Proteome analysis of embryo and endosperm from germinating tomato seeds

Proteome analysis of embryo and endosperm tissues from germinating tomato seed was conducted using 1-DE, 2-DE, and MS. Mobilization of the most abundant proteins, which showed similar profiles in the two tissues, occurred first in the endosperm. CBB R-250 staining of 2-DE gels revealed 352 and 369 major protein spots in the embryo and endosperm, respectively, at 0 h. Of these, 75 major spots were selected, excised, in-gel digested with trypsin, and analyzed by MALDI-TOF-MS and/or LC-ESI-Q/TOF-MS/MS. Peptide MS and MS/MS data were searched against publicly available protein and EST databases, and 47 proteins identified. Embryo-specific proteins included a BAC19.13 homologue, whereas four proteins specific to the endosperm were tomato mosaic virus coat proteins related to defense mechanisms. The most abundant proteins both in the embryo and endosperm were seed storage proteins, i.e., legumins (11 spots), vicilins (11 spots), albumin (2 spots). Housekeeping enzymes, actin-binding profilin, defense-related protein kinases, nonspecific lipid transfer protein, and proteins involved in general metabolism were also identified. The roles of some of the proteins identified in the embryo and endosperm are discussed in relation to seed germination in tomato.     

Structural proteome of human colostral fat globule membrane proteins

Milk fat globule membrane (MFGM) contains proteins derived from the apical membrane of secreting epithelial cells of the mammary gland. Between 2-4% of total human milk protein content is associated with the fat globule fraction, as MFGM proteins. While MFGM proteins have very low classical nutritional value, they play important roles in various cell processes and defence mechanisms for the newborn. To date, fewer than 30 human MFGM proteins have been identified and characterized, either by immunological methods or by Edman sequencing and mass spectrometry. This study aimed to update the structural proteome of human colostral MFGM proteins and to create an annotated two-dimensional electrophoresis (2-DE) MFGM protein database available on-line. More than one hundred 2-DE spots derived from human colostral MFGM proteins were investigated by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and proteins were identified by three different software packages available on the web (PeptIdent, MS-Fit and ProFound); uncertain identifications were solved by nanoelectrospray ionization-ion trap mass spectrometry using SEQUEST software.     

Characterization of pollen tube development in Pinus strobus (Eastern white pine) through proteomic analysis of differentially expressed proteins

The differentially expressed proteins in pollen tubes indicate their specific roles in this stage of male gametophyte development. To isolate these proteins, 2-DE was done using ungerminated pollen and 2-day-old pollen tubes of Pinus strobus. Results show that 645 and 647 protein spots were clearly resolved from pollen grains and pollen tubes, respectively. Thirty-eight protein spots were expressed only in pollen tubes, while 19 increased in intensity. MALDI-TOF MS was used to generate tryptic peptide masses that were submitted to Mascot for identification. Of the differentially expressed proteins, 12% matched with hypothetical proteins, 33% did not hit any protein, and for the 55%, a putative function was assigned based on similarity of sequences with previously characterized proteins. Therefore, pollen tube development can be characterized by the cellular activities that involve metabolism, stress/defense response, gene regulation, signal transduction, and cell wall formation. This study expands our understanding of the changes in protein expression associated with pollen tube development and provides insights into the molecular programs that separate the development of the pollen tubes from pollen grains. This is the first report that describes a global analysis of differentially expressed proteins from the pollen tube of any seed plant.     

Comparative analysis of the proteome of left ventricular heart of arteriosclerosis in rat

Despite the worldwide occurrence of coronary atherosclerotic heart disease (CAHD), the pathogenic mechanisms underlying this disease remain largely unknown. In this study, the experimental model of atherosclerosis in rat (CAHD rat) was established by the injection of vitamin D3 associated with high fat diet for 6 weeks. By using the proteomic approach, we comparatively analyzed the proteome of the control and CAHD rat left ventricular myocardial tissues. We reproducibly separated over 2500 polypeptides by using two-dimensional electrophoresis (2-DE) at pH range of 3-11. Among these proteins, 26 proteins with large amount were identified using micro high performance liquid chromatography mass spectrometer/mass spectrometer (micro-HPLC-MS/MS). Using PDQUEST software to process the 2-DE gel images, 38 protein spots that significantly altered in CAHD were detected. Of these, 12 proteins were identified with high confidence by using 2-DE and matrix-associated laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF-MS). The identification of protein alterations specify to CAHD would clarify the pathogenetic mechanisms involved in the disease and might be of prognostic and therapeutic benefit.     

Black Point is associated with reduced levels of stress, disease- and defence-related proteins in wheat grain

Black Point in wheat is a dark discoloration at the embryo end of the grain, which causes substantial financial losses to wheat growers due to down-grading of otherwise high-grade wheat. There does not appear to be a single cause for Black Point, although evidence suggests that fungal infection is the main link to Black Point symptoms. We sought to identify grain proteins from Black Point-affected and Black Point-free wheat cultivar SUN239V, which is known to be very susceptible to Black Point. The proteomes of both the germ and endosperm-bran components of Black Point-affected and Black Point-free grain were compared using two-dimensional gel electrophoresis (2-DE) with six replicate gels run for each protein sample. Approximately 1478 discrete protein spots were found in 2-DE gels from the germ fraction of the grain, of which 354 were identified by mass spectrometry (MS). Similarly, 1360 discrete protein spots were found from the endosperm-bran fraction, of which 303 were identified by MS. No proteins of fungal or bacterial origin were positively identified, suggesting that, at least in some cases, Black Point is not associated with microbial activity. Of the germ proteins, 252 were differentially expressed in Black Point-affected tissue, with 67 of these proteins identified by MS. Of the endosperm-bran proteins, 317 were differentially expressed in Black Point-affected tissue, with 86 identified. The largest of 12 functional classes to which the differentially abundant proteins were assigned was the 'stress' class, i.e. products of genes associated with stress, disease and defence. Higher levels of these proteins were found in Black Point-free grain, suggesting that protection from the disease might be afforded by increased levels of the 'stress' proteins.     

Comparative proteomic analysis of Arabidopsis thaliana roots between wild type and its salt-tolerant mutant

Two-dimensional electrophoresis (2-DE) showed the variation expression of Arabidopsis thaliana root proteins between wild type and its salt-tolerant mutant obtained from cobalt-60 γ ray radiation. Forty-six differential root protein spots were reproducibly presented on 2-DE maps, and 29 spots were identified by matrix assisted laser desorption ionization-time of flight/time of flight mass spectrometry (MS). Fifteen protein spots corresponding to 10 proteins, and 14 protein spots corresponding to 9 proteins were constitutively up-regulated and down-regulated in the salt-tolerant mutant root. Bioinformatic analysis indicated that those differential proteins might be involved in the regulation of redox homeostasis, nucleotide metabolism, signal transduction, stress response and defense, carbohydrate metabolism, and cell wall metabolism. Peroxidase 22 might be a versatile enzyme and might play dual roles in both cell wall metabolism and regulation of redox homeostasis. Our work provides not only new insights into salt-responsive proteins in root, but also the potential salt-tolerant targets for further dissection of molecular mechanism adapted by plants during salt stress.     

Plant signalling peptides

Biochemical and genetic studies have identified peptides that play crucial roles in plant growth and development, including defence mechanisms in response to wounding by pests, the control of cell division and expansion, and pollen self-incompatibility. The first two signalling peptides to be described in plants were tomato systemin and phytosulfokine (PSK). There is also biochemical evidence that natriuretic peptide-like molecules, immunologically-related to those found in animals, may exist in plants. Another example of signalling peptide is ENOD40, a product of a gene, which became active early in the root nodulation process following Rhizobium infection of legumes. Other predicted bioactive peptides or oligopeptides have been identified by means of genetic, rather then biochemical methods. The Arabidopsis CLAVATA3 protein is required for the correct organization of the shoot apical meristem and the pollen S determinant S-locus cysteine-rich protein (SCR) also called S-locus protein 11, SP11). The plant signalling peptides discovered so far are involved in various processes and play an important role in communication between cells or organs, respectively. This review will focus on these peptides and their role in intercellular signalling.     

Two-dimensional gel electrophoresis pattern (pH 6-11) and identification of water-soluble barley seed and malt proteins by mass spectrometry

A protocol was established for two-dimensional gel electrophoresis (2-DE) of barley seed and malt proteins in the pH range of 6-11. Proteins extracted from flour in a low-salt buffer were focused after cup-loading onto IPG strips. Successful separation in the second dimension was achieved using gradient gels in a horizontal SDS-PAGE system. Silver staining of gels visualized around 380 (seed) and 500 (malt) spots. Thirty-seven different proteins from seeds were identified in 60 spots, among these 46 were visualized also in the malt 2-D pattern. Proteins were identified by peptide mass fingerprinting and by tandem MS sequencing after in-gel digestion by trypsin. In addition, the N-terminal sequence of 10 different proteins from 11 spots was determined after electroblotting to a polyvinylidene difluoride (PVDF) membrane. Five identified proteins (in 9 spots) are involved in glycolysis, 12 in defence against pathogens (21 spots), 4 in storage, folding, and synthesis of proteins, and in nitrogen metabolism (5 spots), 6 in carbohydrate metabolism (11 spots), and 4 in stress and detoxification (9 spots). Six proteins (7 spots) were not grouped in these categories, and 3 were not ascribed a function. The presented 2-D patterns and identifications will be used to describe proteome differences between cultivars and changes during malting.     

Proteomic analysis of the tegument and excretory-secretory products of adult Schistosoma bovis worms

Schistosoma bovis is a ruminant pathogen that is poorly known at a molecular level. With an aim of identifying the parasite proteins involved in host-parasite interplay, we studied two protein extracts that contain, respectively, the proteins excreted/secreted by the adult worm (ES) and the tegumental proteins exposed to the host (TG). The 2-DE, 2-D immunoblot and MS were employed to separate and identify the antigenic proteins and the most abundant non-antigenic proteins in each extract. There were some 400 and 600 spots detected in the ES and the TG extracts, respectively. Ninety-six spots were subjected to MS analysis and 64 of them were identified. Overall, we identified 18 S. bovis proteins located at the host-parasite interface, 16 of which have not been identified previously in this parasite, and one of which -lysozyme- has never been reported in a Schistosoma species. Of the proteins identified, at least 4 can counteract host defence mechanisms. The other proteins are also likely to play some role in the host-parasite relationships. Therefore, studies in grater depth on all these proteins will provide a better understanding of how this parasite interacts with its host and new strategies for anti-schistosome drug or vaccine design.     

Cellular and extracellular proteome analysis of Streptococcus mutans grown in a chemostat

The oral pathogen, Streptococcus mutans, was grown under glucose limitation in a chemostat at pH 7.0 and a dilution rate of 0.1 h(-1) to mimic the conditions prevailing in a healthy human oral cavity in between meal times. Solubilized cellular and extracellular proteins were separated by two-dimensional gel electrophoresis (2-DE) and, following tryptic digestion, 421 protein spots analyzed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry or electrospray ionization-tandem mass spectrometry. Analyses of the mass spectral data showed that the proteins matched the translation products of 200 different open reading frames (ORFs) deduced from contigs of the S. mutans UA159 genome and thus represented proteins derived from approximately 11% of the total ORFs of the bacterium. Of the identified proteins, 172 (including one surface protein) were characterized in the cellular fraction, and the remaining 28 (including two surface proteins) were uniquely identified from the culture fluid. The expression and therefore the existence of 30 proteins previously designated as 'hypothetical' or with no known function was confirmed. 2-DE of whole cell lysates revealed only a single intrinsic membrane protein. This is consistent with proteomic analyses of other Gram-positive bacteria where hydrophilic proteins represent the vast majority of those characterized.     

Comparative proteomics analysis of serum proteins in ulcerative colitis patients

In the present study, we investigated the differentially expressed proteins associated with ulcerative colitis (UC) using proteomic methods. Two-dimensional electrophoresis (2-DE) technology was performed to separate the total proteins of ulcerative tissues from those of the normal tissues of UC patients. PDQuest software was applied to analyze the obtained 2-DE images. Candidate protein spots between the two groups were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and bioinformatics analysis. The well resolution and reproducible 2-DE patterns of UC and normal tissues were established. Of the 12 differentially expressed proteins, 9 were successfully identified, of which 6 proteins were up-regulated including apolipoprotein C-III, haptoglobin, receptor tyrosine kinase, aldehyde reductase, pericentriolar material 1, and heat shock factor protein 2, and 3 were down-regulated including keratin, filamin A-interacting protein 1, and tropomyosin 3. These identified proteins were related to hormonal modulation, immune response, oxidative stress, and signal conduction. The 2-DE protein expression profile of the UC tissues displays an obvious difference from that of the normal controls. Various proteins may be involved in the occurrence of UC.     

Proteome analysis of rat pancreas induced by pancreatectomy

The previous study demonstrated that the streptozotocin (STZ)-induced diabetic mice can be cured by injecting the regenerating pancreatic extract (RPE) of the partially pancreatectomized Wistar-Kyoto rats. In this study, to characterize the complex pattern of protein expression in RPE, the proteins of altered expression level after the pancreatectomy were identified by 2-dimensional electrophoresis (2-DE) and mass spectrometry. Of 76 significantly up- or down-regulated protein spots, 61 were identified by MALDI-TOF/MS. Moreover, the whole RPE was fractionated into 4 groups using an anion-exchange chromatography and each fraction's cell proliferating activity was measured by MTT assay. Compared to the normal pancreatic extract, fraction 3 and 4 of RPE showed the maximal cell proliferating activity. On 2-DE of 3 and 4 fractions, a total of 10 spots, which are differentially expressed after the pancreatectomy, were identified by MS/MS. Of these identified proteins, Reg III which might be functionally associated with well known regenerating factor (Reg I) was found. Taken together, our results demonstrated that the differential protein expression associated with pancreas regeneration could be sought by 2-DE and mass spectroscopy and suggested that the pre-fractionation method combined with in vitro cell proliferation assay is effectively used to pinpoint the active components for pancreas regeneration.     

The expression of tomato prosystemin gene in tobacco plants highly affects host proteomic repertoire

Systemin, an octadecapeptide isolated from tomato, is a primary signal molecule involved in the local and systemic responses to pest attack, elicited by activation of a set of defence genes. It derives from processing of prosystemin, a prohormone of almost 200 amino acids. Prosystemin orthologues have been found in other Solanaceae species but not in tobacco, where are present hydroxyproline-rich peptides functionally but not structurally related to tomato systemin. Molecular events leading to the release of signalling peptides from protein precursors are unknown in plants; the occurrence of a family of signal molecules suggests that initiation of wound response may involve different processing mechanisms. It has been previously shown that the protein product from an engineered tomato prosystemin gene is processed in tobacco, thus suggesting that the components responsible for its post-translational modifications are present in this species. By analyzing analysing the proteome repertoire of transformed tobacco plant leaves with 2-DE, here we demonstrate that the constitutive expression of the tomato prosystemin gene highly affected host protein synthesis. In particular, engineered plants showed a number of differentially synthesized proteins that were identified by PMF MALDI-TOF and microLC-ESI-IT-MS/MS experiments as polypeptide species involved in protection from pathogens and oxidative stress, or in carbon/energy metabolism. Significant differences in over-produced proteins were observed with respect to previous data reported on systemin-engineered tomato plants. Our results strongly support the need of using proteomic approaches during systematic analysis of plant tissues to investigate the principle of substantial equivalence in transgenic plants expressing a transgene coding for a signalling molecule.     

Proteomic analysis of Holm oak (Quercus ilex subsp. ballota [Desf.] Samp.) pollen

This paper presents an analysis of Holm oak pollen proteome, together with an evaluation of the potentiality that a proteomic approach may have in the provenance variability assessment. Proteins were extracted from pollen of four Holm oak provenances, and they were analyzed by gel-based (1- and 2-DE in combination with MALDI-TOF/TOF) and gel-free (nLC-LTQ Orbitrap MS) approaches. A comparison of 1- and 2-DE protein profiles of the four provenances revealed significant differences, both qualitative and quantitative, in abundance (18 bands and 16 spots, respectively). Multivariate statistical analysis carried out on bands and spots clearly showed distinct associations between provenances, which highlight their geographical origins. A total of 100 spots selected from the 402 spots observed on 2-DE gels were identified by MALDI-TOF/TOF. Moreover, a complementary gel-free shotgun approach was performed by nLC-LTQ Orbitrap MS. The identified proteins were classified according to biological processes, and most proteins in both approaches were related to metabolism and defense/stress processes. The nLC-LTQ Orbitrap MS analysis allowed us the identification of proteins belonging to the cell wall and division, transport and translation categories. Besides providing the first reference map of Holm oak pollen, our results confirm previous studies based on morphological observations and acorn proteomic analysis. Moreover, our data support the valuable use of proteomic techniques as phylogenetic tool in plant studies.