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.     

iTRAQ-based proteomic analysis of human umbilical vein endothelial cells with platelet endothelial aggregation receptor-1 knockdown

The disorders of hemostasis and coagulation were believed to be the main contributors to the pathogenesis of pulmonary thromboembolism (PTE), and platelets are the basic factors regulating hemostasis and coagulation and play important roles in the process of thrombosis. This study investigated the proteome of human umbilical vein endothelial cells (HUVECs) with platelet endothelial aggregation receptor-1 (PEAR1) knockdown using the isobaric tags for relative and absolute quantitation (iTRAQ) method and analyzed the role of differential abundance proteins (DAPs) in the regulation of platelets aggregation. Our results showed that the conditioned media-culturing HUVECs with PEAR1 knockdown partially suppressed the adenosine diphosphate (ADP)-induced platelet aggregation. The proteomics analysis was performed by using the iTRAQ technique, and a total of 215 DAPs (124 protein was upregulated and 91 protein were downregulated) were identified. The Gene Ontology (GO) enrichment analysis showed that proteins related to platelet α granule, adenosine triphosphate metabolic process, and endocytosis were significantly enriched. Further, the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis also identified the significant enrichment of endocytosis-related pathways. The real-time polymerase chain reaction assay confirmed that the expression of P2Y₁₂, mitochondrial carrier 2, NADH dehydrogenase (ubiquinone) iron-sulfur protein 3, and ubiquinol-cytochrome c reductase hinge protein are significantly downregulated in the HUVECs with PEAR1 knockdown. In conclusion, our in vitro results implicated that DAPs induced by PEAR1 knockdown might contribute to the platelet aggregation. Proteomic studies by employing GO enrichment and KEGG pathway analysis suggested that the potential effects of DAPs on platelet aggregation may be linked to the balance of ADP synthesis or degradation in mitochondria.     

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.     

Isobaric tag for relative and absolute quantitation based quantitative proteomics reveals unique urinary protein profiles in patients with preeclampsia

Preeclampsia (PE) is one of the most significant pregnancy‐related hypertensive disorders. Currently, there are no useful markers to predict the onset of the condition in pregnant women. To provide further insights into the pathogenesis of PE and identify biomarkers of the condition, we used isobaric tags for relative and absolute quantitation (iTRAQ) proteomics coupled with 2‐D LC‐MS/MS, to analyze urinary protein profiles from 7 PE patients and 7 normotensive pregnant women. A total of 294 proteins were abnormally expressed in PE patients. Of these, 233 were significantly down‐regulated and 61 proteins were significantly up‐regulated. Bioinformatics analysis using the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) database, found that the most differentially expressed proteins (DEPs) were involved in coagulation and complement pathways, the renin‐angiotensin system and cell adhesion molecules (CAMs) pathways. We further validated three of the DEPs, including serotransferrin (TF) and complement factor B (CFB) by immunoblottingand serum paraoxonase/arylesterase 1 (PON1) by ELISA using 14 pairs of urine samples from PE patients and normal pregnant women. Taken together, our results provide the basis for further understanding the pathogenesis of PE and identifying predictive biomarkers.     

Comparative proteomic analysis of tomato (Solanum lycopersicum L.) shoots reveals crosstalk between strigolactone and auxin

As one of the main vegetable crops cultivated in the world, the tomato has advantages of high yield and economic benefits, and plays an important role in promoting farmers' income and social and economic growth. However, lateral branches during the growth process of tomato consume considerable nutrients and reduce the yield of tomato. Phytohormones such as strigolactone and auxin can inhibit the formation of lateral branches. However, the mechanism of their interaction is not particularly clear. To better understand the effects of exogenous strigolactone and auxin on tomato, proteome analyses of tomato shoots treated with exogenous GR24 and indole acetic acid were performed using an integrated approach involving tandem mass tag (TMT) labeling and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). We identified 6685 proteins, of which 5822 contained quantitative information. Many differentially expressed proteins (DEPs) were found in different comparisons, including 415, 148, and 130 DEPs in GR24 vs mock, IAA vs mock, and GR24 + IAA vs mock comparisons, respectively. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that ‘photosynthesis - antenna proteins’ were significantly enriched in three treatments. Our data can help reveal the interaction between strigolactone and auxin in tomato seedlings.     

不同抗性苹果品种应答轮纹病菌胁迫的差异蛋白质组分析

为鉴定不同抗性苹果(Malus domestica)品种响应轮纹病菌胁迫的抗性相关蛋白表达差异, 以抗病品种华月及易感品种金冠为试材, 采用高通量同位素标记定量(IBT)技术结合液相色谱-串联质谱(LC-MS)鉴定技术, 对病原菌处理前后抗、感病品种叶片的蛋白质组差异表达进行分析, 共鉴定出171个差异表达蛋白(DEPs)。GO富集及KEGG通路分析表明, 在细胞组分、分子功能和生物过程3类中共注释到686个GO条目, 其中52个DEPs注释于KEGG通路的18个显著差异途径(P<0.05)。亚细胞定位预测分析表明, 171个DEPs中有170个分别定位于8类细胞器。蛋白功能注释分析表明, 46个DEPs注释于7类抗性相关蛋白, 包括类甜蛋白、过氧化物酶、多酚氧化酶、过敏原蛋白、几丁质酶、内切葡聚糖酶以及主乳胶蛋白。此外, 还对抗性相关蛋白的表达特点及基因定量结果进行了分析。该研究结果可为进一步解析抗、感病苹果品种应答轮纹病菌胁迫的抗性机制提供参考。     

金针菇成熟期和伸长期菌盖的转录组与蛋白组比较分析

菌盖是大型真菌的重要组成部分,也是其产生有性孢子的部位,但是其发育机制仍不明确。本研究以金针菇Flammulina filiformis为材料,采用转录组和蛋白组联合分析的方法,比较分析了金针菇成熟期和伸长期菌盖的差异基因与蛋白,并对其进行GO (gene ontology)功能聚类分析、KEGG (Kyoto encyclopedia of genes and genomes)富集分析和蛋白互作网络分析。本研究筛选到差异表达基因有1 391个,差异表达蛋白147个,均以上调表达为主。GO功能聚类分析结果表明,催化活性(catalytic activity)条目富集基因最多,其次是细胞组分(cell part)、细胞过程(cellular process)和细胞器(organelle)。KEGG富集分析结果表明,差异表达基因和蛋白主要富集在碳水化合物代谢通路(carbohydrate metabolism)和氨基酸代谢通路(amino acid metabolism)等。本研究选取了9个关键的差异表达基因,使用实时荧光定量PCR (real-time quantitative PCR,RT-qPCR)对其表达量进行了验证。RT-qPCR验证结果与转录组测序结果相一致。蛋白互作网络分析表明,水解酶类、结构域类和转录调节类蛋白为互作网络的主要结点。本研究联合转录组、蛋白组测序数据,通过分析差异基因与蛋白,为深入了解金针菇菌盖发育机制提供数据参考。     

Intratympanic steroid treatment can reduce ROS and immune response in human perilymph investigated by in-depth proteome analysis

Intratympanic (IT) steroid treatment is one of the most widely used and effective treatments for inner ear disorders such as sudden sensorineural hearing loss (SNHL). However, a clear mechanism of IT steroids in inner ear recovery has not yet been revealed. Therefore, we investigated proteome changes in extracted human perilymph after steroid treatment. In this study, we applied a tandem mass spectrometry (MS/MS)-based proteomics approach to discover global proteome changes by comparing human perilymph after steroid treatment with non-treated perilymph group. Using liquid chromatography-MS/MS analysis, we selected 156 differentially expressed proteins (DEPs) that were statistically significant according to Student's t-test. Functional annotation analysis showed that upregulated proteins after steroid treatment are related to apoptosis signaling, as well as reactive oxygen species (ROS) and immune responses. The protein–protein interaction (PPI) clusters the proteins associated with these processes and attempts to observe signaling circuitry, which mediates cellular response after IT steroid treatments. Moreover, we also considered the interactome analysis of DEPs and observed that those with high interaction scores were categorized as having equivalent molecular functions (MFs). Collectively, we suggest that DEPs and interacting proteins in human perilymph after steroid treatment would inhibit the apoptotic and adaptive immune processes that may lead to anti-inflammatory effects.     

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.     

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.     

Proteomics-based analysis of potential therapeutic targets in patients with peritoneal dialysis-associated peritonitis

BackgroundPeritoneal dialysis-associated peritonitis (PDAP) is the most common complication in peritoneal dialysis patients. We propose screening for characteristic expressed proteins in the dialysate of PDAP patients to provide clues for the diagnosis of PDAP and its therapeutic targets.MethodsDialysate samples were collected from patients with a first diagnosis of PDAP (n = 15) and from patients who had not experienced peritonitis (Control, n = 15). Data-independent acquisition (DIA) proteomic analysis was used to screen for differentially expressed proteins (DEPs). Co-expression networks were constructed via weighted gene co-expression network analysis (WGCNA) for detection of gene modules. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were used for functional annotation of DEPs and gene modules. Hub proteins were validated using the parallel reaction monitoring (PRM) method.ResultsA total of 142 DEPs in the dialysate of PDAP patients were identified. 70 proteins were upregulated and 72 proteins were downregulated. GO and KEGG analysis showed that DEPs were mainly enriched in cell metabolism, glycolysis/glycogenesis and hypoxia-inducible factor-1 signaling pathway. Subsequently, a co-expression network was constructed and four gene modules were detected. Myeloperoxidase (MPO) and myeloperoxidase (HP) were the key proteins of the blue and turquoise modules, respectively. Additionally, PRM analysis showed that the expression of MPO and HP was significantly upregulated in the PDAP group compared to the non-peritonitis group, which was consistent with our proteomics data.ConclusionMPO and HP were differentially expressed in the dialysate of PDAP patients and may be potential diagnostic and therapeutic targets for PDAP.     

Comprehensive Profiling of Lysine Acetylome in Baculovirus Infected Silkworm (Bombyx mori) Cells

Bombyx mori is one of the key lepidopteran model species, and is economically important for silk production and proteinaceous drug expression. Baculovirus and insect host are important natural biological models for studying host–pathogen interactions. The impact of Bombyx mori nucleopolyhedrovirus (BmNPV) infection on the proteome and acetylome of Bombyx mori ovarian (BmN) cells are explored to facilitate a better understanding of infection‐driven interactions between BmNPV and host in vitro. The proteome and acetylome are profiled through six‐plex Tandem mass tag (TMT) labeling‐based quantitative proteomics. A total of 4194 host proteins are quantified, of which 33 are upregulated and 47 are downregulated in BmN cells at 36 h post‐infection. Based on the proteome, quantifiable differential Kac proteins are identified and functionally annotated to gene expression regulation, energy metabolism, substance synthesis, and metabolism after BmNPV infection. Altogether, 644 Kac sites in 431 host proteins and 39 Kac sites in 22 viral proteins are identified and quantified in infected BmN cells. Our study demonstrates that BmNPV infection globally impacts the proteome and acetylome of BmN cells. The viral proteins are also acetylated by the host acetyltransferase. Protein acetylation is essential for cellular self‐regulation and response to virus infection. This study provides new insights for understanding the host–virus interaction mechanisms, and the role of acetylation in BmN cellular response to viral infection.     

Differential seminal plasma proteome signatures of acute lymphoblastic leukemia survivors

With advances in therapeutic methods, there is a high survival rate among leukemia patients, of an extent more than 80%. However, chemotherapeutic drugs used to treat these patients have adverse effects on their overall health profile including fertility. The primary aim of this study was to identify differentially expressed proteins in seminal plasma of acute lymphoblastic leukemia (ALL) survivors compared to age-matched healthy controls, which can provide molecular basis of idiopathic infertility in such survivors. Differential proteome profiling was performed by 2D–differential in-gel electrophoresis, protein spots were identified by mass spectrometry and selective differentially expressed proteins (DEPs) were validated by western blotting and ELISA method. Out of eight DEPs identified, five proteins (isocitrate dehydrogenase 1, semenogelin 1, lactoferrin, prolactin-inducible protein, and human serum albumin) were upregulated and three (pepsinogen, prostate specific antigen and prostatic acid phosphatase) were downregulated. Expression profiles of these proteins are suggestive of reduction in semen quality in ALL survivors and can further be explored to determine their fertility status.     

Proteomics Analysis of Soybean Seedlings under Short-Term Water Deficit

Soybeans are one of the most important grain crops worldwide. Water deficit, which seriously affects the yield and quality of soybeans, is the main abiotic stress factor in soybean production. As a follow-up study, the droughttolerant soybean variant Heinong 44 was analyzed via proteome analysis. Soybean was exposed to water deficit for 0, 8, and 24 h, and protein samples were extracted for detection of differentially expressed proteins. Protein sequencing of leaf tissues under water stress yielded a total of 549 differentially expressed proteins: 75 and 320 upregulated proteins as well as 70 and 84 downregulated proteins were obtained after 8 and 24 h of water deficit, respectively. Gene Ontology analysis revealed that most of the differentially expressed proteins (DEPs) were involved in catalytic activity, molecular function, and metabolic processes, whereas some of them were involved in photosynthesis, carbon metabolism, and energy metabolism. We also identified some differentially expressed proteins that may be involved in the regulation of water deficit response. Our study provides a theoretical basis for the breeding of drought-resistant soybean varieties.     

Comparative proteome analysis of Aspergillus oryzae 3.042 and A. oryzae 100-8 strains: Towards the production of different soy sauce flavors

Aspergillus oryzae plays a central role in soybean fermentation, particularly in its contribution to the flavor of soy sauce. We present a comparative assessment of the intracellular differences between wild-type strain 3.042 and mutant strain A100-8, at the proteome level. 522 different protein spots were identified by MALDI-TOF MS, with 134 spots being confirmed by MALDI-TOF MS/MS. Of these, 451 were differentially expressed proteins (DEPs). There was at least a two-fold increase for 288 spots, and at least a two-fold decrease for 163 spots, in strain A100-8 when compared to 3.042. Further analysis showed that 63 of the more abundant proteins were involved in glycolysis and the citrate cycle; 43 more abundant proteins and 10 less abundant proteins were related to amino acid biosynthesis and metabolism; two of the more abundant proteins were involved in vitamin biosynthesis; and five of the more abundant proteins and four of the less abundant proteins were related to secondary metabolites. Moreover, quantitative real time PCR showed that the mRNA expression levels of six typical genes we selected were consistent with changes in protein expression. We postulate that there may be a relationship between DEPs and the flavor formation mechanism in A. oryzae.     

利用蛋白质组学研究新德里金属b-内酰胺酶1对鲍曼不动杆菌的影响

【目的】比较临床分离的亲缘关系近的多药耐药鲍曼不动杆菌MDR-ZJ06(blaNDM-1–)和ABC3229(blaNDM-1+)的差异蛋白质组,以期发现新德里金属?-内酰胺酶1(New Delhimetallo-β-lactamase-1,NDM-1)对鲍曼不动杆菌生长代谢的影响。【方法】利用2-DE联合MALDI-TOF MS/MS技术鉴定差异表达蛋白,并在GO注析的基础上,对差异蛋白进行通路分析、功能分类和富集分析,并作出蛋白与蛋白相互作用网络。【结果】发现ABC3299相对于MDR-ZJ06有51个差异表达蛋白,其中11个蛋白表达上调,40个蛋白表达下调,并且这些差异蛋白主要涉及降低碳代谢、氨基酸代谢、脂肪酸代谢和细胞壁合成,增加铁离子转运系统形成。【结论】这个结果揭示了NDM-1可能是通过减缓细菌自身的代谢,增加自身铁的摄取使细菌机体系统地抵抗抗生素从而达到耐药。