Effects of calcium application on apple fruit softening during storage revealed by proteomics and phosphoproteomics

Fruit ripening has been reported to be related to calcium(Ca),but the underlying mechanisms by which Ca regulates this process remain largely unknown.In order to study the changes of proteins and enriched phosphopeptides,we conducted TMT labeling,bio-material-based PTM enrichment based on mass spectrometry in Ca-treated‘Golden Delicious’(GD)apple fruit(Malus×domestica).This dataset presents a comprehensive overview of the critical pathways involved in fruit ripening.A total of 47 proteins and 124 phosphoproteins significantly changed in Ca-treated fruit,which are crucial for regulating the cell wall and cytoskeleton,Ca-mediated signaling and transport,ethylene production,protein fate,especially ubiquitination-based protein degradation,and primary and secondary metabolisms.Our results indicated that Ca inhibited the abundance of polygalacturonase(PG)activity and increased the phosphorylation level of CSLD3.PG and phosphorylation were involved in cell wall degradation,thereby delaying fruit softening.As a secondary messenger,Ca-mediated signaling subsequently triggered downstream mitogen-activated protein kinases(MAPK)cascades and activated the membrane,transport,and ROS signaling.Moreover,MdEIN2,a key enzyme involved in the ubiquitin of protein modification,increased at Ser753 and Ser758 in Ca-treated fruit.Furthermore,diverse primary and secondary metabolisms including glycolysis,fatty acid metabolism,and oxidation respiratory chain were modulated to prevent fruit softening.These results provide basic information from protein and phosphorylation levels for apple fruit ripening during storage,which may be helpful for apple fruit storage control.

Mechanisms of Dangua Fang(丹瓜方)in multi-target and multi-method regulation of glycolipid metabolism based on phosphoproteomics

OBJECTIVE:To explore the mechanism of Dangua Fang(丹瓜方,DGR)in multi-target and multi-method regulation of glycolipid metabolism based on phosphoproteomics.METHODS:Sprague-Dawley rats with normal glucose levels were randomly divided into three groups,including a conventional diet control group(Group A),high-fat-highsugar diet model group(Group B),and DGR group(Group C,high-fat-high-sugar diet containing 20.5 g DGR).After 10 weeks of intervention,the fasting blood glucose(FBG),2 h blood glucose[PBG;using the oral glucose tolerance test(OGTT)],hemoglobin A1c(HbA1c),plasma total cholesterol(TC),and triglycerides(TG)were tested,and the livers of rats were removed to calculate the liver index.Then,hepatic portal TG were tested using the Gross permanent optimization-participatiory action planning enzymatic method and phosphoproteomics was performed using liquid chromatography with tandem mass spectrometry(LC-MS/MS)analysis followed by database search and bioinformatics analysis.Finally,cell experiments were used to verify the results of phosphoproteomics.Phosphorylated mitogen-activated protein kinase kinase kinase kinase 4(MAP4k4)and phosphorylated adducin 1(ADD1)were detected using western blotting.RESULTS:DGR effectively reduced PBG,TG,and the liver index(P<0.05),and significantly decreased HbA1c,TC,and hepatic portal TG(P<0.01),showed significant hematoxylin and eosin(HE)staining,red oil O staining,and Masson staining of liver tissue.The total spectrum was 805334,matched spectrum was 260471,accounting for accounting 32.3%,peptides were 19995,modified peptides were 14671,identified proteins were 4601,quantifiable proteins were 4417,identified sites were 15749,and quantified sites were 14659.Based on the threshold of expression fold change(>1.2),DGR upregulated the modification of 228 phosphorylation sites involving 204 corresponding function proteins,and downregulated the modification of 358 phosphorylation sites involving 358 corresponding function proteins,which included correcting 75 phosphorylation sites involving 64 corresponding function proteins relating to glycolipid metabolism.Therefore,DGR improved biological tissue processes,including information storage and processing,cellular processes and signaling,and metabolism.The metabolic functions regulated by DGR mainly include energy production and conversion,carbohydrate transport and metabolism,lipid transport and metabolism,inorganic ion transport and metabolism,secondary metabolite biosynthesis,transport,and catabolism.In vitro phosphorylation validation based on cell experiments showed that the change trends in the phosphorylation level of MAP4k4 and ADD1 were consistent with that of previous phosphoproteomics studies.CONCLUSION:DGR extensively corrects the modification of phosphorylation sites to improve corresponding glycolipid metabolism-related protein expression in rats with glycolipid metabolism disorders,thereby regulating glycolipid metabolism through a multi-target and multi-method process.

Mass spectrometry-based phosphoproteomics in cancer research

Phosphorylation is one of the most common post translational modifications （PTM）, participating in a large number of processes to regulate cellular functions. Phosphorylation is also one of the key factors in the origin and development of cancer. The rapid development of mass spectrometric-based phosphoproteomic technologies has made it possible for high-throughput identification and quantification of phosphorylation events. In this review, we provide a general introduction and summary of the achievements made in mass spectrometry based phosphoproteomic research, including the approaches for phosphopeptide identification and quantification, as well as instrumentation and data interpretation methods. We also review some discoveries in cancer research made possible by phosphoproteomic analysis technologies.

Phosphoproteomics Reveals the AMPK Substrate Network in Response to DNA Damage and Histone Acetylation

AMP-activated protein kinase(AMPK)is a conserved energy sensor that plays roles in diverse biological processes via phosphorylating various substrates.Emerging studies have demonstrated the regulatory roles of AMPK in DNA repair,but the underlying mechanisms remain to be fully understood.Herein,using mass spectrometry-based proteomic technologies,we systematically investigate the regulatory network of AMPK in DNA damage response(DDR).Our system-wide phosphoproteome study uncovers a variety of newly-identified potential substrates involved in diverse biological processes,whereas our system-wide histone modification analysis reveals a link between AMPK and histone acetylation.Together with these findings,we discover that AMPK promotes apoptosis by phosphorylating apoptosis-stimulating of p53 protein 2(ASPP2)in an irradiation(IR)-dependent manner and regulates histone acetylation by phosphorylating histone deacetylase 9(HDAC9)in an IR-independent manner.Besides,we reveal that disrupting the histone acetylation by the bromodomain BRD4 inhibitor JQ-1 enhances the sensitivity of AMPKdeficient cells to IR.Therefore,our study has provided a resource to investigate the interplay between phosphorylation and histone acetylation underlying the regulatory network of AMPK,which could be beneficial to understand the exact role of AMPK in DDR.

Surviving winter on the Qinghai-Xizang Plateau:Extensive reversible protein phosphorylation plays a dominant role in regulating hypometabolism in hibernating Nanorana parkeri

Changes in protein abundance and reversible protein phosphorylation(RPP)play important roles in regulating hypometabolism but have never been documented in overwintering frogs at high altitudes.To test the hypothesis that protein abundance and phosphorylation change in response to winter hibernation,we conducted a comprehensive and quantitative proteomic and phosphoproteomic analysis of the liver of the Xizang plateau frog,Nanorana parkeri,living on the Qinghai-Xizang Plateau.In total,5170 proteins and 5695 phosphorylation sites in 1938 proteins were quantified.Based on proteomic analysis,674 differentially expressed proteins(438 up-regulated,236 down-regulated)were screened in hibernating N.parkeri versus summer individuals.Functional enrichment analysis revealed that higher expressed proteins in winter were significantly enriched in immune-related signaling pathways,whereas lower expressed proteins were mainly involved in metabolic processes.A total of 4251 modified sites(4147 up-regulated,104 down-regulated)belonging to 1638 phosphoproteins(1555 up-regulated,83 down-regulated)were significantly changed in the liver.During hibernation,RPP regulated a diverse array of proteins involved in multiple functions,including metabolic enzymatic activity,ion transport,protein turnover,signal transduction,and alternative splicing.These changes contribute to enhancing protection,suppressing energy-consuming processes,and inducing metabolic depression.Moreover,the activities of phosphofructokinase,glutamate dehydrogenase,and ATPase were all significantly lower in winter compared to summer.In conclusion,our results support the hypothesis and demonstrate the importance of RPP as a regulatory mechanism when animals transition into a hypometabolic state.

Na_(2)CO_(3)-responsive Photosynthetic and ROS Scavenging Mechanisms in Chloroplasts of Alkaligrass Revealed by Phosphoproteomics

Alkali-salinity exerts severe osmotic,ionic,and high-p H stresses to plants.To understand the alkali-salinity responsive mechanisms underlying photosynthetic modulation and reactive oxygen species(ROS)homeostasis,physiological and diverse quantitative proteomics analyses of alkaligrass(Puccinellia tenuiflora)under Na_(2)CO_(3)stress were conducted.In addition,Western blot,real-time PCR,and transgenic techniques were applied to validate the proteomic results and test the functions of the Na_(2)CO_(3)-responsive proteins.A total of 104 and 102 Na_(2)CO_(3)-responsive proteins were identified in leaves and chloroplasts,respectively.In addition,84 Na_(2)CO_(3)-responsive phosphoproteins were identified,including 56 new phosphorylation sites in 56 phosphoproteins from chloroplasts,which are crucial for the regulation of photosynthesis,ion transport,signal transduction,and energy homeostasis.A full-length Pt FBA encoding an alkaligrass chloroplastic fructosebisphosphate aldolase(FBA)was overexpressed in wild-type cells of cyanobacterium Synechocystis sp.Strain PCC 6803,leading to enhanced Na_(2)CO_(3)tolerance.All these results indicate that thermal dissipation,state transition,cyclic electron transport,photorespiration,repair of photosystem(PS)Ⅱ,PSI activity,and ROS homeostasis were altered in response to Na_(2)CO_(3)stress,which help to improve our understanding of the Na_(2)CO_(3)-responsive mechanisms in halophytes.

Phosphoproteomics Analysis of Endometrium in Women with or without Endometriosis

Background：The molecular mechanisms underlying the endometriosis are still not completely understood.In order to test the hypothesis that the approaches in phosphoproteomics might contribute to the identification of key biomarkers to assess disease pathogenesis and drug targets,we carried out a phosphoproteomics analysis of human endometrium.Methods：A large-scale differential phosphoproteome analysis,using peptide enrichment of titanium dioxide purify and sequential elution from immobilized metal affinity chromatography with linear trap quadrupole-tandem mass spectrometry,was performed in endometrium tissues from 8 women with or without endometriosis.Results：The phosphorylation profiling of endometrium from endometriosis patients had been obtained,and found that identified 516 proteins were modified at phosphorylation level during endometriosis.Gene ontology annotation analysis showed that these proteins were enriched in cellular processes of binding and catalytic activity.Further pathway analysis showed that ribosome pathway and focal adhesion pathway were the top two pathways,which might be deregulated during the development of endometriosis.Conclusions：That large-scale phosphoproteome quantification has been successfully identified in endometrium tissues of women with or without endometriosis will provide new insights to understand the molecular mechanisms of the development of endometriosis.

Comparative phosphoproteomics reveal new candidates in the regulation of spermatogenesis of Drosophila melanogaster

The most common phenotype induced by the endosymbiont Wolbachia in in-sects is cytoplasmic incompatibility,where none or fewer progenies can be produced when Wolbachia-infected males mate with uninfected females.This suggests that some modi-fications are induced in host sperms during spermatogenesis by Wolbachia.To identify the proteins whose phosphorylation states play essential roles in male reproduction in Drosophila melanogaster,we applied isobaric tags for relative and absolute quantitation(iTRAQ)-based proteomic strategy combined with titanium dioxide(TiO2)enrichment to compare the phosphoproteome of Wolbachia-infected with that of uninfected male re-productive systems in D.melanogaster.We identified 182 phosphopeptides,defining 140 phosphoproteins,that have at least a 1.2 fold change in abundance with a P-value of<0.05.Most of the differentially abundant phosphoproteins(DAPPs)were associated with micro-tubule cytoskeleton organization and spermatid differentiation.The DAPPs included pro-teins already known to be associated with spermatogenesis,as well as many not previously studied during this process.Six genes coding for DAPPs were knocked down,respectively,in Wolbachia-free fly testes.Among them,Slmap knockdown caused the most severe dam-age in spermatogenesis,with no mature sperm observed in seminal vesicles.Immunoflu-orescence staining showed that the formation of individualization complex composed of actin cones was completely disrupted.These results suggest that Wolbachia may induce wide changes in the abundance of phosphorylated proteins which are closely related to male reproduction.By identifying phospho-modulated proteins we also provide a signifi-cant candidate set for future studies on their roles in spermatogenesis.

Reversible protein phosphorylation, a central signaling hub to regulate carbohydrate metabolic networks

Plants produce a range of carbohydrates to meet their growth and developmental needs. Protein reversible phosphorylation plays key roles in coordinating multiple metabolic pathways and integrating diverse internal and external cues. Understanding such regulatory metabolism will provide novel resources for breeding and crop management by modulating metabolic pathways for control of growth and stress response. In this review, we summarize the complex, multifaceted functions of protein phosphorylation and their connections to plant metabolism. We focus particularly on carbohydrate metabolic pathways that are controlled by key kinases and discuss how they are linked to downstream changes in physiology, important agronomic traits and crop quality.

Quantitative proteomic and phosphoproteomic analyses of the hippocampus reveal the involvement of NMDAR1 signaling in repetitive mild traumatic brain injury

The cumulative damage caused by repetitive mild traumatic brain injury can cause long-term neurodegeneration leading to cognitive impairment.This cognitive impairment is thought to result specifically from damage to the hippocampus.In this study,we detected cognitive impairment in mice 6 weeks after repetitive mild traumatic brain injury using the novel object recognition test and the Morris water maze test.Immunofluorescence staining showed that p-tau expression was increased in the hippocampus after repetitive mild traumatic brain injury.Golgi staining showed a significant decrease in the total density of neuronal dendritic spines in the hippocampus,as well as in the density of mature dendritic spines.To investigate the specific molecular mechanisms underlying cognitive impairment due to hippocampal damage,we performed proteomic and phosphoproteomic analyses of the hippocampus with and without repetitive mild traumatic brain injury.The differentially expressed proteins were mainly enriched in inflammation,immunity,and coagulation,suggesting that non-neuronal cells are involved in the pathological changes that occur in the hippocampus in the chronic stage after repetitive mild traumatic brain injury.In contrast,differentially expressed phosphorylated proteins were mainly enriched in pathways related to neuronal function and structure,which is more consistent with neurodegeneration.We identified N-methyl-D-aspartate receptor 1 as a hub molecule involved in the response to repetitive mild traumatic brain injury,and western blotting showed that,while N-methyl-D-aspartate receptor 1 expression was not altered in the hippocampus after repetitive mild traumatic brain injury,its phosphorylation level was significantly increased,which is consistent with the omics results.Administration of GRP78608,an N-methyl-D-aspartate receptor 1 antagonist,to the hippocampus markedly improved repetitive mild traumatic brain injury-induced cognitive impairment.In conclusion,our findings suggest that N-methyl-D-aspartate receptor 1 signaling in the hippocampus is involved in cognitive impairment in the chronic stage after repetitive mild traumatic brain injury and may be a potential target for intervention and treatment.

Phosphorylation of SiRAV1 at Ser31 regulates the SiCAT expression to enhance salt tolerance in Setaria italica

Salinity severely affects plant growth and development.Thus,it is crucial to identify the genes functioning in salt stress response and unravel the mechanism by which plants against salt stress.This study used the phosphoproteomic assay and found that 123 of the 4000 quantitative analyzed phosphopeptides were induced by salt stress.The functional annotation of the non-redundant protein database(NR)showed 23 differentially expressed transcription factors,including a phosphopeptide covering the Serine 31 in the RAV(related to ABI3/VP1)transcription factor(named SiRAV1).SiRAV1 was located in the nucleus.Phenotypic and physiological analysis showed that overexpressing SiRAV1 in foxtail millet enhanced salt tolerance and alleviated the salt-induced increases of H_(2)O_(2) accumulation,malondialdehyde(MDA)content,and percent of electrolyte leakage.Further analysis showed that SiRAV1 positively regulated SiCAT expression to modulate the catalase(CAT)activity by directly binding to the SiCAT promoter in vivo and in vitro.Moreover,we found that phosphorylation of SiRAV1 at the Ser31 site positively regulated salt tolerance in foxtail millet via enhancing its binding ability to SiCAT promoter but did not affect its subcellular localization.Overall,our results define a mechanism for SiRAV1 function in salt response where salt-triggered phosphorylation of SiRAV1 at Ser31 enhances its binding ability to SiCAT promoter,and the increased SiCAT expression contributes to salt tolerance in foxtail millet.

Identification of a sensor histidine kinase (BfcK) controlling biofilm formation in Clostridium acetobutylicum

Clostridium acetobutylicum has been extensively exploited to produce biofuels and solvents and its biofilm could dramatically improve the productivities.However,genetic control of C.acetobutylicum biofilm has not been dissected so far.Here,to identify potential genes controlling C.acetobutylicum biofilm formation,over 40 gene candidates associated with extracellular matrix,cell surface,cell signaling or gene transcription,were tried to be disrupted to examine their individual impact.A total of 25 disruptants were finally obtained over years of attempts,for which biofilm and relevant phenotypes were characterized.Most of these disruptants formed robust biofilm still,or suffered both growth and biofilm defect.Only a strain with a disrupted histidine kinase gene(CA_C2730,designated bfcK in this study)abolished biofilm formation without impairing cell growth or solvent production.Further analysis revealed that bfcK could control flagellar biogenesis and cell motility at protein levels.The bfcK also appeared to repress the phosphorylation of a serine/threonine protein kinase(encoded by CA_C0404)that might negatively regulate biofilm formation.Based on these findings,possible bfcK-mediated mechanisms for biofilm formation were proposed.This is a big step toward understanding the biofilm formation in C.acetobutylicum and will help further engineering of its biofilm-based industrial processes.

Protein Phosphorylation and Phosphoproteome:An Overview of Rice

Protein phosphorylation,one of the major post-translational modifications,plays a crucial role in cell signaling,DNA replication,gene expression and differentiation;and alters enzyme activity and other biological activities;and regulates cell proliferation and enlargement,phytohormone biosynthesis and signaling,plant disease resistance,and grain filling and quality during rice seed development.Research work on protein phosphorylation started in the 1950 s with the discovery of phosphorylase a and phosphorylase b which are phospho and dephospho forms of the same enzyme.Over the last decade,rice proteomics has accomplished tremendous progress in setting up techniques to proteome nearly all tissues,organs and organelles.The progress made in this field is evident in number of research works.However,research on rice protein phosphorylation is still at its infancy and there are still many unanswered questions.In this review,the general description of protein phosphorylation,including history,structure,frequency of occurrence and function,are discussed.This work also elucidates the different methods for identification,qualification and finally,the progress in rice phosphoproteome research and perspectives.

用磷酸化蛋白组学分析鉴别磷酸二酯酶功能（英文）

We now know that 11 different families of cyclic nucleotide phosphodiesterases(PDEs) are expressed in mammalian species. Most of these families contain multiple gene products and most of the genes utilize alternative splicing or alternative start sites to encode more than one RNA/protein.However,specific functions for these different PDEs have not yet been identified in most cell types despite the fact that selective inhibitors to most of the PDE families are available. Conventional approaches to study PDE function typically rely on measurements of global cAMP,or general increases in cAMP-dependent protein kinase A(PKA),or exchange protein activated by cAMP(EPAC) activity.Although newer approaches utilizing subcellularly-targeted FRET reporter sensors have helped to define more compartmentalized regulation of cAMP,PKA,and EPAC,they have limited ability to link this regulation to downstream effector molecules and biological functions. To address this problem,we have begun to use an unbiased,mass spectrometry-based approach coupled with treatment using PDE isozyme-selective inhibitors to characterize the phosphoproteomes of the ″ functional pools″ of cAMP that are regulated by specific cAMP-PDEs(the PDE-regulated phosphoproteomes). In MA-10 Leydig cells we find that in order to detect appreciable increased in either phosphorylation or steroid production,one needs to inhibit both PDE 4 and PDE 8 activity.Using this combination of inhibitors,we find large PDE inhibitor-induced changes in many different proteins that modulate steroid trafficking and biosynthesis. The data are consistent with the idea that cAMP serves to coordinate hormone stimulation of steroid production by altering the phosphorylation of many different proteins at multiple points in the overall pathway rather than just controlling a single rate limiting step. It seems quite likely that many of the proteins phosphorylated in this cell type in response to PDE inhibition,will also be regulated by cAMP in many other cell types.Similarly,in Jurkat cells we find multiple,distinct,PDE regulated phosphoproteomes that differ in response to different PDE inhibitors. Here we also find that little phosphorylation occurs unless at least 2 different PDEs are concurrently inhibited in these cells. Inhibition of a single PDE produces little effect. Bioinformatics analyses of these phosphoproteomes suggest differing functional roles,mechanisms of action,and synergistic relationships among the different PDEs that coordinate cAMP-signaling cascades in these cells. In this tissue also,the data strongly imply that phosphorylation of many different substrates contribute to cAMP-dependent regulation of these cells. Overall,the findings illustrate that the approach of using selective,inhibitor-dependent phosphoproteome analysis can provide a generalized methodology for understanding the roles of different PDEs in the regulation of cyclic nucleotide signaling.

Temporal Integrative Omics Reveals an Increase in Nondegradative Ubiquitylation during Primary Hepatocyte Dedifferentiation

Primary hepatocytes(PHCs)are widely used in various fields,but the progressive deterioration of liverspecific features in vitro significantly limits their application.While the transcriptional regulation and whole cell proteome(WCP)of PHCs have been extensively studied,only a small number of studies have addressed the role of posttranslational modifications in this process.To elucidate the underlying mechanisms that induce dedifferentiation,we carried out parallel quantifications of the transcriptome,WCP,ubiquitinome,and phosphoproteome of rat PHCs after 0,6,12,24,and 48 h of in vitro culture.Our data constitute a detailed proteomic analysis of dedifferentiated PHCs including 2196 proteins,2056 ubiquitinated sites,and 4932 phosphorylated peptides.We revealed a low correlation between the transcriptome and WCP during dedifferentiation.A combined analysis of the ubiquitinome with the corresponding WCP indicated that the dedifferentiation of PHCs led to an increase in nondegradative K27 ubiquitination.Functional analysis of the altered phosphoproteins suggested a significant enrichment in ferroptosis.In all,404 proteins with both ubiquitination and phosphorylation were identified to be involved in critical metabolic events.Furthermore,Ptbph Hnqjd,Hnrnpu,and Srrm2 were identified as hub genes.Taken together,our data provide new insights into proteome dynamics during PHC dedifferentiation and potential targets to inhibit the dedifferentiation process.

Transcriptomic,proteomic,and phosphoproteomic analyses reveal dynamic signaling networks influencing long-grain rice development

The LGS1(Large grain size 1)gene,also known as GS2/GL2/Os GRF4,is involved in regulating grain size and quality in rice,but the mechanism governing grain size has not been elucidated.We performed transcriptomic,proteomic,and phosphoproteomic analyses of young rice panicles in Samba(a wild-type cultivar with extra-small grain)and NIL-LGS1(a nearly isogenic line of LGS1 with large grain in the Samba genetic background)at three developmental stages(4–6)to identify internal dynamic functional networks determining grain size that are mediated by LGS1.Differentially expressed proteins formed seven highly functionally correlated clusters.The concordant regulation of multiple functional clusters may be key features of the development of grain length in rice.In stage 5,16 and 24 phosphorylated proteins were significantly up-regulated and down-regulated,and dynamic phosphorylation events may play accessory roles in determining rice grain size by participating in protein–protein interaction networks.Transcriptomic analysis in stage 5 showed that differentially expressed alternative splicing events and dynamic gene regulatory networks based on 39 transcription factors and their highly correlated target genes might contribute to rice grain development.Integrative multilevel omics analysis suggested that the regulatory network at the transcriptional and posttranscriptional levels could be directly manifested at the translational level,and this analysis also suggested a regulatory mechanism,regulation of protein translation levels,in the biological process that extends from transcript to protein to the development of grain.Functional analysis suggested that biological processes including MAPK signaling,calcium signaling,cell proliferation,cell wall,energy metabolism,hormone pathway,and ubiquitin-proteasome pathway might be involved in LGS1-mediated regulation of grain length.Thus,LGS1-mediated regulation of grain size is affected by dynamic transcriptional,posttranscriptional,translational and posttranslational changes.

GreenPhos,a universal method for in-depth measurement of plant phosphoproteomes with high quantitative reproducibility

Protein phosphorylation regulates a variety of important cellular and physiological processes in plants.In-depth profiling of plant phosphoproteomes has been more technically challenging than that of animal phosphoproteomes.This is largely due to the need to improve protein extraction efficiency from plant cells,which have a dense cell wall,and to minimize sample loss resulting from the stringent sample clean-up steps required for the removal of a large amount of biomolecules interfering with phosphopeptide purification and mass spectrometry analysis.To this end,we developed a method with a streamlined workflow for highly efficient purification of phosphopeptides from tissues of various green organisms including Arabidopsis,rice,tomato,and Chlamydomonas reinhardtii,enabling in-depth identification with high quantitative reproducibility of about 11000 phosphosites,the greatest depth achieved so far with single liquid chromatography-mass spectrometry(LC-MS)runs operated in a data-dependent acquisition(DDA)mode.The mainstay features of the method are the minimal sample loss achieved through elimination of sample clean-up before protease digestion and of desalting before phosphopeptide enrichment and hence the dramatic increases of time-and cost-effectiveness.The method,named GreenPhos,combined with single-shot LC-MS,enabled in-depth quantitative identification of Arabidopsis phosphoproteins,including differentially phosphorylated spliceosomal proteins,at multiple time points during salt stress and a number of kinase substrate motifs.GreenPhos is expected to serve as a universal method for purification of plant phosphopeptides,which,if samples are further fractionated and analyzed by multiple LC-MS runs,could enable measurement of plant phosphoproteomes with an unprecedented depth using a given mass spectrometry technology.

基于数据独立采集的磷酸化蛋白质组学全面揭示I型酪蛋白激酶在植物发育中的作用

I型酪蛋白激酶(CKI)是一类真核生物中高度保守的丝氨酸/苏氨酸蛋白激酶,通过介导底物的磷酸化调节生长发育和信号转导:拟南芥蛋白AELs,是植物特异的CK1,具有多种功能,但其内源底物的准确识别和验证极大地限制了对CK1生理功能的系统阐明.本研究通过数据独立采集模式(DIA)开展的磷酸化蛋白质组学研究系统揭示了CKI的内源底物:从AELs过表达及多种突变体的幼苗中提取蛋白并进行质谱检测,与野生型比较分析获取了3985个在AELs过表达材料中丰度高于野生型(Col-0)以及在缺失突变体中丰度低于Col-0的磷酸化肽段(AEL依赖的磷酸化肽段).对应获得1032个磷酸化蛋白。进一步从磷酸化肽段中富集了8个底物识别基序并对其中4个新基序进行了实验验证,用于预测CK1的潜在候选底物,基于富集到的一个参与生殖发育的锌指转录因子,C3H17,我们通过生化和遗传分析进行了底物验证和功能鉴定,证明AEL通过介导C3H17磷酸化以增强蛋白稳定性和转录活性进而调控拟南芥的胚胎发育,基于CK1在真核生物中的高度保守性,进一步利用CK1的底物识别基序搜索了水稻、小鼠和人类的蛋白数据库,获得了远超已知数目的众多候选底物,极大地扩展了对CK1在动植物中发挥的共同和独特功能的理解,为跨物种全面揭示CK1的保守功能提供了重要线索.

Remodeling of the ryanodine receptor isoform 1 channel regulates the sweet and umami taste perception of Rattus norvegicus

Sweet and umami tastes are elicited by sweet and umami receptors on the tongue and palate epithelium,respectively.However,the molecular machinery allowing the taste reaction remains incompletely understood.Through a phosphoproteomic approach,we identified the key proteins that trigger taste mechanisms based on phosphorylation cascades.Ryanodine receptor isoform 1(RYR1)was further verified by sensory and behavioral assays.We propose a model of RYR1-mediated sweet/umami signaling in which the RYR1 channel,which mediates Ca^(2+)release from the endoplasmic reticulum,is closed by dephosphorylation in bud tissue after sweet/umami treatment.The alteration in Ca^(2+)content in the cytosol induces transient membrane depolarization and generates a cell current for taste signal transduction.We demonstrate that RYR1 is a new channel involved in the regulation of sweet/umami signal transduction and propose a“metabolic clock”notion based on sweet/umami sensing.Our study provides a valuable foundation for a system-level understanding of the taste perception mechanism.

Proteomic and phosphoproteomic analysis reveal threonine deficiency increases hepatic lipid deposition in Pekin ducks via reducing STAT phosphorylation

Dietary threonine(Thr)deficiency enhances triglyceride(TG)deposition in the liver of Pekin ducks,which injures hepatic function and impairs growth performance.However,the underlying molecular mechanisms remain unclear.In the present study,we investigated the effects of dietary Thr deficiency on the expressions of proteins and phosphoproteins in liver of Pekin ducks,to identify the underlying molecular changes.A total of 300 one-day-old ducklings were divided into 3 groups with 10 replicates of 10 birds.All ducks were fed corn-wheat-peanut meal diets containing 0.46%,0.71%,and 0.96%Thr,respectively,from 1 to 21 days of age.Growth performance,serum parameters,hepatic TG content,and expression of genes involved in lipid metabolism of Pekin ducks were determined.A Thr deficiency group(Thr-D,0.46%Thr)and a Thr sufficiency group(Thr-S,0.71%Thr)were selected for subsequent proteomic and phosphoproteomic analysis.The results showed that Thr-D reduced the growth performance(P<0.001),and increased the plasma concentrations of cholesterol,high-density lipoprotein cholesterol,low-density lipoprotein cholesterol,and hepatic TG(P<0.05).Thr-D increased gene expression related to fatty acid and TG synthesis(P<0.05).A total of 176 proteins and 259 phosphosites(containing 198 phosphoproteins)were observed to be differentially expressed as a result of Thr-D.The upregulated proteins were enriched in the pathway related to amino acid metabolism,peroxisome.The down-regulated proteins were enriched in linolenic and arachidonic acid metabolism,and the Janus kinase-signal transducer and activator of transcription(JAK-STAT)signaling pathway.The upregulated phos-phoproteins were enriched in the pathways related to fatty acid biosynthesis,fructose and mannose metabolism,and glycolysis/gluconeogenesis.Thr-D reduced the phosphorylation of STAT1 at S729 and STAT3 at S728,and expression of STAT5B.In contrast,Thr-D increased non-receptor tyrosine-protein kinase(TYK2)expression and STAT1 phosphorylation at S649.Taken together,dietary Thr-D increased hepatic TG accumulation by upregulating the expression of genes and proteins,and phosphoproteins related to fatty acid and triglyceride synthesis.Furthermore,these processes might be regulated by the JAK-STAT signaling pathway,especially the phosphorylation of STAT1 and STAT3.