johnson, and M. F. Bean,“Structural characterization of the rat seminal vesicle secretionII protein and gene,” Journal of Biological Chemistry, vol. 265,no. 17, pp. 9896–9903, 1990.

[41] T. Iwamoto, H. Tanaka, T. Osada, T. Shinagawa, Y. Osamura,and C. Gagnon, “Origin of a sperm motility inhibitor fromboar seminal plasma,” Molecular Reproduction and Develop-ment, vol. 36, no. 4, pp. 475–481, 1993.

[42] S. M. Weis and D. A. Cheresh, “Pathophysiological conse-quences of VEGF-induced vascular permeability,” Nature, vol.437, no. 7058, pp. 497–504, 2005.

[43] K. Mann and M. Mann, “The chicken egg yolk plasma andgranule proteomes,” Proteomics, vol. 8, no. 1, pp. 178–191,2008.

[44] B.-B. Gao, L. Stuart, and E. P. Feener, “Label-free quantitativeanalysis of one-dimensional PAGE LC/MS/MS proteome:application on angiotensin II-stimulated smooth muscle cellssecretome,” Molecular and Cellular Proteomics, vol. 7, no. 12,pp. 2399–2409, 2008.

Submit your manuscripts athttp://www.hindawi.com

Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation http://www.hindawi.com

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation http://www.hindawi.com Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttp://www.hindawi.com

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research


International Journal of

Microbiology

Documents

ComparativeProteomicAnalysisofProteinsInvolvedin ...downloads.hindawi.com/archive/2010/726968.pdf · carcinomas of the bladder, prostate, or upper tracts. Very few studies have been