iTRAQ Technique Research Articles

Proteomics analysis to understand the ABA stimulation of wound suberization in kiwifruit

Quick suberin-based healing after wounding played a protective role for plant to prevent further damage. In this study, the stimulative effect of exogenous abscisic acid (ABA) on wound suberization in postharvest kiwifruit was evaluated through suberin staining with toluidine blue O as well as the determination of suberin phenolics and aliphatics in wound tissue. Furthermore, to reveal the regulatory involvement of ABA in wound suberization, comparative quantitative proteomics and transcriptomics analyses based on iTRAQ and qRT-PCR technique were performed. In proteomics levels, a total of 95 protein species consistently showed differential abundance between ABA and control, including 29 down-regulated and 66 up-regulated protein species. The Kyoto Encyclopedia of Genes and Genomes (KEGG) with protein-protein interaction analyses revealed that ABA mainly affected the antioxidant system, phenylpropanoid metabolism and lipid metabolism associated with wound suberization. Based on the data of proteomics analysis, the differential expressions of genes encoding 11 selected protein species were confirmed by qRT-PCR analyses. GSH-Px, MDHAR, SOD, APX, POD, PAL, CCR, PPO, CYP86B1, DGGT and KCS11 were likely to be the key enzymes that involved the response of ABA to stimulate wound suberization by mediating the antioxidant system, phenylpropanoid metabolism and lipid metabolism. Biological significanceKiwifruit is susceptible to physical injury causing postharvest deterioration during harvest, transportation and storage. Therefore, quick healing is important for maintaining the postharvest quality of injured fruit. This work elucidated the potential role of ABA and the proteomic mechanism of its regulation in wound suberization of postharvest kiwifruit.

Proteomic insight into fruit set of cucumber (Cucumis sativus L.) suggests the cues of hormone-independent parthenocarpy

BackgroundParthenocarpy is an excellent agronomic trait that enables crops to set fruit in the absence of pollination and fertilization, and therefore to produce seedless fruit. Although parthenocarpy is widely recognized as a hormone-dependent process, hormone-insensitive parthenocarpy can also be observed in cucumber; however, its mechanism is poorly understood. To improve the global understanding of parthenocarpy and address the hormone-insensitive parthenocarpy shown in cucumber, we conducted a physiological and proteomic analysis of differently developed fruits.ResultsPhysiological analysis indicated that the natural hormone-insensitive parthenocarpy of ‘EC1’ has broad hormone-inhibitor resistance, and the endogenous hormones in the natural parthenocarpy (NP) fruits were stable and relatively lower than those of the non-parthenocarpic cultivar ‘8419 s-1.’ Based on the iTRAQ technique, 683 fruit developmental proteins were identified from NP, cytokinin-induced parthenocarpic (CP), pollinated and unpollinated fruits. Gene Ontology (GO) analysis showed that proteins detected from both set and aborted fruits were involved in similar biological processes, such as cell growth, the cell cycle, cell death and communication. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that ‘protein synthesis’ was the major biological process that differed between fruit set and fruit abortion. Clustering analysis revealed that different protein expression patterns were involved in CP and NP fruits. Forty-one parthenocarpy-specialized DEPs (differentially expressed proteins) were screened and divided into two distinctive groups: NP-specialized proteins and CP-specialized proteins. Furthermore, qRT-PCR and western blot analysis indicated that NP-specialized proteins showed hormone- or hormone-inhibitor insensitive expression patterns in both ovaries and seedlings.ConclusionsIn this study, the global molecular regulation of fruit development in cucumber was revealed at the protein level. Physiological and proteomic comparisons indicated the presence of hormone-independent parthenocarpy and suppression of fruit abortion in cucumber. The proteomic analysis suggested that hormone-independent parthenocarpy is regulated by hormone-insensitive proteins such as the NP-specialized proteins. Moreover, the regulation of fruit abortion suppression may be closely related to protein synthesis pathways.

ITRAQ-based proteomic analysis of the viable but nonculturable state of Vibrio parahaemolyticus ATCC 17802 induced by food preservative and low temperature

Vibrio parahaemolyticus is a significant food-borne pathogen which can cause serious acute gastroenteritis. The viable but nonculturable (VBNC) state is a survival strategy of some bacteria when they are exposed to environmental stresses. In this study, V. parahaemolyticus ATCC 17802 was induced into VBNC state by food preservative at low temperature and oligotrophic condition, and the aim of the study is to compare differentially expressed proteins (DEPs) between the VBNC state and the exponential phase of V. parahaemolyticus ATCC 17802 using isobaric tags for relative and absolute quantitation (iTRAQ) technique. Functional annotations were carried out using protein sequence similarity, GO and KEGG pathway analysis. The results showed that V. parahaemolyticus ATCC 17802 was induced into the VBNC state at 4°C in seawater containing 10 mmol/L potassium sorbate within 40 days. A total of 1139 proteins were identified,1088 proteins were quantified. Of the DEPs under the VBNC state, 36 were significantly down-regulated and 15 up-regulated. The remarkable down-regulated proteins were type III secretion host injection and negative regulator protein, polar flagellin protein, ribosomal proteins, multidrug efflux pump component MtrF, and other key components involved in metabolism. The notable up-regulated proteins were mainly focused on transporter proteins, outer membrane protein, ferritin and amino acid synthase. The majorities of the significant DEPs were associated with translation, structural constituent of ribosome, rRNA binding, siderophore transmembrane transporter activity, receptor activity, and bacterial-type flagellum organization. This study contributes to a better understanding of the adaptation mechanism of the VBNC state of V. parahaemolyticus under food processing and environmental stresses.

Alterations in epididymal proteomics and antioxidant activity of mice exposed to fluoride.

It is well known that high fluoride results in low fertility. Epididymis is the important place for spermatozoa maturation, which is essential for successful fertilization. In the previous studies, fluoride was reported to damage the epididymal structure of mouse and rabbit. However, the mechanism underlying sodium fluoride (NaF)-induced epididymal toxicity has not yet been well elucidated. The aim of this study is to explore the global protein alterations in epididymis of mice exposed to NaF using the iTRAQ technique. Results showed that 211 proteins were differentially expressed in both 25 and 100mg/L NaF groups. Some of them have been proved to be important for reproduction, such as low-density lipoprotein receptor-related protein 2 (Lrp2), cytochrome c, testis-specific (Cyct), sorbitol dehydrogenase (Sord), glutathione S-transferases (GSTs), acrosin, beta-defensin 126, cysteine-rich secretory protein (Crisp) 1, and Crisp2. Gene ontology (GO) analysis suggested cellular process, organelle and catalytic activity account for high percent and number of differentially expressed proteins. 171 pathways were found after the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, among which the representative maps, such as ribosome, focal adhesion, and phagosome, were involved. Different functional categories post-translational modification, protein turnover, chaperones; translation, ribosomal structure and biogenesis; cytoskeleton; energy production and conversion are implicated in the Cluster of Orthologous Groups (COG) of proteins analysis. Subsequently, the effect of NaF on the antioxidant activity in epididymis, especially glutathione and glutathione-related enzymes, was evaluated. Results exhibited high fluoride caused low total antioxidant capacity (T-AOC), high methane dicarboxylic aldehyde (MDA), decreased reduced glutathione (GSH), and the glutathione-related enzymes [GSH peroxidase (GPx), GSH reductase (GR), and GSH S-transferase (GST)] changes in activity, protein, and mRNA expressions. In summary, NaF decreased the antioxidant activity of epididymis, especially glutathione and glutathione-related enzymes, as well as iTRAQ results, providing new explanations for the low sperm quality induced by fluoride.

Differential proteomic profiling of endometrium and plasma indicate the importance of hydrolysis in bovine endometritis

Endometritis is an important disease of dairy cows that leads to significant economic losses in the dairy cattle industry. To investigate the alteration of proteins associated with endometritis in the dairy cow, the isobaric tags for relative and absolute quantification (iTRAQ) technique was applied to quantitatively identify differentially expressed proteins (DEP) in the endometrium and peripheral plasma of Chinese Holstein cows with endometritis. Compared with the normal (control) group, 159 DEP in the endometrium and 137 DEP in the plasma were identified in cows with endometritis. Gene ontology analysis demonstrated that the predominant endometrial DEP were primarily involved in responses to stimulus and stress processes and mainly played a role in hydrolysis in the extracellular region. The predominant plasma DEP were mainly components of the cytosol and non-membrane-bound organelles, and they were involved in the response to stress and regulation of enzyme activity. Protein-protein interaction of tissue DEP revealed that some core seed proteins, such as RAC2, ITGB2, and CDH1 in the same network as CD14, MMP3, and MMP9, had important functions in the cross-talk of pathways related to extracellular proteolysis. In summary, significant enzymatic hydrolase activity in the extracellular region is proposed as a molecular mechanism by which altered proteins may promote inflammation and hence endometritis.

Proteomic analysis of phytase transgenic and non-transgenic maize seeds

Proteomics has become a powerful technique for investigating unintended effects in genetically modified crops. In this study, we performed a comparative proteomics of the seeds of phytase-transgenic (PT) and non-transgenic (NT) maize using 2-DE and iTRAQ techniques. A total of 148 differentially expressed proteins (DEPs), including 106 down-regulated and 42 up-regulated proteins in PT, were identified. Of these proteins, 32 were identified through 2-DE and 116 were generated by iTRAQ. It is noteworthy that only three proteins could be detected via both iTRAQ and 2-DE, and most of the identified DEPs were not newly produced proteins but proteins with altered abundance. These results indicated that many DEPs could be detected in the proteome of PT maize seeds and the corresponding wild type after overexpression of the target gene, but the changes in these proteins were not substantial. Functional classification revealed many DEPs involved in posttranscriptional modifications and some ribosomal proteins and heat-shock proteins that may generate adaptive effects in response to the insertion of exogenous genes. Protein-protein interaction analysis demonstrated that the detected interacting proteins were mainly ribosomal proteins and heat-shock proteins. Our data provided new information on such unintended effects through a proteomic analysis of maize seeds.

Quantitative proteomic analysis of pre- and post-harvest peach fruit ripening based on iTRAQ technique

To further elucidate the molecular mechanism involved in peach (Prunus persica cv. Xiacui) fruit ripening, a differential proteomic analysis was conducted using iTRAQ based strategy for studying peach fruit ripening from day 15 before commercial harvesting (D0) to 15-day storage period at 25 °C. 64 differentially expressed proteins (≥1.5-fold, p ≤ 0.05) were successfully identified. Lipoxygenase (LOX) family members exposed the status of causing disorder metabolism and senescence by increased expressions. Some well-known antioxidant enzymes did not show significant expressions especially in later storage time. Nevertheless, proteins involved in ABA metabolism were up-regulated, demonstrating the important role of ABA in adaptation of plants to various environmental stresses. Glutathione S-transferase, cysteine proteinase inhibitor, transcription factor bHLH135 all showed great potential in response to ROS or other stresses. Proteins associated with flavonoid biosynthesis and amino acid metabolism also revealed critical roles in metabolism during storage.

Plasma protein profiling in patients undergoing coronary artery bypass grafting surgery and clinical significance.

This study was designed to identify the protein profiling in patients with triple vessel coronary artery disease (CAD) undergoing CABG, in order to detect CAD-related differential proteins in these patients. CABG patients with triple vessel disease with/without left main stenosis (n =160) were compared to normal coronary angiographic subjects (n =160). Plasma samples of 20 males and 20 females in each group were analyzed with iTRAQ technique. ELISA test was used to test the chosen proteins from iTRAQ results in plasma samples from a new cohort of the CABG group (n=120, male/femal=61/59) and control (n =120, male/female=60/60). iTRAQ detected 544 proteins with 35 up-regulated and 41 down-regulated (change fold > 1.2 or < 0.83, p < 0.05). Three proteins including platelet factor 4 (PF4), coagulation factor XIII B chain (F13B), and secreted frizzled-related protein 1 (sFRP1) were selected for validation by using ELISA that demonstrated significant up-regulation of PF4 and sFRP1 (p < 0.05). There was a positive correlation between these proteins and CAD (p < 0.05) and myocardial infarction history (p < 0.05). Thus, we for the first time have found 76 proteins differentially expressed in plasma of CABG patients. The thrombotic disease/inflammation progress-related protein PF4 and sFRP1, a member of the Wnt/fz signal-transduction pathway and related to myocardial repair, are significantly up-regulated in triple-vessel disease with/without left main stenosis. PF4 may be developed as a biomarker for the diagnosis of the severity of CAD requiring CABG procedure.

Differential Proteomic Analysis by iTRAQ Reveals the Mechanism of Pyropia haitanensis Responding to High Temperature Stress

Global warming increases sea temperature and leads to high temperature stress, which affects the yield and quality of Pyropia haitanensis. To understand the molecular mechanisms underlying high temperature stress in a high temperature tolerance strain Z-61, the iTRAQ technique was employed to reveal the global proteomic response of Z-61 under different durations of high temperature stress. We identified 151 differentially expressed proteins and classified them into 11 functional categories. The 4 major categories of these are protein synthesis and degradation, photosynthesis, defense response, and energy and carbohydrate metabolism. These findings indicated that photosynthesis, protein synthesis, and secondary metabolism are inhibited by heat to limit damage to a repairable level. As time progresses, misfolded proteins and ROS accumulate and lead to the up-regulation of molecular chaperones, proteases, and antioxidant systems. Furthermore, to cope with cells injured by heat, PCD works to remove them. Additionally, sulfur assimilation and cytoskeletons play essential roles in maintaining cellular and redox homeostasis. These processes are based on signal transduction in the phosphoinositide pathway and multiple ways to supply energy. Conclusively, Z-61 establishes a new steady-state balance of metabolic processes and survives under higher temperature stress.

A MIXED-EFFECTS MODEL FOR INCOMPLETE DATA FROM LABELING-BASED QUANTITATIVE PROTEOMICS EXPERIMENTS.

In mass spectrometry (MS) based quantitative proteomics research, the emerging iTRAQ (isobaric tag for relative and absolute quantitation) and TMT (tandem mass tags) techniques have been widely adopted for high throughput protein profiling. In a typical iTRAQ/TMT proteomics study, samples are grouped into batches, and each batch is processed by one multiplex experiment, in which the abundances of thousands of proteins/peptides in a batch of samples can be measured simultaneously. The multiplex labeling technique greatly enhances the throughput of protein quantification. However, the technical variation across different iTRAQ/TMT multiplex experiments is often large due to the dynamic nature of MS instruments. This leads to strong batch effects in the iTRAQ/TMT data. Moreover, the iTRAQ/TMT data often contain substantial batch-level nonignorable missing entries. Specifically, the abundance measures of a given protein/peptide are often either observed or missing altogether in all the samples from the same batch, with the missing probability depending on the combined batch-level abundances. We term this unique missing-data mechanism as the Batch-level Abundance-Dependent Missing-data Mechanism (BADMM). We introduce a new method- mixEMM-for analyzing iTRAQ/TMT data with batch effects and batch-level nonignorable missingness. The mixEMM method employs a linear mixed-effects model and explicitly models the batch effects and the BADMM. With simulation studies, we showed that, compared with existing approaches that utilize relative abundances and ignore the missing batches under the missing-completely-at-random assumption, the mixEMM method achieves more accurate parameter estimation and inference. We applied the method to an iTRAQ proteomics data from a breast cancer study and identified phosphopeptides differentially expressed between different breast cancer subtypes. The method can be applied to general clustered data with cluster-level nonignorable missing-data mechanisms.

Comparative iTRAQ proteomic profiling of susceptible and resistant apple cultivars infected by Alternaria alternata apple pathotype

Alternaria blotch, caused by the Alternaria alternata apple pathotype (A. alternata AP), is one of serious pathogen of apples. In order to better understand the molecular mechanisms that underlie the defense responses of apple resistance to Alternaria blotch disease, a comparative proteomic approach was applied to analyze of susceptible and resistant apple cultivars response to A. alternata AP infection using iTRAQ (isobaric tags for relative and absolute quantitation) technique. A total of 4225 proteins were identified, and 1226 proteins were quantified. Of the quantified proteins, 280 and 34 expressed differentially (fold change >1.5) at 72 h post-infection (HPI) in the susceptible (“Starking Delicious”) and the resistant (“Jonathan”) apple cultivars, respectively, compared with mock-inoculated controls. Most of the differentially expressed proteins (DEPs) were associated with host plant resistance to pathogens, including signal transduction, stress and defense, and photosynthesis metabolism. Among these proteins, beta-1,3-glucanase(PR2), thaumatin-like protein (PR5), and lipoxygenase were found in both susceptible and resistant hosts. However, endochitinase and (+)-neomenthol dehydrogenase were only detected in the resistant cultivar and increased in abundance in response to the pathogen attack. To study the role of pathogenesis-related (PR) proteins in the early infection process, their expressions at 6, 18, 36, and 72 HPI were analyzed by western blot. It showed that PR5 were accumulated to a high level at 6 HPI in “Jonathan,” while cannot be detected in “Starking Delicious” until 18 HPI. The above results suggested that endochitinase and (+)-neomenthol dehydrogenase, as well as PR5 which exerts function at early stage, play important roles in apple plant against A. alternata AP infestation.

Identification of cold-stress responsive proteins in Anabasis aphylla seedlings via the iTRAQ proteomics technique

ABSTRACTCold stress is one of the major environmental factors that affects plant growth, development, and species distributions. In order to study the molecular mechanism of cold-stress responses in Anabasis aphylla seedlings, we used the isobaric tags for relative and absolute quantification (iTRAQ) technique to identify differential protein expression under cold stress. In total, 211 differentially expressed proteins were identified, including 109 up-accumulated proteins and 102 down-accumulated proteins, these cold-stress response proteins were mainly identified as proteins involved in carbohydrate and energy metabolism, protein metabolism and translation, stress response, transcription-related, amino acid metabolism, signal transduction, and membrane and transport. GO and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses indicated that most of the proteins related to cold-stress response were involved in carbohydrate and energy metabolism, indicating A. aphylla seedlings adapted to cold conditions mainly through changes in energy metabolism pathways. Additionally, the correspondence between mRNA transcript levels and protein abundance levels for nine accumulated proteins were also tested by qRT-PCR, only one protein did not show consistent trends in mRNA and protein levels. These data clarify our current understandings of stress responses in seedlings of species such as A. aphylla and identify the molecular mechanisms of the cold-stress responses.

ITRAQ - based proteomic and physiological analyses of broccoli sprouts in response to the stresses of heat, hypoxia and heat plus hypoxia

Stressful conditions, such as heat and hypoxia stresses, could enhance glucosinolate metabolism, but impacted plant growth and productivity. It is very important to identify the differences in physiological and proteomic changes in broccoli sprouts under the stresses of heat, hypoxia and heat plus hypoxia. Physiological properties, antioxidant enzyme activity and glucosinolates and sulforaphane were studied. The iTRAQ technique was applied to identify the differentially abundant proteins. A total of 145, 92 and 105 proteins in broccoli sprouts showed differential relative abundance under the stresses of heat, hypoxia and heat plus hypoxia, respectively. These proteins were mainly involved in energy, defense/stress, carbohydrate catabolism and protein biosynthesis. Compared with heat- and hypoxia-stress, heat plus hypoxia stress resulted in 102 and 17 differentially abundant proteins, respectively. Heat and heat plus hypoxia stresses might modulate stress-triggered ROS homeostasis and MDA by activating heat shock proteins and antioxidant enzymes as well as by inducing of glucosinolates and sulforaphane. Hypoxia stress induced antioxidant enzymes and glucosinolates metabolism to strengthen the defense system in broccoli sprouts. Heat plus hypoxia stress did not have the synergistic effect of heat and hypoxia both in physiological changes and in proteomic analysis; and it was similar to hypoxia stress.

ITRAQ-based proteomic analysis of defence responses triggered by the necrotrophic pathogen Rhizoctonia solani in cotton

The soil-borne necrotrophic pathogen fungus Rhizoctonia solani is destructive, causing disease in various important crops. To date, little is known about the host defence mechanism in response to invasion of R. solani. Here, an iTRAQ-based proteomic analysis was employed to investigate pathogen-responsive proteins in the disease tolerant/resistant cotton cultivar CRI35. A total of 174 differentially accumulated proteins (DAPs) were identified after inoculation of cotton plants with R. solani. Functional categorization analysis indicated that these DAPs can be divided into 12 subclasses. Notably, a large portion of DAPs are known to function in reactive oxygen species (ROS) metabolism and the expression of several histone-modifying and DNA methylating proteins were significantly induced upon challenge with the fungus, indicating that the redox homeostasis and epigenetic regulation are important for cotton defence against the pathogen. Additionally, the expression of proteins involved in phenylpropanoid biosynthesis was markedly changed in response to pathogen invasion, which may reflect a particular contribution of secondary metabolism in protection against the fungal attack in cotton. Together, our results indicate that the defence response of cotton plants to R. solani infection is active and multifaceted and involves the induction of proteins from various innate immunity-related pathways. SignificanceCotton damping-off is a destructive disease caused by the necrotrophic fungus Rhizoctonia solani. To date, the host defence mechanism involved in the disease protection remains largely unknown. Here, we reported the first proteomic analysis on cotton immune responses against R. solani infection. Employing iTRAQ technique, we obtained a total of 174 differentially accumulated proteins (DAPs) that can be classified into 12 functional groups. Further analysis indicated that ROS homeostasis, epigenetic regulation and phenylpropanoid biosynthesis were tightly associated with the innate immune responses against R. solani infection in cotton. The obtained data provide not only important information for understanding the molecular mechanism involved in plant-R. solani interaction but also application clues for genetic breeding of crops with improved R. solani resistance.

Plasma concentration of fibronectin is decreased in patients with hypertrophic cardiomyopathy

ObjectivesTo confirm the initial iTRAQ-based proteomic findings related to the plasma fibronectin level and hypertrophic cardiomyopathy using an antibody-based assay. MethodsThe iTRAQ technique was used for the discovery proteomic analysis of the pooled plasma samples. The ELISA was used for verification of fibronectin plasma concentration in individual samples. Additional five related plasma proteins were assessed: matrix metalloproteinase 2, matrix metalloproteinase 9, tissue inhibitor of metalloproteinase 1, tissue inhibitor of metalloproteinase 2 and brain natriuretic peptide. ResultsThe plasma fibronectin level in patients with hypertrophic cardiomyopathy was significantly lower in comparison to the healthy subjects [215.5±47.3μg/ml, n=17 vs. 376.7±134.8μg/ml, n=17; p<0.0001]. ConclusionIn this study we present and confirm our initial proteomic findings and we suggest fibronectin as a potential indicator of an extracellular matrix remodeling related to the hypertrophic cardiomyopathy.

Comparative Proteomic Analysis of Buffalo Oocytes Matured in vitro Using iTRAQ Technique.

To investigate the protein profiling of buffalo oocytes at the germinal vesicle (GV) stage and metaphase II (MII) stage, an iTRAQ-based strategy was applied. A total of 3,763 proteins were identified, which representing the largest buffalo oocytes proteome dataset to date. Among these proteins identified, 173 proteins were differentially expressed in GV oocytes and competent MII oocytes, and 146 proteins were differentially abundant in competent and incompetent matured oocytes. Functional and KEGG pathway analysis revealed that the up-regulated proteins in competent MII oocytes were related to chromosome segregation, microtubule-based process, protein transport, oxidation reduction, ribosome, and oxidative phosphorylation, etc., in comparison with GV and incompetent MII oocytes. This is the first proteomic report on buffalo oocytes from different maturation stages and developmental competent status. These data will provide valuable information for understanding the molecular mechanism underlying buffalo oocyte maturation, and these proteins may potentially act as markers to predict developmental competence of buffalo oocyte during in vitro maturation.

Comprehensive Analysis of the Triterpenoid Saponins Biosynthetic Pathway in Anemone flaccida by Transcriptome and Proteome Profiling

Background: Anemone flaccida Fr. Shmidt (Ranunculaceae), commonly known as ‘Di Wu’ in China, is a perennial herb with limited distribution. The rhizome of A. flaccida has long been used to treat arthritis as a tradition in China. Studies disclosed that the plant contains a rich source of triterpenoid saponins. However, little is known about triterpenoid saponins biosynthesis in A. flaccida.Results: In this study, we conducted the tandem transcriptome and proteome profiling of a non-model medicinal plant, A. flaccida. Using Illumina HiSeq 2000 sequencing and iTRAQ technique, a total of 46,962 high-quality unigenes were obtained with an average sequence length of 1,310 bp, along with 1473 unique proteins from A. flaccida. Among the A. flaccida transcripts, 36,617 (77.97%) showed significant similarity (E-value < 1e-5) to the known proteins in the public database. Of the total 46,962 unigenes, 36,617 open reading frame (ORFs) were predicted. By the fragments per kilobases per million reads (FPKM) statistics, 14,004 isoforms/unigenes were found to be upregulated, and 14,090 isoforms/unigenes were down-regulated in the rhizomes as compared to those in the leaves. Based on the bioinformatics analysis, all possible enzymes involved in the triterpenoid saponins biosynthetic pathway of A. flaccida were identified, including cytosolic mevalonate pathway (MVA) and the plastidial methylerythritol pathway (MEP). Additionally, a total of 126 putative cytochrome P450 (CYP450) and 32 putative UDP glycosyltransferases were selected as the candidates of triterpenoid saponins modifiers. Among them, four of them were annotated as the gene of CYP716A subfamily, the key enzyme in the oleanane-type triterpenoid saponins biosynthetic pathway. Furthermore, based on RNA-Seq and proteome analysis, as well as quantitative RT-PCR verification, the expression level of gene and protein committed to triterpenoids biosynthesis in the leaf versus the rhizome was compared.Conclusion: A combination of the de novo transcriptome and proteome profiling based on the Illumina HiSeq 2000 sequencing platform and iTRAQ technique was shown to be a powerful method for the discovery of candidate genes, which encoded enzymes that were responsible for the biosynthesis of novel secondary metabolites in a non-model plant. The transcriptome data of our study provides a very important resource for the understanding of the triterpenoid saponins biosynthesis of A. flaccida.

Proteomic profiling of heat acclimation in cerebrospinal fluid of rabbit

Heat acclimation (AC) is a phenotypic adaptation to the high ambient temperatures. So far, the physiological effects of AC have been well studied, but the molecular mechanisms underlying it, especially the proteomic studies have been rarely reported. Conducting a protein profile of cerebrospinal fluid (CSF) can facilitate the understanding of molecular pathways involved in AC and identifying stress-specific proteins as a laboratory biomarker. In this study we carried out proteomic profiling of the AC in CSF of rabbit, which would allow a deep insight into molecular signals underlying the AC. For this purpose, rabbits were subjected to AC (dry bulb temperature of (36±1)°C, wet bulb temperature of (29±0.5)°C, black-bulb temperature of (40±1.0)°C, 100min per day for 21days, untreated rabbits were used as controls. We adopted a gel-free proteomic approach (iTRAQ) method to identify protein composition in CSF of rabbits with AC. In total, 1310 proteins were identified. Among which 127 were significant up-regulated and 77 were down-regulated. According to the functions, all AC-induced proteins were classified into 8 categories, including plasma protein factors, metabolism-related proteins, energy metabolism-related proteins, cell surface/intercellular matrix proteins, stress related proteins, tumor-related proteins, as well as housekeeping proteins and putative proteins. Meanwhile, a total of 21 pathways were found involved in the developing of AC. Further analysis indicated that proteins mostly close to AC were grouped into two signal pathways, the immune-related signal pathways and the carbohydrate/lipoprotein metabolism-related signal pathways. Our study was first to carry out the whole proteomic picture of AC, and screen out the critical signaling pathways involved in this physical procedure. Biological significanceThis study reported the comparative proteomic analysis of cerebrospinal fluid of rabbits between heat acclimation and normal conditions using the gel-free proteomic mass-spectrometry approach with isotope-labeled samples (iTRAQ) techniques. Mass spectrometry analysis of the proteins from heat acclimated rabbits resulted in the identification of a total of 1310 proteins, among these, 204 proteins were related to the formation of heat acclimation. These proteins were assigned to 8 categories according to their functions. Additionally, 21 pathways involved in infectious diseases, metabolism, immunology, blood circulation, transcriptional regulation and renin-angiotensin were identified by pathway analysis in heat acclimation. This study was the first to use rabbits as a model for unraveling the molecular pathways underlying the establishment of integrative heat acclimation.

Identification of New Epididymal Luminal Fluid Proteins Involved in Sperm Maturation in Infertile Rats Treated by Dutasteride Using iTRAQ.

Background: Spermatozoa become mature and acquire fertilizing capacity during their passage through the epididymal lumen. In this study, we identified new epididymal luminal fluid proteins involved in sperm maturation in infertile rats by dutasteride, a dual 5α-reductase inhibitor, in order to provide potential epididymal targets for new contraceptives and infertility treatment. Methods: Male rats were treated with dutasteride for 28 consecutive days. We observed the protein expression profiles in the epididymal luminal fluids in infertile and normal rats using isobaric tags for relative and absolute quantitation (iTRAQ) technique. The confidence of proteome data was validated by enzyme-linked immunosorbent assays. Results: 1045 proteins were tested, and 23 of them presented different expression profiling in the infertile and normal rats. The seven proteins were down-regulated, and 16 proteins were up-regulated. Among the seven proteins which were significantly down-regulated by dutasteride in the epididymal luminal fluids, there were three β-defensins (Defb2, Defb18 and Defb39), which maybe the key proteins involved in epididymal sperm maturation and male fertility. Conclusions: We report for the first time that dutasteride influences the protein expression profiling in the epididymal luminal fluids of rats, and this result provides some new epididymal targets for male contraception and infertility therapy.

Pharmacoproteomic analysis reveals that metapristone (RU486 metabolite) intervenes E-cadherin and vimentin to realize cancer metastasis chemoprevention.

Metapristone is the most predominant biological active metabolite of mifepristone, and being developed as a novel cancer metastasis chemopreventive agent by us. Despite its prominent metastasis chemopreventive effect, the underlying mechanism remains elusive. Our study, for the first time, demonstrated that metapristone had the ability to prevent breast cancer cells from migration, invasion, and interfere with their adhesion to endothelial cells. To explore the underlying mechanism of metapristone, we employed the iTRAQ technique to assess the effect of metapristone on MDA-MB-231 cells. In total, 5,145 proteins were identified, of which, 311 proteins showed significant differences in metapristone-treated cells compared to the control group (P-value < 0.05). Bioinformatic analysis showed many differentially expressed proteins (DEPs) functionally associated with post-translational modification, chaperones, translation, transcription, replication, signal transduction, etc. Importantly, many of the DEPs, such as E-cadherin, vimentin, TGF-β receptor I/II, smad2/3, β-catenin, caveolin, and dystroglycan were associated with TGF-β and Wnt signaling pathways, which were also linked to epithelial-to-mesenchymal transition (EMT) process. Further validation of the epithelial marker “E-caderin” and mesenchymal marker “vimetin” were carried out using immunoblot and immunofluorescence. These results have revealed a novel mechanism that metapristone-mediated metastasis chemoprevention is through intervening the EMT-related signaling pathways.