In‐depth characterization of the tomato fruit pericarp proteome

Since the genome of Solanum lycopersicum L. was published in 2012, some studies have explored its proteome although with a limited depth. In this work, we present an extended characterization of the proteome of the tomato pericarp at its ripe red stage. Fractionation of tryptic peptides generated from pericarp proteins by off‐line high‐pH reverse‐phase phase chromatography in combination with LC‐MS/MS analysis on a Fisher Scientific Q Exactive and a Sciex Triple‐TOF 6600 resulted in the identification of 8588 proteins with a 1% FDR both at the peptide and protein levels. Proteins were mapped through GO and KEGG databases and a large number of the identified proteins were associated with cytoplasmic organelles and metabolic pathways categories. These results constitute one of the most extensive proteome datasets of tomato so far and provide an experimental confirmation of the existence of a high number of theoretically predicted proteins. All MS data are available in the ProteomeXchange repository with the dataset identifiers PXD004947 and PXD004932.     

Two‐dimensional proteome reference maps for the human pathogenic filamentous fungus Aspergillus fumigatus

The filamentous fungus Aspergillus fumigatus has become the most important airborne fungal pathogen causing life‐threatening infections in immunosuppressed patients. We established a 2‐D reference map for A. fumigatus. Using MALDI‐TOF‐MS/MS, we identified 381 spots representing 334 proteins. Proteins involved in cellular metabolism, protein synthesis, transport processes and cell cycle were most abundant. Furthermore, we established a protocol for the isolation of mitochondria of A. fumigatus and developed a mitochondrial proteome reference map. 147 proteins represented by 234 spots were identified.     

Identification and Analysis of Resistance‐like Genes in the Tomato Genome

Tomato (Solanum lycopersicum L.) is one of the most important vegetable crops in the world. However, the tomato production is severely affected by many diseases. The use of host resistance is believed to be the most effective approach to control the pathogens. In this study, a total of 1003 resistance‐like genes were identified from the tomato genome using individual full‐length search and conserved domain verification approach. Of the predicted resistance genes, serine/threonine protein kinase was the largest class with 384 genes followed by 212 genes encoding receptor‐like kinase, 107 genes encoding receptor‐like proteins, 68 genes encoding coiled‐coil–nucleotide‐binding site (NBS)–leucine‐rich repeat (LRR) and 19 genes encoding Toll interleukin‐1 receptor domain‐NBS‐LRR. Physical map positions established for all predicted genes using the tomato WGS chromosomes SL2.40 information indicated that most resistance‐like genes clustered on certain chromosomal regions. Comparisons of the sequences from the same resistance‐like genes in S. pimpinellifolium and S. lycopersicum showed that 93.5% genes contained single nucleotide polymorphisms and 19.7% genes contained insertion/deletion. The data obtained here will facilitate isolation and characterization of new resistance genes as well as marker‐assisted selection for disease resistance breeding in tomato.     

Major proteome variations associated with cherry tomato pericarp development and ripening

Tomato (Solanum lycopersicum) is a model plant for studying fleshy fruit development. Several genetic and molecular approaches have been developed to increase our knowledge about the physiological basis of fruit growth, but very few data are yet available at the proteomic level. The main stages of fruit development were first determined through the dynamics of fruit diameter and pericarp cell number. Then, total proteins were extracted from pericarp tissue at six relevant developmental stages and separated by two-dimensional gel electrophoresis. Protein patterns were markedly different between stages. Proteins showing major variations were monitored. We identified 90 of 1,791 well-resolved spots either by matrix-assisted laser-desorption ionization time-of-flight peptide mass fingerprinting or liquid chromatography-mass spectrometry sequencing and expressed sequence tag database searching. Clustered correlation analysis results pointed out groups of proteins with similar expression profiles during fruit development. In young fruit, spots linked to amino acid metabolism or protein synthesis were mainly expressed during the cell division stage and down-regulated later. Some spots linked to cell division processes could be identified. During the cell expansion phase, spots linked to photosynthesis and proteins linked to cell wall formation transiently increased. In contrast, the major part of the spots related to C compounds and carbohydrate metabolism or oxidative processes were up-regulated during fruit development, showing an increase in spot intensity during development and maximal abundance in mature fruit. This was also the case for spots linked to stress responses and fruit senescence. We discuss protein variations, taking into account their potential role during fruit growth and comparing our results with already known variations at mRNA and metabolite-profiling levels.     

A comprehensive proteome map of bovine cerebrospinal fluid

Cerebrospinal fluid (CSF) is considered as the most promising body fluid target for the discovery of biomarkers for early diagnosis of neurodegenerative diseases such as Creutzfeldt–Jakob disease in humans and bovine spongiform encephalopathy in cattle. For the recognition of disease‐associated changes in bovine CSF protein patterns, a detailed knowledge of this proteome is a prerequisite. The absence of a high‐resolution CSF proteome map prompted us to determine all bovine CSF protein spots that can be visualised on 2‐D protein gels. Using state‐of‐the‐art 2‐DE technology for proteome mapping of bovine ante mortem CSF combined with sensitive fluorescent protein staining and MALDI‐TOF/TOF MS for protein identification, a highly detailed 2‐DE map of the bovine CSF proteome was established. Besides the proteins mapped by earlier studies, this map contains 66 different proteins, including 58 which were not annotated in bovine 2‐DE CSF maps before.     

Protein pattern changes in tomato under in vitro salt stress

The investigation of salt-induced changes in the proteome would highlight important genes because of a high resolution of protein separation by two-dimensional gel electrophoresis (2-DE) and protein identification by mass spectrometry and database search. Tomato (Lycopersicon esculentum Mill.) is a model plant for studying the mechanisms of plant salt tolerance. Seeds of tomato cv. Shirazy were germinated on water-agar medium. After germination, seedlings were transferred to Murashige and Skoog nutrient medium supplemented with 0, 40, 80, 120, and 160 mM NaCl. After 24 days, leaf and root samples were collected for protein extraction and shoot dry weight measurement. Alterations induced in leaf and root proteins under salt stress treatments were studied by one-dimensional SDS-PAGE. Leaf proteins were also analyzed by 2-DE. With increasing salt concentration in the medium, shoot dry weight decreased. SDS-PAGE showed induction of at least five proteins with mol wts of 30, 62, and 75 kD in roots and 38 and 46 kD in leaves. On the 2-DE gel, more than 400 protein spots were reproducibly detected. At least 18 spots showed significant changes under salt stress. Three of them corresponded to new proteins, while six proteins were up-regulated and five proteins were down-regulated by salt stress. In addition, salinity inhibited the synthesis of four leaf proteins. Ten spots were analyzed by matrix-assistant laser desorption/ionization-time of flight (MALDI-TOF), which led to the identification of some proteins, which could play a physiological role under salt stress. The expression of new proteins(enoyl-CoA hydratase, EGF receptor-like protein, salt tolerance protein, phosphoglycerate mutase-like protein, and M2D3.3 protein) under salt stress indicates that tomato leaf cells respond to salt stress by changes in different physiological processes. All identified proteins are somehow related to various salt stress responses, such as cell proliferation. Published in Russian in Fiziologiya Rastenii, 2007, Vol. 54, No. 4, pp. 526–533. The text was submitted by the authors in English.     

A comprehensive analysis of Trypanosoma brucei mitochondrial proteome

The composition of the large, single, mitochondrion (mt) of Trypanosoma brucei was characterized by MS (2‐D LC‐MS/MS and gel‐LC‐MS/MS) analyses. A total of 2897 proteins representing a substantial proportion of procyclic form cellular proteome were identified, which confirmed the validity of the vast majority of gene predictions. The data also showed that the genes annotated as hypothetical (species specific) were overpredicted and that virtually all genes annotated as hypothetical, unlikely are not expressed. By comparing the MS data with genome sequence, 40 genes were identified that were not previously predicted. The data are placed in a publicly available web‐based database (www.TrypsProteome.org). The total mitochondrial proteome is estimated at 1008 proteins, with 401, 196, and 283 assigned to the mt with high, moderate, and lower confidence, respectively. The remaining mitochondrial proteins were estimated by statistical methods although individual assignments could not be made. The identified proteins have predicted roles in macromolecular, metabolic, energy generating, and transport processes providing a comprehensive profile of the protein content and function of the T. brucei mt.     

Strategic proteome analysis of Candida magnoliae with an unsequenced genome

Erythritol is a noncariogenic, low calorie sweetener. It is safe for people with diabetes and obese people. Candida magnoliae is an industrially important organism because of its ability to produce erythritol as a major product. The genome of C. magnoliae has not been sequenced yet, limiting the available proteome database. Therefore, systematic approaches were employed to construct the proteome map of C. magnoliae. Proteomic analysis with systematic approaches is based on two-dimensional electrophoresis, matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS), tandem mass spectrometry (MS/MS) and database interrogation. First, 24 spots were analyzed using peptide mass fingerprinting along with MALDI-TOF MS with high mass accuracy. Only four spots were reliably identified as carbonyl reductase and its isoforms. The reason for low sequence coverage seemed to be that these identification strategies were based on the presence of the protein database obtained from the publicly accessible genome database and the availability of cross-species protein identification. MS/MS (MS/MS ion search and de novo sequencing) in combination with similarity searches allowed successful identification of 39 spots. Several proteins including transaldolase identified by MS/MS ion searches were further confirmed by partial sequences from the expressed sequence tag database. In this study, 51 protein spots were analyzed and then potentially identified. The identified proteins were involved in glycolysis, stress response, other essential metabolisms and cell structures.     

Analysis of the cytosolic proteome of Halobacterium salinarum and its implication for genome annotation

The halophilic archaeon Halobacterium salinarum (strain R1, DSM 671) contains 2784 protein-coding genes as derived from the genome sequence. The cytosolic proteome containing 2042 proteins was separated by two-dimensional gel electrophoresis (2-DE) and systematically analyzed by a semi-automatic procedure. A reference map was established taking into account the narrow isoelectric point (pI) distribution of halophilic proteins between 3.5 and 5.5. Proteins were separated on overlapping gels covering the essential areas of pI and molecular weight. Every silver-stained spot was analyzed resulting in 661 identified proteins out of about 1800 different protein spots using matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) peptide mass fingerprinting (PMF). There were 94 proteins that were found in multiple spots, indicating post-translational modification. An additional 141 soluble proteins were identified on 2-D gels not corresponding to the reference map. Thus about 40% of the cytosolic proteome was identified. In addition to the 2784 protein-coding genes, the H. salinarum genome contains more than 6000 spurious open reading frames longer than 100 codons. Proteomic information permitted an improvement in genome annotation by validating and correcting gene assignments. The correlation between theoretical pI and gel position is exceedingly good and was used as a tool to improve start codon assignments. The fraction of identified chromosomal proteins was much higher than that of those encoded on the plasmids. In combination with analysis of the GC content this observation permitted an unambiguous identification of an episomal insert of 60 kbp ("AT-rich island") in the chromosome, as well as a 70 kbp region from the chromosome that has integrated into one of the megaplasmids and carries a series of essential genes. About 63% of the chromosomally encoded proteins larger than 25 kDa were identified, proving the efficacy of 2-DE MALDI-TOF MS PMF technology. The analysis of the integral membrane proteome by tandem mass spectrometric techniques added another 141 identified proteins not identified by the 2-DE approach (see following paper).     

Analysis of the budding yeast pH 4–7 proteome in meiosis

Meiosis, the developmental programme generating haploid gametes from diploid precursors, requires two cell divisions and many innovations. In budding yeast, a large number of genes are expressed exclusively during meiosis while others are repressed compared to vegetative growth. Microarray analysis has shown that gene expression during meiosis is highly regulated, and has been used to classify yeast genes according to meiotic temporal expression pattern. In this study, we have begun to investigate the kinetics of meiotic protein expression using a proteomics approach. 2‐D DIGE was used to characterise the temporal protein expression patterns of the budding yeast pH 4–7 proteome in meiosis. More than 1400 meiotic protein spots were visualised and at least 63 spots were temporally regulated during meiosis in a statistically significant manner. Gel spots with significant expression changes were excised and 26 unique proteins were identified using LC‐MS/MS. The identified proteins could be classified into functional categories and the genes encoding a number of these were previously shown to be involved in yeast sporulation and meiosis. This data set was used to assemble the first differential 2‐D PAGE map of budding yeast meiosis, which can be accessed through a web server. This work represents one of the first quantitative proteomic analyses of meiosis in yeast and will provide a valuable resource for future investigations.     

The proteome of Mannheimia succiniciproducens, a capnophilic rumen bacterium

Mannheimia succiniciproducens MBEL55E isolated from bovine rumen is an industrially important bacterium as an efficient succinic acid producer. Recently, its full genome sequence was determined. In the present study, we analyzed the M. succiniciproducens proteome based on the genome information using 2-DE and MS. We established proteome reference map of M. succiniciproducens by analyzing whole cellular proteins, membrane proteins, and secreted proteins. More than 200 proteins were identified and characterized by MS/MS supported by various bioinformatic tools. The presence of proteins previously annotated as hypothetical proteins or proteins having putative functions were also confirmed. Based on the proteome reference map, cells in the different growth phases were analyzed at the proteome level. Comparative proteome profiling revealed valuable information to understand physiological changes during growth, and subsequently suggested target genes to be manipulated for the strain improvement.     

Proteomic analysis of Arabidopsis thaliana (L.) Heynh responses to a generalist sucking pest (Myzus persicae Sulzer)

Herbivorous insects can cause severe cellular changes to plant foliage following infestations, depending on feeding behaviour. Here, a proteomic study was conducted to investigate the influence of green peach aphid (Myzus persicae Sulzer) as a polyphagous pest on the defence response of Arabidopsis thaliana (L.) Heynh after aphid colony establishment on the host plant (3 days). Analysis of about 574 protein spots on 2‐DE gels revealed 31 differentially expressed protein spots. Twenty out of these 31 differential proteins were selected for analysis by mass spectrometry. In 12 of the 20 analysed spots, we identified seven and nine proteins using MALDI‐TOF‐MS and LC‐ESI‐MS/MS, respectively. Of the analysed spots, 25% contain two proteins. Different metabolic pathways were modulated in Arabidopsis leaves according to aphid feeding: most corresponded to carbohydrate, amino acid and energy metabolism, photosynthesis, defence response and translation. This paper has established a survey of early alterations induced in the proteome of Arabidopsis by M. persicae aphids. It provides valuable insights into the complex responses of plants to biological stress, particularly for herbivorous insects with sucking feeding behaviour.     

The citrus fruit proteome: insights into citrus fruit metabolism

Fruit development and ripening are key processes in the production of the phytonutrients that are essential for a balanced diet and for disease prevention. The pathways involved in these processes are unique to plants and vary between species. Climacteric fruit ripening, especially in tomato, has been extensively studied; yet, ripening of non-climacteric fruit is poorly understood. Although the different species share common pathways; developmental programs, physiological, anatomical, biochemical composition and structural differences must contribute to the operation of unique pathways, genes and proteins. Citrus has a non-climacteric fruit ripening behavior and has a unique anatomical fruit structure. For the last few years a citrus genome-wide ESTs project has been initiated and consists of 222,911 clones corresponding to 19,854 contigs and 37,138 singletons. Taking advantage of the citrus database we analyzed the citrus proteome. Using LC-MS/MS we analyzed soluble and enriched membrane fractions of mature citrus fruit to identify the proteome of fruit juice cells. We have identified ca. 1,400 proteins from these fractions by searching NCBI-nr (green plants) and citrus ESTs databases, classified these proteins according to their putative function and assigned function according to known biosynthetic pathways.     

Protein reference mapping of dihydrofolate reductase‐deficient CHO DG44 cell lines using 2‐dimensional electrophoresis

Chinese hamster ovary (CHO) cells are the major mammalian host for producing various therapeutic proteins. Among CHO cells, the dihydrofolate reductase‐deficient CHO DG44 cell line has been used as a popular mammalian host because of the availability of a well‐characterized genetic selection and amplification system. However, this cell line has not been studied at the proteome level. Here, the first detailed proteome analysis of the CHO DG44 cell line is described. A protein reference map of the CHO DG44 cell line was established by analyzing whole cellular proteins using 2‐DE with various immobilized pH gradients (pHs 3–10, 5–8, and 3–6) in the first dimension and a 12% acrylamide gel in the second dimension. The map is composed of over 1400 silver‐stained protein spots. Among them, 179 protein spots, which represent proteins associated with various biological processes and cellular compartments, were identified based on MALDI‐TOF‐MS and MS/MS. This proteome database should be valuable for better understanding of CHO cell physiology and protein expression patterns which may lead to efficient therapeutic protein production.     

Drosophila melanogaster larval hemolymph protein mapping

With the completion of the genome sequence of Drosophila melanogaster the importance of constructing a proteome map is to be considered. Therefore, with the application of recent advances in proteomic analysis approaches, a protein map of D. melanogaster larvae hemolymph proteins was obtained using 2-DE in the range of pH 3-10. After Coomassie colloidal detection of 289 spots, a total of 105 were excised from the gel and digested with trypsin. Identification was done based on a combination of MALDI-TOF/TOF MS and MS/MS spectra. The 99 proteins identified using this approach include a large number of metabolic enzymes, translational apparatus components, and structural proteins. Among these we emphasize the identification of proteins with molecular chaperone properties (heat shock proteins and PPIases) and protein spots involved in defense responses such as antioxidant and immunological defense mechanisms (thioredoxin, prophenoloxidase, and serine proteases), as well as in signal transduction pathways.