Serum proteins differentially expressed in gestational diabetes mellitus assessed using isobaric tag for relative and absolute quantitation proteomics

BACKGROUND As a well-known fact to the public,gestational diabetes mellitus(GDM)could bring serious risks for both pregnant women and infants.During this important investigation into the linkage between GDM patients and their altered expression in the serum,proteomics techniques were deployed to detect the differentially expressed proteins(DEPs)of in the serum of GDM patients to further explore its pathogenesis,and find out possible biomarkers to forecast GDM occurrence.METHODS Subjects were divided into GDM and normal control groups according to the IADPSG diagnostic criteria.Serum samples were randomly selected from four cases in each group at 24-28 wk of gestation,and the blood samples were identified by applying iTRAQ technology combined with liquid chromatography-tandem mass spectrometry.Key proteins and signaling pathways associated with GDM were identified by bioinformatics analysis,and the expression of key proteins in serum from 12 wk to 16 wk of gestation was further verified using enzyme-linked immunosorbent assay (ELISA).RESULTS Forty-seven proteins were significantly differentially expressed by analyzing the serum samples between the GDMgravidas as well as the healthy ones. Among them, 31 proteins were found to be upregulated notably and the rest16 proteins were downregulated remarkably. Bioinformatic data report revealed abnormal expression of proteinsassociated with lipid metabolism, coagulation cascade activation, complement system and inflammatory responsein the GDM group. ELISA results showed that the contents of RBP4, as well as ANGPTL8, increased in the serumof GDM gravidas compared with the healthy ones, and this change was found to initiate from 12 wk to 16 wk ofgestation.CONCLUSION GDM symptoms may involve abnormalities in lipid metabolism, coagulation cascade activation, complementsystem and inflammatory response. RBP4 and ANGPTL8 are expected to be early predictors of GDM.

Identification of protein targets for the antidepressant effects of Kai-Xin-San in Chinese medicine using isobaric tags for relative and absolute quantitation

Kai-Xin-San consists of Ginseng Radix, Polygalae Radix, Acori Tatarinowii Rhizoma, and Poria at a ratio of 3:3:2:2. Kai-Xin-San has been widely used for the treatment of emotional disorders in China. However, no studies have identified the key proteins implicated in response to Kai-Xin-San treatment. In this study, rat models of chronic mild stress were established using different stress methods over 28 days. After 14 days of stress stimulation, rats received daily intragastric administrations of 600 mg/kg Kai-Xin-San. The sucrose preference test was used to determine depression-like behavior in rats, while isobaric tags were used for relative and absolute quantitation-based proteomics to identify altered proteins following Kai-Xin-San treatment. Kai-Xin-San treatment for 2 weeks noticeably improved depression-like behaviors in rats with chronic mild stress. We identified 33 differentially expressed proteins: 7 were upregulated and 26 were downregulated. Functional analysis showed that these differentially expressed proteins participate in synaptic plasticity, neurodevelopment, and neurogenesis. Our results indicate that Kai-Xin-San has an important role in regulating the key node proteins in the synaptic signaling network, and are helpful to better understand the mechanism of the antidepressive effects of Kai-Xin-San and to provide objective theoretical support for its clinical application. The study was approved by the Ethics Committee for Animal Research from the Chinese PLA General Hospital(approval No. X5-2016-07) on March 5, 2016.

Different protein expression patterns in rat spinal nerves during wallerian degeneration assessed using isobaric tags for relative and absolute quantitation proteomics profiling

Sensory and motor nerve fibers of peripheral nerves have different anatomies and regeneration functions after injury. To gain a clear understanding of the biological processes behind these differences, we used a labeling technique termed isobaric tags for relative and absolute quantitation to investigate the protein profiles of spinal nerve tissues from Sprague-Dawley rats. In response to Wallerian degeneration, a total of 626 proteins were screened in sensory nerves, of which 368 were upregulated and 258 were downregulated. In addition, 637 proteins were screened in motor nerves, of which 372 were upregulated and 265 were downregulated. All identified proteins were analyzed using the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis of bioinformatics, and the presence of several key proteins closely related to Wallerian degeneration were tested and verified using quantitative real-time polymerase chain reaction analyses. The differentially expressed proteins only identified in the sensory nerves were mainly relevant to various biological processes that included cell-cell adhesion, carbohydrate metabolic processes and cell adhesion, whereas differentially expressed proteins only identified in the motor nerves were mainly relevant to biological processes associated with the glycolytic process, cell redox homeostasis, and protein folding. In the aspect of the cellular component, the differentially expressed proteins in the sensory and motor nerves were commonly related to extracellular exosomes, the myelin sheath, and focal adhesion. According to the Kyoto Encyclopedia of Genes and Genomes, the differentially expressed proteins identified are primarily related to various types of metabolic pathways. In conclusion, the present study screened differentially expressed proteins to reveal more about the differences and similarities between sensory and motor nerves during Wallerian degeneration. The present findings could provide a reference point for a future investigation into the differences between sensory and motor nerves in Wallerian degeneration and the characteristics of peripheral nerve regeneration. The study was approved by the Ethics Committee of the Chinese PLA General Hospital, China(approval No. 2016-x9-07) in September 2016.

Isobaric Tag for Relative and Absolute Quantitation-Based Proteomics for Investigating the Effect of Guasha on Lumbar Disc Herniation in Rats

Objective:The objective of the study is to examine the possible mechanism by which Guasha(scraping therapy)affects the expression profiles of proteins in a lumbar disc herniation(LDH)rat model using isobaric tags for relative and absolute quantitation(iTRAQ)-based proteomics.Methods:Thirty-six rats were used in this study.LDH rats were subjected to noncompressive LDH surgeries.Rats were randomly divided into the model and Guasha groups.Guasha was applied on alternate days for a total of nine times(three courses).At the end of each course,six rats were randomly selected from each group and their blood samples were collected.iTRAQ labeling was used to examine the mechanism of action of Guasha against LDH.The molecular functions,cellular components,and biological processes were analyzed using gene ontology analysis.The Ingenuity Pathway Analysis database was used to identify canonical pathways involving these proteins.Results:Compared to the model group,198,182,and 170 proteins were identified as differentially expressed at the three respective Guasha treatment courses.Pathways,including focal adhesion kinase signaling,acute-phase response signaling,and the LXR/RXR activation pathway,were closely related to the effects of Guasha in LDH rats.Furthermore,Rac1,Orm1,and Hpx were validated by western blotting,and the results were consistent with the protein expression levels observed using the iTRAQ method.Conclusion:Guasha could not only regulate the pathological changes related to LDH,but also achieve therapeutic effects by stimulating physiological changes.Our results offer a better understanding of the effects of Guasha on LDH.

Inflammation and apoptosis accelerate progression to irreversible atrophy in denervated intrinsic muscles of the hand compared with biceps: proteomic analysis of a rat model of obstetric brachial plexus palsy

In treating patients with obstetric brachial plexus palsy,we noticed that denervated intrinsic muscles of the hand become irreversibly atrophic at a faster than denervated biceps.In a rat model of obstetric brachial plexus palsy,denervated intrinsic musculature of the forepaw entered the irreversible atrophy far earlier than denervated biceps.In this study,isobaric tags for relative and absolute quantitation were examined in the intrinsic musculature of forepaw and biceps on denervated and normal sides at 3 and 5 weeks to identify dysregulated proteins.Enrichment of pathways mapped by those proteins was analyzed by Kyoto Encyclopedia of Genes and Genomes analysis.At 3 weeks,119 dysregulated proteins in denervated intrinsic musculature of the forepaw were mapped to nine pathways for muscle regulation,while 67 dysregulated proteins were mapped to three such pathways at 5 weeks.At 3 weeks,27 upregulated proteins were mapped to five pathways involving inflammation and apoptosis,while two upregulated proteins were mapped to one such pathway at 5 weeks.At 3 and 5 weeks,53 proteins from pathways involving regrowth and differentiation were downregulated.At 3 weeks,64 dysregulated proteins in denervated biceps were mapped to five pathways involving muscle regulation,while,five dysregulated proteins were mapped to three such pathways at 5 weeks.One protein mapped to inflammation and apoptotic pathways was upregulated from one pathway at 3 weeks,while three proteins were downregulated from two other pathways at 5 weeks.Four proteins mapped to regrowth and differentiation pathways were upregulated from three pathways at 3 weeks,while two proteins were downregulated in another pathway at 5 weeks.These results implicated inflammation and apoptosis as critical factors aggravating atrophy of denervated intrinsic muscles of the hand during obstetric brachial plexus palsy.All experimental procedures and protocols were approved by the Experimental Animal Ethics Committee of Fudan University,China(approval No.DF-325)in January 2015.

基于iTRAQ结合质谱筛选HBV肝纤维化的差异表达蛋白

目的:采用核素标记相对和绝对定量(isobaric rags for relative and absolute quantitation,iTRAQ)蛋白组学技术分析乙型肝炎病毒(hepatitis B virus,HBV)肝纤维化患者和健康人血清中的差异表达蛋白.方法:肝纤维患者与正常人血清各30例,去除血清中14种高丰度蛋白,运用液相色谱基质辅助激光解析/电离-飞行时间质谱技术(matrix-assisted laser desorption/ionization time-of-fight mass spectrometry,MALDI-TOF-MS)筛选并鉴定差异表达蛋白,并对差异表达蛋白进行生物学分析.结果:共鉴定血清蛋白274个,符合条件的差异蛋白20个.其中,在肝纤维患者血清中有11个蛋白表达上调,7个蛋白表达下调.20种差异表达蛋白参与48种生物学过程、8种细胞组分和12种分子途径;蛋白功能交互网图显示APOC3、CLU、C4B、CRP和APOE在网图中处于功能网络交叉点.结论:处于差异蛋白功能交互网中交叉点位置的载脂蛋白C-Ⅲ(apolipoprotein C-Ⅲ,APOC3)、聚集素(clusterin,CLU)、补体C4B(complement C4-B,C4B)、C-反应蛋白(c-reactive protein,CRP)、载脂蛋白E(apolipoprotein E,APOE),可能在HBV肝纤维化的发生发展过程中发挥重要作用.

Proteomic analysis of trans-hemispheric motor cortex reorganization following contralateral C7 nerve transfer

Nerve transfer is the most common treatment for total brachial plexus avulsion injury. After nerve transfer, the movement of the injured limb may be activated by certain movements of the healthy limb at the early stage of recovery, i.e., trans-hemispheric reorganization. Pre- vious studies have focused on functional magnetic resonance imaging and changes in brain-derived neurotrophic factor and growth asso- ciated protein 43, but there have been no proteomics studies. In this study, we designed a rat model of total brachial plexus avulsion injury involving contralateral C7 nerve transfer. Isobaric tags for relative and absolute quantitation and western blot assay were then used to screen differentially expressed proteins in bilateral motor cortices. We found that most differentially expressed proteins in both cortices of upper limb were associated with nervous system development and function （including neuron differentiation and development, axonogenesis, and guidance）, microtubule and cytoskeleton organization, synapse plasticity, and transmission of nerve impulses. Two key differentially expressed proteins, neurofilament light （NFL） and Thy-1, were identified. In contralateral cortex, the NFL level was upregulated 2 weeks after transfer and downregulated at 1 and 5 months. The Thy-1 level was upregulated from 1 to 5 months. In the affected cortex, the NFL level increased gradually from 1 to 5 months. Western blot results of key differentially expressed proteins were consistent with the proteom- ic findings. These results indicate that NFL and Thy-1 play an important role in trans-hemispheric organization following total brachial plexus root avulsion and contralateral C7 nerve transfer.

Effects of Space Flight on Expression of Key Proteins in Rice Leaves

As a unique form of abiotic stress, the environmental conditions of outer space are expected to induce changes in plant genomes, proteomes and metabolic pathways. However, the effect of outer space conditions on the overall physiology of plants at the protein level has yet to be reported. To investigate the effects of outer space conditions on the growth-and development-related physiological processes and metabolic pathways of rice different stages, the seeds of rice variety DN423 were sent into orbit for 12.5 d aboard the SJ-10 Returning Satellite, and then the seedlings of both treated and control rice were compared at the three-leaf stage(TLS) and tillering stage(TS). In addition to comparing plant growth and reactive oxygen species(ROS) levels, seedling proteomes were also compared using isobaric tags for relative and absolute quantitation(i TRAQ). Space flight increased TLS plant height by 20%, reduced and increased ROS levels of the TLS and TS seedlings, respectively, and affected the expression of 36 and 323 proteins in TLS and TS leaves, respectively. Furthermore, the functions of the differentially abundant proteins were mainly associated with metabolism, energy, and protein synthesis and degradation. These results suggested that the exposure of seeds to outer space conditions affects the subsequent abundance of key signaling proteins, gene expression, and the processes of protein synthesis and degradation, thereby affecting metabolic processes and promoting adaptation to the abiotic stress of outer space. As such, the present study sheds light on the effects of space flight on plants and contributes to a more comprehensive understanding of extraterrestrial biology.

Lactobacillus gasseri LA39 promotes hepatic primary bile acid biosynthesis and intestinal secondary bile acid biotransformation

A growing body of evidence has linked the gut microbiota to liver metabolism.The manipulation of intestinal microflora has been considered as a promising avenue to promote liver health.However,the effects of Lactobacillus gasseri LA39,a potential probiotic,on liver metabolism remain unclear.Accumulating studies have investigated the proteomic profile for mining the host biological events affected by microbes,and used the germ-free(GF)mouse model to evaluate host-microbe interaction.Here,we explored the effects of L.gasseri LA39 gavage on the protein expression profiles of the liver of GF mice.Our results showed that a total of 128 proteins were upregulated,whereas a total of 123 proteins were downregulated by treatment with L.gasseri LA39.Further bioinformatics analyses suggested that the primary bile acid(BA)biosynthesis pathway in the liver was activated by L.gasseri LA39.Three differentially expressed proteins(cytochrome P450 family 27 subfamily A member 1(CYP27A1),cytochrome P450 family 7 subfamily B member 1(CYP7B1),and cytochrome P450 family 8 subfamily B member 1(CYP8B1))involved in the primary BA biosynthesis pathway were further validated by western blot assay.In addition,targeted metabolomic analyses demonstrated that serum and fecalβ-muricholic acid(a primary BA),dehydrolithocholic acid(a secondary BA),and glycolithocholic acid-3-sulfate(a secondary BA)were significantly increased by L.gasseri LA39.Thus,our data revealed that L.gasseri LA39 activates the hepatic primary BA biosynthesis and promotes the intestinal secondary BA biotransformation.Based on these findings,we suggest that L.gasseri LA39 confers an important function in the gut‒liver axis through regulating BA metabolism.

Analyzing proteins in colonic tissues from mice with ulcerative colitis using the iTRAQ technology

Objective The aim of the study was to investigate the expression of proteins in colonic tissues of mice with ulcerative colitis(UC) by using isobaric tags for relative and absolute quantitation(iTRAQ), probe into the pathogenesis of UC, and find potential biomarkers of UC. Methods Forty C57 mice were randomly divided into the control and model groups(20 mice in each group). The mice in the model group were administered dextran sulphate sodium(DSS) for 7 consecutive days ad libitum to induce acute colitis, and the colon tissue was extracted on the 8 th day after the successful establishment of the UC model. Proteins were identified by the i TRAQ and tandem mass spectrometry techniques,and the identified proteins were analyzed by bioinformatics. Results A total of 4019 proteins were identified among the two groups. Among them, 317 significant differentially expressed proteins(DEPs) were detected according to the screening criteria for selecting DEPs, i.e. fold change ratios ≥ 1.5 or ≤ 0.67 and P-values < 0.05, of which 156 were upregulated and 161 were downregulated. In the Gene Ontology(GO) analysis, the DEPs were classified into 48 functional categories, which contained biological process, cellular component, and molecular function. Based on the 317 DEPs, the KEGG pathway analysis identified 160 vital pathways.Conclusion DEPs in colonic tissues of mice with UC were screened using the iTRAQ technique, which laid a foundation for further studies regarding the pathogenesis of UC.

基于iTRAQ方法分析副溶血弧菌活的非可培养状态的差异表达上调蛋白

采用低温、寡营养和食品防腐剂处理诱导副溶血弧菌进入活的非可培养状态(viable but nonculturable state,VBNC),并利用同位素标记相对与绝对定量(isobaric rags for relative and absolute quantitation,i TRAQ)蛋白组学技术分析处于VBNC状态和对数生长期的副溶血弧菌的差异表达蛋白,结合液质联用技术对蛋白进行鉴定,对差异蛋白进行GO富集分析和Pathway通路分析。结果表明,副溶血弧菌于山梨酸钾浓度为10 mmol/L的3%Na Cl寡营养培养基中,4℃条件下40~45 d即可进入到VBNC状态。通过i TRAQ蛋白组学技术共鉴定到135880个蛋白,其中定量的蛋白有1088个;在VBNC状态显著下调蛋白36个,上调蛋白15个。差异表达的上调蛋白主要是外膜蛋白、转运蛋白、铁蛋白和其他参与代谢的关键蛋白,表明VBNC状态的副溶血弧菌根据环境应激提高这些蛋白的表达以保持活性。该研究结果为更深入探讨VBNC的分子形成机制提供了理论基础。

Cardiac proteomics reveals the potential mechanism of microtubule associated protein 4 phosphorylation-induced mitochondrial dysfunction

Background:Our previous work suggested that microtubule associated protein 4(MAP4)phosphorylation led to mitochondrial dysfunction in MAP4 phosphorylation mutant mice with cardiomyopathy,but the detailed mechanism was still unknown.Thus,the aim of this study was to investigate the potential mechanism involved in mitochondrial dysfunction responsible for cardiomyopathy.Methods:The present study was conducted to explore the potential mechanism underlying the mitochondrial dysfunction driven by MAP4 phosphorylation.Strain of mouse that mimicked constant MAP4 phosphorylation(S737 and S760)was generated.The isobaric tag for relative and absolute quantitation(iTRAQ)analysis was applied to the heart tissue.Gene Ontology(GO),Kyoto Encyclopedia of Genes and Genomes(KEGG),and protein-protein interaction(PPI)were all analyzed on the basis of differential expressed proteins(DEPs).Results:Among the 72 cardiac DEPs detected between the two genotypes of mice,12 were upregulated and 60 were downregulated.GO analysis showed the biological process,molecular function,and cellular component of DEPs,and KEGG enrichment analysis linked DEPs to 96 different biochemical pathways.In addition,the PPI network was also extended on the basis of DEPs as the seed proteins.Three proteins,including mitochondrial ubiquitin ligase activator of NF-κB 1,reduced form of nicotinamide adenine dinucleotide(NADH)-ubiquinone oxidoreductase 75 kDa subunit,mitochondrial and growth arrest,and DNA-damage-inducible proteins-interacting protein 1,which play an important role in the regulation of mitochondrial function,may correlate with MAP4 phosphorylationinduced mitochondrial dysfunction.Western blot was used to validate the expression of the three proteins,which was consistent with iTRAQ experiments.Conclusions:These findings revealed that the DEPs caused by MAP4 phosphorylation in heart tissue using iTRAQ technique and may provide clues to uncover the potential mechanism of MAP4 phosphorylation-induced mitochondrial dysfunction.

Effects of electroacupuncture on rats with cognitive impairment:An iTRAQ-based proteomics analysis

Objective The study explores the effects of electroacupuncture(EA)at the governing vessel(GV)on proteomic changes in the hippocampus of rats with cognitive impairment.Methods Healthy male rats were randomly divided into 3 groups:sham,model and EA.Cognitive impairment was induced by left middle cerebral artery occlusion in the model and EA groups.Rats in the EA group were treated with EA at Shenting(GV24)and Baihui(GV20)for 7 d.Neurological deficit was scored using the Longa scale,the learning and memory ability was detected using the Morris water maze(MWM)test,and the proteomic profiling in the hippocampus was analyzed using protein-labeling technology based on the isobaric tag for relative and absolute quantitation(iTRAQ).The Western blot(WB)analysis was used to detect the proteins and validate the results of iTRAQ.Results Compared with the model group,the neurological deficit score was significantly reduced,and the escape latency in the MWM test was significantly shortened,while the number of platform crossings increased in the EA group.A total of 2872 proteins were identified by iTRAQ.Differentially expressed proteins(DEPs)were identified between different groups:92 proteins were upregulated and 103 were downregulated in the model group compared with the sham group,while 142 proteins were upregulated and 126 were downregulated in the EA group compared with the model group.Most of the DEPs were involved in oxidative phosphorylation,glycolipid metabolism and synaptic transmission.Furthermore,we also verified 4 DEPs using WB technology.Although the WB results were not exactly the same as the iTRAQ results,the expression trends of the DEPs were consistent.The upregulation of heat-shock proteinβ1(Hspb1)was the highest in the EA group compared to the model group.Conclusion EA can effect proteomic changes in the hippocampus of rats with cognitive impairment.Hspb1 may be involved in the molecular mechanism by which acupuncture improves cognitive impairment.

Quantitative Proteomic Studies on TNBC in Premenopausal Patients

The molecular mechanism of triple-negative breast cancer（TNBC） remains unclear, and there has been no effective targeted therapy for it. A better understanding of the mechanisms of TNBC is urgently needed to identify new therapeutic targets. In this study, eight cases of premenopausal TNBC patients were collected, and a comparative proteomic analysis of their breast cancer tissues and matched paraneoplastic ones was performed via isobaric tags for relative and absolute quantitation（iTRAQ） technology coupled with two-dimensional liquid chromatography-tandem mass spectrumetry（2D LC-MS/MS）. The researches result in the identification of 1254 nonredundant proteins, of which 1243 proteins reached the strict quantitative standard. The quantitative comparison reveal that among the 214 proteins, 81 proteins significantly increased and 133 proteins decreased in TNBC tissues compared to corresponding ones in control. The Gene Ontology（GO） annotations and pathway analysis show their distributions in GO and the marked functions, as well as the closely related signal transduction pathways involved in extra cellular matrix （ECM）-receptor interaction, protein digestion and absorption, renin-angiotensin system, complement and coagulation cascades and focal adhesion. This pilot study will lay a foundation for further searching for therapeutic targets of TNBC and exploring the molecular mechanism, which can also be extended as a part of a large scale biomarker discovery plan.