In‐depth proteome mining of cultured Catharanthus roseus cells identifies candidate proteins involved in the synthesis and transport of secondary metabolites

Madagascar periwinkle (Catharanthus roseus) is the major source of terpenoid indole alkaloids, such as vinblastine or vincristine, used as natural drugs against various cancers. In this study, we have extensively analyzed the proteome of cultured C. roseus cells. Comparison of the proteomes of two independent cell lines with different terpenoid indole alkaloid metabolism by 2D‐DIGE revealed 358 proteins that differed quantitatively by at least a twofold average ratio. Of these, 172 were identified by MS; most corresponded to housekeeping proteins. Less abundant proteins were identified by LC separation of tryptic peptides of proteins from one of the lines. We identified 1663 proteins, most of which are housekeeping proteins or involved in primary metabolism. However, 63 enzymes potentially involved in secondary metabolism were also identified, of which 22 are involved in terpenoid indole alkaloid biosynthesis and 16 are predicted transporters putatively involved in secondary metabolite transport. About 30% of the proteins identified have an unclear or unknown function, indicating important gaps in knowledge of plant metabolism. This study is an important step toward elucidating the proteome of C. roseus, which is critical for a better understanding of how this plant synthesizes terpenoid indole alkaloids.     

Conserved and unique features of the maize (Zea mays L.) root hair proteome

Root hairs are unicellular extensions of specialized epidermis cells. Under limiting conditions, they significantly increase the water and nutrient uptake capacity of plants by enlarging their root surface. Thus far, little is known about the initiation and growth of root hairs in the monocot model species maize. To gain a first insight into the protein composition of these specialized cells, the 2573 most abundant proteins of maize root hairs attached to four-day-old primary roots of the inbred line B73 were identified by combining 1DE with nanoLC-MS/MS in a shotgun proteomic experiment. Among the identified proteins, homologues of 252 proteins have been previously associated with root hair formation and development in other species. Comparison of the root hair reference proteome of the monocot species maize with the previously published root hair proteome of the dicot species soybean revealed conserved, but also unique, protein functions in root hairs of these two major groups of flowering plants.     

A comparative proteome approach to decipher the mechanism of rice adaptation to phosphorous deficiency

Mineral deficiency limits crop production in most soils and in Asia alone, about 50% of rice lands are phosphorous deficient. In an attempt to determine the mechanism of rice adaptation to phosphorous deficiency, changes in proteome patterns associated with phosphorous deficiency have been investigated. We analyzed the parental line Nipponbare in comparison to its near isogenic line (NIL6‐4) carrying a major phosphorous uptake QTL (Pup1) on chromosome 12. Using 2‐DE, the proteome pattern of roots grown under 1 and 100 μM phosphorous were compared. Out of 669 proteins reproducibly detected on root 2‐DE gels, 32 proteins showed significant changes in the two genotypes. Of them, 17 proteins showed different responses in two genotypes under stress condition. MS resulted in identification of 26 proteins involved in major phosphorous deficiency adaptation pathways including reactive oxygen scavenging, citric acid cycle, signal transduction, and plant defense responses as well as proteins with unknown function. Our results highlighted a coordinated response in NIL in response to phosphorous deficiency which may confer higher adaptation to nutrient deficiency.     

Quantitative Proteomics of Presynaptic Mitochondria Reveal an Overexpression and Biological Relevance of Neuronal MitoNEET in Postnatal Brain Development

Although it has been recognized that energy metabolism and mitochondrial structure and functional activity in the immature brain differs from that of the adult, few studies have examined mitochondria specifically at the neuronal synapse during postnatal brain development. In this study, we examined the presynaptic mitochondrial proteome in mice at postnatal day 7 and 42, a period that involves the formation and maturation of synapses. Application of two independent quantitative proteomics approaches – SWATH‐MS and super‐SILAC – revealed a total of 40 proteins as significantly differentially expressed in the presynaptic mitochondria. In addition to elevated levels of proteins known to be involved in ATP metabolic processes, our results identified increased levels of mitoNEET (Cisd1), an iron‐sulfur containing protein that regulates mitochondrial bioenergetics. We found that mitoNEET overexpression plays a cell‐type specific role in ATP synthesis and in neuronal cells promotes ATP generation. The elevated ATP levels in SH‐SY5Y neuroblastoma cells were associated with increased mitochondrial membrane potential and a fragmented mitochondrial network, further supporting a role for mitoNEET as a key regulator of mitochondrial function.     

Proteomic profiling of the mitochondrial inner membrane of rat renal proximal convoluted tubules

The proximal convoluted tubule is the primary site of renal fluid, electrolyte, and nutrient reabsorption, processes that consume large amounts of adenosine‐5′‐triphosphate. Previous proteomic studies have profiled the adaptions that occur in this segment of the nephron in response to the onset of metabolic acidosis. To extend this analysis, a proteomic workflow was developed to characterize the proteome of the mitochondrial inner membrane of the rat renal proximal convoluted tubule. Separation by LC coupled with analysis by MS/MS (LC‐MS/MS) confidently identified 206 proteins in the combined samples. Further proteomic analysis identified 14 peptides that contain an N‐?‐acetyl‐lysine, seven of which are novel sites. This study provides the first proteomic profile of the mitochondrial inner membrane proteome of this segment of the rat renal nephron. The MS data have been deposited in the ProteomeXchange with the identifier PXD000121.     

iTRAQ‐Based Quantitative Proteomic Analysis of Duck Eggshell During Biomineralization

The avian egg is a valuable model for the calcitic biomineralization process as it is the fastest calcification process occurring in nature and is a clear example of biomineralization. In this study, iTRAQ MS/MS is used to detect and study for the first time: 1) the overall duck eggshell proteome; 2) regional differences in the proteome between the inner and outer portions of the duck eggshell. The new reference protein datasets allow us to identify 179 more eggshell proteins than solely using the current release of Ensembl duck annotations. In total, 484 proteins are identified in the entire duck eggshell proteome. Twenty‐eight novel proteins of unknown function that are involved in eggshell formation are also identified. Among the identified eggshell proteins, 54 proteins show differential abundances between the inner, partially mineralized eggshell (obtained 16 h after ovulation) compared to the overall complete eggshell (normally expulsed eggshell). At least 64 of the abundant matrix proteins are common to eggshell of 4 different domesticated bird species (chicken, duck, quail, turkey) and zebra finch. This study provides a new resource for avian eggshell proteomics, and augments the inventory of eggshell matrix proteins that will lead to a deeper understanding of calcitic biomineralization.     

Analysis of Proteome Profile in Germinating Soybean Seed,and Its Comparison with Rice Showing the Styles of Reserves Mobilization in Different Crops

Background

Seed germination is a complex physiological process during which mobilization of nutrient reserves happens. In different crops, this event might be mediated by different regulatory and metabolic pathways. Proteome profiling has been proved to be an efficient way that can help us to construct these pathways. However, no such studies have been performed in soybean germinating seeds up to date.

Results

Proteome profiling was conducted through one-dimensional gel electrophoresis followed by liquid chromatography and tandem mass spectrometry strategy in the germinating seeds of soybean (glycine max). Comprehensive comparisons were also carried out between rice and soybean germinating seeds. 764 proteins belonging to 14 functional groups were identified and metabolism related proteins were the largest group. Deep analyses of the proteins and pathways showed that lipids were degraded through lipoxygenase dependent pathway and proteins were degraded through both protease and 26S proteosome system, and the lipoxygenase could also help to remove the reactive oxygen species during the rapid mobilization of reserves of soybean germinating seeds. The differences between rice and soybean germinating seeds proteome profiles indicate that each crop species has distinct mechanism for reserves mobilization during germination. Different reserves could be converted into starches before they are totally utilized during the germination in different crops seeds.

Conclusions

This study is the first comprehensive analysis of proteome profile in germinating soybean seeds to date. The data presented in this paper will improve our understanding of the physiological and biochemical status in the imbibed soybean seeds just prior to germination. Comparison of the protein profile with that of germinating rice seeds gives us new insights on mobilization of nutrient reserves during the germination of crops seeds.     

Quantitative analysis of cellular proteome alterations in human influenza A virus‐infected mammalian cell lines

Over the last years virus–host cell interactions were investigated in numerous studies. Viral strategies for evasion of innate immune response, inhibition of cellular protein synthesis and permission of viral RNA and protein production were disclosed. With quantitative proteome technology, comprehensive studies concerning the impact of viruses on the cellular machinery of their host cells at protein level are possible. Therefore, 2‐D DIGE and nanoHPLC‐nanoESI‐MS/MS analysis were used to qualitatively and quantitatively determine the dynamic cellular proteome responses of two mammalian cell lines to human influenza A virus infection. A cell line used for vaccine production (MDCK) was compared with a human lung carcinoma cell line (A549) as a reference model. Analyzing 2‐D gels of the proteomes of uninfected and influenza‐infected host cells, 16 quantitatively altered protein spots (at least ±1.7‐fold change in relative abundance, p<0.001) were identified for both cell lines. Most significant changes were found for keratins, major components of the cytoskeleton system, and for Mx proteins, interferon‐induced key components of the host cell defense. Time series analysis of infection processes allowed the identification of further proteins that are described to be involved in protein synthesis, signal transduction and apoptosis events. Most likely, these proteins are required for supporting functions during influenza viral life cycle or host cell stress response. Quantitative proteome‐wide profiling of virus infection can provide insights into complexity and dynamics of virus–host cell interactions and may accelerate antiviral research and support optimization of vaccine manufacturing processes.     

Analysis of the Outer Membrane Proteome and Secretome of Bacteroides fragilis Reveals a Multiplicity of Secretion Mechanisms

Bacteroides fragilis is a widely distributed member of the human gut microbiome and an opportunistic pathogen. Cell surface molecules produced by this organism likely play important roles in colonization, communication with other microbes, and pathogenicity, but the protein composition of the outer membrane (OM) and the mechanisms used to transport polypeptides into the extracellular space are poorly characterized. Here we used LC-MS/MS to analyze the OM proteome and secretome of B. fragilis NCTC 9343 grown under laboratory conditions. Of the 229 OM proteins that we identified, 108 are predicted to be lipoproteins, and 61 are predicted to be TonB-dependent transporters. Based on their proximity to genes encoding TonB-dependent transporters, many of the lipoprotein genes likely encode proteins involved in nutrient or small molecule uptake. Interestingly, protease accessibility and biotinylation experiments indicated that an unusually large fraction of the lipoproteins are cell-surface exposed. We also identified three proteins that are members of a novel family of autotransporters, multiple potential type I protein secretion systems, and proteins that appear to be components of a type VI secretion apparatus. The secretome consisted of lipoproteins and other proteins that might be substrates of the putative type I or type VI secretion systems. Our proteomic studies show that B. fragilis differs considerably from well-studied Gram-negative bacteria such as Escherichia coli in both the spectrum of OM proteins that it produces and the range of secretion strategies that it utilizes.     

Cell wall proteomic of Brachypodium distachyon grains: A focus on cell wall remodeling proteins

Cell walls play key roles during plant development. Following their deposition into the cell wall, polysaccharides are continually remodeled according to the growth stage and stress environment to accommodate cell growth and differentiation. To date, little is known concerning the enzymes involved in cell wall remodeling, especially in gramineous and particularly in the grain during development. Here, we investigated the cell wall proteome of the grain of Brachypodium distachyon. This plant is a suitable model for temperate cereal crops. Among the 601 proteins identified, 299 were predicted to be secreted. These proteins were distributed into eight functional classes; the class of proteins that act on carbohydrates was the most highly represented. Among these proteins, numerous glycoside hydrolases were found. Expansins and peroxidases, which are assumed to be involved in cell wall polysaccharide remodeling, were also identified. Approximately half of the proteins identified in this study were newly discovered in grain and were not identified in the previous proteome analysis conducted using the culms and leaves of B. distachyon. Therefore, the data obtained from all organs of B. distachyon infer a global cell wall proteome consisting of 460 proteins. At present, this is the most extensive cell wall proteome of a monocot species.     

Proteomics Analysis of Soybean Seedlings under Short-Term Water Deficit

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

Comprehensive proteomic datasets for studying adipocyte–macrophage cell–cell communication

Cellular communication is a fundamental process in biology. The interaction of adipocytes with macrophages is a key event in the development of common diseases such as type 2 diabetes. We applied an established bilayer cell coculture system and comprehensive MS detection to analyse on a proteome‐wide scale the paracrine interaction of murine adipocytes and macrophages. Altogether, we identified 4486 proteins with at least two unique peptides, of which 2392 proteins were informative for 3T3‐L1 adipocytes and 2957 proteins for RAW 264.7 macrophages. Further, we observed over 12000 phosphorylation sites, of which we could assign 3200 informative phosphopeptides with a single phosphosite for adipocytes and 4514 for macrophages. Using protein set enrichment and phosphosite analyses, we deciphered regulatory protein pathways involved in cellular stress and inflammation, which can contribute to metabolic impairment of cells including insulin resistance and other disorders. The generated datasets provide a holistic, molecular pathway‐centric view on the interplay of adipocytes and macrophages in disease processes and a resource for further studies.     

Two‐dimensional proteome reference map for the radiation‐resistant bacterium Deinococcus geothermalis

2‐DE reference maps for Deinococcus geothermalis cytosolic and cell envelope proteomes were constructed. In total, 403 spots were identified as 299 different proteins. Unique in the proteomes were four subunits of V‐type ATPase and Deinococcus specific proteins constituting one‐fourth of cell envelope proteome. The cytoplasmic proteome included enzymes of the central carbon metabolism, chaperones, enzymes of protein and DNA repair, and oxidative stress. A total of 34 abundant proteins with unknown function may relate to the extreme stress tolerance of D. geothermalis.     

Proteome analysis of soybean roots subjected to short-term drought stress

Drought is one of the most important constraints on the growth and productivity of many crops, including soybeans. However, as a primary sensing organ, the plant root response to drought has not been well documented at the proteomic level. In the present study, we carried out a proteome analysis in combination with physiological analyses of soybean roots subjected to severe but recoverable drought stress at the seedling stage. Drought stress resulted in the increased accumulation of reactive oxygen species and subsequent lipid peroxidation. The proline content increased in drought-stressed plants and then decreased during the period of recovery. The high-resolution proteome map demonstrated significant variations in about 45 protein spots detected on Comassie briliant blue-stained 2-DE gels. Of these, 28 proteins were identified by mass spectrometry; the levels of 5 protein spots were increased, 21 were decreased and 2 spots were newly detected under drought condition. When the stress was terminated by watering the plants for 4 days, in most cases, the protein levels tended towards the control level. The proteins identified in this study are involved in a variety of cellular functions, including carbohydrate and nitrogen metabolism, cell wall modification, signal transduction, cell defense and programmed cell death, and they contribute to the molecular mechanism of drought tolerance in soybean plants. Analysis of protein expression patterns revealed that proteins associated with osmotic adjustment, defense signaling and programmed cell death play important roles for soybean plant drought adaptation. The identification of these proteins provides new insight that may lead to a better understanding of the molecular basis of the drought stress responses.     

Integrative analysis of the mitochondrial proteome in yeast

In this study yeast mitochondria were used as a model system to apply, evaluate, and integrate different genomic approaches to define the proteins of an organelle. Liquid chromatography mass spectrometry applied to purified mitochondria identified 546 proteins. By expression analysis and comparison to other proteome studies, we demonstrate that the proteomic approach identifies primarily highly abundant proteins. By expanding our evaluation to other types of genomic approaches, including systematic deletion phenotype screening, expression profiling, subcellular localization studies, protein interaction analyses, and computational predictions, we show that an integration of approaches moves beyond the limitations of any single approach. We report the success of each approach by benchmarking it against a reference set of known mitochondrial proteins, and predict approximately 700 proteins associated with the mitochondrial organelle from the integration of 22 datasets. We show that a combination of complementary approaches like deletion phenotype screening and mass spectrometry can identify over 75% of the known mitochondrial proteome. These findings have implications for choosing optimal genome-wide approaches for the study of other cellular systems, including organelles and pathways in various species. Furthermore, our systematic identification of genes involved in mitochondrial function and biogenesis in yeast expands the candidate genes available for mapping Mendelian and complex mitochondrial disorders in humans.