Objective
We sought to examine changes in the intraamniotic proteomic environment after the administration of antenatal corticosteroids to women with impending preterm delivery.
Study Design
Amniotic fluid samples were collected at the time of clinically indicated amniocentesis before and within 7 days of administration of antenatal corticosteroids for impending preterm delivery (n = 12). Proteins differentially expressed before and after corticosteroids were identified by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry. They were isolated, characterized, and quantified by fast protein liquid chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, in-gel tryptic digestion, immunodepletion assays, enzyme-linked immunosorbent assay, and electrospray ionization tandem mass spectrometry.
Results
Five protein peaks of interest were identified and characterized, all of which were significantly decreased after antenatal corticosteroid administration. These included 2 isoforms of transthyretin, albumin, prothrombin fragment 2, and lumican.
Conclusion
Four proteins, identified and characterized in amniotic fluid, were differentially expressed with antenatal corticosteroid administration. These data may provide additional insight into the molecular mechanisms by which antenatal corticosteroids prevent neonatal complications.
Antenatal administration of glucocorticoids significantly reduces neonatal mortality and the incidence of respiratory distress syndrome (RDS), intraventricular hemorrhage, and necrotizing enterocolitis in neonates delivering <34 weeks’ gestation. Ever since Liggins and Howie demonstrated that antenatal corticosteroids increase pulmonary surfactant in newborn lambs and reduces the incidence of RDS in premature infants, investigators have been trying to understand how these agents work to promote fetal lung development. Several mechanisms have been proposed, including: (1) acceleration of type II pneumocyte maturation with up-regulation of surfactant messenger RNA and protein levels and increased phospholipid synthesis; (2) increased antioxidant activity; and (3) differential regulation of pulmonary growth factors and apoptosis resulting in improved developmental structural changes. Despite these observations, the precise mechanism(s) by which antenatal corticosteroids act to promote cellular differentiation and maturation within the fetal lungs and other organ systems is not well understood. For example, administration of glucocorticoids to pneumocytes in vitro promotes cellular differentiation and surfactant production, but this effect is seen also in the presence of a glucocorticoid receptor inhibitors, suggesting that a nongenomic or glucocorticoid receptor–independent mechanism is involved. To improve our understanding of the effects of antenatal corticosteroids on fetal lung maturation in vivo, this study uses proteomic analysis by surface-enhanced laser desorption/ionization (SELDI) time-of-flight (TOF) mass spectrometry to examine changes in the intraamniotic proteome after clinically indicated antenatal corticosteroid administration in patients with impending preterm delivery.
SELDI-TOF mass spectrometry is a high-throughput proteomic tool for the identification of discriminatory protein biomarkers in complex body tissues and fluids for specific diseases. This same technology has been previously applied to amniotic fluid (AF) by our group and other investigators to discover discriminatory protein biomarkers for intraamniotic infection/inflammation (IAI), fetal aneuploidy, and preeclampsia. This is the first reported attempt on the use of proteomic analysis to identify proteins that are differentially expressed in the AF of women before and after antenatal corticosteroid administration.
Materials and Methods
Study design
Samples of AF were collected at Seoul National University Hospital, Korea, at the time of clinically indicated amniocentesis to document fetal lung maturity and/or exclude IAI both prior to and within 7 days of administration of antenatal corticosteroids for impending preterm delivery for maternal or fetal indications (n = 12). The research protocol was approved by the institutional review board at Seoul National University Hospital, Korea, and written consent was obtained from all patients. Inclusion criteria included: (1) singleton gestation; (2) well-dated pregnancy by first-trimester ultrasound or firm menstrual dating consistent with a second-trimester ultrasound; (3) clinically indicated amniocentesis to document fetal lung maturity or exclude IAI because of impending preterm delivery; (4) gestational age at initial amniocentesis <34 weeks; (5) no antenatal corticosteroids prior to initial amniocentesis; and (6) a follow-up amniocentesis performed to exclude IAI or document fetal lung maturity within 2-7 days of antenatal corticosteroid administration or sampling of AF at the time of indicated cesarean delivery within the same time period. An aliquot of each of the AF samples was sent immediately for biochemical and microbiologic examination to exclude IAI, including aerobic and anaerobic culture and white blood cell count. The remainders of the AF samples were stored at –80°C until further analysis. Patients were excluded from the study if there was clinical evidence of IAI or if either of their AF samples showed evidence of IAI (eg, positive culture or white blood cell count >20/mm 3 ).
After initial amniocentesis, all subjects received a full course of antenatal corticosteroids (dexamethasone, 6 mg intramuscularly every 12 hours for 4 doses) and were managed according to standardized clinical protocols. The timing and route of delivery was dictated by standard obstetric indications. Maternal/fetal demographic information, laboratory results, and clinical outcomes were abstracted from the medical records.
AF proteomic profiling using SELDI-TOF mass spectrometry
Before AF samples were analyzed by SELDI-TOF mass spectrometry (ProteinChip system model series 4000; Ciphergen Biosystems, Fremont, CA), the ProteinChip Array (Ciphergen Biosystems) was calibrated as follows: superoxide dismutase (double charged) 7795.7 Da, skeletal myoglobin 8475.7 Da, superoxide dismutase 15,591.4 Da, myoglobin 16,951.51 Da, β-lactoglobulin A 18,363.34 Da, peroxidase 43,240.0 Da, and albumin 66,410.0 Da.
After each spot of the Q10 ProteinChip Array was equilibrated, 2 μL of each AF sample was loaded with 8 μL of binding buffer into each spot and incubated in a humidity chamber for 1 hour at room temperature. The Q10 ProteinChip Array was selected because previous studies in our laboratory have shown that it gives the most discriminatory AF proteomic profile. After washing and air drying, 1 μL of a saturated solution of 5 mg of sinapinic acid in 50% acetonitrile/0.5% trifluoroacetic acid was loaded to each spot. The ProteinChip arrays were then analyzed by a ProteinChip Reader using ProteinChip software (Ciphergen Express Client 3.0). After the peak calibration, peak alignment, and normalization of each proteomic profile obtained from the analysis, AF samples taken before antenatal corticosteroid administration were matched to and compared with the AF samples taken from the same patient after corticosteroid administration. In this way, patients served as their own controls. The proteomic “fingerprint” (peaks of interest) that was able to discriminate the AF profiles before and after antenatal corticosteroid administration was then identified.
Purification and isolation of the protein peaks of interest
AF samples were purified by centrifugation and by passage through a 0.45-μm syringe filter. Filtered AF was then loaded onto a fast protein liquid chromatography (FPLC) column (1.0-mL HiTrap Q; GE Healthcare Corp, Waukesha, WI). FPLC elution fractions were separated and concentrated. Thereafter, 1 μL of each FPLC fraction was loaded onto spots on Au ProteinChips. The prepared ProteinChips were read using the ProteinChip Reader as described above. The FPLC fractions containing the proteins of interest were then collected separately and concentrated.
To isolate the proteins of interest, 80 μL of concentrated FPLC fractions containing each of the peaks of interest were mixed with sample buffer and boiled. The mixture was electrophoresed on a 10% sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gel, which was stained with Coomasie blue-250. The gel band corresponding to the molecular weight of the protein of interest was excised, washed, and dehydrated by incubation with 100% acetonitrile.
The elution method was performed to confirm that the protein contained within the dehydrated SDS-PAGE gel was the same as the protein of interest identified by SELDI-TOF mass spectrometry. The dehydrated gel was then incubated with elution solution and sonicated. After the prepared solution was applied to a NP20 chip (Ciphergen Biosystems) and the mass/charge intensity obtained, the gel band whose mass/charge intensity was identical to that of the peak of interest was selected for further identification and characterization.
Identification of the proteins of interest by in-gel tryptic digestion and ESI-tandem mass spectrometry
Excised pieces of SDS-PAGE gel containing the proteins of interest were subjected to overnight in-gel trypsin digestion at 37°C using the method of Jensen et al. The gel pieces incubated with the digestion buffer were then desalted, centrifuged, and the supernatant was collected.
Tandem mass spectrometry (MS/MS) of peptides generated by in-gel tryptic digestions of pooled AF samples was performed by nano-electrospray ionization on a Q-TOF-2 mass spectrometer (Micromass, Manchester, UK). The source temperature was 80°C. A potential of 1 kV was applied to the precoated borosilicate nanoelectrospray needles (EconoTip; New Objective Inc, Woburn, MA) in the ion source combined with a nitrogen back-pressure. The mass spectrometer was operated in automatic data-dependent MS/MS mode. Finally, an MS/MS spectrum was recorded to confirm the sequence of the precursor ion using collision-induced dissociation with the relative collision energy dependant on molecular weight. The cone voltage was 40 V. The quadrupole analyzer was used to select precursor ions for fragmentation in the hexapole collision cell. The collision gas was Ar at a pressure of 6-7 × 10-5 mbar and the collision energy was 20-30 V. Product ions were analyzed using an orthogonal TOF analyzer fitted with a reflector, a microchannel plate detector, and a time-to-digital converter. The data were processed using a Mass Lynx (Waters, Manchester, UK) on a Windows NT PC system (Microsoft Corp, Redmond, WA).
To identify the proteins of interest, all MS/MS spectra recorded on tryptic digest peptides derived from spot and amino acid sequences deduced using a peptide de novo sequencing program were searched against protein sequences from NCBI Expressed Sequence Tags databases using the MASCOT search program (Matrix Science Ltd, London, UK) and NCBI Basic Local Alignment Search Tool.
Validation of proteins of interest by immunodepletion
Immunodepletion methodology was used to confirm the identity of the proteins of interest identified by MS/MS. In brief, 10 mg of cyanogen bromide-activated sepharose 4B beads were loaded into an Eppendorf tube (Eppendorf AG, Hamburg, Germany), washed, and swelled with 1 mmol/L of HCl. After centrifuged, the beads were washed and 40 μL of binding buffer was added at room temperature for 30 minutes followed by 10 μL of 1 of the antibodies against the protein of interest. The mixture was then incubated at 4°C overnight, centrifuged, and the supernatant removed. Thereafter, binding of antibody to bead was blocked and the mixture was washed. The antibody-labeled beads were then incubated with a solution containing the unidentified protein of interest at room temperature for 3 hours, and supernatant was collected after a brief spin. The supernatant was loaded onto a Q10 ProteinChip array and the ProteinChip analysis repeated with a view to confirming that the proteomic peak of interest had been selectively depleted by the antibody.
After all the supernatant had been removed, the beads were recovered, washed twice with phosphate-buffered saline, mixed with 20 μL of elution buffer, and vortexed for 5 minutes at room temperature. The resultant supernatant was collected, loaded onto a NP20 chip, and analyzed by SELDI-TOF mass spectrometry to confirm that the protein bound to the bead-antibody mixture was indeed the putative proteins of interest.
Quantification of proteins of interest by enzyme-linked immunosorbent assay
The proteins of interest were quantified using specific enzyme-linked immunosorbent assays (ELISAs) according to the instructions of the manufacturers. ELISA assays were performed on individual (not pooled) undiluted AF samples by providers who were blinded to the identity of the samples and the corresponding clinical information ([for albumin: antigen – ab7473; Abcam, Cambridge, MA; capture antibody – ab18081; Abcam; detecting antibody – ab18078-B, Abcam], [for transthyretin: antigen – P1742; Sigma-Aldrich, St Louis, MO; capture antibody – A0002; Dako, Glostrup, Denmark; detecting antibody – LF-MA0174-B; ABfrontier, Seoul, Korea], [for prothrombin fragment 2: capture antibody – ab22700; Abcam; detecting antibody – SEG; Bioinfra, Seoul, Korea], [for lumican: ELISA kit for human lumican – E1496H; USCN, Wuhan, China]). Intraassay coefficients of variation were <10% each.
Statistical analysis
Proportions were compared with the use of Fisher’s exact test and Pearson χ 2 test. The Kruskal-Wallis analysis of variance test and the Mann-Whitney U test were used for comparison of continuous variables in skewed datasets. One-way analysis of variance and t test were used for comparison of continuous variables normally distributed. Wilcoxon signed rank test was used to select the peaks of interest and to examine the difference in AF concentrations of identified proteins of interest between before and after the antenatal corticosteroids use. A probability value of < .05 was considered significant.
Results
Study population
The clinical characteristics of the 12 study subjects are shown in the Table . The mean (±SD) gestational age at initial amniocentesis was 29.7 ± 3.1 weeks and the mean interval from initial to follow-up amniocentesis was 6.2 ± 1.3 (range, 2–7) days. Amniocenteses were undertaken in 6 cases with preterm premature rupture of membranes, 4 cases with preterm labor and intact membranes, 1 case with preeclampsia, and 1 case with advanced cervical examination (suspected cervical insufficiency). The mean gestational age at delivery was 36.4 ± 3.7 weeks’ gestation.
| Case no. | GA at initial amniocentesis, wk | GA at follow-up amniocentesis, wk | Interval from initial to follow-up amniocentesis, d | Interval from corticosteroids to follow-up amniocentesis, d | Indication for amniocentesis | GA at delivery, wk |
|---|---|---|---|---|---|---|
| 1 | 26.9 | 27.4 | 4 | 4 | Preeclampsia | 27.4 |
| 2 | 25.0 | 26.0 | 7 | 7 | ACE | 36.9 |
| 3 | 32.6 | 33.6 | 7 | 7 | PTL | 41.1 |
| 4 | 26.0 | 27.0 | 7 | 7 | PTL | 40.1 |
| 5 | 33.7 | 34.1 | 3 | 2 | PTL | 36 |
| 6 | 30.3 | 31.1 | 6 | 6 | PTL | 37.9 |
| 7 | 26.7 | 27.6 | 6 | 5 | pPROM | 39.4 |
| 8 | 32.7 | 33.7 | 7 | 7 | pPROM | 38.1 |
| 9 | 33.4 | 34.4 | 7 | 7 | pPROM | 37.9 |
| 10 | 31.6 | 32.4 | 6 | 6 | pPROM | 34.1 |
| 11 | 28.3 | 29.3 | 7 | 7 | pPROM | 34 |
| 12 | 29.6 | 30.6 | 7 | 7 | pPROM | 34.1 |
Stay updated, free articles. Join our Telegram channel
Full access? Get Clinical Tree
