Systematic proteomics analysis of lysine acetylation reveals critical features of placental proteins in pregnant women with preeclampsia

Abstract Preeclampsia (PE) is a dangerous hypertensive disorder that occurs during pregnancy. The specific aetiology and pathogenesis of PE have yet to be clarified. To better reveal the specific pathogenesis of PE, we characterized the proteome and acetyl proteome (acetylome) profile of placental tissue from PE and normal‐term pregnancy by label‐free quantification proteomics technology and PRM analysis. In this research, 373 differentially expressed proteins (DEPs) were identified by proteome analysis. Functional enrichment analysis revealed significant enrichment of DEPs related to angiogenesis and the immune system. COL12A1, C4BPA and F13A1 may be potential biomarkers for PE diagnosis and new therapeutic targets. Additionally, 700 Kac sites were identified on 585 differentially acetylated proteins (DAPs) by acetylome analyses. These DAPs may participate in the occurrence and development of PE by affecting the complement and coagulation cascades pathway, which may have important implications for better understand the pathogenesis of PE. In conclusion, this study systematically analysed the reveals critical features of placental proteins in pregnant women with PE, providing a resource for exploring the contribution of lysine acetylation modification to PE.


| INTRODUC TI ON
Preeclampsia (PE) is a placenta-induced hypertensive disorder of pregnancy that affects 3-5% of pregnancies, and it is one of the main causes of morbidity and mortality in mothers and foetuses. 1,2 When left untreated, PE can lead to significant heart, liver, kidney, brain and lungs dysfunction in pregnant women, as well as a series of foetal complications (such as neonatal growth restriction, neonatal respiratory distress syndrome, autism spectrum disorder, cerebral palsy or even death). 2,3 Some scholars have proposed a two-stage model of PE: stage 1 is poorly perfused placenta in the first trimester, wherein the failure of trophoblast invasion leads to dysfunctional spiral artery remodelling, and hypoxia may cause oxidative stress and imbalance of reactive oxygen species in the placenta of PE; stage 2 is a maternal syndrome in the last second and third trimesters characterized by angiogenesis imbalance, inflammatory cytokines and immune cell alterations. 4,5 The placental tissue is directly exposed to the uterine decidua, which is involved in maternal-foetal immune tolerance and pregnancy maintenance. 6 Although its exact pathogenesis and triggering conditions are still not fully clarified, it is widely believed that the placenta plays a central role in PE formation. 7 The most common cause of PE includes placental hypoxia, oxidative stress, disturbed angiogenesis, increased proinflammatory cytokines, complement dysregulation and endothelial dysfunction. 5,8,9 Recent research has found that the immune system and mitochondrial dysfunction play an essential role in the pathophysiology of PE. 10,11 Interestingly, hypoxia leads to placental chromatin modification, which reduces placental histone acetylation and placental acetyl-CoA, suggesting that acetylation modification may be critical to the pathogenesis of PE. 3 Proteins posttranslational modifications (PTMs) play a crucial role in regulating various biological processes, which can modulate the structure, activity and function of proteins by introducing new functional groups. 12,13 Lysine acetylation (Kac) is an essential posttranslational modification discovered in histones in the 1960s. 14 This modification regulates many biological functions (such as chromatin remodelling, gene expression regulation, protein stability, cytoskeletal dynamics and cellular metabolic state) and is closely linked to cardiovascular diseases. [15][16][17] However, there is still a lack of available studies on the role of Kac modification in PE. Previous researchers have used iTRAQ proteomics to identify differentially expressed proteins (DEPs) in PE and normal pregnancy placental tissues to look for potential biomarkers of PE. 6 Quantitative analysis and PTMs of proteins are essential for understanding biological complexity. 12,18 To better understand the pathogenesis of PE, we integrated proteome and acetyl proteome (acetylome) systematically to analyse the vital features of placental proteins in pregnant women with PE and provided a landscape of acetylation in PE, which may contribute to developing valuable novel biomarkers for PE.

| Sample collection
In this study, six pregnant women with PE and six normal-term pregnant women admitted to the Department of Obstetrics and Gynecology, Shenzhen People's Hospital, from February 2020 to July 2020, were recruited and divided into the PE group and NC group.
The inclusion criteria of the PE group were ACOG gestational hypertension and preeclampsia guidance. 19 All the pregnant women were without infection, coronary heart disease, chronic kidney disease, tuberculosis or other diseases affecting placental protein content. The study was approved by the Medical Ethics Committee of Shenzhen People's Hospital. All pregnant women signed written informed consent before inclusion in the study. Placental tissue near the maternal side's central area was collected immediately in an aseptic environment after delivery. Calcification, necrosis and vascular areas were excluded from sampling. The residual maternal blood and amniotic fluid were washed with PBS, and the excess water was removed with sterile filter paper. Finally, the tissue samples were put into centrifuge tube and transferred to −80°C refrigerator for further processing.

| Sample processing
Protein extraction and trypsin digestion were carried out using the published method. 20 Placental tissue samples were removed from a −80°C refrigerator and ground into cell powder with liquid nitrogen. After that, a four-fold volume of lysis buffer (1% Triton-100, 1% protease inhibitor, 3 μM TSA and 50 mM NAM) was added to each centrifuge tube containing cell powder. Then, the suspension was sonicated on ice with a high-intensity ultrasound processor. The remaining debris was removed by centrifugation for 10 min (12000 g at 4℃). Subsequently, we used a BCA protein assay kit (Beyotime) to determine the protein concentration in the supernatant. After treatment with trichloroacetic acid (TCA), the protein precipitation was dispersed with 200 mM tetraethylammonium bromide (TEAB) and then digested overnight with trypsin at a ratio of 1:50 (trypsin: protein). Upon addition of dithiothreitol (DTT; final concentration of 5 mM), samples were reduced for 30 min at 56°C, followed by addition of iodoacetamide (final concentration of 11 mM) and incubation for 15 min at room temperature in the dark.

| Affinity enrichment of Kac peptides
Compared with the total proteome, the acetylome added an IP enrichment process. Before Kac enrichment, pan anti-acetyllysine antibody beads (Lot number: PTM-104, PTM Bio) were prewashed with PBS. The peptides were dissolved in IP buffer (100 mM NaCl, 1 mM EDTA, 50 mM Tris-HCl, 0.5% NP-40, pH 8.0), and then the supernatant was incubated with antibody beads at 4°C overnight with mild shaking. After incubation, the antibody beads were washed four times with IP buffer and twice with ddH 2 O. Finally, we used 0.1% trifluoroacetic acid to elute the bound Kac peptides from antibody beads. The eluted fractions after vacuum drying were desalted with C18 ZipTips (Merck Millipore) and then analysed by LC-MS/MS. 21

| LC-MS/MS analysis
The peptides from total protein digestion or enrichment of Kac peptides were dissolved in 0.1% formic acid and 2% acetonitrile before being injected into a nanoElute UPLC system (Bruker). For the peptides used for proteome quantification, the gradient elution was set to solvent B (0.1% formic acid and 100% acetonitrile) increase from 6% to 24% over 70 min, 24% to 35% in 14 min, a linear increase to 80% in 3 min and then maintained in 80% for the last 3 min; the flow rate was constant at 450 nL/min. 22 For the Kac peptides used for acetylome quantification, the gradient elution was set to solvent B increase from 7% to 22% over 40 min, 22% to 30% in 14 min and climbing to 80% in 3 min and then maintained in 80% for the last 3 min; the flow rate was constant at 350 nL/min. Peptides were ionized with 2.0 kV electrospray voltage from a capillary ion source on a timsTOF Pro mass spectrometer (MS) (Bruker Daltonics). Precursor ions were detected in the TOF MS at a resolution of 30000. The data acquisition used the parallel accumulation serial fragmentation (PASEF) mode. 23 Dynamic exclusion was set to 30 s to reduce repeated sequencing of precursor ions.

| Database search
The MaxQuant search engine (v.1.6.15.0) was used to process MS/ MS data. 24LC-MS/MS was searched against the SwissProt database (Homo sapiens, containing 20366 sequences) concatenated with the reverse decoy database. 25 Trypsin/P was specified as a cleavage enzyme allowing up to four missed cleavages and five modifications per peptide, and the minimum peptide length was set as 7. Cysteine carbamidomethylation was defined as fixed modification; protein N-terminal acetylation and methionine oxidation were defined as variable modifications. False discovery rate (FDR) thresholds for peptides, proteins and modification sites were adjusted to 1%. These common parameters were used for proteome and acetylome database search analysis. In addition, the identification of the acetylome used the following additional settings: Kac was defined as variable modification and modified peptide score was set at >40.

| Parallel Reaction Monitoring analysis
Tissue collection and processing were the same as described above.
For the total proteome, the tryptic peptides of each sample were separated on an EASY-nLC 1000 UPLC system (Thermo Fisher Scientific). Subsequently, the eluted peptides were analysed using

| Bioinformatics and statistical analysis
The differences between PE and NC were calculated using fold change (FC) as a criterion. FC>1.5 and FC<1/1.5 were considered as up-regulated and down-regulated proteins, respectively. p < 0.05 was the critical value for identifying DEPs and differentially acety-

| Clinical characteristics
The clinical characteristics of women with PE and NC were given in Table 1. We analysed critical biometric data such as maternal age, gestational age, maternal BIM, systolic blood pressure, diastolic blood pressure, 24 h proteinuria and baby birth weight. The results showed no significant difference in maternal age, body mass index (BMI) or baby weight between the two groups (p > 0.05).
Simultaneously, systolic blood pressure, diastolic blood pressure and 24-h proteinuria in patients with PE were significantly higher than those in NC.

| Proteome profiling of PE placental tissues
An overview of the experimental procedure is shown in Figure 1A.
A total of 6460 proteins were identified in label-free quantification proteomics analysis, of which 5457 proteins had a quantifiable level. Among these quantifiable proteins, 373 proteins showed statistically significant changes between PE and NC, including 127 proteins up-regulated and 246 down-regulated ( Figure 1B). It is noteworthy that up-regulated proteins include COL12A1, ANG, FLT1 and C4BPA, and the down-regulated proteins include F13A1, ITGAM and ITGB2 ( Figure 1B). Hierarchical clustering analysis of DEPs is depicted in Figure 1C, demonstrating a moderate distinction between NC and PE.

| Function enrichment analysis of DEPs
To further clarify the functions of these DEPs, we performed

| Profiles of DAPs and motif analysis of Kac sites in PE placental tissues
We corrected the quantification values corresponding to the acety-  13,31,32 whether these different types of enzymes that regulate acetylation can affect the pathophysiology of PE remains to be further studied.

| Subcellular localization and functional enrichment analysis of DAPs
Protein acetylation modification regulates many critical cellular processes and has vital implications for protein transport and cellular localization. 15 Kac was first discovered in histones. 14 In addition to histones, acetylated proteins were found in the nucleus, cytoplasm, mitochondria and other cell compartments. 13 Consistent with previous studies, DAPs were mainly distributed in the cytoplasm, nucleus and mitochondria 31,33,34 ( Figure 4D). Kac widely exists in mitochondria and is closely associated with the regulation of energy generation and fatty acid, amino acid and sugar metabolism in these organelles. 17,31 Placental mitochondrial dysfunction may be associated with a series of pregnancy disorders, including PE. 35 To further investigate the possible biological functions of these acetylated

| Protein-protein networks of DAPs
To fully describe protein function, understanding the proteinprotein interaction (PPI) is very important. 36 Here, we designed a PPI network using Cytoscape. 29

| Verification of the potential biomarkers in PE by PRM analysis
PRM is a "targeted" mass spectrometry technique capable of verifying many targeted proteins and has been used in recent years to assess quantitative differences between biological samples. 26,40 Previous studies have utilized the LFQ-PRM approach to identify and validate ovarian cancer-related protein changes in patient urine samples to search for urine biomarkers. 41 Here, we performed PRM-based   and PE, and found that DEPs were involved in various physiological processes, such as angiogenesis, oxidative stress and placental development. 43 In another study, researchers used label-free proteomics to identify and quantify placental proteins in a rat model of spontaneous hypertension and gestational hypertension, and found that these proteins were associated with inflammation and trophoblast invasion. 44 Here, we found that DEPs were distributed in mitochondria and involved in multiple biological processes associated with immunity and angiogenesis. Simultaneously, most DAPs were related to the structural constituent of the cytoskeleton and The activation of neutrophils produces elastase, which may lead to vascular injury and is closely related to vascular dysfunction in PE. 11 In our research, ITGB2 and ITGAM participated in a variety of biological processes related to immunity. Related research has reported that ITGB2 and ITGAM genes may be closely related to the pathogenesis of PE and are considered as new biomarkers of PE, which may be used for PE diagnosis. 48,49 To sum up, the immune system and angiogenesis may affect the pathogenesis of PE.
Strikingly, collagen type XII alpha 1 chain (COL12A1) has the greatest fold change between PE and NC, which was an interesting finding in this study. Increased DNA methylation and gene expression at COL12A1 are associated with elevated maternal blood pressure during pregnancy, as reported earlier. 50 Furthermore, additional research supports that COL12A1 gene is up-regulated in PE placental tissue. 51 Importantly, hypertension is a major characteristic of PE, which suggests that COL12A1 may be a potential candidate biomarker for the assessment of PE.
PE arises from abnormal placentation, which is associated with insufficient cytotrophoblast (CTB) cells fusion, syncytiotrophoblast (STB) dysfunction and inadequate trophoblast invasion. 52,53 In addition, the differentiation of CTB is also related to changes in However, there were some limitations to consider in the current study. The generalizability of our findings may be limited by the current sample size. Although the results seem convincing, these findings need to be confirmed in larger prospective studies. In addition, the sample of early-onset PE and late-onset PE should be further separated to explore the distinction between the two phenotypes of PE.

| CON CLUS ION
In this study, systematic analysis of Kac revealed the critical fea-

CO N FLI C T O F I NTE R E S T
The authors confirm that they have no conflicts of interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
The raw data have been deposited in the OMIX (https://ngdc.cncb. ac.cn/omix) under the accession number OMIX546.