Resurrection plant Ramonda serbica is a suitable model to investigate vegetative desiccation tolerance. However, the detailed study of these mechanisms at the protein level is hampered by the severe tissue water loss, high amount of phenolics and polysaccharide, and possible protein modifications and aggregations during the extraction and purification steps. When applied to R. serbica leaves, widely used protein extraction protocols containing polyvinylpolypyrrolidone and ascorbate, as well as the phenol/SDS/buffer-based protocol recommended for recalcitrant plant tissues failed to eliminate persistent contamination and ensure high protein quality. Here we compared three protein extraction approaches aiming to establish the optimal one for both hydrated and desiccated R. serbica leaves. To evaluate the efficacy of these protocols by shotgun proteomics, we also created the first R. serbica annotated transcriptome database, available at http://www.biomed.unipd.it/filearrigoni/Trinity_Sample_RT2.fasta . The detergent-free phenol-based extraction combined with dodecyl-β-D-maltoside-assisted extraction enabled high-yield and high-purity protein extracts. The phenol-based protocol improved the protein-band resolution, band number, and intensity upon electrophoresis, and increased the protein yield and the number of identified peptides and protein groups by LC-MS/MS. Additionally, dodecyl-β-D-maltoside enabled solubilisation and identification of more membrane-associated proteins. The presented study paves the way for investigating the desiccation tolerance in R. serbica, and we recommend this protocol for similar recalcitrant plant material.
- MeSH
- chromatografie kapalinová metody MeSH
- elektroforéza v polyakrylamidovém gelu MeSH
- listy rostlin chemie MeSH
- Magnoliopsida chemie MeSH
- proteomika metody MeSH
- rostlinné proteiny izolace a purifikace MeSH
- tandemová hmotnostní spektrometrie metody MeSH
- voda chemie MeSH
- vysoušení MeSH
- Publikační typ
- časopisecké články MeSH
Peptide spectral libraries enable targeted identification and quantitation of low-abundance proteins in a complex plant proteome. Here we describe parallel protein and peptide fractionation techniques to improve plant proteome coverage and facilitate construction of spectral libraries.
Proteins were obtained from effluent of a starch manufacture by using different isolation temperatures (40, 60, 80, and 100 °C). The proteins, remaining in effluent after treatment of potato juice at 80 and 100 °C differed significantly in composition and in structural stability as well as in trypsin inhibitory and antifungal activities in comparison with the variants of 40 and 60 °C. The protein samples of 80 °C exhibited the highest antifungal activity and its average value of IC50 against five strains of two Fusarium species was determined in average at 0.18 mg ml-1. The 80 °C protein samples consisted predominantly of low-molecular proteins (7-17 kDa) identified as potato tuber protease inhibitors I and II. Predominantly, protease inhibitors II were identified for the protein samples obtained by 100 °C and here we identified 7 spots in comparison with 12 identified for the 80 °C samples. Samples of 40 and 60 °C with low antifungal activities represent high variability of detected and identified proteins. We identified various representatives of aspartic, cysteine, and serine protease inhibitors in both types of samples. These samples also contained Kunitz-type protease inhibitors that were not found in the 80 and 100 °C samples which documented thermal unstableness of Kunitz-type protease inhibitors. Functional stability at high temperatures and antifungal activity of isolated potato protease inhibitors I and II support the potential of this fraction usage in food, feed, pharmaceutical, or agricultural industry and offer new products for starch manufactures. At the same time, utilization of the stable protein fraction of waste deproteinized potato water promotes exploitation of potato starch production resources.
- MeSH
- 2D gelová elektroforéza MeSH
- antifungální látky farmakologie MeSH
- Fusarium účinky léků MeSH
- hlízy rostlin chemie MeSH
- mikrobiální testy citlivosti MeSH
- proteomika MeSH
- rostlinné proteiny chemie izolace a purifikace farmakologie MeSH
- škrob MeSH
- Solanum tuberosum chemie MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
- stabilita proteinů MeSH
- tandemová hmotnostní spektrometrie MeSH
- teplota MeSH
- Publikační typ
- časopisecké články MeSH
Protein phosphorylation was repeatedly shown to be the most dynamic post-translational modification mediated by a huge orchestra of protein kinases and phosphatases. Upon landing on a stigma, pollen grain dehydration and activation are accompanied by changes in protein phosphorylation together with the translation activation of stored mRNAs. To enable studies of the total phosphoproteome, it is usually necessary to apply various enrichment techniques. In this chapter, one of these protocols that worked previously well on tobacco mature pollen is presented in more detail. The method comprises of three basic steps: (1) picking flowers from the flowering tobacco plants (Nicotiana tabacum cv. Samsun), and collection of the shed pollen grains; (2) extraction of total proteins by TCA/acetone; (3) phosphoprotein enrichment by MOAC with aluminum hydroxide matrix. Taken together this protocol describes how to isolate phosphoproteins out of tobacco mature pollen.
- MeSH
- chromatografie afinitní MeSH
- fosfoproteiny chemie izolace a purifikace metabolismus MeSH
- hydroxid hlinitý chemie MeSH
- posttranslační úpravy proteinů MeSH
- pyl metabolismus MeSH
- rostlinné proteiny chemie izolace a purifikace metabolismus MeSH
- tabák metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
Protein sample preparation is a critical and an unsustainable step since it involves the use of tedious methods that usually require high amount of solvents. The development of new materials offers additional opportunities in protein sample preparation. This work explores, for the first time, the potential application of carboxylate-terminated carbosilane dendrimers to the purification/enrichment of proteins. Studies on dendrimer binding to proteins, based on protein fluorescence intensity and emission wavelengths measurements, demonstrated the interaction between carboxylate-terminated carbosilane dendrimers and proteins at all tested pH levels. Interactions were greatly affected by the protein itself, pH, and dendrimer concentration and generation. Especially interesting was the interaction at acidic pH since it resulted in a significant protein precipitation. Dendrimer-protein interactions were modeled observing stable complexes for all proteins. Carboxylate-terminated carbosilane dendrimers at acidic pH were successfully used in the purification/enrichment of proteins extracted from a complex sample. Graphical Abstract Images showing the growing turbidity of solutions containing a mixture of proteins (lysozyme, myoglobin, and BSA) at different protein:dendrimer ratios (1:0, 1:1, 1:8, and 1:20) at acidic pH and SDS-PAGE profiles of the corresponsing supernatants. Comparison of SDS-PAGE profiles for the pellets obtained during the purification of proteins present in a complex sample using a conventional "no-clean" method based on acetone precipitation and the proposed "greener" method using carboxylate-terminated carbosilane dendrimer at a 1:20 protein:dendrimer ratio.
- MeSH
- chemická precipitace MeSH
- dendrimery chemie MeSH
- koncentrace vodíkových iontů MeSH
- kyseliny karboxylové chemie MeSH
- muramidasa chemie izolace a purifikace MeSH
- myoglobin chemie izolace a purifikace MeSH
- rostlinné proteiny izolace a purifikace MeSH
- rozpouštědla MeSH
- sekundární struktura proteinů MeSH
- semena rostlinná chemie MeSH
- sérový albumin hovězí chemie izolace a purifikace MeSH
- silany chemie MeSH
- simulace molekulární dynamiky MeSH
- slivoň švestka chemie MeSH
- vazba proteinů MeSH
- Publikační typ
- časopisecké články MeSH
Here, we describe a method for the combined metabolomic, proteomic, transcriptomic and genomic analysis from one single sample as a major step for multilevel data integration strategies in systems biology. While extracting proteins and DNA, this protocol also allows the separation of metabolites into polar and lipid fractions, as well as RNA fractionation into long and small RNAs, thus allowing a broad range of transcriptional studies. The isolated biomolecules are suitable for analysis with different methods that range from electrophoresis and blotting to state-of-the-art procedures based on mass spectrometry (accurate metabolite profiling, shot-gun proteomics) or massive sequencing technologies (transcript analysis). The low amount of starting tissue, its cost-efficiency compared with the utilization of commercial kits, and its performance over a wide range of plant, microbial, and algal species such as Chlamydomonas, Arabidopsis, Populus, or Pinus, makes this method a universal alternative for multiple molecular isolation from plant tissues.
- MeSH
- Arabidopsis genetika metabolismus MeSH
- borovice genetika metabolismus MeSH
- Chlamydomonas reinhardtii genetika metabolismus MeSH
- DNA rostlinná izolace a purifikace MeSH
- genomika metody MeSH
- metabolomika metody MeSH
- Populus genetika metabolismus MeSH
- proteomika metody MeSH
- reprodukovatelnost výsledků MeSH
- RNA rostlin izolace a purifikace MeSH
- rostlinné proteiny izolace a purifikace MeSH
- rostliny * genetika metabolismus MeSH
- systémová biologie metody MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Telomerase maintains the ends of eukaryotic chromosomes, and its activity is an important parameter correlating with the proliferative capacity of cells. We have investigated cell cycle-specific changes in telomerase activity using cultures of Desmodesmus quadricauda, a model alga naturally synchronized by light/dark entrainment. A quantitative telomerase assay revealed high activity in algal cultures, with slight changes during the light period. Significantly increased telomerase activity was observed at the end of the dark phase, when cell division was complete. In contrast to other models, a natural separation between nuclear and cellular division typical for the cell cycle in D. quadricauda made this observation possible.
- MeSH
- biotest MeSH
- buněčné dělení MeSH
- Chlorophyta cytologie enzymologie účinky záření MeSH
- fotoperioda MeSH
- kinetika MeSH
- kultivované buňky MeSH
- rostlinné proteiny izolace a purifikace metabolismus MeSH
- světlo MeSH
- telomerasa izolace a purifikace metabolismus MeSH
- telomery metabolismus účinky záření MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Biochemical characteristics of patatin proteins purified by ion-exchange and affinity chromatography from tubers of 20 potato cultivars were studied to evaluate their genotype differences with respect to utility groups, table potato cultivars (TPCs) and processing potato cultivars (PPCs). Both groups of cultivars showed similar values of protein content in dry matter (3.98-7.39%) and of patatin relative abundance (5.40-35.40%). Three mass levels (∼40.6, 41.8, and 42.9 kDa) of purified patatins were found by MALDI-TOF MS within all cultivars. Differences among mass levels corresponding with the mass of sugar antenna (∼1.2 kDa) confirmed the previous concept of different glycosylation extentsin patatin proteins. It was showed that the individual types of patatin varying in their masses occur in the patatin family in a ratio specific for each of the cultivars, with the lowest mass type being the major one. Electrophoretic analyses demonstrated wide cultivar variability in number of patatin forms. Especially 2D-PAGE showed 17-23 detected protein spots independently on the utility group. Specific lipid acyl hydrolase (LAH) activity of purified patatins from the individual tested cultivars varied between 0.92 and 5.46 μmol/(min mg). Patatin samples within most of the TPCs exhibited higher values of specific LAH activity than samples of PPCs. It may be supposed that individual patatin forms do not have similar physiological roles.
- MeSH
- druhová specificita MeSH
- glykosylace MeSH
- hlízy rostlin chemie MeSH
- karboxylesterhydrolasy chemie izolace a purifikace metabolismus MeSH
- molekulová hmotnost MeSH
- protein - isoformy chemie izolace a purifikace metabolismus MeSH
- rostlinné proteiny analýza chemie izolace a purifikace metabolismus MeSH
- Solanum tuberosum chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
Rapid changes of protein phosphorylation play a crucial role in the regulation of many cellular processes. Being post-translationally modified, phosphoproteins are often present in quite low abundance and tend to co-exist with their unphosphorylated isoform within the cell. To make their identification more practicable, the use of enrichment protocols is often required. The enrichment strategies can be performed either at the level of phosphoproteins or at the level of phosphopeptides. Both approaches have their advantages and disadvantages. Most enriching strategies are based on chemical modifications, affinity chromatography to capture peptides and proteins containing negatively charged phosphate groups onto a positively charged matrix, or immunoprecipitation by phospho-specific antibodies.In this article, the most up-to-date enrichment techniques are discussed, taking into account their optimization, and highlighting their advantages and disadvantages. Moreover, these methods are compared to each other, revealing their complementary nature in providing comprehensive coverage of the phosphoproteome.
- MeSH
- barvení a značení MeSH
- chromatografie afinitní MeSH
- fosfoproteiny chemie izolace a purifikace metabolismus MeSH
- fosforylace MeSH
- hmotnostní spektrometrie MeSH
- imunoprecipitace MeSH
- peptidové fragmenty chemie izolace a purifikace metabolismus MeSH
- posttranslační úpravy proteinů MeSH
- proteom chemie izolace a purifikace metabolismus MeSH
- proteomika MeSH
- rostlinné proteiny chemie izolace a purifikace metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
The crystal structures of both isoforms of the aminoaldehyde dehydrogenase from pea (PsAMADH) have been solved recently [Tylichováet al. (2010) J Mol Biol396, 870-882]. The characterization of the PsAMADH2 proteins, altered here by site-directed mutagenesis, suggests that the D110 and D113 residues at the entrance to the substrate channel are required for high-affinity binding of ω-aminoaldehydes to PsAMADH2 and for enzyme activity, whereas N162, near catalytic C294, contributes mainly to the enzyme's catalytic rate. Inside the substrate cavity, W170 and Y163, and, to a certain extent, L166 and M167 probably preserve the optimal overall geometry of the substrate channel that allows for the appropriate orientation of the substrate. Unconserved W288 appears to affect the affinity of the enzyme for the substrate amino group through control of the substrate channel diameter without affecting the reaction rate. Therefore, W288 may be a key determinant of the differences in substrate specificity found among plant AMADH isoforms when they interact with naturally occurring substrates such as 3-aminopropionaldehyde and 4-aminobutyraldehyde.
- MeSH
- aldehydoxidoreduktasy chemie genetika izolace a purifikace metabolismus MeSH
- aldehydy metabolismus MeSH
- aminokyseliny aromatické metabolismus MeSH
- aminokyseliny dikarboxylové metabolismus MeSH
- betain analogy a deriváty metabolismus MeSH
- biokatalýza MeSH
- cirkulární dichroismus MeSH
- hrách setý enzymologie MeSH
- izoenzymy metabolismus MeSH
- katalytická doména MeSH
- kinetika MeSH
- mutageneze cílená MeSH
- mutantní proteiny chemie izolace a purifikace metabolismus MeSH
- propylaminy metabolismus MeSH
- rekombinantní proteiny chemie izolace a purifikace metabolismus MeSH
- rostlinné proteiny chemie genetika izolace a purifikace metabolismus MeSH
- sekundární struktura proteinů MeSH
- substrátová specifita MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
