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in vivo 20: 479-490 (2006)

The Normal Human Amniotic Fluid Supernatant Proteome

GEORGE T. TSANGARIS1, AGELIKI KOLIALEXI2, PANAGIOTIS M. KARAMESSINIS1, ATHANASIOS K. ANAGNOSTOPOULOS1, ARIS ANTSAKLIS3, MICHAEL FOUNTOULAKIS1 and ARIADNI MAVROU2

1Division of Biotechnology, Center of Basic Research, Foundation for Biomedical Research of the Academy of Athens, Athens; 2Medical Genetics and 31st Department of Obstetrics & Gynecology, Athens University School of Medicine, Athens, Greece

Abstract. Proteomic analysis combining two-dimentional diagnostic markers. The application of this new technology electrophoresis (2DE) and mass spectrometry (MS) has the seems to have an important impact on the recognition of potential for a wide range of applications in biological and mechanisms of - related disorders and fetal medical sciences, as screening in tissues obtained from genetic abnormalities (1). healthy and diseased conditions can determine drug targets and Amniotic fluid (AF) is known to contain large amounts diagnostic markers. Conventionally, amniotic fluid (AF) samples of whose expression reflects the genotypic are routinely used for prenatal diagnosis of a wide range of fetal constitution of the and regulates feto-maternal abnormalities. Proteomics have already been applied in the physiological interaction (2). An intricate balance of analysis of tissues from with Down’s syndrome, in order to proteins is required throughout pregnancy and in cases of detect differences in their protein profile as compared to the pregnancy complications or fetal genetic abnormalities, this normal profiles and to determine possible diagnostic tools. A balance may be disturbed (3-5). Identification of these detailed protein 2DE for the normal human AF has not been changes therefore may be used for the detection of a reported. In the present study, the 2D protein database of the particular type of pathology. normal human AF supernatant (AFS) was constructed. Ten AFS Proteomic studies of AF have been recently used for the samples from women carrying normal fetuses were analysed by detection of rupture of amnion and the diagnosis of intra- 2DE. A mean of 412 spots per gel were analyzed and protein amniotic infection (6). Furthermore, distinct proteomic identification was carried out by MALDI-MS and MALDI-MS- profiles in amniotic fluid supernatant (AFS) of normal fetuses MS. A 2D protein map comprising of 136 different products and fetuses with abnormal karyotypes were identified, but was constructed. The majority of the identified proteins are these proteins have not as yet been characterized (7). regulatory proteins, enzymes, secreted proteins, carriers and We recently reported a 2-D database of normal human immunoglobulins. Twelve hypothetical proteins were also amniotic fluid cells comprising 432 different gene products included. The normal AFS proteome map is a valuable tool for (8), but a complete protein map of all human amniotic fluid the study of aberrant protein expression and the search for proteins components, including soluble fragment does not as yet exist. as possible markers for the prediction of abnormal fetuses. In the present study, the detailed 2-D protein database of normal human AFS consisting of 136 different proteins is Proteomic analysis, which combines two-dimensional reported. This map can be used as a reference for the electrophoresis (2DE) and mass spectrometry (MS) has identification of differentially expressed proteins that are wide applications in protein screening in tissues obtained secreted in the amniotic fluid in cases of aneuploidies or from healthy and diseased states for the discovery of novel pregnancy complications. Materials and Methods

Ten ml of human AF samples were obtained after written Correspondence to: George Th. Tsangaris, Genomics and informed consent from 10 women in the 16th-18th week of Proteomics Research Units, Division of Biotechnology, Center of pregnancy undergoing prenatal diagnosis for advanced maternal Basic Research, Foundation for Biomedical Research of the age. Within 1-2 hours the samples were centrifuged at 1000 xg for Academy of Athens, Athens, Greece. Tel: 0030 210 6597075, 10 min and the supernatant was removed and stored at –80ÆC e-mail: [email protected] until further processing. All women were normotensive, free of any medical history and under no medication. By conventional Key Words: Amniotic fluid supernatant, proteomics, two- cytogenetic analysis it was shown that they all carried dimensional protein database, mass spectrometry. chromosomally normal female fetuses.

0258-851X/2006 $2.00+.40 479 in vivo 20: 479-490 (2006)

2D gel electrophoresis. AFS were precipitated using 3 volumes of Results 100% ice cold acetone at –20ÆC overnight and the pellet was resuspended in 0.5 ml buffer consisting of 20 mM Tris-HCl (pH 8.5) The protein profiles of all ten AFS analyzed were to a large 7 M urea, 2 M thiourea, 4% CHAPS, 10 mM 1,4-dithioerythritol, extent similar. A mean of 412 spots per gel was detected, 1 mM EDTA, 1 Ìl/ml of a mixture of inhibitors (1 mM resulting in the identification of 136 different gene products phenylmethylsulfonyl fluoride, 1 tablet completeì (Roche Diagnostics, Mannheim, Germany) per 50ml of suspension buffer) with an identification rate of 90%. A 2-D gel analysis of normal AFS when 1mg of total protein was applied is provided and phosphatase inhibitors (0.2 mM Na2VO3 and 1 mM NaF). After centrifugation at 14,000 xg, the protein content in the in Figure 1. Small variations observed were possibly due to supernatants was determined using the Coomassie blue method (9) allelic differences or to technical artifacts. and was quantitated with Agilent 2100 Bioanalyzer equipped with (ALBU) was the most abundant protein, Protein 200 Plus LabChip Kitì (Agilent Technologies Inc, represented in the 2-D gel by a mean of 158 spots per gel Waldbronn, Germany). (57%), followed by alpha-1-antitrypsin (A1AT), serotransferrin Normalized proteins were used for 2DE as previously described (10): 1 mg protein was applied on immobilized pH 3-10 nonlinear (TRFE) and immunoglobulin heavy chain represented by 14 gradient strips (18 cm). Focusing started at 250 V after which the spots each. Ig kappa chain C region and (FINC) voltage was gradually increased to 6000 V with 3 V/min and was were represented by an average of 8 spots per gel and Vitamin continued for 18 h. The second-dimensional separation was D-binding protein (VTDB) by 7 spots. The SWISS-PROT performed in 12% SDS-polyacrylamide gels (180x200 x 1.5 mm). accession numbers, abbreviations and full names, theoretical The gels were run at 50 mA per gel in an ETTAN apparatus and approximate Mr and pI values, as well as data from the (Amersham Biosciences, Uppsala, Sweden), fixed with 50% MS analysis are listed in Table I. ethanol containing 5% phosphoric acid, stained with colloidal Coomassie blue and scanned using the GS-800 Calibrated A total of 76 (56%) proteins were identified by PMF and 60 Densitometer (Bio-rad Laboratories, Hercules, CA, USA). (44%) by PSD-MS-MS. Two proteins (O14502 and Q63061) were identified by one matching peptide after PSD-MS-MS MALDI-MS and MALDI MS-MS analysis. The MALDI-MS analysis, 11 by 2 (6/11 by PMF analysis and 5/11 by PSD-MS- analysis was performed as described by Fountoulakis and Gasser MS). Seventeen proteins were identified by 3 matching (11). Spots were excised, placed into 96-well microtiter plates, peptides (7 PSD-MS-MS and 10 after PMF) and the remaining destained with 100 Ìl of 30% acetonitrile in 50 mM ammonium 107 proteins using 4 to 24 matching peptides. bicarbonate and dried in a speedvac evaporator. Each dried gel Functional analysis indicated that 18 of the identified piece was rehydrated with 4 Ìl of 3 mM Tris-HCl, pH 9.0, proteins were regulatory (13.23%), 15 enzymes (11.02%), 15 containing 50 ng trypsin (Promega, Madison, WI, USA). Proteins binding proteins (11.02%), 11 were related to transportation were extracted using 4 Ìl of 50% acetonitrile, containing 0.3% trifluoroacetic acid. A peptide mixture (1.5 Ìl) was simultaneously (8.08%) and 9 were carriers of various substrates (6.61%) applied with 1 Ìl of matrix solution, consisting of 0.025% (Figure 2A). ·-cyano-4-hydroxycinnamic acid (Sigma, St. Louis, MO, USA), According to data obtained from databases, 29 (21.32%) of standard peptides des-Arg-bradykinin (Sigma, 904.4681 Da) and the identified proteins were reported as secreted, 16 (11.76%) adrenocorticotropic fragment 18-39 (Sigma, 2465.1989 in the extracellular space and 11 (8.08%) were membranic Da) in 65% ethanol, 35% acetonitrile and 0.03% trifluoroacetic (Figure 2B). Seventy proteins with unknown localization and acid. The samples were analyzed for PMF with MALDI-MS in a 58 with unknown function, including 13 hypothetical proteins, time-of-flight mass spectrometer (Ultraflex, Bruker Daltonics, Bremen, Germany). Matching peptide and protein searches were were also identified. performed automatically as described by Berndt et al. (12) and each spectrum was interpreted by the Mascot Software (Matrix Discussion Sciences Ltd, London, UK). For peptide identification, the mono- isotopic masses were used and a mass tolerance of 0.0025% A 2-D database of normal human AFS proteins consisting of (25ppm) was allowed. Unmatched peptides or peptides with up 136 different gene products was constructed. Only samples to one miscleavage site were not considered. The peptide masses from with female fetuses were used in order to were compared with the theoretical peptide masses of all available proteins from all species using SWISS-PROT, IPI, and avoid the identification of proteins related to the sex of the MSDB databases. The probability score identified by the software fetus. The introduction of internal peptide standards to correct was used as the criterion of the identification. Samples not the measured peptide masses permitted use of very narrow identified by PMF (probalilty significance of p<0.05) were windows of mass tolerance (0.0025%), increasing the automatically selected for post source decay (PSD) MS-MS confidence of identification. Additionally, the inclusion of analysis with MALDI-MS-MS. The peptide masses chosen for homologous mammalian proteins in the protein search list PSD-MS-MS analysis had a signal intensity of >600 counts and increased the confidence of identification up to 90%, allowing were excluded from the trypsin autodigest, matrix and keratin peaks. The resulting PSD spectra were also interpreted by the for the identification of 15 proteins from other species based Mascot Software and Mascot probability based scores of p<0.02 on the high homology of the corresponding proteins. In some were considered significant. cases the identified proteins could not be assigned, most likely

480 Tsangaris et al: The Amniotic Fluid Supernatant Proteome

Figure 1. Two-dimensional gel analysis of normal human amniotic fluid supernatant stained with Coomassie blue. because neither the human nor a highly homologous are of fetal or maternal origin. The remaining 116 have been counterpart from other species was included in the database, previously reported in the human AFS (Table I). Existing data or since number of matching peptides generated from the indicate that 35 are expressed during pregnancy in various digestion of low-molecular-mass proteins was insufficient. maternal and fetal tissues, 23 have been previously detected Since proteins can alter throughout pregnancy, this work only in AF and 58 are found in the placenta, AF or fetal and presents a reference map of the 17th week of gestation. Seven embryonic tissues. Further analysis revealed that 15/58 known out of 136 proteins identified in AFS have also been reported proteins are expressed and secreted from the fetal liver, 7 from in AF cells and their presence in AFS is possibly due to cell the placenta, 4 from fetal membranes, 3 from fetal kidney, 4 damage during manipulations. Seventy proteins with unknown from the deciduas, 2 from the small intestine and one each localization and 58 proteins with unknown function, including from membrane-decidual contact, fetal lung and fetal 13 hypothetical proteins, were identified. parenchymal cells respectively (Table II). Fifteen proteins Twenty proteins identified in the present study are reported detected in the AFS are known to bind and carry other for the first time in human AF, but it is not known as yet if they molecules and 11 are related to the transportation of iron, fatty

481 in vivo 20: 479-490 (2006)

Table I. Protein database of normal human amniotic fluid supernatant.

Name Protein Accession Full Identifi- pI Protein Pepti-Score Cove- Function Locali- number name cation MW des rage zation

1AOJA eps8 sh3 domain, chain A - Mouse PSD 2.26 7029.57 4 39** 60 2ROYB 2ROYB 2ROYB , chain B - Human PMF 5.29 13082.56 5 71* 57 AAA52002 Alpha-1 type III (fragment)- PSD 36978.00 5 70** 23 Human AAB03452 Immunoglobulin heavy chain variable PSD 7.94 11492.44 4 33** 61 region (fragment) - Human AAB48817 Anti-DNA immunoglobulin light chain PSD 9.39 11126.30 4 39** 34 IgG (Fragment) - Mus musculus (Mouse) AAB49121 Anti-DNA immunoglobulin heavy chain PSD 9.88 12676.25 5 45** 75 IgG (Fragment) - Mus musculus (Mouse) AAD20496 Immunoglobulin heavy chain variable PMF 9.48 13393.49 3 57* 22 region (fragment) - Human AAH14308 BC014308 NID: - Human PSD 8.00 23321.79 6 74** 44 AAH60271 BC060271 NID: - Mus musculus PSD 40926.84 9 39** 29 [RIKEN cDNA 2610207F23 (Q6PAJ0)] AAK84191 Anti- immunoglobulin PMF 8.70 12053.85 5 58* 49 heavy chain variable region (Fragment) - Human AAO40400 Immunoglobulin heavy chain (Fragment)- PSD 9.50 3313.58 3 39** 89 Mus musculus (Mouse) AAQ05354 AF471179 NID: - Human PMF 5.04 13392.51 2 59* 34 AAQ05379 AF471204 NID: - Human PMF 4.99 13201.33 3 57* 52 AAS54981 AY539809 NID: - Human PMF 23283.50 5 78* 37 BAA37169 IgG kappa chain (Fragment)- PMF 6.92 23689.59 4 60* 31 Human BAA75038 Immunogloblin heavy chain variable PSD 7.74 12973.38 5 39** 64 region (Fragment)- Human BAA82105 Anti-Entamoeba histolytica immuno- PMF 8.26 23575.69 5 71* 39 kappa light chain (Fragment)- Human BAC01670 BAC01670 BAC01670 Immunoglobulin kappa light chain VLJ PMF 6.71 29116.22 4 62* 30 region (Fragment) - Human BAC01671 Immunoglobulin kappa light chain VLJ PMF 7.53 29329.33 4 60* 30 region (Fragment) - Human BAC01750 Immunoglobulin kappa light chain VLJ PMF 6.73 28806.21 6 76* 36 region (Fragment) - Human BAC02031 Immunoglobulin heavy chain VHDJ PMF 5.17 12478.08 2 57* 42 region (Fragment) - Human BAC02120 Immunoglobulin heavy chain VHDJ PSD 6.86 14323.94 3 41** 45 region (Fragment) - Human BAC85395 AK130614 NID: - Human PMF 6.75 51893.59 5 57* 17 BAC86226 AK125633 NID: - Human PSD 8.58 49942.10 5 48** 17 CAA00975 APOA1 protein (fragment) - PMF 5.27 28061.47 7 66* 28 Human CAC10254 Immunoglobulin heavy chain PMF 8.30 40579.99 6 67* 22 (Fragment) - Human CAC29446 T-cell receptor alpha chain (Fragment) - PSD 8.18 3241.67 3 33** 100 Human CAD42145 Sequence 5 from Patent WO0250290 PMF 7.18 39172.21 5 44* 21 (Fragment) - Human CAD88056 Sequence 7 from Patent WO03014151- PSD 9.04 24977.76 7 68** 36 Human CAE98494 AX884380 NID: - Human PSD 11124.85 5 39** 39 CAF01148 CAF01148 CAF01148 AX887010 NID: - Human PSD 8611.56 7 39** 64 CAF01562 AX887424 NID: - Human PSD 6044.21 5 42** 69 CAF14612 CAF14612 CAF14612 AX969504 NID: - Human PSD 18034.93 5 43** 43 CAF15299 CAF15299 AX970191 NID: - Human PSD 16797.37 7 40** 57 CAF16207 AX971099 NID: - Human PSD 10832.26 6 43** 54 continued

482 Tsangaris et al: The Amniotic Fluid Supernatant Proteome

Table I. continued

Name Protein Accession Full Identifi- pI Protein Pepti-Score Cove- Function Locali- number name cation MW des rage zation

CHLR1 Q92998 G02071 CHL1 potential helicase (CHLR1) PSD 10.02 11865.28 5 42** 46 DNA Nucleus [helicase - human (fragment)] binding, enzyme, (hydrolase activity) IPI: 00402619 IPI00402619 Tax_Id,9606 hypothetical protein PMF 10934.73 2 57* 35 XP_379500 JN0387 JN0387 JN0387 Somatotropin - sei whale PSD 5.33 18896.87 5 41** 27 (tentative sequence) (fragments) O14502 Alpha-2-HS (fragment) - PSD 5.22 38142.80 1 49** 6 Regula- Extracellular Cercopithecus aethiops (Green monkey) tion space (Grivet) VAM4 VAM4_HUMANO75379 Vesicle-associated membrane protein PMF 6.15 16287.45 4 59* 28 Structural Membrane, 4 (VAMP-4) (Tax_Id,9606 Splice protein, Golgi isoform 2 of O75379 Vesicule-associated transport, network membrane protein 4, IPI00219648) cellular and trafficking secretory transport granules LHX6 LHX6_MOUSE O88706 LIM/homeobox protein Lhx6 (Lhx6) PMF 9.33 41889.62 5 61* 18 Nuclear (MMLHX6 NID: - Mus musculus, (probably) CAA04011) O95370 CD44 isoform RC (CD44) (Tax_Id,9606 PMF 8.81 15966.72 4 57* 33 CD44 isoform RC, IPI00002541) adhesion PH4H PH4H_HUMAN P00439 Phenylalanine-4-hydroxylase (PAH) PSD 6.15 52342.65 4 45** 16 Enzyme (Phe-4-monooxygenase) (EC 1.14.16.1) (WHHUF) CERU CERU_HUMAN P00450 precursor (EC 1.16.3.1) PMF 5.44 122982.91 12 95* 18 Enzyme Extracellular (Ferroxidase) (ferro- space xidase activity) PLMN PLMN_HUMAN P00747 Plasminogen precursor (EC 3.4.21.7) PMF 7.04 93247.19 9 88* 15 Enzyme, Secreted [Contains: ] coagulation/ hemostasis, CFAB CFAB_HUMAN P00751 Complement factor B [Precursor] PSD 6.00 27548.89 4 57** 31 Enzyme Secreted Glycine-rich beta glycoprotein (peptidase (EC 3.4.21.47) (C3/C5 convertase) family), ( factor B) (Glycine-rich beta proliferation glycoprotein)(GBG, PBF2) (HSMHCBA § and differen- NID: - Human) (CAA25077) tiation ANT3 ANT3_HUMAN P01008 -III precursor (ATIII) PMF 6.32 53025.03 11 90* 27 Serine Secreted (PRO0309) protease inhibitor A1AT A1AT_HUMAN P01009 Alpha-1-antitrypsin precursor PMF 5.37 46878.08 12 175* 44 Inhibitor Secreted (Alpha-1 protease inhibitor) of serine (Alpha-1-antiproteinase) AACT AACT_HUMAN P01011 Alpha-1-antichymotrypsin precursor PMF 5.33 47791.60 9 83* 28 Inhibitor Extracellular (ACT) activity, protein binding CO3 CO3_HUMAN P01024 Complement C3 precursor PMF 6.02 188585.48 13 129* 13 Signal [Contains: C3a anaphylatoxin] transduction, receptor binding, immune response KNG KNG_HUMAN P01042 Kininogen precursor PMF 6.34 72983.52 7 71* 13 Binding, Secreted (Alpha-2-thiol cell proteinase inhibitor) adhesion [Contains: Bradykinin] continued

483 in vivo 20: 479-490 (2006)

Table I. continued

Name Protein Accession Full Identifi- pI Protein Pepti-Score Cove- Function Locali- number name cation MW des rage zation

PRL PRL_HUMAN P01236 [Precursor] (PRL) PMF 6.13 25159.80 5 57* 35 Receptor Secreted (HSPROL3 NID: - Human, CAA25214) binding, KAC KAC_HUMAN P01834 Ig kappa chain C region PSD 5.58 11772.73 3 108** 48 Immune Extracellular response, space antigen binding GC1 GC1_HUMAN P01857 Ig gamma-1 chain C region PMF 8.46 36596.36 9 124* 46 Binding Membrane fraction CA11 CA11_HUMAN P02452 Collagen alpha 1(I) chain precursor PSD 5.60 139824.95 5 82** 4 Structural, Extracellular development CA13 CA13_HUMAN P02461 Collagen alpha 1(III) chain precursor PSD 5.99 139724.44 5 86** 7 Structural Extracellular CAA25879 CAA25879 P02461 HSPCAIII NID: - Human PMF 5.34 27904.47 7 70* 31 APA1 APA1_HUMAN P02647 Apolipoprotein A-I precursor (Apo-AI) PMF 5.56 30758.93 9 79* 34 Binding, Secreted transport, carrier A2GL A2GL_HUMAN P02750 Leucine-rich alpha-2-glycoprotein PMF 6.45 38382.20 6 67* 24 Unknown/ Secreted precursor (LRG) unspecified FINC FINC_HUMAN P02751 Fibronectin precursor (FN) PMF 5.45 266034.43 19 141* 12 Cell Secreted; (Cold-insoluble globulin) (CIG) adhesion, extracellular structural matrix VAHU VAHU P02753 Plasma retinol-binding protein precursor PMF 5.48 23195.28 6 68* 39 Carrier Secreted [Precursor] - human (PRBP) (RBP) (RBP4) AMBP AMBP_HUMAN P02760 AMBP protein precursor PSD 5.95 39886.32 9 87** 22 Trypsin Secreted [Contains: Alpha-1- inhibitor, (Protein HC) (Complex-forming calcium glycoprotein heterogeneous in charge) channel (Alpha-1 microglycoprotein) inhibitor, anti-inflam- matory response, cell adhesion A1AG A1AG_HUMAN P02763 Alpha-1-acid glycoprotein 1 precursor PMF 4.93 23724.84 5 63* 30 Inflam- Secreted (AGP 1) ( 1) (OMD 1) matory response A2HS A2HS_HUMAN P02765 Alpha-2-HS-glycoprotein precursor PMF 5.43 40098.01 6 59* 24 Regulatory, Secreted (Fetuin-A) (Alpha-2-Z-globulin) transport (Ba-alpha-2-glycoprotein) TTHY TTHY_HUMAN P02766 Transthyretin precursor (Prealbumin) PMF 5.52 15991.09 4 76* 47 Binding Secreted (TBPA) (TTR) (ATTR) protein, transport ALBU ALBU_HUMAN P02768 precursor PMF 5.92 71317.25 23 251* 49 Trans- Secreted porter, binding activity, osmoregulation VTDB VTDB_HUMAN P02774 -binding protein precursor PMF 5.40 54525.63 8 102* 26 Carrier, Secreted (DBP) (Group-specific component) transport (Gc-globulin) (VDB) TRFE TRFE_HUMAN P02787 Serotransferrin precursor () PMF 6.81 79280.47 17 170* 27 Transport Secreted (Siderophilin) (Beta-1-metal binding globulin) carrier (PR) A1BG A1BG_HUMAN P04217 Alpha-1B-glycoprotein precursor PMF 5.58 54808.79 9 122* 26 Unknown/ Secreted (Alpha-1-B glycoprotein) unspecified K2C1 K2C1_HUMAN P04264 Keratin, type II cytoskeletal 1 PMF 8.16 66018.01 9 94* 23 Structural, membrane, (Cytokeratin 1) (K1) (CK 1) receptor cytoskeleton (67 kDa cytokeratin) activity IC1 IC1_HUMAN P05155 Plasma protease C1 inhibitor PMF 6.09 55347.48 8 67* 17 Endopepti- Secreted precursor (C1 Inh) (C1Inh) dase inhibitor activity, circulation continued

484 Tsangaris et al: The Amniotic Fluid Supernatant Proteome

Table I. continued

Name Protein Accession Full Identifi- pI Protein Pepti-Score Cove- Function Locali- number name cation MW des rage zation

GELS GELS_HUMAN P06396 precursor, plasma PMF 5.90 86043.34 9 62* 16 Cytoske- Secreted, (Actin-depolymerizing factor) leton cytosol, (ADF) (Brevin) (AGEL) extracellular region APA4 APA4_HUMAN P06727 Apolipoprotein A-IV precursor PMF 5.28 45343.46 11 118* 31 Regulatory, Secreted (Apo-AIV) transport carrier IBP1 IBP1_HUMAN P08833 Insulin-like growth factor binding protein PSD 5.11 28911.69 4 133** 18 Signal 1 precursor (IGFBP-1) (IBP-1) trans- Secreted (IGF-binding protein 1) duction, (Placental protein 12) (PP12) regulation of cell life, proliferation/ growth PAEP PAEP_HUMAN P09466 Glycodelin precursor (GD) (Pregnancy- PMF 5.05 18609.49 4 59* 27 Deve- Secreted associated endometrial alpha-2 globulin) lopment, (PEG) (PAEG) (Placental protein 14) immunity/ (Progesterone-associated defense endometrial protein) (Progestagen-associated endometrial protein) (Tax_Id,9606 Splice isoform 2 of P09466 Glycodelin precursor, IPI00218735) SPRC SPRC_HUMAN P09486 SPARC precursor (Secreted protein acidic PSD 4.73 35465.03 2 62** 20 Regulate Extracellular and rich in cysteine) (Osteonectin) (ON) , space (Basement membrane protein BM-40 ) binding SUIS SUIS_HUMAN P14410 Sucrase-isomaltase, intestinal [Contains: PMF 5.40 210565.50 10 95* 8 Enzyme Membrane Sucrase (EC 3.2.1.48); Isomaltase (EC 3.2.1.10)] AMPN AMPN_HUMAN P15144 Aminopeptidase N (hAPN) (Alanyl PSD 5.31 51666.79 7 44** 17 ∂nzyme Membrane aminopeptidase) (Microsomal aminopeptidase) (aminope- protein. A (EC 3.4.11.2) (HSA427985 NID: - Human, ptidase) soluble form CAD20931) has also been detected A1AH A1AH_HUMAN P19652 Alpha-1-acid glycoprotein 2 precursor PMF 5.03 23872.74 7 60* 39 Secreted (AGP 2) (Orosomucoid 2) (OMD 2) CA15 CA15_HUMAN P20908 Collagen alpha 1(V) chain [Precursor] PSD 4.99 184189.38 7 50** 6 Structural Extracellular (COL5A1) (HUMCA1V NID: - Human, BAA14323) CSR1 CSR1_HUMAN P21291 Cysteine and glycine-rich protein 1 PMF 21409.08 7 64* 38 Develop- Nuclear (Cysteine-rich protein 1) (CRP1) ment, (Tax_Id,9606 cysteine and glycine-rich zinc ion protein 1, IPI00219674) binding FBRL FBRL_HUMAN P22087 Fibrillarin (34 kDa nucleolar PMF10.18 33877.46 4 61* 16 RNA Nuclear scleroderma antigen) (Tax_Id,9606 binding Fibrillarin, IPI00025039) CP7A CP7A_HUMAN P22680 Cytochrome P450 7A1 (Cholesterol PMF 8.48 58250.69 3 55* 8 Enzyme Membrane- 7-alpha-monooxygenase) (CYPVII) bound (EC 1.14.13.17) (Cholesterol 7-alpha-hydroxylase) (Tax_Id,9606 Cytochrome P450 7A1, IPI00305356) FBL1 FBL1_HUMAN P23142 -1 precursor PMF 5.11 81314.98 8 57* 14 Structural Extracellular constituent, matrix cell adhesion and migration ZA2G ZA2G_HUMAN P25311 Zinc-alpha-2-glycoprotein precursor PMF 5.57 34078.98 10 96* 36 Carrier Secreted (Zn-alpha-2-glycoprotein) (Zn-alpha-2-GP) activity, lipid catabolism continued

485 in vivo 20: 479-490 (2006)

Table I. continued

Name Protein Accession Full Identifi- pI Protein Pepti-Score Cove- Function Locali- number name cation MW des rage zation

PDA3_ PDA3_HUMAN P30101 Protein disulfide-isomerase A3 precursor PMF 5.98 57145.90 24 218* 52 Enzyme, Endoplasmic (EC 5.3.4.1) (Disulfide isomerase signal reticulum ER-60) (ERp60) transduction, / stress AKT2 AKT2_HUMAN P31751 RAC-beta serine/threonine-protein PMF 5.98 56132.25 3 60* 9 Enzyme (EC 2.7.1.37) (RAC-PK-beta) (kinase (Protein kinase Akt-2)(Protein kinase B, activity) beta) (PKB beta) (Tax_Id,9606 RAC-beta serine/threonine-protein kinase, IPI00012870) K1CI K1CI_HUMAN P35527 Keratin, type I cytoskeletal 9 PMF 8.16 62177.91 11 124* 35 Epidermis Structural (Cytokeratin 9) (K9) (CK 9) development MYHB MYHB_HUMAN P35749 Myosin heavy chain, smooth muscle isoform PSD 5.06 13154.71 5 44** 37 Muscle (SMMHC) (MYH11) (HUU91323 NID: - contraction, Human, AAC35212) structural protein K22E K22E_HUMAN P35908 Keratin, type II cytoskeletal 2 epidermal PMF 8.07 66110.50 10 93* 24 Epidermis (Cytokeratin 2e) (K2e) (CK 2e) development, structural PEDF PEDF_HUMAN P36955 Pigment epithelium-derived factor PMF 5.97 46484.36 7 63* 22 Serine Secreted precursor (PEDF) (EPC-1) protease inhibitor, development P2Y2 P2Y2_HUMAN P41231 P2Y purinoceptor 2 (P2Y2) (P2U PMF 9.72 43060.38 4 62* 14 Receptor Membrane purinoceptor 1) (P2U1) (ATP receptor) activity (Purinergic receptor) (Tax_Id,9606 P2Y purinoceptor 2, IPI00298653) FRAP FRAP_HUMAN P42345 FKBP12-rapamycin complex-associated PSD 6.34 19009.42 4 39** 45 Regulation protein (FK506-binding protein 12-rapamycin of cell cycle complex-associated protein 1) (Rapamycin target protein) (RAPT1) (HUMRAPT1A NID: - Human, AAC41713) AFAM AFAM_HUMAN P43652 Afamin precursor (Alpha-albumin) PMF 5.64 70962.74 8 70* 14 Unknown Secreted (Alpha-Alb) LUM LUM_HUMAN P51884 precursor ( PMF 6.16 38746.92 8 71* 28 Structural, Secreted; lumican) (KSPG lumican) collagen extracellular binding matrix RETB RETB_PANTR P61641 Plasma retinol-binding protein PSD 5.76 23337.39 5 76** 36 Carrier Secreted precursor (PRBP) (RBP) PDCL Q9UNX1 (PDCL) PDCL Phosducin-like protein (Fragment) - PSD 4.74 16745.24 6 38** 56 Signaling Human (PDCL) protein MMP-3 MMP-3 PW0052 Pro--3 - human PSD 8.04 9453.85 2 39** 35 (fragment) (MMP-3) FABE FABE_HUMAN Q01469 Fatty acid-binding protein, PSD 6.60 15496.68 5 41** 56 Carrier Cytoplasm epidermal (E-FABP) (Fatty acid binding protein homolog - human, I56326) (Psoriasis-associated fatty acid-binding protein homolog) (PA-FABP) ITH4 ITH4_HUMAN Q14624 Inter-alpha-trypsin inhibitor heavy chain PMF 6.51 103522.03 11 76* 12 Endo- Extracellular H4 precursor (ITI heavy chain H4) peptidase region inhibitor Q5SRH3Epidermal growth factor receptor pathway PMF 4.56 84133.91 3 57* 4 Calcium substrate 15 (EPS15 protein) (Tax_Id,9606 ion binding EPS15 protein, IPI00385325) Q5T0H7 Gelsolin (Amyloidosis, Finnish type) (GSN) PMF 5.58 80875.62 4 62* 9 Cyto- (Tax_Id,9606 gelsolin isoform b, IPI00377087) skeleton Q62600 Nitric-oxide synthase 3 - rat (fragment) PSD 6.06 7356.67 4 36** 73 Enzyme (EC 1.14.13.39) (EC-NOS) (NOS, type III) (NOSIII), Endothelial NOS (eNOS), Constitutive NOS (cNOS) continued

486 Tsangaris et al: The Amniotic Fluid Supernatant Proteome

Table I. continued

Name Protein Accession Full Identifi- pI Protein Pepti- Score Cove- Function Locali- number name cation MW des rage zation

Q63061 Hypothetical protein - Rattus PSD 8.03 4762.24 1 39** 77 norvegicus (Rat). Q6N093 Hypothetical protein DKFZp686I04196 PMF 36489.00 3 57* 12 [Fragment] ((DKFZp686I04196) (Tax_Id,9606 Ig gamma-2 chain C region, IPI00399007) Q6P3X8 PiggyBac transposable element PMF 8.82 68880.65 4 58* 10 derived 2 (PGBD2 ) (Tax_Id,9606 PiggyBac transposable element derived 2, IPI00335939) Q6P5P6 Phosphoribosylpyrophosphate synthetase PMF 5.92 35387.18 3 57* 10 Enzyme isoform I [Fragment] (Q15244), LOC221823 (ligase B protein ((Q6P5P6), (Tax_Id,9606 activity) phosphoribosyl pyrophosphate synthetase 1-like 1, IPI00218371) BAC85950 Q6ZVB2 AK124788 NID: - Human PMF 4.23 16285.70 2 57* 27 (Hypothetical protein FLJ42798) Q71S09 Vascular endothelial growth factor 41 PSD 5195.60 3 34** 68 Growth (VEGF41) (AF323587 NID: - factor Human, AAQ14857) activity, cell growth and/or maintenance Q7LDG7 Calcium and DAG-regulated guanine PMF 70059.67 3 59* 8 Kinase nucleotide exchange factor I (Calcium-and activity, diacylglycerol-regulated guanine nucleotide signal exchange factor I) (F25B3.3 kinase like protein) transduction (Guanine exchange factor MCG7 isoform 2) (Tax_Id,9606 Calcium and DAG-regulated guanine nucleotide exchange factor I, IPI00376502) Q7TMH9 Hypothetical protein (Fragment) - PSD 31553.65 9 47** 42 Mus musculus (Mouse). Fbxo28 protein [Fragment] (Fbxo28) KIAA1582 KIAA1582 Q86UE5 KIAA1582 protein [Fragment] PMF 62005.75 2 57* 6 (Tax_Id,9606 Hypothetical protein FLJ31859, IPI00043407) Q86V94 Hypothetical protein - Human PSD 6.96 21174.51 6 42** 41 Immune Membrane response Q86YG0 Similar to alpha-fetoprotein - PSD 5.97 48641.19 6 56** 19 Carrier, Extracellular Human transport Q8C4U1 Hypothetical protein (Fragment) - Mus PSD 11.91 12830.79 4 42** 45 musculus (Mouse). Mus musculus 10 days neonate cerebellum cDNA, RIKEN full-length enriched library, clone:B930078G14 product:hypothetical protein, full insert sequence. [Fragment] TGLY TGLY_HUMAN Q8IUE0 Homeobox protein TGIF2LY PSD 21028.69 4 37** 21 Trans- Nuclear (TGFB-induced factor 2-like protein, cription Y-linked) (TGF(beta)induced transcription factor 2-like protein)(TGIF-like on the Y) (AF332223 NID: - Human, AAK13475) Q8IYE1 Similar to hypothetical protein FLJ25467 - PSD 8.93 80804.38 7 35** 16 Human Q8TCL7 Hypothetical protein (Fragment) - PSD 8.09 27386.92 7 37** 24 Enzyme Human Hypothetical protein DKFZp761M1710 [Fragment] (ubiquitin-protein ligase activity) Q95LT7 Hypothetical protein - Macaca fascicularis PSD 5.78 12503.95 2 36** 20 (Crab eating macaque) (Cynomolgus monkey) continued

487 in vivo 20: 479-490 (2006)

Table I. continued

Name Protein Accession Full Identifi- pI Protein Pepti- Score Cove- Function Locali- number name cation MW des rage zation

Q95ND8 Pro alpha 1(I) collagen (Fragment) - PMF 5.46 32134.45 5 59* 22 Structural, Cytoplasm Bos taurus (Bovine). transport PKC1 PKC1_HUMAN Q96AC1 Pleckstrin homology domain containing PMF 7.21 72864.19 4 60* 9 Regulatory, Extracellular, family C member 1 (Kindlin 2) binding, cytoplasmic (Mitogen-inducible gene 2 protein) cytoskeleton (Mig-2) (Tax_Id,9606 Splice isoform 2 organization of Q96AC1 Pleckstrin homology domain and biogenesis containing family C member 1, IPI00383500) CCD2 CCD2_HUMAN Q96LB3 Coiled-coil domain containing protein 2 PSD 5.56 36679.55 9 49** 40 Enzyme, Intracellular (Capillary morphogenesis protein-1) (partici- vesicular (CMG-1) (Hypothetical protein pates in compart- FLJ22621 - Human, Q9H643). protein- ment protein binding, protein multimerization) Q96NL6 Q96NL6 Q96NL6 Hypothetical protein FLJ30655 - PSD 5.83 81275.92 16 43** 23 Human Q99769 Hypothetical protein - Human PSD 6.24 26787.14 3 46** 27 ERC1 Q8C7Y1 Q99MI1 ERC protein 1(Rab6-interacting protein 2) PSD 9.2 43626.30 11 44** 39 Regulatory, Cytoplasmic (ERC1_) (CAZ-associated structural protein 2) protein and MOUSE (CAST2 ) - Mus musculus (Mouse). binding membrane- associated Q9BED9 AlphaA-crystallin (Fragment) - PSD 11.16 2640.29 2 41** 100 Nycticebus coucang (Slow loris). ANGT ANGT_ Q9GLN8 Angiotensinogen precursor PSD 5.87 53361.59 2 169** 17 Regulate Secreted PANTR [Contains: Angiotensin I (Ang I); volume Angiotensin II (Ang II); and mineral balance of body fluids Q9GZP4 AD039 (Hypothetical protein OTTHUMP00000044843) (Thioredoxin family Trp26) (HT014) PMF 24390.83 3 57* 17 (Tax_Id,9606 AD039, IPI00015351) Q9H5H1 Hypothetical protein FLJ23441 - PSD 5.49 29031.57 3 38** 22 Ligase Cytoplasm Human activity, protein biosynthesis CAI22058 Q9NVS3 Hypothetical protein FLJ10547 - PSD 53616.61 9 39** 26 Human Q9NVY1 Hypothetical protein FLJ10440 - PSD 8.78 46593.05 9 39** 32 Human Q9NZY1 HSPC054 (Tax_Id,9606 HSPC054, PMF 9.13 10716.37 2 57* 35 IPI00023043) RSG3 RSG3_RAT Q9QYJ2 Ras GTPase-activating protein 3 PMF 5.38 14363.02 4 57* 33 Regulator (GAP1(IP4BP)) (Ins P4-binding protein) (R-ras GAP) (Fragment) Q9TS41 Prolactin - Papio (baboons). PSD 5.85 23177.66 7 41** 48 Binding, Secreted signal transduction Q9Y4H7 Human leucocyte antigen DRB1 PSD 6.34 12014.78 4 51** 56 Receptor Membrane (Fragment) - Human activity, immune response CAN6 CAN6_HUMAN Q9Y6Q1 6 (Calpamodulin)(CalpM) PMF 6.62 75383.95 4 61* 7 Not known (Calpain-like protease X-linked) (Tax_Id,9606 Calpain 6, IPI00002547)

Proteins are designated with their abbreviated names or the SWISS-PROT accession numbers or the accession numbers of the other databases. The codes can be deduced from the column "Name". The theoretical Mr and pI values, the matching peptides and the probability of a random identification (Score), as well the annotated subcellular location and function are listed. Score is -10*log(P), were P is the probability that the observed match is a random event (MASCOT, http:/www.matrixscience.com). Scores >57 indicate p<0.05(*), scores >33 indicate, p<0.05 (**).

488 Tsangaris et al: The Amniotic Fluid Supernatant Proteome

Figure 2. Function (A) and location (B) of amniotic fluid supernatant proteins. The annotated location for each protein is given in Table I. acids and retinol. This group includes FABE, known to be NOSIII) and 3 proteins related to (PEDF, related to the early development of the nervous system and the VEGF41, EPS15) were also detected , indicating that the differentiation of the epidermis development (13) and IBP1, endometrium blood vessel growth and stability are under the the insulin-like growth factor binding protein 1 which regulates control of angiogenic and anti-angiogenic factors (16-19). placental and fetal development (14). It has been proposed With regards to the organization of the , that IBP1 levels in maternal serum during pregnancy may be osteonectin (SPARC), fibronectin (FINC), matrix used as a predictive marker to evaluate embryonic metalloproteinase 3 (MMP-3, PW0052) and plasminogen development and fetal growth (15). (PLMN) were identified. FINC, expressed in high levels in The identification of 11 immunoglobins and 6 proteins with fetal tissues, plays a critical role in the regulation of cell known immunoregulatory effect emphasizes the role of the adhesion in maternal-fetal junctions (20), while MMP-3, AFS in the immune protection of the fetus. Two proteins produced by the fetal membranes, regulates the host response related to the regulation of the blood pressure (ANGT, to intrauterine infections (21). Recent studies showed that

489 in vivo 20: 479-490 (2006)

Table II. Protein identification in AFS expressed and secreted from fetal tissues. 8 Tsangaris G, Weitzdörfer R, Pollak D, Lubec G and Fountoulakis M: The amniotic fluid cell proteome. Electrophoresis 26: 1168- Origin Protein 1173, 2005. 9 Bradford M: A rapid and sensitive method for the quantitation of Fetal liver TTHY, CERU, A1AG, ITH4, RETB, microgram quantities of protein utilizing the principle of protein- VAHU, AACT, APA1, ZA2G, TRFE, dye binding. Anal Biochem 72: 248-254, 1976. A2HS, P2Y2, IBP1, ALBU, AMBP 10 Langen H, Roeder D, Juranville J-F and Fountoulakis M: Effect of Placenta ANGT, AMPN, FABE, PLMN, proteinapplication mode and acrylamide concentration on the Q71S09, PAEP, CAN6 resolution of protein spotsseparated by two-dimensional gel Fetal membranes PW0052, SPRC, AMPN, FBL1 electrophoresis. Electrophoresis 18: 2085-2090, 1997. Desidua PRL, IBP1, PLMN, LUM 11 Fountoulakis M and Gasser R: Proteomic analysis of the cell Fetal kidney AMBP, P2Y2, MYHB envelope fraction of Escherichia coli. Amino Acids 24: 19-41, 2003. Small intestine APA1, APA4 12 Berndt P, Hobohm U and Langen H: Reliable automatic protein Fetal membrane-decidual contact FINC identification from matrix-assisted laser desorption/ionization mass Fetal lung Q5SRH3 spectrometric peptide fingerprints. Electrophoresis 20: 3521-3526, Fetal parenchymal cells O14502 1999. 13 Larque E, Demmelmair H and Koletzko B: Perinatal supply and metabolism of long-chain polyunsaturated fatty acids: importance for the early development of the nervous system. Ann NY Acad SPARC, also present in AF cells, increases the secretion of Sci 967: 299-310, 2002. matrix metalloproteinases. Plasminogen activator inhibitor 1 14 Fang Q, Wang YX and Zhou Y: Insulin-like growth factor binding (PAI-1) inhibited the synthesis of laminin, fibronectin and protein 1and human embryonic development during 6-10 thrombospondin, inducing membrane rupture and delivery (22, gestational weeks. Chin Med J (Engl) 117: 488-491, 2004. 23). The data in the present map for the human amniotic fluid 15 Clapp JF 3rd, Schmidt S, Paranjape A and Lopez B: Maternal insulin-like growth factor-I levels (IGF-I) reflect placental mass supernatant proteome, include 136 different gene products and and neonatal fat mass Am J Obstet Gynecol 190: 730-736, 2004. can be used as a supplement to the AF cell proteome profile 16 Malamitsi-Puchner A, Tziotis J, Tsonou A, Protonotariou E, recently published (8). Hopefully, this will contribute to the Sarantakou A and Creatsas G: Changes in serum levels of vascular understanding of the physiology of pregnancy and elucidate the endothelial growth factor inmales and females throughout life. J pathogenesis of gestation. In the area of prenatal diagnosis, Soc Gynecol Investig 7: 309-312, 2000. 17 Tamada H, Tsubutani D, Kawate N, Inaba T, Matsuyama S, comparative proteome profiling of amniotic fluid supernatant Imakawa K, Sakai S, Christenson RK and Sawada T: Detection of from normal and abnormal pregnancies could be used to transforming growth factor-alpha and epidermal growth factor identify disease markers and possibly allow for therapeutic receptor mRNA and immunohistochemical localization of their interventions. proteins in the ovine uterus during the early implantation period. Histochem J 34: 383-390, 2002. References 18 Tombran-Tink J and Barnstable CJ: Therapeutic prospects for PEDF: more than a promising . Trends Mol 1 Oh J, Fountoulakis M, Juranville J-F, Rosner M, Hengstschlager Med 9: 244-250, 2003. M and Lubec G: Proteomic determination of metabolic enzymes 19 Hefler LA, Tempfer CB, Moreno RM, O'Brien WE and Gregg of the amnion cell: basis for a possible diagnostic tool? Proteomics AR: Endothelial-derived nitric oxide and angiotensinogen: blood 4: 1145-1158, 2004. pressure and metabolism during mouse pregnancy. Am J Physiol 2 Brenner B: Haemostatic changes in pregnancy. Thromb Res 114: Regul Integr Comp Physiol 280: R174-182, 2001. 409-414, 2004 20 Ma Y, Lockwood CJ, Bunim AL, Giussani DA, Nathanielsz PW 3 Dekker GA and Sibai B: Etiology and pathogenesis of and Guller S: Cell type-specific regulation of fetal fibronectin preeclampsia: current concepts. Am J Obstet Gynecol 179: 1359- expression in amnion: conservation of glucocorticoid 1375, 1998. responsiveness in human and nonhuman primates. Biol Reprod 4 Page NM, Kemp CF, Butlin DV and Lowry PJ: Placental peptides 62: 1812-1817, 2000. as markers of gestational disease. Reproduction 123: 487-495, 21 Park KH, Chaiworapongsa T, Kim YM, Espinoza J, Yoshimatzu J, 2002. Edwin S, Gomez R, Yoon BH and Romero R: Matrix 5 Keelan, JA, Blumenstein M, Heliwell RL, Sato TA, Marvin KW metalloproteinase 3 in parturition, premature rupture of the and Mitchell MD: Cytokines, prostaglandins and parturition – a membranes, and microbial invasion of the amniotic cavity. J review. Placenta 24(Suppl A): S33-46, 2003 Perinat Med 31: 12-22, 2003. 6 Vuadens F, Benay C, Crettaz D, Gallot D, Sapin V, Schneider P, 22 Kamihagi K, Katayama M, Ouchi R and Kato I: Bienvenut WV, Lemery D, Quadroni M, Dastugue B and Tissot Osteonectin/SPARC regulates cellular secretion rates of JD: Identification of biologic markers of the premature rupture of fibronectin and laminin extracellular matrix proteins.Biochem fetal membranes: proteomic approach. Proteomics 3: 1521-1525, Biophys Res Commun 200: 423-428, 1994. 2003. 23 Strandjord TP, Sage EH and Clark JG: SPARC participates in the 7 Wang TH, Chang YL, Peng HH, Wang ST, Lu HW, Teng SH, branching morphogenesis of developing fetal rat lung. Am J Respir Chang SD and Wang HS: Rapid detection of fetal aneuploidy Cell Mol Biol 13: 279-287, 1995. using proteomics approaches on amniotic fluid supernatant. Prenat Received March 10, 2006 Diagn 25: 559-566, 2005. Accepted April 12, 2006

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