Search

Abstract Background Two-dimensional electrophoresis (2-DE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) are widely used in plant proteomics research. However, these two techniques cannot be simultaneously satisfied by traditional protein extraction methods when investigate cotton leaf proteome. Results Here, we evaluated the efficiency of three different protein extraction methods for 2-DE and LC-MS/MS analyses of total proteins obtained from cotton leaves. The protein yield of the borax/PVPP/phenol (BPP) method (0.14%) was significantly lower than the yields of the trichloroacetic acid/acetone (TCA) precipitation method (1.42%) and optimized TCA combined with BPP (TCA-B) method (0.47%). The BPP method was failed to get a clear 2-DE electrophoretogram. Fifty pairs of protein spots were randomly selected from the 2-DE gels of TCA- and TCA-B-extracted proteins for identification by MALDI TOF/TOF, and the results of 42 pairs were consistent. High-throughput proteomic analysis showed that 6339, 9282 and 9697 unique proteins were identified from the total cotton leaf proteins extracted by the TCA, BPP and TCA-B methods, respectively. Gene Ontology (GO) analysis revealed that the proteins specifically identified by TCA method were primarily distributed in the plasma membrane, while BPP and TCA-B methods specific proteins distributed in the cytosol, indicating the sub-cellular preference of different protein extraction methods. Further, ATP-dependent zinc metalloprotease FTSH 8 could be observed in the 2-DE gels of TCA and TCA-B methods, and could only be detected in the LC-MS/MS results of the BPP and TCA-B methods, showing that TCA-B method might be the optimized choice for both 2-DE and LC-MS/MS. Conclusion Our data provided an improved TCA-B method for protein extraction that is compatible with 2-DE and LC-MS/MS for cotton leaves and similar plant tissues which is rich in polysaccharides and polyphenols.

Background: Truffles are symbiotic fungi that develop underground in association with plant roots, forming ectomycorrhizae. They are primarily known for the organoleptic qualities of their hypogeous fruiting bodies. Primarily, Tuber magnatum Pico is a greatly appreciated truffle species mainly distributed in Italy and Balkans. Its price and features are mostly depending on its geographical origin. However, the genetic variation within T. magnatum has been only partially investigated as well as its adaptation to several environments. Results: Here, we applied an integrated omic strategy to T. magnatum fruiting bodies collected during several seasons from three different areas located in the North, Center and South of Italy, with the aim to distinguish them according to molecular and biochemical traits and to verify the impact of several environments on these properties. With the proteomic approach based on two-dimensional electrophoresis (2-DE) followed by mass spectrometry, we were able to identify proteins specifically linked to the sample origin. We further associated the proteomic results to an RNA-seq profiling, which confirmed the possibility to differentiate samples according to their source and provided a basis for the detailed analysis of genes involved in sulfur metabolism. Finally, geographical specificities were associated with the set of volatile compounds produced by the fruiting bodies, as quantitatively and qualitatively determined through proton transfer reaction-mass spectrometry (PTR-MS) and gas-chromatography-mass spectrometry (GC-MS). In particular, a partial least squares-discriminant analysis (PLS-DA) model built from the latter data was able to return high confidence predictions of sample source. Conclusions: Results provide a characterization of white fruiting bodies by a wide range of different molecules, suggesting the role for specific compounds in the responses and adaptation to distinct environments. [ABSTRACT FROM AUTHOR]

Background: Two-dimensional electrophoresis (2-DE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) are widely used in plant proteomics research. However, these two techniques cannot be simultaneously satisfied by traditional protein extraction methods when investigate cotton leaf proteome. Results: Here, we evaluated the efficiency of three different protein extraction methods for 2-DE and LC-MS/MS analyses of total proteins obtained from cotton leaves. The protein yield of the borax/PVPP/phenol (BPP) method (0.14%) was significantly lower than the yields of the trichloroacetic acid/acetone (TCA) precipitation method (1.42%) and optimized TCA combined with BPP (TCA-B) method (0.47%). The BPP method was failed to get a clear 2-DE electrophoretogram. Fifty pairs of protein spots were randomly selected from the 2-DE gels of TCA- and TCA-B-extracted proteins for identification by MALDI TOF/TOF, and the results of 42 pairs were consistent. High-throughput proteomic analysis showed that 6339, 9282 and 9697 unique proteins were identified from the total cotton leaf proteins extracted by the TCA, BPP and TCA-B methods, respectively. Gene Ontology (GO) analysis revealed that the proteins specifically identified by TCA method were primarily distributed in the plasma membrane, while BPP and TCA-B methods specific proteins distributed in the cytosol, indicating the sub-cellular preference of different protein extraction methods. Further, ATP-dependent zinc metalloprotease FTSH 8 could be observed in the 2-DE gels of TCA and TCA-B methods, and could only be detected in the LC-MS/MS results of the BPP and TCA-B methods, showing that TCA-B method might be the optimized choice for both 2-DE and LC-MS/MS. Conclusion: Our data provided an improved TCA-B method for protein extraction that is compatible with 2-DE and LC-MS/MS for cotton leaves and similar plant tissues which is rich in polysaccharides and polyphenols. [ABSTRACT FROM AUTHOR]

Background: Haliotis diversicolor is commercially important species. The trochophore and veliger are distinct larval stages in gastropod development. Their development involves complex morphological and physiological changes. We studied protein changes during the embryonic development of H. diversicolor using two dimensional electrophoresis (2-DE) and label-free methods, tandem mass spectrometry (MS/MS), and Mascot for protein identification. Results: A total of 150 2-DE gel spots were identified. Protein spots showed upregulation of 15 proteins and downregulation of 28 proteins as H. diversicolor developed from trochophore to veliger larvae. Trochophore and veliger larvae were compared using a label-free quantitative proteomic approach. A total of 526 proteins were identified from both samples, and 104 proteins were differentially expressed (> 1.5 fold). Compared with trochophore larvae, veliger larvae had 55 proteins upregulated and 49 proteins downregulated. These differentially expressed proteins were involved in shell formation, energy metabolism, cellular and stress response processes, protein synthesis and folding, cell cycle, and cell fate determination. Compared with the 5 protein (fructosebisphosphate aldolase, 14-3-3e, profilin, actin-depolymerizing factor (ADF)/cofilin) and calreticulin) expression patterns, the mRNA expression exhibited similar patterns except gene of fructose-bisphosphate aldolase. Conclusion: Our results provide insight into novel aspects of protein function in shell formation, torsion, and nervous system development, and muscle system differentiation in H. diversicolor larvae. "Quality control" proteins were identified to be involved in abalone larval development. [ABSTRACT FROM AUTHOR]

Background: The evolutionary diversification of gene families through gene creation (and loss) is a dynamic process believed to be critical to the evolution of functional novelty. Previous identification of a closely related family of eight annotated metalloprotease genes of the M17 Merops family in the Drosophila sperm proteome (termed, S perm-L eucylA minoP eptidases, S-LAPs 1-8) led us to hypothesize that this gene family may have experienced such a diversification during insect evolution. Results: To assess putative functional activities of S-LAPs, we (i) demonstrated that all S-LAPs are specifically expressed in the testis, (ii) confirmed their presence in sperm by two-dimensional gel electrophoresis and mass spectrometry, (iii) determined that they represent a major portion of the total protein in sperm and (iv) identified aminopeptidase enzymatic activity in sperm extracts using LAP-specific substrates. Functionally significant divergence at the canonical M17 active site indicates that the largest phylogenetic group of S-LAPs lost catalytic activity and likely acquired novel, as yet undetermined, functions in sperm prior to the expansion of the gene family. Conclusions: Comparative genomic and phylogenetic analyses revealed the dramatic expansion of the S-LAP gene family during Drosophila evolution and copy number heterogeneity in the genomes of related insects. This finding, in conjunction with the loss of catalytic activity and potential neofunctionalization amongst some family members, extends empirical support for pervasive "revolving door" turnover in the evolution of reproductive gene family composition and function. [ABSTRACT FROM AUTHOR]

Background: Two-dimensional gel electrophoresis (2D-PAGE) has proven over the years to be a reliable and efficient method for separation of hundreds of proteins based on charge and mass. Nevertheless, the complexity of even the simplest proteomes limits the resolving power of 2D-PAGE. This limitation can be partially alleviated by sample prefractionation using a variety of techniques. Results: Here, we have used Vivapure Ion Exchange centrifugal adsorber units to rapidly prefractionate total fission yeast protein lysate based on protein charge. Three fractions were prepared by stepwise elution with increasing sodium chloride concentrations. Each of the fractions, as well as the total lysate, were analyzed by 2D-PAGE. This simple prefractionation procedure considerably increased the resolving power of 2D-PAGE. Whereas 308 spots could be detected by analysing total protein lysate, 910 spots were observed upon prefractionation. Thorough gel image analysis demonstrated that prefractionation visualizes an additional set of 458 unique fission yeast proteins not detected in whole cell lysate. Conclusions: Prefractionation with Vivapure Q spin columns proved to be a simple, fast, reproducible, and cost-effective means of increasing the resolving power of 2D-PAGE using standard laboratory equipment. [ABSTRACT FROM AUTHOR]

Background: The mesocotyl connects the coleoptilar node and the basal part of the seminal root of maize (Zea mays) seedling. The mesocotyl pushes the shoot of the seedling out of the soil during seed germination; thus, its growth is highly related to deep-sowing tolerance. Although many studies on the maize mesocotyl have been carried out at physiological and molecular levels, the proteomic changes associated with cellular and physiological activities during mesocotyl growth are still unknown. Results: In the present study, the maize hybrid Zhengdan 958 was used to study mesocotyl growth and accompanying protein changes. The dark-grown etiolated mesocotyls exhibited a slow-fast-slow feature, with significant changes in the levels of indole-3-acetic acid (IAA) and cellulose and the activity of peroxidase (POD). In particular, POD activity increased with mesocotyl growth, showing higher activity at the mature (lower) end of the mesocotyl. For the proteomic analysis, soluble proteins were extracted from etiolated mesocotyls dark-grown for 48 h, 84 h, and 132 h, corresponding to the initial, rapid, and slow growth periods, respectively, and subjected to separation by two-dimensional gel electrophoresis (2-DE). As a result, 88 differentially abundant proteins (DAPs) were identified using MALDI-TOF-TOF analysis. At 48 h, most DAPs were stress proteins, heat shock proteins and storage proteins; at 84 h, oxidation/reduction proteins, carbohydrate biogenesis-related proteins and cytoskeleton-related proteins were highly accumulated; at 132 h, the most striking DAPs were those involved in the synthesis and modification of the cell wall and the biogenesis of carbohydrates. Gene ontology (GO) analysis showed that changes in the abundance and proportion of DAPs were consistent with cellular and physiological activities and biological processes during mesocotyl growth. The accumulation of nine DAPs of interest was verified by immunoblotting and RT-qPCR. Conclusions: The present study revealed that the protein patterns in 2-D gels differed greatly with mesocotyl growth. At different growth periods, a specific set of DAPs participate in various biological processes and underlie the cellular and physiological activities of the mesocotyl. These results contributed to the understanding of mesocotyl growth and the cultivation of maize lines with deep-sowing tolerance. [ABSTRACT FROM AUTHOR]

Background: Pre-slaughter stress (PSS) impairs animal welfare and meat quality. Dark, firm and dry (DFD) are terms used to designate poor quality meats induced by PSS. Protein phosphorylation can be a potentially significant mechanism to explain rapid and multiple physiological and biochemical changes linked to PSS-dependent muscle-to-meat conversion. However, the role of reversible phosphorylation in the response to PSS is still little known. In this study, we report a comparative phosphoproteomic analysis of DFD and normal meats at 24 h post-mortem from the longissimus thoracis (LT) bovine muscle of male calves of the Rubia Gallega breed. For this purpose, two-dimensional gel electrophoresis (2-DE), in-gel multiplex identification of phosphoproteins with PRO-Q Diamond phosphoprotein-specific stain, tandem (MALDI-TOF/TOF) mass spectrometry (MS), novel quantitative phosphoproteomic statistics and bioinformatic tools were used. Results: Noticeable and statistically significant differences in the extent of protein phosphorylation were detected between sample groups at the qualitative and quantitative levels. Overall phosphorylation rates across significantly changed phosphoproteins were about three times higher in DFD than in normal meat. Significantly changed phosphoproteins involved a variable number of isoforms of 13 myofibrillar and sarcoplasmic nonredundant proteins. However, fast skeletal myosin light chain 2 followed by troponin T, F-actin-capping and small heat shock proteins showed the greatest phosphorylation change, and therefore they were the most important phosphoproteins underlying LT muscle conversion to DFD meat in the Rubia Gallega breed. Conclusions: This is the first study reporting global meat phosphoproteome changes in response to PSS. The results show that reversible phosphorylation is a relevant mechanism underlying PSS response and downstream effects on meat quality. This research opens up novel horizons to unravel the complex molecular puzzle underlying muscle-to-meat conversion in response to PSS. [ABSTRACT FROM AUTHOR]