Proteomic Analysis of Peritoneal Fluid in Women with Endometriosis Simone Ferrero,*,†,‡ David J. Gillott,†,§ Valentino Remorgida,‡ Paola Anserini,‡ Kit-Yi Leung,| Nicola Ragni,‡ and Jurgis G. Grudzinskas†,§ Reproductive Physiology Laboratory, St. Bartholomew’s School of Medicine & Dentistry, Queen Mary University of London, United Kingdom, Department of Obstetrics and Gynaecology, San Martino Hospital and University of Genoa, Italy, The London Bridge Fertility, Gynaecology, and Genetics Centre, London, United Kingdom, and William Harvey Research Institute, Barts and The London, Queen Mary University of London, United Kingdom Received December 19, 2006
This study aims to evaluate differences in the expression of proteins present in the peritoneal fluid (PF) of women with and without endometriosis. PF samples were subjected to two-dimensional gel electrophoresis; protein spots of interest were identified by liquid chromatography tandem mass spectrometry. Several molecules had aberrant expression in PF of women with endometriosis; they may be useful for a better understanding of the pathogenesis of this disease. Keywords: endometriosis • peritoneal fluid • tandem mass spectrometry • proteomics • two-dimensional gel electrophoresis
Introduction Endometriosis is a common, benign, oestrogen-dependent, chronic gynecological disorder associated with pelvic pain and infertility. It is defined as the presence of endometrial glands and stroma outside the uterus, mainly on the pelvic peritoneum, on the ovaries, and in the rectovaginal septum, more rarely in the bowel, diaphragm, pericardium, pleura, and even the brain. It is estimated to affect at least 5% of women of reproductive age. The precise aetiology of endometriosis remains unknown despite years of research. The most widely accepted theory on the pathogenesis of endometriosis is Sampson’s theory1,2 suggesting that this disorder originates from retrograde menstruation of viable endometrial tissue through the fallopian tubes into the peritoneal cavity where it implants on peritoneal surface or pelvic organs. However, as retrograde menstruation is observed in almost all cycling women,3 other factors are likely to promote the development and progression of endometriosis. Several studies provided evidence that endometriosis develops as result of specific molecular characteristics of the peritoneal environment and in particular of peritoneal fluid (PF).4-7 PF is often seen in the pelvic cavity, particularly in the vesico-uterine fold and in the cul-de-sac in proximity with the uterus, fallopian tubes, and ovaries. * Corresponding author. Simone Ferrero, M.D., Reproductive Physiology Laboratory, St. Bartholomew’s School of Medicine & Dentistry, QMW College, 48-53 St. Bartholomew’s Close, St. Bartholomew’s Hospital, West Smithfield, London, EC1A 7BE, U.K. Phone, 00393477211682; fax, 0039010511525; e-mail,
[email protected]. † St. Bartholomew’s School of Medicine & Dentistry, Queen Mary University of London. ‡ San Martino Hospital and University of Genoa. § The London Bridge Fertility, Gynaecology, and Genetics Centre. | William Harvey Research Institute, Barts and The London, Queen Mary University of London.
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Journal of Proteome Research 2007, 6, 3402-3411
Published on Web 08/03/2007
Several previous studies applied proteomics technology to the study of endometriosis.8 Since the 1990s, two-dimensional electrophoresis has been used to investigate molecules involved in the pathogenesis of endometriosis.9 More recently, Tabibzadeh et al.10 compared 2D-PAGE of PF of women with and without endometriosis; however, the gels exhibited a limited number of protein spots (approximately 73), and the identity of the majority of protein spots with abnormal expression in endometriosis was not determined by either immunoblotting or mass spectrometry. Fowler et al.11 investigated the effects of endometriosis on the proteome of human eutopic endometrium by using 2D-PAGE and mass spectrometry; several dysregulated proteins were identified, including molecular chaperones, proteins involved in cellular redox state, molecules involved in protein and DNA formation/breakdown, and secreted proteins. In a similar study, Zhang et al.12 observed abnormal expression of proteins involved in cell cycle, signal transduction, and immunological function. Recently, ProteinChip technology has been applied to the study of protein expressed by endometrium, endometriotic tissues, and normal peritoneum obtained from women with and without endometriosis.13 By using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) combined with semiquantitative computerized analysis and immunoblotting, we have previously demonstrated that the PF of women with endometriosis, when compared with that of control subjects, contains higher levels of a particular isoform of Hp β chain14 and lower levels of an isoform of vitamin D binding protein.15 The present study was designed to search for other specific proteins present in the PF of women with endometriosis using 2D-PAGE combined with liquid chromatography tandem mass spectrometry. 10.1021/pr060680q CCC: $37.00
2007 American Chemical Society
Proteomic Analysis of PF in Women with Endometriosis
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Figure 1. Section of a rectovaginal endometriotic nodule, hemeatoxylin eosin staining demonstrates the endometriotic glands surrounded by stroma.
Materials and Methods Study Population. Subjects were recruited from women scheduled for laparoscopy who were premenopausal and had a cycle length between 26 and 32 days. Exclusion criteria included the following: menstrual bleeding in the day of surgery, signs of pelvic inflammatory disease, pregnancy, breastfeeding, abdominal surgery in the 6 months prior to surgery, hysterosalpingography in the 2 months prior to the surgical procedure, hormonal treatment or intrauterine device in the 3 months prior to the surgical procedure. PF samples were collected from women with endometriosis and control subjects who underwent surgery for infertility or tubal sterilization. No woman included in the control group had previously been surgically treated for endometriosis or had detectable endometriotic lesion at the time of surgery. Transvaginal ultrasound was performed within the month before surgery, and patients with uterine leiomyomas were excluded from the study because our preliminary data suggest that uterine leiomyomas may be associated with pathological changes in the amount and type of protein that predominate in the PF.16 The extent of endometriosis was scored according to the revised staging system designed by the former American Fertility Society17 (rAFS), now the American Society for Reproductive Medicine (ASRM).18 In addition, women with only one type of endometriotic lesion were classified in three subgroups for further analysis: women with superficial endometriotic lesions, women with endometriotic ovarian cysts, and women with deep infiltrating disease (lesions deeper than 5 mm under the peritoneal surface19). The phase of the menstrual cycle (proliferative and secretory) was determined according to cycle history of the patients and to serum hormones levels (LH, FSH, 17-β estradiol, and progesterone).
The study was approved by the Institutional Review Board. Written informed consent was obtained from all participants before surgery. Histological Diagnosis of Endometriosis. All specimens excised at surgery were histopathologically evaluated; the diagnosis of endometriosis was based on the presence of both ectopic endometrial glands and stroma (Figure 1). Only women with histologically confirmed diagnosis of endometriosis in at least one of the lesions excised at surgery were included in the study; patients with endometriosis suspected at laparoscopy by not confirmed at histology were excluded from the study. Specimens removed at surgery were immediately fixed in 4% formaldehyde for 12 h and then embedded in paraffin. Samples were cut in slices 4 µm thick and stained with hematoxylineosin for microscopical evaluation. Peritoneal Fluid Collection. PF samples were collected and processed as previously described.14,15 Briefly, all visible PF was aspirated from the cul-de-sac and the vesico-uterine fold at the beginning of laparoscopy; samples were not used if bleeding into the pelvic cavity from the abdominal stab punctures was observed. PF samples collected from different sites in the pelvis (i.e., cul-de-sac and anterior vesico-uterine fold) were mixed before centrifuging at 600g for 10 min; the supernatant was collected and stored at -80 °C until assayed. Two-Dimensional Gel Electrophoresis. PF samples were subjected to 2D-PAGE combining isoelectric focusing in the first dimension and sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the second dimension. Proteins with isoelectric point (pI) ranging from 4 to 7 and molecular weights (Mr) ranging from 10 to 190 kDa were evaluated. The protein concentration of PF samples was determined using the bicinchoninic acid method adapted to microwells (Perbio Science U.K. Ltd, Cheshire, U.K.) with bovine serum albumin as a standard. Journal of Proteome Research • Vol. 6, No. 9, 2007 3403
research articles 2D-PAGE was performed according to the technique of Gorg et al.20 with minor modifications. PF samples were diluted 1:10 in lysis buffer (9 M urea, 2% CHAPS, 65 mM DTT, 2% IPG buffer, pH 4-7, and 0.05% bromophenol blue). The volume of the solubilized sample containing 90 µg of proteins was mixed with a reswelling buffer (8 M urea, 2% CHAPS, 13 mM DTT, and 2% IPG buffer, pH 4-7) in order to obtain a final volume of 250 µL which was pipetted into the IPG strip holder channels. Precast IPG Dry Strips (pH 4-7, 13 cm, Amersham Pharmacia Biotech, Chalfont St. Giles, U.K.) were lowered onto the mixture with the gel side down and overlayered with mineral oil (DryStrip Cover fluid, Amersham Pharmacia Biotech); the strips were rehydrated overnight at room temperature. IPG strips were subjected to isoelectric focusing by using the Multiphor II apparatus (Amersham Pharmacia Biotech). Strips were prevented from dehydratation and oxidation by covering with mineral oil. The following focusing protocol was used: gradient from 0 to 300 V for 1 min, 300 V for 6 h, gradient from 300 to 3500 V for 5 h, and 3500 V for 8 h and 12 min. The temperature was maintained at 20 °C. The focused strips were prepared for second-dimension separation by incubation in equilibration buffer (6 M urea, 2% SDS, 50 mM Tris-HCl, pH 6.8, 30% glycerol, 65 mM DTT, and 0.01% bromophenol blue) on a rocking table for 15 min. For SDS-PAGE, the equilibrated strips were transferred to a 12% polyacrylamide gel; no stacking gel was used. Electrophoresis was carried out with a gel running buffer containing 25 mM Tris base, 192 mM glycine, and 0.1% (w/v) SDS.21 The running parameter was set as constant power of 5 W per gel at 20 °C until the bromophenol blue front had reached the bottom of the gel. Molecular weight markers (Amersham Pharmacia Biotech) were run on the gels (0.5 µL) alongside. Analytical gels were silver-stained according to Blum22 with minor modifications. The gels were soaked overnight in methanol/acetic acid/water (40:10:50). Then the gels were washed three times for 20 min in water, soaked in 0.02% sodium thiosulphate for 60 s, followed by three times 20 s washing in water. The gels were stained for 20 min in 0.2% silver nitrate and 0.0076% formaldehyde, washed three times in water, and developed in 3% sodium carbonate, 0.019% formaldehyde, and 0.0005% sodium thiosulphate for 5-10 min. Development was stopped with 0.5% glycine for 10 min. After three times of 20 min washes in water, the stained gels were captured wet as high-resolution (600 dots per inch) digital images and saved as tiff files using the UVP GDS8000 system (UVP, Upland, CA). Analysis of Protein Expression. Computer-assisted gel analysis was performed using the Phoretix 2D version 5.1 software (Nonlinear Dynamics, Newcastle, U.K.). This software has been shown to be an objective and accurate method of gel analysis.23 In the current study, we wanted to explore the absence of spots in one study group compared with the other or to detect a difference in spot intensity between the two study groups. The difference in protein spot intensity was determined only for spots consistently present (g90% of the cases) in PF samples of study and control group. After automatic spot detection, the images were edited manually. All protein spots were matched to a virtual reference gel based on all the PF gels. Background subtraction was automatically performed by the software using the mode of average on boundary. The intensity and area of each spot were calculated. The spot volumes were normalized to reduce the 3404
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variation in spot intensity between gels that is caused by differences in protein loading and degree of silver-stain development rather than by differential protein expression. Gels were compared for differences in proteins expression by using the normalized percent volume (%Vo) ) (spot volume/volume of all spots in the gel) × 100. The investigators performed blind studies with respect to the clinical status of the patients for both two-dimensional gels preparation and computerized analysis. Validation of Two-Dimensional Gel Electrophoresis Technique. The reproducibility of the technique was evaluated running one PF sample on six separate gels; the gels were processed by the same operator on different days. 1. Reproducibility of Normalized Percent Volume. A set of 150 spots with %Vo ranging from 0.017 to 1.932 were analyzed (Figure 2). The standard deviation (SD) for %Vo was between 0.002 and 0.095 with an average SD of 0.018. The coefficient of variation (CV%) ranged between 1.018% and 40.086%, and the average CV was 10.728%; 96 spots (64%) had a CV < 10% and 135 (90%) had CV < 20%. 2. Reproducibility of Mr and pI determination. A set of 70 spots ranging from 23.1 to 185.9 kDa and from pI 4.58 to 6.36 were analyzed (Figure 3). The SD for Mr was between 0.104 and 1.975 kDa with an average SD of 0.722 kDa. The SD for pI was ranging from 0.045 to 0.357 kDa with an average SD of 0.144 kDa. Enzymatic Digestion. Preparative gels were stained with a protocol compatible with subsequent digestion, elution, and mass spectrometry. The spots of interest were manually excised from gels using a self-made plunger. After excision, preparative gels were scanned and saved as tiff files; this additional step was performed to compare the position of the spot in analytical and preparative gels. In-gel reduction, alkylation, and digestion with trypsin were performed prior to analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS). Cysteine residues were reduced with DTT (Sigma-Aldrich, Gillingham, U.K.) and derivatized by treatment with iodoacetamide (Sigma-Aldrich) to form stable carbamidomethyl (CAM) derivatives. Trypsin (Roche, Lewes, U.K.) digestion was carried out overnight at room temperature after an initial incubation for 1 h at 37 °C. Liquid Chromatography Tandem Mass Spectrometry. Peptides were extracted from the gel pieces by a series of acetonitrile (VWR International, Poole, U.K.) and ammonium bicarbonate (Sigma) washes. The extract was pooled and lyophilized. Each sample was then resuspended in 50 mM ammonium bicarbonate and analyzed by LC-MS/MS. Chromatographic separations were performed using CapLC system (Waters Corporation, Manchester, U.K.). Peptides were resolved by reversed-phase chromatography on a 75 µm C18 column. A gradient of acetonitrile in 0.05% formic acid was delivered to elute the peptides at a flow rate of 200 nL/min. Peptides were ionized by electrospray ionization using a Z-spray source fitted to a QTof-micro (Water Corporation). The instrument was set to run in automated switching mode, selecting precursor ions based on their intensity, for sequencing by collision-induced fragmentation. The MS/MS analyses were conducted using collision energy profiles that were chosen based on the m/z and the charge state of the peptide. Database Searching. The mass spectral data were processed into peak lists and searched against the Swiss-Prot Database using Mascot software (Matrix Science, London, U.K.). Carbamidomethyl (C) and oxidation (M) were set as variable modi-
Proteomic Analysis of PF in Women with Endometriosis
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Figure 2. Silver-stained two-dimensional gel of peritoneal fluid. Protein spots circled in blue were used to determine the reproducibility of normalized percent volume.
Figure 3. Silver-stained two-dimensional gel of peritoneal fluid. Protein spots circled in red were used to determine the reproducibility of Mr and pI.
fications within the searching parameters. A high level of confidence can be assigned to these proteins identities with multiple matched peptides from each protein. Peptides with Mascot score of more than 30 were considered significant hits. Statistical Analysis. Demographic characteristics of the study population were compared using the Student’s t test and the
χ2 test. The comparison of protein spots expression between women with and without endometriosis was performed by the nonparametric Mann-Whitney test. Comparisons between multiple groups were performed using the one-way analysis of variance and Kruskal-Wallis one-way analysis of variance on ranks according to data distribution; when significative differJournal of Proteome Research • Vol. 6, No. 9, 2007 3405
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Table 1. Demographic Characteristics of the Patients endometriosis (n ) 97)
Age (years, mean ( SD) Previous live birth [n (%)]
control group (n ) 50)
32.2 ( 5.4 29 (29.9%)
33.9 ( 5.8 27 (54.0%)
Indications for surgery [n (%)]a -infertility 37 (38.1%) -dysmenorrhoea 43 (44.3%) -deep dyspareunia 26 (26.8%) -chronic pelvic pain 27 (27.8%) -ovarian cyst 48 (49.5%) -tubal sterilization 0 (0.0%)
24 (48.0%) 0 (0.0%) 0 (0.0%) 0 (0.0%) 0 (0.0%) 26 (52.0%)
Phase of the menstrual cycle [n (%)] -proliferative 48 (49.5%) -secretory 49 (50.5%)
25 (50.0%) 25 (50.0%)
a
p
0.078 0.004
