Comprehensive analyses of the proteome and ubiquitome revealed mechanism of high temperature accelerating petal abscission in tree peony

Tree peony(Paeonia suffruticosa Andrews)is a well-known ornamental plant with high economic value,but the short fluorescence is a key obstacle to its ornamental value and industry development.High temperature accelerates flower senescence and abscission,but the associated mechanisms are poorly understood.In this study,the tandem mass tag(TMT)proteome and label-free quantitative ubiquitome from tree peony cut flowers treated with 20℃for 0 h(RT0),20℃or 28℃for 60 h(RT60 or HT60)were examined based on morphological observation,respectively.Totally,6970 proteins and 1545 lysine ubiquitinated(Kub)sites in 844 proteins were identified.Hydrophilic residues(such as glutamate and aspartate)neighboring the Kub sites were in preference,and 36.01%of the Kub sites were located on the protein surface.The differentially expressed proteins(DEPs)and Kub-DEPs in HT60 vs RT60 were mainly enriched in ribosomal protein,protein biosynthesis,secondary metabolites biosynthesis,flavonoid metabolism,carbohydrate catabolism,and auxin biosynthesis and signaling revealed by GO and KEGG analysis,accompanying the increase of endogenous abscisic acid(ABA)accumulation and decrease of endogenous indoleacetic acid(IAA)level.Additionally,the expression patterns of six enzymes(SAMS,ACO,YUC,CHS,ANS and PFK)putatively with Kub modifications were analyzed by proteome and real-time quantitative RT-PCR.The cell-free degradation assays showed PsSAMS and PsACO proteins could be degraded via the 26 S proteasome system in tree peony flowers.Finally,a working model was proposed for the acceleration of flower senescence and abscission by high temperature.In summary,all results contributed to understanding the mechanism of flower senescence induced by high temperature and prolonging fluorescence in tree peony.

Identification of transition factors in myotube formation from proteome and transcriptome analyses

Muscle fibers are the main component of skeletal muscle and undergo maturation through the formation of myotubes.During early development,a population of skeletal muscle satellite cells(SSCs)proliferate into myoblasts.The myoblasts then undergo further differentiation and fusion events,leading to the development of myotubes.However,the mechanisms involved in the transition from SSCs to myotube formation remain unclear.In this study,we characterized changes in the proteomic and transcriptomic expression profiles of SSCs,myoblasts(differentiation for 2 d)and myotubes(differentiation for 10 d).Proteomic analysis identified SLMAP and STOM as potentially associated with myotube formation.In addition,some different changes in MyoD,MyoG,Myosin7 and Desmin occurred after silencing SLMAP and STOM,suggesting that they may affect changes in the myogenic marker.GO analysis indicated that the differentiation and migration factors SVIL,ENSCHIG00000026624(AQP1)and SERPINE1 enhanced the transition from SSCs to myoblasts,accompanied by changes in the apoptotic balance.In the myoblast vs.myotube group,candidates related to cell adhesion and signal transduction were highly expressed in the myotubes.Additionally,CCN2,TGFB1,MYL2 and MYL4 were identified as hub-candidates in this group.These data enhance our existing understanding of myotube formation during early development and repair.

Phosphorus Starvation Tolerance in Rice Through Combined Physiological,Biochemical,and Proteome Analyses

Phosphorus(P) deficiency limits the growth,development,and productivity of rice.To better understand the underlying mechanisms in P-deficiency tolerance and the role of Pup1 QTL in enhancing P use efficiency(PUE) for the development of P-efficient rice cultivars,a pair of contrasting rice genotypes(Pusa-44 and NIL-23) was applied to investigate the morpho-physio-biochemical and proteomic variation under P-starvation stress.The rice genotypes were grown hydroponically in a PusaRich medium with adequate P(16 mg/kg,+P) or without P(0 mg/kg,-P) for 30 d.P-starvation manifested a significant reductions in root and shoot biomass,shoot length,leaf area,total chlorophyll,and P,nitrogen and starch contents,as well as protein kinase activity.The stress increased root-to-shoot biomass ratio,root length,sucrose content,and acid phosphatase activity,particularly in the P-tolerant genotype(NIL-23).Comparative proteome analysis revealed several P metabolism-associated proteins(including OsCDPKs,OsMAPKs,OsCPKs,OsLecRK2,and OsSAPks) to be expressed in the shoot of NIL-23,indicating that multiple protein kinases were involved in P-starvation/deficiency tolerance.Moreover,the up-regulated expression of OsrbcL,OsABCG32,OsSUS5,OsPoll-like B,and ClpC2 proteins in the shoot,and OsACA9,OsACA8,OsSPS2F,OsPP2C15,and OsBiP3 in the root of NIL-23,indicated their role in P-starvation stress control through the Pup1 QTL. Thus,our findings indicated that-P stress-responsive proteins,in conjunction with morpho-physio-biochemical modulations,improved PUE and made NIL-23 a P-deficiency tolerant genotype due to the introgression of the Pup1 QTL in the Pusa-44 background.

The sperm-interacting proteome in the bovine isthmus and ampulla during the periovulatory period

Background Spermatozoa interact with oviduct secretions before fertilization in vivo but the molecular players of this dialog and underlying dynamics remain largely unknown.Our objectives were to identify an exhaustive list of sperm-interacting proteins(SIPs)in the bovine oviduct fluid and to evaluate the impact of the oviduct anatomical region(isthmus vs.ampulla)and time relative to ovulation(pre-ovulatory vs.post-ovulatory)on SIPs number and abundance.Methods Pools of oviduct fluid(OF)from the pre-ovulatory ampulla,pre-ovulatory isthmus,post-ovulatory ampulla,and post-ovulatory isthmus in the side of ovulation were collected from the slaughterhouse.Frozen-thawed bull sperm were incubated with OF or phosphate-buffered saline(control)for 60 min at 38.5℃.After protein extraction and digestion,sperm and OF samples were analyzed by nanoLC-MS/MS and label-free protein quantification.Results A quantitative comparison between proteins identified in sperm and OF samples(2333 and 2471 proteins,respectively)allowed for the identification of 245 SIPs.The highest number(187)were found in the pre-ovulatory isthmus,i.e.,time and place of the sperm reservoir.In total,41 SIPs(17%)were differentially abundant between stages in a given region or between regions at a given stage and 76 SIPs(31%)were identified in only one region×stage condition.Functional analysis of SIPs predicted roles in cell response to stress,regulation of cell motility,fertilization,and early embryo development.Conclusion This study provides a comprehensive list of SIPs in the bovine oviduct and evidences dynamic spatiotemporal changes in sperm-oviduct interactions around ovulation time.Moreover,these data provide protein candidates to improve sperm conservation and in vitro fertilization media.

Subinhibitory Levels of Fluoroquinolones Result in Enrichment of the Membrane Proteome of Staphylococcus aureus

Staphylococcus aureus is a common marine foodborne pathogen.In this study,antibiotics ciprofloxacin and enrofloxacin were used to induce drug-resistance in S.aureus.The differentially expressed proteins(DEPs)were analyzed and compared with those in the bacteria cultured without antibiotics.The primary proteomic alterations were in the levels of cell membrane components and proteins related to lysine and folic acid biosynthesis,which were all significantly up-regulated.The minimal inhibitory concentrations(MIC)for both test drugs were elevated to 10μg m L^(-1)following serial passaging.These results indicated that,for both ciprofloxacin and enrofloxacin,drug-resistance were developed even in the subinhibitory levels and the primary response was a major alteration in the cell membrane proteome.These changes were similar to those observed in S.aureus cultured with super-MIC levels of these antibiotics.The current study provides a theoretical basis for in-depth study of the related changes of marine foodborne pathogens in subinhibitory concentrations that are commonly found in situ.

Comprehensive and deep profiling of the plasma proteome with protein corona on zeolite NaY

Proteomic characterization of plasma is critical for the development of novel pharmacodynamic biomarkers.However,the vast dynamic range renders the profiling of proteomes extremely challenging.Here,we synthesized zeolite NaY and developed a simple and rapid method to achieve comprehensive and deep profiling of the plasma proteome using the plasma protein corona formed on zeolite NaY.Specifically,zeolite NaY and plasma were co-incubated to form plasma protein corona on zeolite NaY(NaY-PPC),followed by conventional protein identification using liquid chromatography-tandem mass spectrometry.NaY was able to significantly enhance the detection of low-abundance plasma proteins,minimizing the“masking”effect caused by high-abundance proteins.The relative abundance of middleand low-abundance proteins increased substantially from 2.54%to 54.41%,and the top 20 highabundance proteins decreased from 83.63%to 25.77%.Notably,our method can quantify approximately 4000 plasma proteins with sensitivity up to pg/mL,compared to only about 600 proteins identified from untreated plasma samples.A pilot study based on plasma samples from 30 lung adenocarcinoma patients and 15 healthy subjects demonstrated that our method could successfully distinguish between healthy and disease states.In summary,this work provides an advantageous tool for the exploration of plasma proteomics and its translational applications.

Global and Comparative Proteome Analysis of Nitrogen-Stress Responsive Proteins in the Root, Stem and Leaf of Brassica napus

Nitrogen(N)is one of the basic nutrients and signals for plant development and deficiency of it would always limit the productions of crops in the field.Quantitative research on expression of N-stress responsive proteins on a proteome level remains elusive.In order to gain a deep insight into the proteins responding to nitrogen stress in rapeseed(Brassica napus L.),comparative proteomic analysis was performed to investigate changes of protein expression profiles from the root,stem and leaf under different N concentrations,respectively.More than 200 differential abundance proteins(DAPs)were detected and categorized into groups according to annotations,including“binding and catalytic activity”,“involved in primary metabolism and cellular processes”,“stress-response”and so on.Variation in chlorophyll(Chl)content and antioxidant activities further revealed that oxidative stress raised with the increase of N concentration.Bioinformatics analysis based on the expression level of total proteins suggested these DAPs might play important roles in adaptation to N-stress conditions.Generally,these results provides a new aspect into N-stress responding proteins in Brassica plants.

Commentary on: Mobley AJ, Lam YW, Lau KM, Pais VM, Lesperance JO, Steadman B, et al. Monitoring the serological Proteome Colon, the latest modality in prostate cancer detection. J Urol 2004; 172: 331-7.

This manuscript examines the utility, utilizing the Ciphergen Protein Biosystem II, to develop a fingerprint for the diagnosis of prostate cancer. The investigators compared samples from control individuals as well as those with prostate cancer. In doing so, they utilize several chip platforms on which to examine the resulting

Comparative analysis between endometrial proteomes of pregnant and non-pregnant ewes during the peri-implantation period

Background： Early pregnancy failure has a profound impact on both human reproductive health and animal production. 2/3 pregnancy failures occur during the peri-implantation period; however, the underlying mechanism（s） remains unclear. Well-organized modification of the endometrium to a receptive state is critical to establish pregnancy Aberrant endometrial modification during implantation is thought to be largely responsible for early pregnancy loss. Result： In this study, using well-managed recipient ewes that received embryo transfer as model, we compared the endometrial proteome between pregnant and non-pregnant ewes during implantation period. After embryo transfer, recipients were assigned as pregnant or non-pregnant ewes according to the presence or absence of an elongated conceptus at Day 17 of pregnancy. By comparing the endometrial proteomic profiles between pregnant and non-pregnant ewes, we identified 94 and 257 differentially expressed proteins （DEPs） in the endometrial caruncular and intercaruncular areas, respectively. Functional analysis showed that the DEPs were mainly associated with immune response, nutrient transport and utilization, as well as proteasome-mediated proteolysis. Conclusion： These analysis imply that dysfunction of these biological processes or pathways of DEP in the endometrium is highly associated with early pregnancy loss. In addition, many proteins that are essential for the establishment of pregnancy showed dysregulation in the endometrium of non-pregnant ewes. These proteins, as potential candidates, may contribute to early pregnancy loss.

Screening of Serum Biomarkers for Distinguishing between Latent and Active Tuberculosis Using Proteome Microarray

Objective To identify potential serum biomarkers for distinguishing between latent tuberculosis infection（LTBI） and active tuberculosis（TB）. Methods A proteome microarray containing 4,262 antigens was used for screening serum biomarkers of 40 serum samples from patients with LTBI and active TB at the systems level. The interaction network and functional classification of differentially expressed antigens were analyzed using STRING 10.0 and the TB database, respectively. Enzyme-linked immunosorbent assays（ELISA） were used to validate candidate antigens further using 279 samples. The diagnostic performances of candidate antigens were evaluated by receiver operating characteristic curve（ROC） analysis. Both antigen combination and logistic regression analysis were used to improve diagnostic ability. Results Microarray results showed that levels of 152 Mycobacterium tuberculosis（Mtb）-antigenspecific IgG were significantly higher in active TB patients than in LTBI patients（P 〈 0.05）, and these differentially expressed antigens showed stronger associations with each other and were involved in various biological processes. Eleven candidate antigens were further validated using ELISA and showed consistent results in microarray analysis. ROC analysis showed that antigens Rv2031 c, Rv1408, and Rv2421 c had higher areas under the curve（AUCs） of 0.8520, 0.8152, and 0.7970, respectively. In addition, both antigen combination and logistic regression analysis improved the diagnostic ability. Conclusion Several antigens have the potential to serve as serum biomarkers for discrimination between LTBI and active TB.

Proteome analysis of alfalfa roots in response to water deficit stress

To evaluate the response of alfalfa to water deficit （WD） stress, WD-induced candidates were investigated through a proteomic approach. Alfalfa seedlings were exposed to WD stress for 12 and 15 days respectively, followed by 3 days re-watering. Water deficit increased H202 content, lipid peroxidation, DPPH （1,1-diphenyl-2-picrylhydrazyl）-radical scavenging activity, and the free proline level in alfalfa roots. Root proteins were extracted and separated by two-dimentional polyacrylamide gel electrophoresis （2-DE）. A total of 49 WD-responsive proteins were identified in alfalfa roots; 25 proteins were reproducibly found to be up-regulated and 24 were down-regulated. Two proteins, namely cytosolic ascorbate peroxidase （APx2） and putative F-box protein were newly detected on 2-DE maps of WD-treated plants. We identified several proteins including agamous-like 65, albumin b-32, inward rectifying potassium channel, and auxin-independent growth promoter. The identified proteins are involved in a variety of cellular functions including calcium signaling, abacisic acid （ABA） biosynthesis, reactive oxygen species （ROS） regulation, transcription/translation, antioxidant/detoxification/stress defense, energy metabolism, signal transduction, and storage. These results indicate the potential candidates were responsible for adaptive response in alfalfa roots.

Proteome Analysis of Inhibitory Effect of Gadolinium on Sinorhizobium fredii

The inhibitory effect of gadolinium on Sinorhizobium fredii USDA 205 was studied on a global scale using twodimensional gel electrophoresis and MALDI-TOF MS. The results indicated that 22 proteins were significantly affected by 1 mmol · L^-1 Gd^3 ＋ treatment when compared with an untreated control. Among these proteins, nine were up-regulated and thirteen were down-regulated. The differently expressed proteins were classified into 8 functional categories based on their functions, including transporters, proteins for cellular defence, and proteins involved in metabolism.

Molecular mechanisms of growth depression in broiler chickens(Gallus Gallus domesticus)mediated by immune stress:a hepatic proteome study

Background:Immunological stress decreases feed intake,suppresses growth and induces economic losses.However,the underlying molecular mechanism remains unclear.Label-free liquid chromatography and mass spectrometry(LC-MS)proteomics techniques were employed to investigate effects of immune stress on the hepatic proteome changes of Arbor Acres broilers(Gallus Gallus domesticus)challenged with Escherichia coli lipopolysaccharide(LPS).Results:Proteomic analysis indicated that 111 proteins were differentially expressed in the liver of broiler chickens from the immune stress group.Of these,28 proteins were down-regulated,and 83 proteins were up-regulated in the immune stress group.Enrichment analysis showed that immune stress upregulated the expression of hepatic proteins involved in defense function,amino acid catabolism,ion transport,wound healing,and hormone secretion.Furthermore,immune stress increased valine,leucine and isoleucine degradation pathways.Conclusion:The data suggests that growth depression of broiler chickens induced by immune stress is triggered by hepatic proteome alterations,and provides a new insight into the mechanism by which immune challenge impairs poultry production.

Desiccation tolerance in bryophytes： the rehydration proteomes of Bryum argenteum provide insights into the resuscitation mechanism

Bryum argenteum Hedw. is a desiccation tolerant bryophyte and belongs to one of the most important components of the biological soil crusts （BSCs） found in the deserts of Central Asia. Limited information is available on rehydration-responsive proteins in desiccation tolerant plants. As a complement to our previous research analyzing the rehydration transcriptome, we present a parallel quantitative proteomic effort to study rehydration-responsive proteins. Bryophyte gametophores were desiccated （Dry） and rehydrated for 2 h （R2） and 24 h （R24）. Proteins from Dry, R2 and R24 gametophores were labeled by isobaric tags for relative and absolute quantitation （iTRAQ） to determine the relative abundance of rehydration-responsive proteins. A total of 5503 non-redundant protein sequences were identified and 4772 （86.7%） protein sequences were annotated using Gene Ontology （GO） terms and Pfam classifications. Upon rehydration 239 proteins were elevated and 461 proteins were reduced as compared to the desiccated protein sample. Differentially up-regulated proteins were classified into a number of categories including reactive oxygen species scavenging enzymes, detoxifying enzymes, Late Embryogenesis Abundant （LEA） proteins, heat shock proteins, proteasome components and proteases, and photosynthesis and translation related proteins. Furthermore, the results of the correlation between transcriptome and proteome revealed the discordant changes in the expression between protein and mRNA.

Comparative Analyses of Proteome Complement Between Worker Bee Larvae of High Royal Jelly Producing Bees (A. m. ligustica) and Carniolian Bees (A. m. carnica)

This study is to compare the protein composition of the high royal jelly producing bee （A. m. ligustica） with that of Carniolian bee （A. m. carnica） during their worker larval developmental stage. The experiment was carried out by two- dimensional gel electrophoresis. The results showed that significant higher numbers of total proteins （283） were detected in larvae of high royal jelly producing bees （Jelly bee） than those of Carniolian bees （152） on 2-d-old larvae. Among them, 110 proteins were presented on both strains of bee larvae, whereas 173 proteins were specific to larvae of Jelly bees, and 42 proteins were exclusive to Carniolian larvae. However, on the 4th d, a significant higher number of total proteins （290） were detected in larvae of Jelly bees than those of Carniolian bees （240）, 163 proteins resolved to both bee larvae, and 127 proteins were specific to Jelly bees and 77 proteins to Camiolian bees. Until the 6th d, also a significant higher number of total proteins （236） were detected in larvae of Jelly bees than those of Carniolian bees （180）, 132 proteins were constantly expressed in two bee larvae, whereas 104 and 48 proteins are unique to Jelly bee and Carniolian bee larvae, respectively. We tentatively concluded that the metabolic rate and gene expression of Jelly bees larvae is higher than those of Carniolian bees based proteins detected as total proteins and proteins specific to each stage of two strains of bee larvae. Proteins constantly expressed on 3 stages of larval development with some significant differences between two bee strains, and proteins unique to each stage expressed differences in term of quality and quantity, indicating that larval development needed house keeping and specific proteins to regulate its growth at different development phage, but the expression mold is different between two strains of larval development.

Effect of fasting on protein metabolism in muscle tissue of Larimichthys crocea revealed by transcriptome and proteome

The large yellow croaker(Larimichthys crocea)is an important mariculture fish in China.Farmed large yellow croaker undergo periods of fasting to adapt to the environment or to improve meat quality.To better understand the physiological responses of their muscle tissues to fasting stresses,we analyzed the transcriptomes and proteome s of both normally-fed and fasting fish groups and identified 7578 differentially expressed genes(DEGs)and 297 differentially expressed proteins(DEPs)among them.Gene ontology and KEGG analysis showed that the enriched biological pathways were mainly involved in various synthetic and catabolic pathways,especially the protein metabolism.Based on the omics data,nine DEGs related to muscle composition(CAN3,MYL3,and TNNC2),growth(MSTN and MYF5),autophagy(TSC2 and ULK1),and the ubiquitin proteasome pathway(PRS6 B and UCHL3)were examined using qPCR.In response to fasting stress,MYL3 and TNNC2 were significantly downregulated,while genes associated with autophagy and the ubiquitin proteasome pathway were significantly upregulated.In re sponse to fasting stres s,MYL3,TNNC2,and MYF5 positively correlated with muscle growth were significantly downregulated,while inhibiting growth MSTN and genes associated with autophagy and the ubiquitin proteasome pathways were significantly upregulated.These results clarify the effects of fasting on metabolic changes in their muscle components and growth at the molecular level.

iTRAQ-based quantitative proteome characterization of wheat grains during filling stages

Using isobaric tags for relative and absolute quantification （iTRAQ） and associated analytic technologies, we have cataloged and compared 7 069 unique wheat proteins expressed during four substages of the filling stage. Among them, 859 are differentially expressed, showing at least a 2-fold difference in concentration across substages. Differentially expressed proteins （DEPs） includind high-molecular weight giutenin subunit （W5AIU1）, low-molecular weight glutenin subunit （QSW3V4）, gliadin/avenin-like seed protein （D2KFG9）, and avenin-like protein （W5DVL2）, all of which have previously been identified as important for nutritional quality and bread-making properties, and all of which were found to increase at the latter stages of development. We have applied statistical techniques to group the proteins into hierarchical clusters, and have consulted databases to infer functional and other relationships among the identified proteins.

Profile Analysis of the Proteome of the Egg of the High Royal Jelly Producing Bees (Apis mellifera L.)

The protein composition of the egg development in the high royal jelly producing bees （Apis mellifera L.） was investigated. This pioneer study was to separate and quantify the proteins in the egg of the high royal jelly producing worker bees （Apis mellifera L.） by using two-dimensional gel electrophoresis along with their three-day development. The results showed that 160, 195, and 176 proteins, with a wide range of molecular weight （17-80 KDa） and relatively narrow scope of pI （4. 00-8.40） could be detected on day 1, day 2, and day 3, respectively, during the developmental process of the egg. Meanwhile 44 protein spots were constantly detected along with the egg development. Among them 36% were in the uptrend along with the egg development, 14% were in the downtrend, and 39% were of the largest expressed volume on day 2. In addition, the specific proteins were expressed on day 1, day 2, and day 3 （89, 77, and 80, respectively）. Besides the coexistent and specific proteins, 24 proteins were expressed on day 1 and day 2, but silenced on day 3, 49 proteins were expressed on day 2 and day 3, but silenced on day 1, only 3 proteins were expressed on day 1 and day 3, but silenced on day 2. The result indicates that egg development is a sequential and complex gene controlled process, where the eggs of day 2 express the most active proteins. The coexistent proteins suggest that it is conservative and indispensable for this event. These specific proteins suggest that the different developmental stage needs specific proteins to regulate it.

iTRAQ-based analysis of 17β-estradiol induced proteome in Chinese tongue sole Cynoglossus semilaevis

The phenomenon of sex dimorphism prevails among many fi sh species. It has attracted the general research interest due to its close relationship to fi sh growth and thus aquaculture productivity. In Chinese tongue sole ( Cynoglossus semilaevis ), 17β-estradiol (E2) can be used reportedly to induce the feminization of fi sh, although the detailed regulatory network remained elusive. We treated the tongue sole fry before sex diff erentiation with E2 (10 and 30 μg/L) to identify potential targets of E2 in Chinese tongue sole. The E2 treatment indeed induced genetic male fi sh sex-reversal to phenotypic female. Using an iTRAQ-based comparative proteomic analysis, 409 proteins that diff erentially expressed after E2 induction were identifi ed, including 259 up-regulated and 150 down-regulated proteins. Furthermore, 19 diff erentially expressed proteins identifi ed in the comparative proteomic analysis were selected to assess their transcription and eight of them exhibited the same tendency. Functions of the eight proteins included mainly nucleotide and protein binding. Interestingly, a far upstream element-binding protein 3-like isoform exhibited the signifi cant upregulation both at transcription and translation levels after E2 treatment. This work identifi ed a set of candidate proteins for E2 response and deepened our understanding of E2 regulatory mechanism.