2.1 Study design and inclusion criteria
Samples were all from the Department of Obstetrics and Gynecology, Wuhan University Zhongnan Hospital. The specific demographic data are shown in Table 1. The diagnostic criteria of CHD are detailed in the expert consensus [6]. This research program was approved by the ethics committee of Wuhan University Zhongnan Hospital (project number: 2020085, in supplement S2) and conducted in compliance with ethical guidelines of the Helsinki Declaration. The informed consent was informed and signed by all voluntary participants.
Table 1
Basic information characteristics of included patients
|
CHD group
|
Control group
|
P value
|
DIA training sets(N=15)
|
|
|
|
Maternal age(Year)
|
27.50±3.02
|
30.50±5.32
|
0.352
|
Faternal age(Year)
|
29.11±2.47
|
31.67±2.34
|
0.067
|
Gestational age(week)
|
24.69±5.82
|
21.00±3.36
|
0.147
|
NT (cm)
|
0.16±0.32
|
0.22±0.91
|
0.175
|
NIPT / Down's screening
|
|
|
0.221
|
Low-risk
|
9
|
5
|
-
|
High-risk
|
0
|
1
|
-
|
PRM validation sets(N=14)
|
|
|
|
Maternal age(Year)
|
29.12±4.58
|
29.00±3.95
|
0.948
|
Fateral age(Year)
|
29.11±2.47
|
31.5±2.51
|
0.088
|
Gestational age(week)
|
24.84±5.76
|
22.37±2.08
|
0.491
|
NT(cm)
|
0.16±0.032
|
0.19±0.073
|
0.864
|
NIPT / Down's screening
|
|
|
0.63
|
Low-risk
|
9
|
0
|
-
|
High-risk
|
4
|
2
|
-
|
Elisa validation sets(N=52)
|
|
|
|
Maternal age(Year)
|
32±4.98
|
31.46±6.376
|
0.666
|
Faternal age(Year)
|
30.58±5.278
|
31.5±7.355
|
0.213
|
Gestational age(week)
|
24.334±4.550
|
20.931±2.744
|
0.023
|
NT(cm)
|
0.149±0.073
|
0.174±0.071
|
0.158
|
NIPT / Down's screening
|
|
|
0.741
|
Low-risk
|
23
|
21
|
-
|
High-risk
|
1
|
3
|
-
|
Notes: N is the number of participants. Each participant had amniotic fluid and serum samples in this study. P<0.05 means statistically significant.
|
Inclusion criteria for the CHD group: (1)fetal CHD was diagnosed by echocardiography; (2)The pregnant women naturally conceived were between 18 and 45 years old; (3) Couples are not close relatives. The inclusion criteria for the control group: (1)meet the above(2)(3); (2)The pregnant women were only older (≥35) or the false positive of NIPT/screening for Down Syndrome or NT ≥ 0.25cm, whose prenatal diagnosis results were normal. Exclusion criteria: (1) Fetus with extracardiac malformation; (2)Pregnant women have suffered from rheumatic heart disease, myocarditis, other non-structural heart diseases, hepatitis, abnormal renal and liver function, diabetes, or other acute or chronic infectious diseases.
Sample collection and preservation: (1) AF(2ml) were taken from pregnant women before amniocentesis, and corresponding maternal peripheral blood (2ml) was taken at the same time. AF supernatant and GS were taken out after centrifuge (4° C, 1000rpm/min,10min) and then stored at - 80 ° C. (2) Fetal heart tissue: the heart tissue (2 ~ 4mm3) of induced labor fetus is taken. One part of the myocardial tissue was fixed in 4% formaldehyde for 48 hours before paraffin embedding, while the other was frozen in the refrigerator at - 80 ° C prepared for RT-PCR.
2.2 DIA
2.2.1 LC-MS / MS
GS was taken to remove the high peak protein (Thermo Fisher company, No:85165, kit column), so the total protein solution of AF can be obtained. All samples were enzymolized and desalted with an equal amount of protein. The peptides were redissolved with mobile phase A and then mixed evenly. Agilent 1100 HPLC system was used to separate the components in mobile phase pH=10. The peptide mixture was loaded by Agilent Zorbax Extend - C18 capture column (2.1× 150 mm, C18, 5 µm). The flow rate was 250nL/min, and the linear gradient was 63 min (0-10 min, 2%B; 10-10.1 min, 2-5%B; 10.1-37 min, 5-20%B; 37-48 min, 20-40%B; 48-48.01 min, 40-90%B; 48.01-58 min, 90%B; 58-58.01 min, 90-2%B; 58.01-63 min, 2%B; Mobile phase A=2%ACN and B=90%ACN). Mobile phase A and B ammonia was used to adjust pH to 10. Samples were collected for 8 to 50 minutes, one tube was collected every one minute in turn, and then the samples were collected in this order until the end of the gradient. A total of 10 components were obtained. After collecting, the samples were freeze-dried in a vacuum and drained. Before mass spectrometry detection, each sample and IRT standard (Biognosys, ThermoFisher) were mixed in the ratio of 1:10, and the coefficient of variation of IRT was less than 10%.
DDA mode database construction LC-MS/MS analyses are performed on a Q-extraction HF mass spectrometer (Thermo, USA). The peptide mixture was loaded by capillary C18 capture column. The analysis column was C18 reversed-phase column (ChromXP Eksigent). The flow rate was 300nL/min, and the linear gradient was 90 min. Full mass spectrometry scans were obtained in the mass range of 350-1650m/z with a mass resolution of 120,000 and an AGC target value of 3E6. All MS/MS spectra were collected using data-dependent high-energy collision pyrolysis in positive ion mode, and the collision energy was set to 27. MS/MS resolution was set as 30000, automatic gain control was set as 2E5, and the maximum injection time was 80 MS. The dynamic exclusion time was set to 40 s.
2.2.2 spectrum matching and database building
The machine signal is transformed into peptide and protein sequence information by matching the mass spectrum output with the theoretical spectrum generated by the FASTA library. Then the spectrum library is established by combining the sequence information, peptide retention time, and fragment ion information to facilitate the subsequent DIA analysis. Finally, the original LC-MS / MS files are imported into Spectronaut pulsar software to match and build the database. Database: UniProt-reviewed-Homo sapiens (Human)-20200817. The specific parameters of DDA and DIA are shown in supplement S4.
2.3 PRM
All samples were pre-scanned with the same amount of mix to correct the retention time of peptides. Based on the results of qualitative analysis, the identified target peptides were screened, and the trusted peptides were retained. PRM mode detection: The flow rate was 300nL/min, and the linear gradient was 90 min. MS/MS resolution was set as 30000 and an AGC target value of 1e5. Isolation Window 1.2m /z and the maximum injection time were 100 MS. The trusted peptides suitable for PRM analysis were introduced into the mass spectrometry software Xcalibur. After three times of PRM detection, the PRM original file data were PRM original file's data were analyzed by Skyline 3.7.0 software, and the target proteins and the target peptides were quantified.
2.4 Bioinformatics analysis
These identified proteins are annotated and analyzed(Including GO, pathway, PPI), which were based on the databases of UniProt((https://www.uniprot.org/), KEGG((https://www.kegg.jp/kegg/pathway.html), GO ((http://geneontology.org/)and KOG / COG((http://cwmda.com/service_detail/90/122). At the same time, correlation analysis, expression pattern clustering heat map, Venn map were done for the difference comparison group data. Differential protein interaction analysis was performed by STRING ( https://www.string-db.org/), and the correlation and interaction network was drawn by Cytoscape 3.6.1 software.
2.5 ELISA
POSTN (A102870-96T, Shanghai Fusheng Industrial Co., Ltd, China) and PAPPA (HM11110, Bioswap, China) in GS and AF were measured using commercially available ELISA kits. The experimental operation has been executed according to the corresponding instructions.
2.6 Immunohistochemistry (IHC)
IHC was performed on paraffin-embedded tissue sections. After being dewaxed, hydrated, repaired with EDTA buffer, and blocked with 5% BSA, tissues were incubated overnight at 4 ° C with primary antibodies to detect POSTN (ab219057, Abcam, USA). After incubation with HRP Goat Anti-Rabbit IgG (SAB4591, Bioswamp, China), slides were visualized by DAB-Substrate (Beyotime, China) and photographed by the Aperio ePathology Scanner (Leica, Germany). ImageJ software analyzed protein expression.
2.7 RNA isolation, cDNA synthesis, and RT-PCR
Total RNA was isolated by Invitrogen™ TRIzol™ Reagent (Thermo Fisher Scientific, USA), and cDNA was synthesized from the total RNA using PrimeScript™RT Master Mix (Takara, Japan). RT-PCR was performed on the Bio-Rad CFX96 (Bio-Rad Laboratories, USA) using SYBR Premix Ex Taq™(Takara, Japan). The sequences of primers are in supplement S4. The relative expression of the mRNA was calculated by the 2-△△Ct.
2.8 Statistical analysis
Data analysis and the ROC curve analysis were processed by SPSS 22.0 (SPSS Inc, Chicago, IL). Metrological data were presented as means ± SD. An independent sample T-test was adopted when normal distribution and homogeneity of variance were met. Otherwise, Mann-Whitney U was performed. Fisher's exact test was utilized to compare the distribution of GO categories or KEGG pathways between the target protein set and the overall protein set and to perform enrichment analysis on the GO categories or KEGG pathways of the target protein set. P < 0.05 is considered statistically significant.