Abstract
Chorionic villi samples are widely used for prenatal diagnosis of various fetal disorders. Although, our knowledge regarding the molecular level of these disorders is extensive, little is known about the implicated proteins. In the present study, two dimensional electrophoresis (2-DE) followed by mass spectrometry (MS) was applied to reveal the proteomic profile of the CV cells. This proteomic technique was previously used successfully in the cases of amniotic fluid, follicular fluid and maternal blood, but has not yet been applied to CV. Therefore, 2-DE was combined with matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF/MS) to characterise the proteome of normal CV cultured cells. Two hundred eighty two-individual gene products were identified including cytoplasmic and nuclear proteins. Although the majority of the proteins were enzymes, structural, signalling and carrier molecules were also isolated. 2D protein map elucidates 282 protein molecules expressed in the CV cells that can be used as a reference for future comparison to various pathological conditions.
- chorionic villus sampling
- trophoblast
- pregnancy
- proteomics
- 2-DE protein database
- mass spectrometry
- MALDI-MS
Abbreviations: CVS: chorionic villus sampling, PMF: peptide mass fingerprint, PSD: post source decay, MALDI: matrix-assisted laser desorption/ionization, IPG: immobilized pH gradient, pI: isoelectric point.
Current prenatal screening methodology can be divided into invasive and non-invasive approaches. The most commonly used invasive techniques are amniocentesis and chorionic villi sampling, while non-invasive procedures include ultrasound examination and screening of maternal blood. CVS remains the method of choice when prenatal diagnosis is required during the early stages of pregnancy, between the 10th to 15th weeks of gestation.
The screening of cultured CV cells by karyotyping, fluoresensce in situ hybridization (FISH), PCR, reverse transcription PCR (RT-PCR) and quantitative fluorescence PCR (QF-PCR) analyzes the nucleotides, DNA, RNA or/and their derivative structures (chromosomes), but diagnostic tests based on protein findings are limited. Currently, proteomic technology is being used in an attempt to identify protein biomarkers linked to pregnancy and contribute to the comprehension of the underlying pathophysiology and the discovery of molecules that mark the fetal genetic diseases or pregnancy complications (1-3). Since proteomic strategies investigate multiple molecules simultaneously, they can potentially lead to the discovery of a ≪panel≫ of markers with sufficient sensitivity and specificity for clinical application. The goal is to identify biomarkers that differ from the “normal” proteomic profile and are specific for aneuploid or disease-affected pregnancy.
Proteomic approaches have previously been applied to study Down and Turner syndromes by analysing tissues from fetuses, amniotic fluid and maternal plasma (4-7). Proteins, such as serotransferin, lumican, plasma retinol-binding protein, apolipoprotein A-I, alpha-1-microglobulin, collagen alpha 1 (I), collagen alpha 1 (III), collagen alpha 1 (V), basement membrane-specific heparin sulfate proteoglycan core protein and insulin binding protein 1 were identified demonstrating a potential link to these pathological phenotypes, further evaluation is required to prove the diagnostic potential of these molecules and their possible future use as biomarkers. Moreover, proteomic analysis has been performed on the placental villous tissue of early pregnancy loss (8) as well as, on the human follicular fluid of recurrent spontaneous abortions (9). However, a detailed two-dimensional (2D) protein database of the normal human CV does not exist. There is an emerging medical need for such a tool, considering the implication of CV in prenatal screening and its importance for diagnosis during the early stages of pregnancy. In the present study the proteomic analysis and the identification of the proteomic profile of cultured normal chorionic villi cells are reported.
Materials and Methods
Materials and reagents. Immobilized pH-gradient (IPG) strips, IPG buffers and acrylamide/piperazine-di-acrylamide (PDA) solution (37.5:1 w/v) were purchased from Biorad Laboratories (Hercules, CA, USA). 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) was obtained from Roche Diagnostics (Mannheim, Germany), urea from AppliChem (Darmstadt, Germany), thiourea from Fluka (Buchs, Switzerland), and 1,4-dithioerythritol (DTE) and EDTA from Merck (Darmstadt, Germany). All the reagents were stored at 4°C, except for CHAPS which was stored at 23°C.
Cell cultures. Chorionic villi samples were obtained from 20 women, during the 11th-13th week of gestation. CVS was performed due to irregular findings during the first trimester tests of pregnancy. Written informed consent was obtained from all women. Samples were subjected to conventional cytogenetic and molecular analysis to certify the normal state of the fetuses. The tissue samples were dissected and cultured in Amniomax culture medium (Invitrogen, Carlsbad, CA, USA) at 37±0.5°C, 5% CO2 for 9-10 days. When the cells reached confluence they were harvested by trypsinization and centrifuged at 1000 × g for 10 min. The cell pellet was washed using 0.9% NaCl and recentrifuged at 1000 × g for 10 min.
Two-dimensional electrophoresis (2-DE). The cell pellets were homogenized in TRI reagent as recommended by the manufacturer (Ambion/Applied Biosystems, Austin, TX, USA) and the RNA and DNA was removed. The protein fractions were resuspended in urea lysis buffer (20 mM Tris, 7 M urea, 2 M thiourea, 4% CHAPS, 10 mM 1,4-DTE, 1 mM EDTA) and a mixture of protease inhibitors [1 mM phenylmethylsulfonyl fluoride (PMSF) and 1 tablet complete™ (Roche Diagnostics, Basel, Switzerland)] and pooled together.
The protein concentration was determined by Bradford colorimetric assay (10) using a Biorad protein assay (Biorad Laboratories). Isoelectric focusing was performed, as previously described (11). Triplicate samples of 1.0 mg total protein were applied on immobilized 3-10 pI or 4-7 pI non-linear gradient strips (17 cm) in sample cups at their basic and acidic ends. Focusing was initially performed at 250 V for 30 min and the voltage was gradually increased to 5000 V at 3 V/min and remained constant for an additional 16 h.
The second-dimensional separation was performed in 12% SDS-polyacrylamide gels (180×200×1.5 mm), running at 40 mA per gel in a Protean apparatus (Biorad Laboratories). Fixation was performed in 50% methanol, containing 10% acetic acid for 2 h; the gels were stained overnight with colloidal Coomassie blue (Novex, San Diego, CA, USA), washed twice with H2O and scanned in a densitometer (GS-800 Calibrated Densitometer, Biorad Laboratories).
Peptide mass fingerprint (PMF). PMF analysis was essentially performed as described previously (12). Briefly, all the spots on the gels were annotated semi-automatically using Melanie 4.02 software (GeneBio, Geneva, Switzerland), excised with a Proteiner SPII robot (Bruker Daltonics, Bremen, Germany) and placed into 96-well microtiter plates. The excised spots were destained using 180 μl of 100 mM ammonium bicarbonate in 30% acetonitrile (ACN) and the gel piece was dried in a MaxiDry Plus speed vacuum concentrator (Heto-Holten A/S, Allerød, Denmark). The dried gel piece was rehydrated with 5 μL of 20 μg/mL recombinant trypsin (Proteomics grade, Roche Diagnostics, Basel, Switzerland) solution. After 16 h at room temperature, 10 μL of 50% acetonitrile containing 0.3% trifluroacetic acid (TFA) were added and the gel pieces were incubated for 15 min with gentle shaking. Sample application to a target plate and analysis as well as peptide matching and protein searching were carried out as described previously (12). Briefly, tryptic peptide mixtures (1 μL) were applied on an anchor chip MALDI plate with 1 μL of matrix solution, consisting of 0.08% α-Cyano-4-hydroxycinnamic acid (CHCA) (Sigma-Aldrich, Taufkirchen, Germany), and the internal standard peptides des-Argbradykinin (Sigma, 904.4681 Da) and adrenocorticotropic hormone fragment 18-39 (Sigma, 2465.1989 Da) in 65% ethanol, 50% ACN and 0.1% TFA. The peptide mixtures were analysed in a MALDITOF mass spectrometer (Ultraflex II, Bruker Daltonics). Laser shots (n=400) of intensity between 40% and 60% were collected and summarized and the peak list was created using the FlexAnalysis v2.2 software (Bruker). Peak list was created with Flexanalysis v2.2 software (Bruker). Smoothing was applied with the Savitzky-Golay algorithm (width 0.2mz, cycle number 1). A signal to noise (S/N) threshold ratio of 2.5 was allowed. The SNAP (Sophisticated Numerical Annotation Procedure) (Bruker) algorithm was used for peak picking. Tryptic autodigest as well as commonly occurring keratin contaminant peaks were filtered out by the software prior to the protein identification process. Peptide matching and protein searches were performed automatically with Mascot Server 2 (Matrix Science, London, UK). Peptide masses were compared with the theoretical peptide masses of all available proteins from Homo sapiens in the Swiss-Prot database. Stringent criteria were used for protein identification with a maximum allowed mass error of 10 ppm and a minimum of four matching peptides. Mascot score ≥55 indicating a probability score with p<0.05 was used as the criterion for affirmative protein identification. Monoisotopic masses were used, and one missed trypsin cleavage site was calculated for proteolytic products.
Results
All visible spots of all 2D gels were mapped by the 2D ImageMaster software (Amersham Biosciences). The protein gels of each pI range were almost identical, revealing the reproducibility of the applied methodology. On the 3-10 pI gels an average of 956 spots per gel were detected (Figure 1A), while ~691 spots were identified on each 4-7 pI gel (Figure 1B). All the detected spots were excised from the gels and the protein contained in the spots were identified by MALDI-MS on the basis of peptide mass matching (13), following in-gel digestion with trypsin. The peptide masses were matched to the theoretical peptide masses of all proteins from all databases, resulted in 250 and 130 discrete identifications for the 3-10 (Figure 2) and the 4-7 pI range gels respectively.
After comparing and combining the above results, 282 individual molecules were identified from both pI ranges, with an identification rate of 80%. The introduction of internal peptide standards to correct the measured peptide masses allowed the use of very narrow windows of mass tolerance (0.0025%), thus increasing the confidence of identification.
The abbreviated and full names of the proteins, their Swiss-Prot accession numbers, the theoretical molecular weight and pI values, as well as data from the mass spectrometry analysis, i.e. the Mascot scores and the protein amino acid sequence coverage by the matching peptides are listed in Table I. A minimal number of 5 matching peptides was used for protein identification. In some cases, mainly for proteins of low molecular mass that usually deliver few peptides, the identification was based on 4 matching peptides. Only human proteins were considered for the searching procedure, increasing the confidence of the identification. Thus, Mascot scores ≥55 indicated that the identification may be considered as unambiguous.
The subcellular localization of the identified proteins was analyzed using publicly accessible data bases (Figure 3A). According to the Swiss-Prot database 25% of the identified proteins were located in both nucleus and cytoplasm, 5% predominantly in the nucleus and 65% were exclusively cytoplasmic. The latter can be divided into mitochondrial (21%), endoplasmic reticulum proteins (10%), cytoskeletal (10%), cytosolic (7%), ribosomal (5%), and inner-membrane proteins (7%). The remaining are either of unknown localization, secreted or extracellular.
Functional analysis (Figure 3B) according to the publicly available UniProtKB database indicated that 118 of the identified proteins were enzymes (28.3%), 60 were regulatory (14.4%), 43 were structural (10.3%), 36 were carriers (8.6%), 30 chaperons (7.2%), 26 RNA-associated (6.2%), 19 implicated in signaling (4.6%) and the rest of them in other biological functions, with smaller percentages.
Discussion
The majority of the 282 proteins identified in this study were enzymes, as expected; however, structural, signalling and carrier molecules were also isolated. Interestingly, one of the molecules identified in this study is Y-box-binding protein 2, the expression of which is normally restricted to germ cells and placental trophoblasts. More specifically, it is observed in oocytes and testicular germ cells in the stage of spermatogonia to spermatocyte, as well as, in placental trophoblasts and it is up-regulated in various carcinomas and germ cell tumors (13, 14). Another interesting molecule was stomatin-like protein 2 (SLP-2), an unusual stomatin homologue of unknown functions, probably implicated in cell motility, proliferation and the cell cycle (15). Recently, the involvement of SLP-2 in human endometrial adenocarcinoma and the effects of SLP-2 on endometrial adenocarcinoma cell growth were demonstrated (16). Another group of proteins identified in CV includes transcription factors or molecules that are involved in transcription such as protein DJ-1 and transgelin. These proteins might also be involved in the regulation of the stability and/or translation of germ cell mRNAs, as well as, in postranlational modification. Further analysis is needed to characterize the role and normal function of these molecules in the chorionic villi. Interestingly, collagen alpha 1 that was identified in the CV proteomic maps, seems to play a critical role in Down syndrome since its chains (I), (III) and (V) have been found to be upregulated in amniotic fluid cells from Down syndrome cases (6). The collagen alpha 1(VI) chain was identified in this study. Comparison of the expression profiles of normal and pathological CV may lead to a more general conclusion about the role of collagen alpha 1 in Down syndrome.
Our previous studies on normal human amniotic fluid cells (AFCs) reported the identification of 432 different gene products (17). Comparative analysis of the present CV and previous AFC proteomic maps indicated that 158 proteins were common, while 124 proteins were found to be expressed only in the CV proteome. In particular, two members of the cystatin (CST or CYT) superfamily of cysteine protease inhibitors, CYTB and CYTN, were expressed only in CV. Cystatins are reversible, competitive inhibitors of cysteine proteases. Their inhibitory profiles, as well as their affinities for target enzymes, vary according to the different cysteine proteases (18). Although in recent studies the overexpression of CYTC was detected in the serum of pregnant women with clinical preeclampsia (19), there are no data regarding the expression of CYTB and CYTN in CV. Another interesting protein overexpressed in CV but not in AFCs is gelsolin (GELS), a calcium-activated actin linked to a number of pathological conditions such as inflammation, cancer and amyloidosis (20). The tight regulation of gelsolin by calcium is crucial for its activation, leading to apoptosis (21). Interestingly, in amniotic fluid supernatant (AFS) from women carrying fetuses with Klinefelter syndrome (47,XXY karyotype) GELS was found to be downregulated (22). Another protein found to be expressed only in CV was prohibitin 2 (PHB2), while PHB was expressed in both CV and AFCs. Prohibitins are ubiquitously expressed proteins, highly conserved throughout evolution and are essential for cell proliferation and embryonic development (23, 24). They are primarily localized in mitochondria but are also found in the nucleus and in the cytoplasmic membrane (25). In mouse models, embryos lacking the PHB genes fail to develop beyond embryonic day 8.5, while depletion of PHB1 or PHB2 impairs proliferation of endothelial cells and mouse embryonic fibroblasts. Furthermore, although prohibitins have a predominantly mitochondrial function, they might also demonstrate other functions outside mitochondria, serving as a negative regulator of E2F-mediated transcription (26). Thus, prohibitins might play an essential role during human pregnancy, even from the early stages, but their exact contribution must be further elucidated. By the obtained results it is becoming obvious that the proteomic approach followed in the present study is providing a pool of information regarding the expression profile of the CV.
The proteomic analysis of chorionic villous cultured cells, isolated from first trimester pregnancies offers the first 2D protein map of CV that will supply valid and interesting leads for future studies. It also provides a reference for future comparison with various fetal pathological conditions which may help in the discovery of diagnostic markers.
- Received April 13, 2011.
- Revision received July 7, 2011.
- Accepted July 8, 2011.
- Copyright © 2011 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved