Abstract
21 min readIntrauterine growth restriction (IUGR) has been associated with increased perinatal morbidity and mortality and increased morbidity and metabolic abnormalities later in life. IUGR is characterized as the failure of a fetus to achieve his or her genetic growth potential in utero. Altered protein expression profiles associated with IUGR may be informative on the pathologic mechanisms of this condition and might reveal potential markers for postnatal complications. The aim of this study was to compare protein profiles of umbilical cord plasma from IUGR and appropriate for gestational age full-term neonates. Blood samples from doubly clamped umbilical cord at delivery from 10 IUGR and 10 appropriate for gestational age full-term neonates were analyzed by two-dimensional electrophoresis and MS. Prominent changes of the α2-HS glycoprotein/fetuin-A were observed in IUGR cases. Specifically we showed that these changes occur primarily at the level of post-translational modifications of the protein. Using a combination of mass spectrometry and classical biochemical assays, single and heavy chain forms of fetuin-A were found to lack the normally present O-linked sialic acids in IUGR neonates. Fetuin A is a glycoprotein that has been associated with promotion of in vitro cell replication, fetal growth and osteogenesis, and protection from Gram-negative bacterial endotoxins. Prominent defects in glycosylation/sialylation of fetuin-A revealed by our study might be responsible for impaired function of fetuin-A, leading to deficient fetal growth, especially osteogenesis, and/or to the development of complications frequently seen later in the lives of IUGR neonates. Intrauterine growth restriction (IUGR) has been associated with increased perinatal morbidity and mortality and increased morbidity and metabolic abnormalities later in life. IUGR is characterized as the failure of a fetus to achieve his or her genetic growth potential in utero. Altered protein expression profiles associated with IUGR may be informative on the pathologic mechanisms of this condition and might reveal potential markers for postnatal complications. The aim of this study was to compare protein profiles of umbilical cord plasma from IUGR and appropriate for gestational age full-term neonates. Blood samples from doubly clamped umbilical cord at delivery from 10 IUGR and 10 appropriate for gestational age full-term neonates were analyzed by two-dimensional electrophoresis and MS. Prominent changes of the α2-HS glycoprotein/fetuin-A were observed in IUGR cases. Specifically we showed that these changes occur primarily at the level of post-translational modifications of the protein. Using a combination of mass spectrometry and classical biochemical assays, single and heavy chain forms of fetuin-A were found to lack the normally present O-linked sialic acids in IUGR neonates. Fetuin A is a glycoprotein that has been associated with promotion of in vitro cell replication, fetal growth and osteogenesis, and protection from Gram-negative bacterial endotoxins. Prominent defects in glycosylation/sialylation of fetuin-A revealed by our study might be responsible for impaired function of fetuin-A, leading to deficient fetal growth, especially osteogenesis, and/or to the development of complications frequently seen later in the lives of IUGR neonates. Intrauterine growth restriction (IUGR) 1The abbreviations used are: IUGR, intrauterine growth restriction; UC, umbilical cord; AGA, appropriate for gestational age; 2-D, two-dimensional; 2-DE, two-dimensional electrophoresis; DTE, dithioerythritol; A2AP, α2-antiplasmin; ALBU, serum albumin; FETUA, α2-HS glycoprotein/fetuin-A; FIBG, fibrinogen γ chain; TTHY, transthyretin; PNGase, peptide-N-glycosidase; HS, Heremans-Schmid; EndoHf, endo-β-N-acetylglucosaminidase Hf. 1The abbreviations used are: IUGR, intrauterine growth restriction; UC, umbilical cord; AGA, appropriate for gestational age; 2-D, two-dimensional; 2-DE, two-dimensional electrophoresis; DTE, dithioerythritol; A2AP, α2-antiplasmin; ALBU, serum albumin; FETUA, α2-HS glycoprotein/fetuin-A; FIBG, fibrinogen γ chain; TTHY, transthyretin; PNGase, peptide-N-glycosidase; HS, Heremans-Schmid; EndoHf, endo-β-N-acetylglucosaminidase Hf. is the failure of a fetus to reach his or her genetic growth potential in utero; this situation leads to reduced fetal size and low birth weight at the time of delivery. IUGR is associated with an increased risk of perinatal morbidity and mortality (1Marsal K. Intrauterine growth restriction.Curr. Opin. Obstet. Gynecol. 2002; 14: 127-135Crossref PubMed Scopus (116) Google Scholar). Furthermore individuals born with IUGR develop abnormalities characteristic of the metabolic syndrome (obesity, dyslipidemia, hypertension, impaired glucose tolerance, and type 2 diabetes mellitus) and its cardiovascular complications in later life. Fetal growth is controlled by maternal, placental, and/or fetal factors, which consequently may also be involved in the pathogenesis of IUGR (2Bamberg C. Kalache K.D. Prenatal diagnosis of fetal growth restriction.Semin. Fetal Neonatal Med. 2004; 9: 387-394Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Umbilical cord (UC) blood drawn from the doubly clamped umbilical cord at delivery reflects the fetal blood compartment. Along these lines, the cord blood levels of various growth factors and hormones have been related to size at birth by several groups (1Marsal K. Intrauterine growth restriction.Curr. Opin. Obstet. Gynecol. 2002; 14: 127-135Crossref PubMed Scopus (116) Google Scholar, 3Shaarawy M. el-Mallah S.Y. Leptin and gestational weight gain: relation of maternal and cord blood leptin to birth weight.J. Soc. Gynecol. Investig. 1999; 6: 70-73Crossref PubMed Scopus (28) Google Scholar, 4Ong K. Kratzsch J. Kiess W. Costello M. Scott C. Dunger D. Size at birth and cord blood levels of insulin, insulin-like growth factor I (IGF-I), IGF-II, IGF-binding protein-1 (IGFBP-1), IGFBP-3 and the soluble IGF-II/mannose-6-phosphate receptor in term human infants.J. Clin. Endocrinol. Metab. 2000; 85: 4266-4269Crossref PubMed Scopus (201) Google Scholar, 5Bader A. Riskin A. Vafsi O. Tamir A. Peskin B. Israel N. Merksamer R. Dar H. David M. Alpha-fetoprotein in the early neonatal period—a large study and review of the literature.Clin. Chim. Acta. 2004; 349: 15-23Crossref PubMed Scopus (104) Google Scholar). Further investigation of differential protein expression levels in UC blood in various pathologic states will hopefully produce information on the cause and mechanisms of prenatal disorders and might reveal possible markers for postnatal complications and disease progression. Proteomics provides information about protein expression levels, post-translational modifications, subcellular localization, and interactions (6Fountoulakis M. Proteomics: current technologies and applications in neurological disorders and toxicology.Amino Acids. 2001; 21: 363-381Crossref PubMed Scopus (110) Google Scholar, 7Görg A. Weiss W. Dunn M.J. Current two-dimensional electrophoresis technology for proteomics.Proteomics. 2004; 4: 3665-3685Crossref PubMed Scopus (1531) Google Scholar). Proteomics techniques have been applied in the investigation of the proteome of various biological systems, including human body fluids, i.e. plasma, urine, amniotic fluid, etc., in normal and diseased states (6Fountoulakis M. Proteomics: current technologies and applications in neurological disorders and toxicology.Amino Acids. 2001; 21: 363-381Crossref PubMed Scopus (110) Google Scholar, 8Rohlff C. Proteomics in molecular medicine: applications in central nervous systems disorders.Electrophoresis. 2000; 21: 1227-1234Crossref PubMed Scopus (93) Google Scholar, 9Brichory F. Beer D. Le Naour F. Giordano T. Hanash S. Proteomics-based identification of protein gene product 9.5 as a tumor antigen that induces a humoral immune response in lung cancer.Cancer Res. 2001; 61: 7908-7912PubMed Google Scholar, 10Petricoin E.F. Ardekani A.M. Hitt B.A. Levine P.J. Fusaro V.A. Steinberg S.M. Mills G.B. Simone C. Fishman D.A. Kohn E.C. Liotta L.A. Use of proteomic patterns in serum to identify ovarian cancer.Lancet. 2002; 359: 572-577Abstract Full Text Full Text PDF PubMed Scopus (2841) Google Scholar, 11Watanabe H. Hamada H. Yamada N. Sohda S. Yamakawa-Kobayashi K. Yoshikawa H. Arinami T. Proteome analysis reveals elevated serum levels of clusterin in patients with preeclampsia.Proteomics. 2004; 4: 537-543Crossref PubMed Scopus (73) Google Scholar, 12Tsangaris G. Th. Karamessinis P.M. Kolialexi A. Garbis S.D. Antsaklis A. Mavrou A. Fountoulakis M. Proteomic analysis of amniotic fluid in pregnancies with Down syndrome.Proteomics. 2006; 6: 4410-4419Crossref PubMed Scopus (96) Google Scholar). Through this approach, proteins involved in cellular functions and pathways affected by disease (13Shiio Y. Donohoe S. Yi E.C. Goodlett D.R. Aebersold R. Eisenman R.N. Quantitative proteomic analysis of Myc oncoprotein function.EMBO J. 2002; 21: 5088-5096Crossref PubMed Scopus (170) Google Scholar, 14Bouwmeester T. Bauch A. Ruffner H. Angrand P.O. Bergamini G. Croughton K. Cruciat C. Eberhard D. Gagneur J. Ghidelli S. Hopf C. Huhse B. Mangano R. Michon A.M. Schirle M. Schlegl J. Schwab M. Stein M.A. Bauer A. Casari G. Drewes G. Gavin A.C. Jackson D.B. Joberty G. Neubauer G. Rick J. Kuster B. Superti-Furga G. A physical and functional map of the human TNF-α/NF-κB signal transduction pathway.Nat. Cell Biol. 2004; 6: 97-105Crossref PubMed Scopus (855) Google Scholar) as well as putative disease biomarkers and drug targets (10Petricoin E.F. Ardekani A.M. Hitt B.A. Levine P.J. Fusaro V.A. Steinberg S.M. Mills G.B. Simone C. Fishman D.A. Kohn E.C. Liotta L.A. Use of proteomic patterns in serum to identify ovarian cancer.Lancet. 2002; 359: 572-577Abstract Full Text Full Text PDF PubMed Scopus (2841) Google Scholar) may be identified. In the present study, we used 2-DE followed by MS to compare UC plasma protein expression profiles from appropriate for gestational age (AGA) and IUGR full-term neonates. Multiple proteins were differentially expressed in IUGR plasma, including the human α2-HS glycoprotein/fetuin-A. We showed that in IUGR the differential expression of fetuin-A is primarily at the level of post-translational modifications of the protein. Specifically using a combination of mass spectrometry and classical biochemical assays, fetuin-A was found to be differentially glycosylated/sialylated in the plasma of IUGR compared with AGA neonates. Collectively our results establish the potential importance of fetuin-A in the pathophysiology of IUGR and generate new hypotheses regarding the role of post-translational modifications in the action of the protein and its effects during fetal and later life. IPG strips (18 cm) and IPG buffer, pH 4–7 linear, were purchased from Amersham Biosciences. Acrylamide/piperazine solution was obtained from Biosolve (Valenswaard, The Netherlands), and the other reagents and solutions for the polyacrylamide gel preparation were supplied from Bio-Rad. Protease inhibitors mixture and proteomics grade trypsin and chymotrypsin were obtained from Roche Diagnostics. PNGase F and neuraminidase were from Sigma, and EndoHf was from New England BioLabs. The colloidal Coomassie Blue staining kit was purchased from Novex (San Diego, CA). Goat polyclonal antibody against human fetuin-A and rabbit polyclonal antibody against transthyretin were from Santa Cruz Biotechnology (Santa Cruz, CA) and horseradish peroxidase-conjugated anti-goat immunoglobulins or anti-rabbit immunoglobulins were purchased from Sigma. The ECL Western blotting detection system was purchased from Pierce. All other chemicals and reagents were from Sigma. The study was approved by the Ethics Committee of the Aretaieion University Hospital, Athens, Greece and was performed after obtaining written informed consent from the mothers of the fetuses. Ten asymmetric IUGR and 10 AGA full-term singleton neonates, as well as their mothers, all of Greek origin, were included in the study (Table I). Offspring of mothers with gestational pathology and with a birth weight below the 10th customized centile were characterized as IUGR. The Gestation Related Optimal Weight (15Gardosi J. Chang A. Kaylan B. Sahota D. Symonds E.M. Customised antenatal growth charts.Lancet. 1992; 339: 283-287Abstract PubMed Scopus (629) Google Scholar, 16Gardosi J. Mongelli M. Wilcox M. Chang A. An adjustable fetal weight standard.Ultrasound Obstet. Gynecol. 1995; 6: 168-174Crossref PubMed Scopus (467) Google Scholar) computer-generated program was used to calculate the customized centile for each pregnancy, taking into consideration significant determinants of birth weight, such as maternal height and booking weight, ethnic group, parity, gestational age, and gender.Table IClinical profile of participating mothers and full-term IUGR and AGA infantsAGA (n = 10) mean ± S.D.IUGR (n = 10) mean ± S.D.Maternal age (years)29 ± 529 ± 3Gestational age (weeks)38.6 ± 1.238.5 ± 1.33Birthweight (g)3276 ± 3562359 ± 197Mode of delivery (n (%) VD/ECS)aVaginal delivery/elective cesarean section.2 (20)/8 (80)3 (30)/7 (70)Parity (n (%) first/other)3 (30)/7 (70)4 (40)/6 (60)Gender (n (%) male/female)7 (70)/3 (30)7 (70)/3 (30)a Vaginal delivery/elective cesarean section. Open table in a new tab The cause of intrauterine growth restriction was identified in each one of the 10 IUGR neonates included in the study. The personal, family, and perinatal histories of each parturient; maternal ultrasounds; and Doppler studies (performed every 10–15 days, starting from the 32nd gestational week) of the uterine, umbilical, and middle cerebral artery were all evaluated. In four cases, IUGR resulted from preeclampsia. In the remaining six cases, parturients suffered from pregnancy-induced hypertension or chronic diseases (anemia, hepatitis B, or thyroiditis) and had small and infarcted placentas despite exclusion of intrauterine infection. Two mothers reported smoking four to five cigarettes per day. Blood flow studies were within the normal ranges in all cases, whereas amniotic fluid volume and placental weights were reduced (the latter ranging from 255 to 400 g). In the AGA group, mothers were healthy, one smoked up to two cigarettes per day, and another reported daily consumption of one cup of coffee. Placentas were normal in appearance and weight. Tests for congenital infections were negative in all women of both groups, and their offspring had no symptoms of intrauterine infection or signs of genetic syndromes. One- and 5-min Apgar scores were ≥7 and ≥8 in all IUGR and AGA cases, respectively. Blood was drawn from the doubly clamped UC (mixed arteriovenous blood) at delivery, reflecting fetal state. Blood was collected in pyrogen-free tubes and was immediately centrifuged in 1000 × g for 30 min. The supernatant plasma was kept frozen at −80 °C until assay. The protein concentration in plasma samples was determined using the Bradford method with a Bio-Rad protein assay reagent kit. UC plasma samples were analyzed with 2-D gel electrophoresis. In detail, 750 μg of total protein were diluted in sample buffer consisting of 50 mm Tris-HCl (pH 8.5), 7 m urea, 2 m thiourea, 2% CHAPS, 0.4% dithioerythritol (DTE), 0.2% IPG buffer (pH 4–7 linear), and 10 μl of a protease inhibitor mixture to a final volume of 250 μl. Protein samples were applied on immobilized pH 4–7 linear gradient IPG strips (18 cm) previously rehydrated for 16 h in rehydration buffer (same as sample buffer but with 8 m urea and without thiourea). Sample application was done using the cup loading method at the basic and acidic ends of the strips. Focusing was performed at 250 V for 30 min after which the voltage was gradually increased to 5000 V for 15 h and kept to 5000 V for 10 h (PROTEAN IEF Cell, Bio-Rad). After focusing, strips were equilibrated for 20 min in 50 mm Tris-HCl (pH 8.8), 6 m urea, 30% glycerol, 2% SDS, and 0.5% DTE followed by a 20-min incubation in the same buffer containing 4% iodoacetamide instead of DTE. In the case of immunoblot analysis the second equilibration step was omitted. The second dimension was performed in 12% SDS-polyacrylamide gels (180 × 200 × 1.5 mm). The gels were run at 40 mA/gel in an ETTAN DALT apparatus (Amersham Biosciences). In the case of immunoblot under non-reducing conditions, sample buffer without thiourea and DTE was utilized, the IPG strips were rehydrated in the absence of DTE, and prior to the second dimension, the equilibration step with DTE was omitted. 2-D gels were fixed in 50% methanol containing 5% phosphoric acid for 2 h, stained with colloidal Coomassie Blue, and scanned using the GS-800 calibrated densitometer (Bio-Rad). All gel images were analyzed using PDQuest 7.2.0 image processing software (Bio-Rad). Six 2-D gels from the IUGR and six from the AGA group were analyzed. Gel images from each group were edited, and spots were matched manually. A unique identification number was assigned to matching spots on different gels. Normalization of the spot intensities was conducted according to the total optical density in the gel (i.e. the normalized intensity was the percentage of the intensity of each spot over the sum of intensities of all detected spots in the gel). The mean value of percentage and S.E. were calculated for each spot in each group and then compared using the two-sided Student's t test. Protein spots whose expression was found to be different between the two groups at the significance level of p < 0.05 were selected for further analysis. 2-D gels were initially subjected to fluorescence staining with Pro-Q Emerald 488 glycoprotein dye (Molecular Probes) according to the manufacturer's instructions. The gels were then stained with SYPRO Ruby fluorescence dye (Molecular Probes) for protein detection. Gel images were obtained by the use of a Typhoon 9200 laser scanner (Amersham Biosciences). MALDI-TOF-MS peptide analysis and protein identification were performed as described previously (12Tsangaris G. Th. Karamessinis P.M. Kolialexi A. Garbis S.D. Antsaklis A. Mavrou A. Fountoulakis M. Proteomic analysis of amniotic fluid in pregnancies with Down syndrome.Proteomics. 2006; 6: 4410-4419Crossref PubMed Scopus (96) Google Scholar, 17Fountoulakis M. Gasser R. Proteomic analysis of the cell envelope fraction of Escherichia coli.Amino Acids. 2003; 24: 19-41Crossref PubMed Scopus (61) Google Scholar). The Coomassie Blue-stained gel spots of interest were detected by the use of Melanie 4.02 software, excised from the gels with the use of Proteiner SPII (Bruker Daltonics, Bremen, Germany), and placed into 96-well microtiter plates. Protein spots were destained with 150 μl of 30% acetonitrile in 50 mm ammonium bicarbonate, washed with 150 μl of ultrapure water, and dried in a speed vacuum concentrator (MaxiDry Plus, Heto, Allered, Denmark). Each dried gel piece was digested with 50 ng of one of the following proteases, trypsin, chymotrypsin, or Asp-N, in the appropriate enzyme buffer according to the manufacturer's protocol. After 16 h at room temperature, peptides were extracted by adding 10 μl of 50% acetonitrile containing 0.3% trifluoroacetic acid to each gel piece. The peptide mixture (1.5 μl) was simultaneously applied on the sample target with 1 μl of matrix solution consisting of 0.025% α-cyano-4-hydroxycinnamic acid (Sigma) and the internal standard peptides des-Arg-bradykinin (Sigma, 904.4681 Da) and adrenocorticotropic hormone fragment 18–39 (Sigma, 2465.1989 Da) in 65% ethanol, 35% acetonitrile, and 0.03% trifluoroacetic acid. Sample peptide mixtures were analyzed with a matrix-assisted laser desorption tandem time-of-flight mass spectrometer (Ultraflex II MALDI-TOF-TOF-MS, Bruker Daltonics). The peak list was created with Flexanalysis version 2.2 software (Bruker Daltonics). The signal to noise ratio was calculated by SNAP algorithm, and a threshold ratio of 2.5 was allowed. Peptide matching and protein searches were performed automatically by the use of Mascot software (Matrix Sciences Ltd., London, UK). For peptide identification monoisotopic masses were used, and a mass tolerance of 0.0025% (25 ppm) was allowed. All extraneous peaks, such as trypsin autodigests, matrix, and keratin peaks, were not considered for the protein search. Cysteine carbamidomethylation and methionine oxidation were set as fixed and variable modifications, respectively. One miscleavage was allowed. The peptide masses were compared with the theoretical peptide masses of all available proteins from Homo sapiens using the Swiss-Prot database. The probability score with p < 0.05 identified by the software was used as the criterion for the affirmative protein identification. Neuraminidase, PNGase F, and EndoHf digestions were performed overnight at 37 °C on UC plasma samples with the appropriate enzyme buffer. The buffer used for PNGase F digestion was 20 mm sodium bicarbonate, pH 8, containing 0.02% SDS, 10 mm 2-mercaptoethanol, and 1.5% Triton X-100. For neuraminidase digestion, the buffer used was 100 mm sodium acetate and 2 mm CaCl2, pH 5. For EndoHf digestion, the buffer utilized was 50 mm sodium citrate, pH 5.5, 0.05% SDS, and 0.1% 2-mercaptoethanol. Equal protein amounts from UC plasma samples were separated by 10% SDS-PAGE or 2-D gel electrophoresis (as described above) under either non-reducing or reducing conditions. After electrophoresis, proteins were transferred to Hybond-ECL nitrocellulose membrane (Amersham Biosciences) by electroblotting, and blots were blocked with 5% nonfat milk in TBS, 0.1% Tween at room temperature for 2 h. After washing, membranes were incubated overnight at 4 °C with the appropriate dilution (1:500) of polyclonal antibody against human fetuin-A or a 1: 500 dilution of polyclonal antibody against transthyretin (Santa Cruz Biotechnology) in the same buffer without Tween 20. After washing with TBS, 0.1% Tween, membranes were incubated with horseradish peroxidase-conjugated anti-goat immunoglobulins (anti-rabbit immunoglobulins in the case of transthyretin) as secondary antibodies at room temperature for 2 h. Bound antibody was detected by the ECL Western blotting detection system. 2-DE was used to analyze protein profiles of UC plasma from 10 AGA and 10 IUGR full-term neonates. Gels corresponding to six cases per category were analyzed by the use of image analysis software. Three hundred eighty spots were matched, and their expression levels were compared. Several differences between UC plasma from AGA and IUGR neonates were detected (Fig. 1, A and B). Specifically 20 protein spots were expressed at statistically significant different levels in the two groups (Fig. 1A); these were excised from the 2-D gels for identification by MALDI-TOF-MS peptide fingerprinting. Sixteen of these protein spots were positively identified (Table II). As shown, the protein spots corresponding to α2-HS glycoprotein/fetuin-A (FETUA), α2-antiplasmin (A2AP), antithrombin-III, serum albumin (ALBU), transthyretin (TTHY), apolipoprotein E, fibrinogen γ chain (FIBG), and complement factor H were significantly down-regulated, whereas serotransferrin was significantly up-regulated in IUGR compared with the AGA UC plasma samples. Notably differences in these spots were also observed in 2-DE gels corresponding to the remaining four IUGR and 4AGA samples, which were not included in the image analyses.Table IIDifferentially expressed proteins in IUGRSpot no.SymbolProtein nameAccession no.Theoretical pI/molecular mass (kDa)MALDI-TOF-MSExpression level AGA group (mean ± S.E.)Expression level IUGR group (mean ± S.E.)Ratio = IUGR/AGApScoreSequence coverageMatching peptidesUnique peptidesUnmatched peptides%%%0208FETUAα2-HS glycoprotein precursor (fetuin-A)P027655.4/39.3591854150.85 ± 0.080.38 ± 0.070.450.0011204FETUAα2-HS glycoprotein precursor (fetuin-A)P027655.4/39.363175430.10 ± 0.010.06 ± 0.020.600.042303A2APα2-Antiplasmin precursorP086975.9/54.559124570.08 ± 0.010.04 ± 0.010.500.0152305A2APα2-Antiplasmin precursorP086975.9/54.57126108750.17 ± 0.010.11 ± 0.010.650.012508TRFESerotransferrin precursorP027877/77202402826520.05 ± 0.020.15 ± 0.033.000.044202ANT3Antithrombin-III precursorP010086.3/52.68225109180.27 ± 0.040.12 ± 0.030.440.0155001ALBUSerum albumin precursorP027685.9/69.3150362522620.50 ± 0.020.27 ± 0.050.540.0035007TTHYTransthyretin precursorP027665.4/15.9124507781.00 ± 0.110.32 ± 0.170.320.0085008APOEApolipoprotein E precursorP026495.5/36.1124451311570.33 ± 0.080.12 ± 0.040.360.0355010ALBUSerum albumin precursorP027685.9/69.355171212570.18 ± 0.020.06 ± 0.020.330.0065011ALBUSerum albumin precursorP027685.9/69.3101411413570.10 ± 0.010.06 ± 0.010.600.045102FIBGFibrinogen γ chain precursorP026795.3/51.5168612219540.27 ± 0.020.13 ± 0.040.480.0095104FIBGFibrinogen γ chain precursorP026795.3/51.5189682321630.25 ± 0.040.09 ± 0.030.360.0156101FIBGFibrinogen γ chain precursorP026795.3/51.5128581616700.30 ± 0.040.18 ± 0.060.600.046902CFAHComplement factor H precursorP086036.3/139295343835260.13 ± 0.030.06 ± 0.010.460.047208ALBUSerum albumin precursorP027685.9/69.369301414700.13 ± 0.020.07 ± 0.010.540.03 Open table in a new tab In the case of fetuin-A, an interesting expression pattern was observed. Indeed fetuin-A was represented in all samples by multiple spots that can be distinguished, according to the pI, in two major groups, one acidic (Fig. 1, C and D, left ellipse referred to as group A) and one basic (Fig. 1, C and D, right ellipse referred to as group B). Besides the pI differences, these two groups differed also in molecular weight with group B spots being of higher molecular weight compared with spots of group A. Interestingly in both groups, an additional row of fetuin-A protein spots with lower molecular weight was detected in eight of 10 IUGR UC plasma samples (Fig. 1D, shown with arrows, referred to as Al and Bl) but in none of the 10 AGA samples. The differential expression of fetuin-A was corroborated by Western blot of UC plasma samples using fetuin-A-specific antibody. As shown in Fig. 2A (lanes 6, 8, 9, 14, and 15), in Western blot analysis, fetuin-A was represented by two protein bands in IUGR UC plasma samples compared with only one band in the AGA samples (Fig. 2A, lanes 1–5 and 10–13). In addition, 2-D Western blot analysis confirmed the identity of all spots of groups A and B including the IUGR-related Al and Bl rows of spots as fetuin-A (Fig. 2, B and C). The difference between the two groups (A and B) of fetuin-A protein spots was initially addressed. Fetuin-A is produced in a single chain form consisting of the A-chain, the B-chain, and a connecting peptide between them (16Gardosi J. Mongelli M. Wilcox M. Chang A. An adjustable fetal weight standard.Ultrasound Obstet. Gynecol. 1995; 6: 168-174Crossref PubMed Scopus (467) Google Scholar) (Fig. 3A, left panel). The mature protein is a two-chain form generated after cleavage between the connecting peptide and the B-chain; the two generated parts, A-chain with connecting peptide and B-chain, are held together with a disulfide bond (17Fountoulakis M. Gasser R. Proteomic analysis of the cell envelope fraction of Escherichia coli.Amino Acids. 2003; 24: 19-41Crossref PubMed Scopus (61) Google Scholar, 18Jahnen-Dechent W. Trindl A. Godovac-Zimmermann J. Muller-Esterl W. Posttranslational processing of human α2-HS glycoprotein (human fetuin). Evidence for the production of a phosphorylated single-chain form by hepatoma cells.Eur. J. Biochem. 1994; 226: 59-69Crossref PubMed Scopus (0) Google Scholar, 19Lee C.C. Bowman B.H. Yang F.M. Human α2-HS-glycoprotein: the A and B chains with a connecting sequence are encoded by a single mRNA transcript.Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 4403-4407Crossref PubMed Scopus (85) Google Scholar, 20Nawratil P. Lenzen S. Kellermann J. Haupt H. Schinke T. Muller-Esterl W. Jahnen-Dechent W. Limited proteolysis of human α2-HS glycoprotein/fetuin. Evidence that a chymotryptic activity can release the connecting peptide.J. Biol. Chem. 1996; 271: 31735-31741Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar) (Fig. 3A, right panel). The A-chain and the connecting peptide form the heavy chain, whereas the B-chain is the light chain of fetuin-A. By the use of mass spectrometry, two peptides of the B-chain of fetuin-A, with peaks at m/z 2016.0 and 2285.2, corresponding to amino acids 341–361 (TVVQPSVGAAAGPVVPPCPGR) and 341–363 (TVVQPSVGAAAGPVVPPCPGRIR), were identified solely in the basic (B) group of spots (Fig. 3C). In contrast, peptides from the A-chain and the connecting peptide (heavy chain) were identified in both A and B groups of spots. These results suggested that the spots of group B probably are isoforms of the single chain form of the protein, whereas the spots of group A represent isoforms of the two-chain form, which lacks the B-chain, possibly due to the reducing experimental conditions. To investigate this hypothesis, 2-D Western blot analysis under non-reducing conditions was conducted; in this case, only one group of fetuin-A spots was detected in either AGA (Fig. 2D) or IUGR (Fig. 2E). The pI range of this group (4.6–5.0) is the same as that of group B (Fig. 2, B and C). This result confirmed that groups B and A correspond to fetuin-A single and heavy chain forms, respectively. The IUGR-related fetuin-A isoforms may correspond to different proteolytic cleavage and/or post-translational modifications. To address the former, several different proteases, namely trypsin, chymotrypsin, and Asp-N, were utilized as a means to increase the peptide sequence coverage received during the MS analysis. With these enzymes, 75 and 70% sequence coverage was achieved for the upper constitutively expressed and lower IUGR-related (Al and
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