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

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.     

Expression changes of proteins associated with the development of preeclampsia in maternal plasma: A case‐control study

Defective deep placentation, involving abnormal transformation of the spiral arteries in the junctional zone of the myometrium, is known to cause significant obstetric complications, such as preeclampsia (PE), fetal growth restriction, and placental infarction leading to fetal death. Serological biomarkers to predict and diagnose PE would help antenatal care and reduce obstetric complications. To discover candidate PE biomarkers, we first performed global proteomic profiling of three pairs of plasma samples obtained from pregnant women in the early second trimester, who subsequently developed PE, and controls to identify candidate proteins that were abundant in the patients. We further evaluated the changes in the expression of PE‐representing proteins in stored plasma samples of a cohort that subsequently developed PE and their matched controls by MRM‐MS analysis. We identified that both complement C1s subcomponent (C1S) and protein AMBP were elevated in the plasma samples of the PE cohort before the manifestation of clinical disease. We propose that these proteins may be involved in the remodeling process of the spiral arteries even before PE manifestation. These proteins can serve as potential plasma biomarkers to predict the pregnant women having an increased risk of developing PE.     

A proteomic approach identifies early pregnancy biomarkers for preeclampsia: Novel linkages between a predisposition to preeclampsia and cardiovascular disease

Preeclampsia (PE) is a common, potentially life‐threatening pregnancy syndrome triggered by placental factors released into the maternal circulation, resulting in maternal vascular dysfunction along with activated inflammation and coagulation. Currently there is no screening test for PE. We sought to identify differentially expressed plasma proteins in women who subsequently develop PE that may perform as predictive biomarkers. In seven DIGE experiments, we compared the plasma proteome at 20 wk gestation in women who later developed PE with an appropriate birth weight for gestational age baby (n=27) or a small for gestational age baby (n=12) to healthy controls with uncomplicated pregnancies (n=57). Of the 49 differentially expressed spots associated with PE‐appropriate for gestational age, PE‐small for gestational age or both (p<0.05, false discovery rate corrected), 39 were identified by LC‐MS/MS. Two protein clusters that accurately (>90%) classified women at risk of developing PE were identified. Immunoblots confirmed the overexpression of fibrinogen γ chain and α‐1‐antichymotrypsin in plasma prior to PE. The proteins identified are involved in lipid metabolism, coagulation, complement regulation, extracellular matrix remodeling, protease inhibitor activity and acute‐phase responses, indicating novel synergism between pathways involved in the pathogenesis of PE. Our findings are remarkably similar to recently identified proteins complexed to high‐density lipoprotein and linked to cardiovascular disease.     

Quantitative proteomics identified 3 oxidative phosphorylation genes with clinical prognostic significance in gastric cancer

The aim of the present study was to explore the underlying mechanisms involved in gastric cancer (GC) formation using data‐independent acquisition (DIA) quantitative proteomics analysis. We identified the differences in protein expression and related functions involved in biological metabolic processes in GC. Totally, 745 differentially expressed proteins (DEPs) were found in GC tissues vs. gastric normal tissues. Despite enormous complexity in the details of the underlying regulatory network, we find that clusters of proteins from the DEPs were mainly involved in 38 pathways. All of the identified DEPs involved in oxidative phosphorylation were down‐regulated. Moreover, GC possesses significantly altered biological metabolic processes, such as NADH dehydrogenase complex assembly and tricarboxylic acid cycle, which is mostly consistent with that in KEGG analysis. Furthermore the higher expression of UQCRQ, NDUFB7 and UQCRC2 were positively correlated with a better prognosis, implicating these proteins may as novel candidate diagnostic and prognostic biomarkers.     

A Proteomic Analysis of Placental Trophoblastic Cells in Preeclampsia–Eclampsia

To explore the proteomic changes of placental trophoblastic cells in preeclampsia–eclampsia (PE), placental trophoblastic cells from normally pregnant women and women with hypertension during gestational period were prepared by laser capture microdissection (LCM), and proteins isolated from these cells were subjected to labeling and proteolysis with isotope-coded affinity tag reagent. A qualitative and quantitative analysis of the proteome expression of placental trophoblastic cells was made using two-dimensional liquid chromatography tandem mass spectrometry (2D LC–MS/MS). A total of 831 proteins in placental trophoblastic cells were identified by combined use of LCM technique and 2D LC–MS/MS. The result was superior to that of conventional two-dimensional electrophoresis method. There were marked differences in 169 proteins of placental trophoblastic cells between normally pregnant women and women with PE. Of 70 (41.4 %) proteins with more than twofold differences, 31 proteins were down-regulated, and 39 were up-regulated in placental trophoblastic cells of the woman with PE. Laminin expression in placenta trophoblastic cells of women with PE was significantly down-regulated as confirmed by Western blot analysis. These findings provide insights into the proteomic changes in placental trophoblastic cells in response to PE and may identify novel protein targets associated with the pathogenesis of PE.     

A high‐confidence reference dataset of differentially expressed proteins in elongating cotton fiber cells

In our previous study, we used a comparative proteomic approach based on 2DE to profile dynamic proteomes of cotton fibers and found 235 protein spots differentially expressed during the elongation process ranging from 5 to 25 days post‐anthesis. Of them, only 106 differentially expressed proteins (DEPs) were identified by MS due to database limitations at the time. In the present work, we successfully identified the remaining 129 DEPs from the same experimental system using high‐resolution MS with an updated database. Bioinformatic analysis revealed that proteins involved in carbohydrate and protein metabolism, transport, and redox homeostasis are the most abundant, and glycolysis was found to be the most significantly regulated process during fiber elongation. Our high‐confidence reference dataset, composed of 235 DEPs, provides a valuable resource for future studies on the molecular mechanism of cotton fiber elongation.     

Motile hepatocellular carcinoma cells preferentially secret sugar metabolism regulatory proteins via exosomes

Exosomes are deliverers of critically functional proteins, capable of transforming target cells in numerous cancers, including hepatocellular carcinoma (HCC). We hypothesize that the motility of HCC cells can be featured by comparative proteome of exosomes. Hence, we performed the super‐SILAC‐based MS analysis on the exosomes secreted by three human HCC cell lines, including the non‐motile Hep3B cell, and the motile 97H and LM3 cells. More than 1400 exosomal proteins were confidently quantified in each MS analysis with highly biological reproducibility. We justified that 469 and 443 exosomal proteins represented differentially expressed proteins (DEPs) in the 97H/Hep3B and LM3/Hep3B comparisons, respectively. These DEPs focused on sugar metabolism‐centric canonical pathways per ingenuity pathway analysis, which was consistent with the gene ontology analysis on biological process enrichment. These pathways included glycolysis I, gluconeogenesis I and pentose phosphate pathways; and the DEPs enriched in these pathways could form a tightly connected network. By analyzing the relative abundance of proteins and translating mRNAs, we found significantly positive correlation between exosomes and cells. The involved exosomal proteins were again focusing on sugar metabolism. In conclusion, motile HCC cells tend to preferentially export more sugar metabolism‐associated proteins via exosomes that differentiate them from non‐motile HCC cells.     

Novel insights into plasma biomarker candidates in patients with chronic mountain sickness based on proteomics

Chronic mountain sickness (CMS) is a progressive incapacitating syndrome induced by lifelong exposure to hypoxia. In the present study, proteomic analysis was used to identify the differentially expressed proteins (DEPs) and then evaluate the potential plasma biomarkers between CMS and non-CMS groups. A total of 145 DEPs were detected in CMS Han Chinese people who live in the plateau (CMS-HPu), among which 89 were significantly up-regulated and 56 were significantly down-regulated. GO enrichment analysis showed that various biological processes were enriched, including the hydrogen peroxide metabolic/catabolic process, reactive oxygen species (ROS) metabolic, and acute inflammatory response. Protein–protein interaction analysis showed that antioxidant activity, the hydrogen peroxide catabolic process and peroxidase activity were primarily mapped in interaction proteins. Nine modules showed significantly clustering based on WGCNA analysis, with two being the most significant, and GO analysis showed that proteins of both modules were primarily enriched in oxidative stress-related biological processes. Four DEPs increased in CMS patients were evaluated as the candidate biomarkers, and three showed significant AUC: hemoglobin β chain (HB-β), thioredoxin-1 (TRX1), and phosphoglycerate kinase 1 (PGK1). The present study provides insights into the pathogenesis of CMS and further evaluates the potentially biomarkers for its prevention and treatment of it.     

An integrative proteome analysis of different seedling organs in tolerant and sensitive wheat cultivars under drought stress and recovery

Roots, leaves, and intermediate sections between roots and leaves (ISRL) of wheat seedlings show different physiological functions at the protein level. We performed the first integrative proteomic analysis of different tissues of the drought‐tolerant wheat cultivar Hanxuan 10 (HX‐10) and drought‐sensitive cultivar Chinese Spring (CS) during a simulated drought and recovery. Differentially expressed proteins (DEPs) in the roots (122), ISRLs (146), and leaves (163) showed significant changes in expression in response to drought stress and recovery. Numerous DEPs associated with cell defense and detoxifications were significantly regulated in roots and ISRLs, while in leaves, DEPs related to photosynthesis showed significant changes in expression. A significantly larger number of DEPs related to stress defense were upregulated in HX‐10 than in CS. Expression of six HSPs potentially related to drought tolerance was significantly upregulated under drought conditions, and these proteins were involved in a complex protein–protein interaction network. Further phosphorylation analysis showed that the phosphorylation levels of HSP60, HSP90, and HOP were upregulated in HX‐10 under drought stress. We present an overview of metabolic pathways in wheat seedlings based on abscisic acid signaling and important protein expression patterns.     

Proteomic analysis of proteins differentially expressed in uterine lymphocytes obtained from wild‐type and NOD mice

Non‐obese diabetic (NOD) mice exhibit impaired fertility and decreased litter size when compared to wild type (WT) mice. However, it is unclear why allogeneic pregnant NOD mice are prone to spontaneous embryo loss. Herein, two‐dimensional gel electrophoresis (2‐DE) and mass spectrometry (MS) were used to detect differentially expressed proteins in the uterine lymphocytes isolated from these mice and WT BALB/c controls. We found 24 differentially expressed proteins. The differential expression of 10 of these proteins was further confirmed by Western blot analysis. Out of the 24 identified proteins, 20 were expressed in uterine lymphocytes of WT mice at a level at least 2 times higher than in NOD mice, whereas 4 were down‐regulated. Western blot analysis confirmed that 8 proteins were up‐regulated and 2 proteins were down‐regulated in WT mice compared with NOD mice, consistent with the results of 2‐DE and MS. Additionally, most of the highly expressed proteins in WT uterine lymphocytes were expressed at a significantly lower level in the corresponding splenic group (17/20). These results suggest that up‐regulated expression of these proteins may be specific to uterine lymphocytes. Reported functions of the highly expressed proteins affect key functions during pregnancy, including cell movement, cell cycle control, and metabolisms. Finally, we analyzed the constitutional ratio of CD3⁺ and CD49b⁺ cells in the isolated lymphocytes by flow cytometry. Our results suggest that the differentially expressed proteins may participate in the modulation of embryo implantation and early‐stage development of embryos, and subsequently influence pregnancy outcome. J. Cell. Biochem. 108: 447–457, 2009. © 2009 Wiley‐Liss, Inc.     

Quantitative proteomics analysis of human vitreous in rhegmatogenous retinal detachment associated with choroidal detachment by data-independent acquisition mass spectrometry

The prognosis of rhegmatogenous retinal detachment (RRD) with choroidal detachment (RRDCD) is often poor and complicated. This study focused on the identification of the characteristic proteins and signal pathways associated with the etiology of RRDCD and to provide guidance for diagnosis and treatment of RRDCD. In this study, vitreous humor samples were obtained from 16 RRDCD patients, 14 with RRD, 12 with idiopathic epiretinal macular membrane (IEMM), and 5 healthy controls from donated corpse eyes. Data-independent acquisition mass spectrometry and bioinformatics analysis were employed to identify differentially expressed proteins (DEPs). In the vitreous humor, 14,842 peptides were identified. Patients with RRDCD had 249 DEPs (93 upregulated and 156 downregulated), with 89 in patients with RRD and 61 in patients with IEMM. Enrichment analysis of the GO and Kyoto Encyclopedia of Genes and Genomes DEP databases indicated functional clusters related to inflammation and immunity, protein degradation and absorption, cell adhesion molecules (CAMs), the hedgehog signaling pathway, and lipid metabolism. Weighted gene co-expression network analysis showed that DEPs with positive co-expression of RRDCD participated in immune-related pathways led by the complement and coagulation cascade, whereas DEPs with negative co-expression of RRDCD participated in protein degradation and absorption, CAMs, and the hedgehog signaling pathway. In summary, our study provides important clues and the theoretical basis for exploring the pathogenesis, progression, and prognosis of ocular fundus disease.

     

Comparative proteomic analysis of rat aorta in a subtotal nephrectomy model

Although accelerated atherosclerosis and arteriosclerosis are the main causes of cardiovascular morbidity and mortality in chronic kidney disease (CKD) patients, the molecular pathogenesis remains largely obscure. Our study of the aortic function in a typical CKD model of subtotal nephrectomy (SNX) rats demonstrated phenotypes that resemble CKD patients with aortic stiffness. The 2‐DE analysis of rat aortas followed by MS identified 29 up‐regulated and 53 down‐regulated proteins in SNX rats. Further Western blot and immunohistochemistry analyses validated the decreased HSP27 and increased milk fat globule epidermal growth factor‐8 (MFG‐E8) in SNX rats. Functional classification of differential protein profiles using KOGnitor revealed that the two major categories involved in aortic stiffness are posttranslational modification, protein turnover, chaperones (23%) and cytoskeleton (21%). Ingenuity Pathway Analysis highlighted cellular assembly and organization, and cardiovascular system development and function as the two most relevant pathways. Among the identified proteins, the clinical significance of the secreted protein MFG‐E8 was confirmed in 50 CKD patients, showing that increased serum MFG‐E8 level is positively related to aortic stiffness and renal function impairment. Drug interventions with an inhibitor of the angiotensin converting enzyme, enalapril, in SNX rats improved aortic stiffness and decreased MFG‐E8 depositions. Together, our studies provide a repertoire of potential biomarkers related to the aortic stiffness in CKD.     

Identification of serum biomarkers for premature ovarian failure

Premature ovarian failure (POF) is defined when a female achieves menopause before the age of 40. Although many conditions are known to be causative for POF, the most common one is idiopathic. This study was undertaken to investigate the pathogenesis of POF using proteomic tools. Two-dimensional electrophoresis (2-DE) analysis was performed to screen for proteins differentially expressed in patients with POF. Using liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS), we identified 11 significant proteins differentially expressed in the serum of POF patients: 5 proteins with expression increased more than two folds, 5 proteins with expression decreased more than two folds, and 1 protein expressed specifically in the serum of patients with POF. The results of the 2-DE analysis were further validated by Western blotting and ELISA analyses, which 5 reproductive system-related proteins (Ceruloplasmin, Complement C3, Fibrinogen α, Fibrinogen β, and SHBG) were selected. The different expression levels for these proteins were confirmed and demonstrated the possibility of using them as biomarkers to screen POF. These pre-clinical data provide plausible translational implications for targeting the pathogenesis of POF for each protein.     

Arbuscular mycorrhizal fungi effect growth and photosynthesis of Phragmites australis (Cav.) Trin ex. Steudel under copper stress

• Arbuscular mycorrhizal fungi (AMF) is an effective way to remove heavy metals’ inhibition on plants, however, few relevant research attempts have been made to determine the contribution of AMF to the physiological and biochemical changes related to the enhanced copper tolerance of Phragmites australis under metal‐stressed conditions.
• In this study, the effects of AMF inoculation on P. australis under different concentrations of copper stress were investigated according to the changes in the parameters related to growth and development, and photosynthetic charateristics. Then, differentially expressed proteins (DEPs) were evaluated by the Isobaric Tag for Relative and Absolute Quantification (iTRAQ) system, which could accurately quantify the DEPs by measuring peak intensities of reporter ions in tandem mass spectrometry (MS/MS) spectra.
• It was found that AMF inoculation may relieve the photosynthesis inhibition caused by copper stress on P. australis and thus promote growth. Proteomic analysis results showed that under copper stress, the inoculation of R. irregularis resulted in a total of 459 differently‐expressed proteins (200 up‐regulated and 259 down‐regulated) in root buds. In addition, the photosynthetic changes caused by AMF inoculation mainly involve the up‐regulated expression of transmembrane protein–pigment complexes CP43 (photosystem II) and FNR (ferredoxin‐NADP+ oxidoreductase related to photosynthetic electron transport).
• These results indicate that AMF could effectively improve the growth and physiological activity of P. australis under copper stress, and thus provides a new direction and instructive evidence for determining the mechanisms by which AMF inoculation enhances the copper tolerance of plants.

     

Proteomic analysis of amniotic fluid to identify potential targets predicting preterm delivery

Preterm delivery is a common complication of pregnancy which leads to significant neonatal mortality and morbidity. Identifying predictive markers linked to spontaneous preterm delivery (SPTD) is important for effective treatment and prevention of PTD. To explore potential biomarkers related to SPTD, we performed proteomics analysis in amniotic fluid (AF). In total, we enrolled 30 pregnant women with singleton gestation who underwent clinically indicated amniocentesis at 15–24 weeks of gestation. LC-MS analysis was used to analyze the AF samples of 10 women with SPTD < 34 weeks after cervix cerclage (Preterm group), 10 women with term delivery (TD) ≥ 34 weeks after cervix cerclage (Term group), and 10 women who delivered at term (Normal group). ELISA validation was performed for candidate proteins in a second independent cohort. As a result, we identified 44 differentially expressed proteins (DEPs, P < 0.05) via proteomic analysis, and based on that, 9 primary pathways were also determined in SPTD. Results of the ELISA assay confirmed that the increased concentration of Serpin A1, decreased concentrations of Renin and IGFBP4 were significantly associated with SPTD at ≤34 weeks.     

蛹虫草原基分化的差异蛋白质组学研究

蛹虫草子实体形成及发育的蛋白分子机制尚不清楚,本研究引入SWATH非标记定量蛋白质组学技术,对蛹虫草Cordyceps militaris 905菌株的菌丝体（mycelium,My）、原基（primordium,Po）、生长期子实体（developmental fruiting body,DF）和成熟期子实体（mature fruiting body,MF）进行了比较蛋白质组学分析。经搜库比对,从蛹虫草的My、Po、DF和MF中依次鉴定蛋白1 136个、1 090个、1 018个和997个（global FDR 1%）,经维恩分析后获得C. militaris 905蛹虫草表达蛋白1 578个。在此基础上,SWATH非标记技术定量蛋白1 109个。本研究获得了蛹虫草Po期与My期、DF期与Po期、MF期与DF期的差异表达蛋白,依次为115个、352个和104个,并对菌丝体分化形成原基的差异表达蛋白进行了重点解析。GO注释结果表明,Po期与My期差异表达蛋白以有机含氮类化合物代谢为主,其中AMP（活性成分虫草素合成的中间产物）从头生物合成途径富集最为显著。约1/5的差异表达蛋白参与氧化还原反应,还原酶活性的蛋白在原基中几乎都上调表达,而氧化功能的蛋白受到抑制,表明蛹虫草原基分化可能受到氧化应激的诱导。蛋白互作网络分析结果进一步表明,氧化还原反应与核苷类物质代谢相关联,可能通过影响AMP从头生物合成途径来调控虫草素的生物合成。对蛹虫草子实体系统的蛋白质组学研究和解析有利于揭示子实体形成的蛋白分子机制,为蛹虫草的基础和栽培研究提供了理论支撑。     

Proteomic tracking of serum protein isoforms as screening biomarkers of ovarian cancer

Epithelial ovarian cancer is the fourth leading cause of cancer death among women. Due to the asymptomatic nature and poor survival characteristic of the disease, screening for specific biomarkers for ovarian cancer is a major health priority. Differentially expressed proteins in the serum of ovarian cancer patients have the potential to be used as cancer-specific biomarkers. In this study, proteomic methods were used to screen 24 serum samples from women with high-grade ovarian cancer and compared to a control group of 11 healthy women. Affigel-Blue treated serum samples were processed either by linear (pH 4-7) or narrow range (pH 5.5-6.7) IEF strips for the first dimension. Proteins separated in first dimension were resolved by 8-16% gradient SDS-PAGE. Protein spots were visualized by SYPRO Ruby staining, imaged by FX-imager and compared and analyzed by PDQuest software. Twenty-two protein spots were consistently differentially expressed between normal and ovarian cancer patients by resolving proteins in a linear pH strip of 4-7 for the first dimension. Six of the protein spots, significantly up-regulated in grade 3 ovarian cancer patients (p < 0.05), were identified by MALDI-TOF MS and Western blotting as the isoforms of haptoglobin precursor. When serum proteins were resolved on narrow pH range strips (5.5-6.7), 23 spots were consistently differentially expressed between normal and grade 3 ovarian cancer patients. Of these, 4 protein spots significantly down regulated in grade 3 ovarian cancer patients (p < 0.05) were identified by MALDI-TOF MS and Western blotting, as isoforms of transferrin precursor. Increased expression of serum haptoglobin and transferrin was also identified in peritoneal tumor fluid obtained from women diagnosed with grade 2/3 ovarian cancer (n = 7). Changes in the expression of haptoglobin and transferrin in the serum of women with different pathological grades of ovarian cancer was examined by one-dimensional Western blotting method. Serum samples collected from women suffering from benign, borderline, grade 1, grade 2 and grade 3 cancer (n = 4 for haptoglobin and n = 5 for transferrin in each group) were analyzed and compared to the serum of normal healthy women. The mean serum haptoglobin expression in grade 3 ovarian cancer patients was fourfold higher than in the control subjects (p < 0.05). On the other hand, transferrin expression in grade 3 ovarian cancer patients was decreased by twofold than in normal healthy women (p < 0.05). Haptoglobin expression in the serum of cancer patients (n = 7) decreased following chemotherapy (six cycles of taxol/carboplatin). Concomitant with the decrease of haptoglobin, transferrin expression remained constant in four patients, but increased in three out of seven patients included in the study. Changes in serum expression of haptoglobin correlated with the change of CA 125 levels before and after chemotherapy. In conclusion, proteomic profiling of differentially expressed proteins in the sera of normal women compared to women with ovarian cancer can greatly facilitate the discovery of a panel of biomarkers that may aid in the detection of ovarian cancer with greater specificity.