Whole-cell Extracts Research Articles

Postrecruitment Function of Yeast Med6 Protein during the Transcriptional Activation by Mediator Complex.

Med6 protein (Med6p) is a hallmark component of evolutionarily conserved Mediator complexes, and the genuine role of Med6p in Mediator functions remains elusive. For the functional analysis of Saccharomyces cerevisiae Med6p (scMed6p), we generated a series of scMed6p mutants harboring a small internal deletion. Genetic analysis of these mutants revealed that three regions (amino acids 33–42 (Δ2), 125–134 (Δ5), and 157–166 (Δ6)) of scMed6p are required for cell viability and are located at highly conserved regions of Med6 homologs. Notably, the Med6p-Δ2 mutant was barely detectable in whole-cell extracts and purified Mediator, suggesting a loss of Mediator association and concurrent rapid degradation. Consistent with this, the recombinant forms of Med6p having these mutations partially (Δ2) restore or fail (Δ5 and Δ6) to restore in vitro transcriptional defects caused by temperature-sensitive med6 mutation. In an artificial recruitment assay, Mediator containing a LexA-fused wild-type Med6p or Med6p-Δ5 was recruited to the lexA operator region with TBP and activated reporter gene expression. However, the recruitment of Mediator containing LexA-Med6p-Δ6 to lexA operator region resulted in neither TBP recruitment nor reporter gene expression. This result demonstrates a pivotal role of Med6p in the postrecruitment function of Mediator, which is essential for transcriptional activation by Mediator.

Def1 interacts with TFIIH and modulates RNA polymerase II transcription

The DNA damage response is an essential process for the survival of living cells. In a subset of stress-responsive genes in humans, Elongin controls transcription in response to multiple stimuli, such as DNA damage, oxidative stress, and heat shock. Yeast Elongin (Ela1-Elc1), along with Def1, is known to facilitate ubiquitylation and degradation of RNA polymerase II (pol II) in response to multiple stimuli, yet transcription activity has not been examined. We have found that Def1 copurifies from yeast whole-cell extract with TFIIH, the largest general transcription factor required for transcription initiation and nucleotide excision repair. The addition of recombinant Def1 and Ela1-Elc1 enhanced transcription initiation in an in vitro reconstituted system including pol II, the general transcription factors, and TFIIS. Def1 also enhanced transcription restart from TFIIS-induced cleavage in a pol II transcribing complex. In the Δdef1 strain, heat shock genes were misregulated, indicating that Def1 is required for induction of some stress-responsive genes in yeast. Taken together, our results extend the understanding of the molecular mechanism of transcription regulation on cellular stress and reveal functional similarities to the mammalian system.

Growth arrest and morphological changes triggered by emodin on Trypanosoma cruzi epimastigotes cultivated in axenic medium

Emodin is an anthraquinone obtained from Rheum palmatum rootstocks. Here we tested the cytotoxic effects of emodin on Trypanosoma cruzi epimastigotes, as well as the morphological changes that were induced by this compound in the parasite. Emodin was permeable and blocked in vitro cell division of T. cruzi epimastigotes in axenic medium, causing growth arrest in a dose-dependent but reversible manner. Emodin-exposed epimastigotes underwent duplication of organelles, such as the nucleus, kinetoplast and flagellum, but were incapable of completing cytokinesis. Neither elongation of the parasite body nor appearance of the regular longitudinal cleavage furrow was displayed, suggesting that emodin is most likely affecting components of the parasite cytoskeleton. Moreover, drug-treated parasites acquired alterations such as protuberances, folds and indentations on their membrane surface. Since emodin has been shown to be a potent protein kinase CK2 inhibitor, and we have previously described an association between tubulin and CK2 in T. cruzi epimastigotes (De Lima et al. Parasitology132, 511–523, 2006), we also measured the indirect effect of the drug on tubulin. Incubation of epimastigotes with axenic medium containing emodin hindered the endogenous phosphorylation of tubulin in whole-cell parasite extracts. All our results suggested that the parasite CK2 may be important for the maintenance of the morphology and for the regulation of mitosis-cytokinesis transition in T. cruzi epimastigotes.

Correction for Travesa et al., "Repression of G1/S Transcription Is Mediated via Interaction of the GTB Motifs of Nrm1 and Whi5 with Swi6".

AUTHOR CORRECTION: Volume 33, no. 8, p. 1476–1486, 2013, https://doi.org/10.1128/MCB.01333-12. Page 1484, Fig. 6B and C: The lanes of the immunoblot showing the interaction between the Whi5 GTB motif and Swi6 were intentionally duplicated in panels B and C to allow comparison between samples. To clarify this, the legend should be changed as follows. In line 5, “and… SWI6ΔC cell extracts” should read “from whole-cell extracts (WCE) from strains expressing wild-type SWI6 or SWI6ΔC. Those strains also carried SWI4-TAP or MBP1-TAP, respectively (see panel C).” In line 7, “and” should read “from.” In lines 7 and 8, “cell extracts” should read “strains. The Swi6 data from panel B are duplicated in the SWI4-TAP lanes to allow direct comparison with the other samples. All samples shown in panels B and C were derived from the same SDS-PAGE gel.” These changes do not affect the conclusions derived from this experiment.

Selective Affinity Enrichment of Nitrotyrosine-Containing Peptides for Quantitative Analysis in Complex Samples.

Protein tyrosine nitration by oxidative and nitrate stress is important in the pathogenesis of many inflammatory or aging-related diseases. Mass spectrometry analysis of protein nitrotyrosine is very challenging because the non-nitrated peptides suppress the signals of the low-abundance nitrotyrosine (NT) peptides. No validated methods for enrichment of NT-peptides are currently available. Here we report an immunoaffinity enrichment of NT-peptides for proteomics analysis. The effectiveness of this approach was evaluated using nitrated protein standards and whole-cell lysates in vitro. A total of 1881 NT sites were identified from a nitrated whole-cell extract, indicating that this immunoaffinity-MS method is a valid approach for the enrichment of NT-peptides, and provides a significant advance for characterizing the nitrotyrosine proteome. We noted that this method had higher affinity to peptides with N-terminal nitrotyrosine relative to peptides with other nitrotyrosine locations, which raises the need for future study to develop a pan-specific nitrotyrosine antibody for unbiased, proteome-wide analysis of tyrosine nitration. We applied this method to quantify the changes in protein tyrosine nitration in mouse lungs after intranasal poly(I:C) treatment and quantified 237 NT sites. This result indicates that the immunoaffinity-MS method can be used for quantitative analysis of protein nitrotyrosines in complex samples.

Nitrogen-fixing rhizobial strains isolated from Desmodium incanum DC in Argentina: Phylogeny, biodiversity and symbiotic ability

Desmodium spp. are leguminous plants belonging to the tribe Desmodieae of the subfamily Papilionoideae. They are widely distributed in temperated and subtropical regions and are used as forage plants, for biological control, and in traditional folk medicine. The genus includes pioneer species that resist the xerothermic environment and grow in arid, barren sites. Desmodium species that form nitrogen-fixing symbiosis with rhizobia play an important role in sustainable agriculture. In Argentina, 23 native species of this genus have been found, including Desmodium incanum. In this study, a total of 64 D. incanum-nodulating rhizobia were obtained from root nodules of four Argentinean plant populations. Rhizobia showed different abiotic-stress tolerances and a remarkable genetic diversity using PCR fingerprinting, with more than 30 different amplification profiles. None of the isolates were found at more than one site, thus indicating a high level of rhizobial diversity associated with D. incanum in Argentinean soils. In selected isolates, 16S rDNA sequencing and whole-cell extract MALDI TOF analysis revealed the presence of isolates related to Bradyrhizobium elkanii, Bradyrhizobium japonicum, Bradyrhizobium yuanmingense, Bradyrhizobium liaoningense, Bradyrhizobium denitrificans and Rhizobium tropici species. In addition, the nodC gene studied in the selected isolates showed different allelic variants.Isolates were phenotypically characterized by assaying their growth under different abiotic stresses. Some of the local isolates were remarkably tolerant to high temperatures, extreme pH and salinity, which are all stressors commonly found in Argentinean soils. One of the isolates showed high tolerance to temperature and extreme pH, and produced higher aerial plant dry weights compared to other inoculated treatments. These results indicated that local isolates could be efficiently used for D. incanum inoculation.

Steroids hydroxylation catalyzed by the monooxygenase mutant 139-3 from Bacillus megaterium BM3

The search of new substrates with pharmaceutical and industrial potential for biocatalysts including cytochrome P450 enzymes is always challenging. Cytochrome P450 BM3 mutant 139-3, a versatile biocatalyst, exhibited hydroxylation activities towards fatty acids and alkanes. However, there were limited reports about its hydroxylation activity towards steroids. Herein, an Escherichia coli–based whole-cell extract containing the recombinant 139-3 protein was used as the biocatalyst to screen 13 steroids. Results revealed that 139-3 was able to specifically hydroxylate androstenedione (1) at 1α-position, generating a hydroxylated steroid 1α-OH-androstenedione (1a). To investigate whether C-1α hydroxylation catalyzed by BM3 mutant 139-3 could be industrially used, an optimization of catalyzing conditions was performed. Accordingly, the BM3 mutant 139-3 enzyme was observed to display maximum activity at 37°C, under pH 7.0 for 4h, with 37% transformation rate. Moreover, four 139-3 variants were generated by random mutagenesis with the aim of improving its activity and expanding substrate scope. Surprisingly, these mutants, sharing a common mutated site R379S, lost their activities towards androstenedione (1). These data clearly indicated that arginine residue located at site 379 played key role in the hydroxylation activities of 139-3. Overall, these new findings broadened the substrate scope of 139-3 enzyme, thereby expanding its potential applications as a biocatalyst on steroids hydroxylation in pharmaceutical industry.

Nocardia xestospongiae sp. nov., isolated from a marine sponge in the Andaman Sea.

A marine sponge-derived actinomycete, strain ST01-07T, was isolated from Xestospongia sp. collected from the Andaman Sea. The strain was characterised taxonomically using a polyphasic approach. The strain contained meso-diaminopimelic acid in the peptidoglycan, whole-cell sugars were arabinose, galactose, glucose, mannose and ribose. Mycolic acids that co-migrated with those from Nocardia araoensis NBRC 100135T were observed in whole-cell extracts. MK-8(H4ω-cycl) was the predominant menaquinone. Major cellular fatty acids were C17 : 1ω8c, C16 : 0 and C17 : 0. The diagnostic phospholipids in the cell consisted of diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylinositol. Phylogenetic analysis based on the 16S rRNA gene sequence showed that strain ST01-07T belonged to the genus Nocardia and was most closely related to N. araoensis IFM 0575T (98.71 % 16S rRNA gene sequence similarity), Nocardia niwae W9241T (98.56 %), Nocardia beijingensis AS4.1521T (98.41 %) and Nocardia arthritidis IFM 10035T (98.36 %). In addition, low DNA-DNA relatedness values (13.6±0.1% to 40.1±0.6%) confirmed that strain ST01-07T represents a novel species of the genus Nocardia, for which the name Nocardia xestospongiae sp. nov., is proposed. The type strain is ST01-07T (=BCC 45622T=NBRC 109069T).

Functional analyses of putative PalS (Palindromic Self-recognition) motifs in bacterial Hfq

The bacterial protein Hfq has been linked to nucleic acid metabolism and signaling, however its explicit role has been elusive. Recently it was proposed that the C-termini of Hfq subunits in Hfq6 complexes could be involved in functional interactions with other Hfq hexamers and/or nucleic acids. To test the proposed model of the native Hfq complex experimentally, we genetically engineered chimeric Hfq6 complexes, in which C-termini of bacterial Hfq subunits were substituted with a sequence derived from human histone H2B (hH2B) that includes multiple functionally significant amino acids whose modifications have been linked to carcinogenesis. We demonstrate that this substitution results in an enhanced formation of dodecameric assemblies by the Hfq–hH2B hybrid – a result pointing to the possibility of a (functional) homology between these motifs in proteins from distant kingdoms. We hypothesize that these putative Palindromic Self-recognition (PalS) motifs could act as proteins' ‘cohesive ends’ that could allow the protein complexes carrying such motifs to interact dynamically and dissociate–reassociate in response to stress and/or growth phase-specific changes. We provide experimental support to the latter hypothesis and demonstrate that in E. coli the dodecameric Hfq assemblies are formed in a growth stage-specific manner. We describe a refined system – consisting solely of purified Hfq, polynucleotide phosporylase (PNP) and ADP – that allows reconstitution in vitro of characteristic ‘SDS-insensitive’ Hfq6–Hfq6 assemblies observed in experiments with whole-cell extracts obtained from exponentially-growing cells. We also optimized conditions for the extraction of intact native dodecameric Hfq complexes.

Immunoproteomic Analysis of Antibody Response of Rabbit Host Against Heat-Killed Francisella tularensis Live Vaccine Strain.

Francisella tularensis, the causative agent of tularemia, has attained the status of one of the high priority agents that could be used in the act of bioterrorism. Currently, there is no licensed vaccine for this highly infectious intracellular pathogen. Being a listed 'Category A' agent of the U.S. Center for Disease Control and Prevention (CDC), vaccines and therapeutics are immediately required against this pathogen. In this study, an immunoproteomic approach based on the techniques of 2-dimensional gel electrophoresis (2DE) and immunoblotting combined with mass spectrometry (MS) was used for elucidation of immunogenic components and putative vaccine candidates. Whole-cell soluble protein extract of F. tularensis LVS (Ft LVS) was separated by 2DE, and immunoblots were developed with sera raised in rabbit after immunization with heat-killed Ft LVS. A total of 28 immunoreactive proteins were identified by tandem mass spectrometry. Rabbit immunoproteome of F. tularensis was compared with those previously reported using sera from human patients and in murine model. Out of 28 immunoreactive proteins identified in this study, 12 and 17 overlapping proteins were recognized by human and murine sera, respectively. Nine proteins were found immunogenic in all the three hosts, while eight new immunogenic proteins were found in this study. Identified immunoreactive proteins may find application in design and development of protein subunit vaccine for tularemia.

Proteomic analysis of a carbapenem-resistant Klebsiella pneumoniae strain in response to meropenem stress.

Antibiotic resistance has become a major problem in treating bacterial infections. The aim of this study was to elucidate the effects of meropenem on a blaKPC-2-harbouring multidrug-resistant clinical strain of Klebsiella pneumoniae through a proteomics approach in order to attain a deeper understanding of bacterial resistance strategies. Analysis was performed by two-dimensional gel electrophoresis of whole-cell extracts of bacteria exposed to a sublethal concentration of meropenem compared with the untreated control. Differentially expressed proteins were identified by matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF). Based on Quantity One® software and MALDI-TOF analysis, 16 overexpressed proteins were identified in meropenem-treated bacteria. These proteins were primarily enzymes involved in defence against oxidative stress as well as glycolytic enzymes. LysM domain/BON superfamily protein was found overexpressed by >12-fold. STRING-10 was used to determine protein-protein interaction among the overexpressed proteins and to predict their functional associations. This study demonstrated that treatment with meropenem resulted in upregulation of various proteins involved in defence and repair mechanisms along with enzymes of energy metabolism. These overexpressed proteins may play an important role in bacterial resistance mechanisms against carbapenems, however their role in resistance needs to be further validated. High expression of lysine M domain/BON superfamily protein may indicate its possible involvement in modulating the bacterial response to antibiotic stress, but its actual role requires more investigation. These findings may also help in the development of newer therapeutic agents or diagnostic markers against carbapenem resistance.

RNA polymerase II components and Rrn7 form a preinitiation complex on the HomolD box to promote ribosomal protein gene expression in Schizosaccharomyces pombe

In Schizosaccharomyces pombe, ribosomal protein gene (RPG) promoters contain a TATA box analog, the HomolD box, which is bound by the Rrn7 protein. Despite the importance of ribosome biogenesis for cell survival, the mechanisms underlying RPG transcription remain unknown. In this study, we found that components of the RNA polymerase II (RNAPII) system, consisting of the initiation or general transcription factors (GTFs) TFIIA, IIB, IIE, TATA-binding protein (TBP) and the RNAPII holoenzyme, interacted directly with Rrn7 in vitro, and were able to form a preinitiation complex (PIC) on the HomolD box. PIC complex formation follows an ordered pathway on these promoters. The GTFs and RNAPII can also be cross-linked to HomolD-containing promoters in vivo. In an in vitro reconstituted transcription system, RNAPII components and Rrn7 were necessary for HomolD-directed transcription. The Mediator complex was required for basal transcription from those promoters in whole cell extract (WCE). The Med17 subunit of Mediator also can be cross-linked to the promoter region of HomolD-containing promoters in vivo, suggesting the presence of the Mediator complex on HomolD box-containing promoters. Together, these data show that components of the RNAPII machinery and Rrn7 participate in the PIC assembly on the HomolD box, thereby directing RPG transcription.

Chytridiomycosis of Marine Diatoms-The Role of Stress Physiology and Resistance in Parasite-Host Recognition and Accumulation of Defense Molecules.

Little is known about the role of chemotaxis in the location and attachment of chytrid zoospores to potential diatom hosts. Hypothesizing that environmental stress parameters affect parasite-host recognition, four chytrid-diatom tandem cultures (Chytridium sp./Navicula sp., Rhizophydium type I/Nitzschia sp., Rhizophydium type IIa/Rhizosolenia sp., Rhizophydium type IIb/Chaetoceros sp.) were used to test the chemotaxis of chytrid zoospores and the presence of potential defense molecules in a non-contact-co-culturing approach. As potential triggers in the chemotaxis experiments, standards of eight carbohydrates, six amino acids, five fatty acids, and three compounds known as compatible solutes were used in individual and mixed solutions, respectively. In all tested cases, the whole-cell extracts of the light-stressed (continuous light exposure combined with 6 h UV radiation) hosts attracted the highest numbers of zoospores (86%), followed by the combined carbohydrate standard solution (76%), while all other compounds acted as weak triggers only. The results of the phytochemical screening, using biomass and supernatant extracts of susceptible and resistant host-diatom cultures, indicated in most of the tested extracts the presence of polyunsaturated fatty acids, phenols, and aldehydes, whereas the bioactivity screenings showed that the zoospores of the chytrid parasites were only significantly affected by the ethanolic supernatant extract of the resistant hosts.

Proteomic Analysis of Chorion-Derived Mesenchymal Stem Cells: Combination of 2D Nano-HPLC in Tandem with ESI Mass Spectrometry

The human placenta plays an important role in foetus development and growth and is also well known as the reservoir of the mesenchymal stem cells (MSCs), which are characterized by significantly higher plasticity in comparison to adult stem cells as well as by the ability to differentiate into multiple cell types. Therefore, they are promising candidates for stem-cell therapy. To understand that mechanism the description of their proteomic profile is required. In this context, the goal of the present study was to isolate and analyse the proteome from chorionic membrane of the human placenta. The whole-cell protein extracts were analysed by LC MS/MS utilizing bottom-up proteomic approach. To maximize the number of identified proteins, two different nanoLC approaches were used. In the first approach only one-dimensional pre-concentration setup (C18 reverse-phase column) was used; whereas, the second approach utilized the two-dimensional LC in a salt plug setup (first dimension—SCX column and second dimension—C18 reverse-phase column). The method validation was accomplished using foetal membranes of six donors. Identification of 334, respectively, 500 proteins as part of chorionic MSCs proteome is reported here, supported by gene ontology classification and systematic protein family ordering. Complete characterization of chorionic stem-cell proteome profile will contribute to identification and understanding of the molecular pathways involved in the cell processes such as self-renewal, proliferation and differentiation.

Isolation of Newly Transcribed RNA Using the Metabolic Label 4-Thiouridine.

Isolation of newly transcribed RNA is an invaluable approach that can be used to study the dynamic life of RNA in cellulo. Traditional methods of whole-cell RNA extraction limit subsequent gene expression analyses to the steady-state levels of RNA abundance, which often masks changes in RNA synthesis and processing. This chapter describes a methodology with low cytotoxicity that permits the labeling and isolation of nascent pre-mRNA in cell culture. The resulting isolate is suitable for use in a series of downstream applications aimed at studying changes in RNA synthesis, processing, or stability.

Recombinant Sox Enzymes from Paracoccus pantotrophus Degrade Hydrogen Sulfide, a Major Component of Oral Malodor.

Hydrogen sulfide (H2S) is emitted from industrial activities, and several chemotrophs possessing Sox enzymes are used for its removal. Oral malodor is a common issue in the dental field and major malodorous components are volatile sulfur compounds (VSCs), including H2S and methyl mercaptan. Paracoccus pantotrophus is an aerobic, neutrophilic facultatively autotrophic bacterium that possesses sulfur-oxidizing (Sox) enzymes in order to use sulfur compounds as an energy source. In the present study, we cloned the Sox enzymes of P. pantotrophus GB17 and evaluated their VSC-degrading activities for the prevention of oral malodor. Six genes, soxX, soxY, soxZ, soxA, soxB, and soxCD, were amplified from P. pantotrophus GB17. Each fragment was cloned into a vector for the expression of 6×His-tagged fusion proteins in Escherichia coli. Recombinant Sox (rSox) proteins were purified from whole-cell extracts of E. coli using nickel affinity chromatography. The enzyme mixture was investigated for the degradation of VSCs using gas chromatography. Each of the rSox enzymes was purified to apparent homogeneity, as confirmed by SDS-PAGE. The rSox enzyme mixture degraded H2S in dose- and time-dependent manners. All rSox enzymes were necessary for degrading H2S. The H2S-degrading activities of rSox enzymes were stable at 25–80°C, and the optimum pH was 7.0. The amount of H2S produced by periodontopathic bacteria or oral bacteria collected from human subjects decreased after an incubation with rSox enzymes. These results suggest that the combination of rSox enzymes from P. pantotrophus GB17 is useful for the prevention of oral malodor.

Helicobacter pyloriAntigens Inducing Early Immune Response in Infants

To identify the Helicobacter pylori antigens operating during early infection in sera from infected infants using proteomics and immunoblot analysis. Two-dimensional (2D) large and small gel electrophoresis was performed using H. pylori strain 51. We performed 2D immunoglobulin G (IgG), immunoglobulin A (IgA), and immunoglobulin M (IgM) antibody immunoblotting using small gels on sera collected at the Gyeongsang National University Hospital from 4–11-month-old infants confirmed with H. pylori infection by pre-embedding immunoelectron microscopy. Immunoblot spots appearing to represent early infection markers in infant sera were compared to those of the large 2D gel for H. pylori strain 51. Corresponding spots were analyzed by matrix-assisted laser desorption/ionization time of flight-mass spectrometry (MALDI-TOF-MS). The peptide fingerprints obtained were searched in the National Center for Biotechnology Information (NCBI) database. Eight infant patients were confirmed with H. pylori infection based on urease tests, histopathologic examinations, and pre-embedding immunoelectron microscopy. One infant showed a 2D IgM immunoblot pattern that seemed to represent early infection. Immunoblot spots were compared with those from whole-cell extracts of H. pylori strain 51 and 18 spots were excised, digested in gel, and analyzed by MALDI-TOF-MS. Of the 10 peptide fingerprints obtained, the H. pylori proteins flagellin A (FlaA), urease β subunit (UreB), pyruvate ferredoxin oxidoreductase (POR), and translation elongation factor Ts (EF-Ts) were identified and appeared to be active during the early infection periods. These results might aid identification of serological markers for the serodiagnosis of early H. pylori infection in infants.

Cell cycle-regulated ubiquitination of tankyrase 1 by RNF8 and ABRO1/BRCC36 controls the timing of sister telomere resolution.

Timely resolution of sister chromatid cohesion in G2/M is essential for genome integrity. Resolution at telomeres requires the poly(ADP-ribose) polymerase tankyrase 1, but the mechanism that times its action is unknown. Here, we show that tankyrase 1 activity at telomeres is controlled by a ubiquitination/deubiquitination cycle depending on opposing ubiquitin ligase and deubiquitinase activities. In late S/G2 phase, the DNA damage-responsive E3 ligase RNF8 conjugates K63-linked ubiquitin chains to tankyrase 1, while in G1 phase such ubiquitin chains are removed by BRISC, an ABRO1/BRCC36-containing deubiquitinase complex. We show that K63-linked ubiquitin chains accumulate on tankyrase 1 in late S/G2 to promote its stabilization, association with telomeres, and resolution of cohesion. Timing of this posttranslational modification coincides with the ATM-mediated DNA damage response that occurs on functional telomeres following replication in G2. Removal of ubiquitin chains is controlled by ABRO1/BRCC36 and occurs as cells exit mitosis and enter G1, ensuring that telomere cohesion is not resolved prematurely in S phase. Our studies suggest that a cell cycle-regulated posttranslational mechanism couples resolution of telomere cohesion with completion of telomere replication to ensure genome integrity.

Modified filter-aided sample preparation (FASP) method increases peptide and protein identifications for shotgun proteomics.

Mass spectrometry (MS)-based protein identification depends mainly on protein extraction and digestion. Although sodium dodecyl sulfate (SDS) can preclude enzymatic digestion and interfere with MS analysis, it is still the most widely used surfactant in these steps. To overcome these disadvantages, a SDS-compatible proteomic technique for SDS removal prior to MS-based analyses was developed, namely filter-aided sample preparation (FASP). Herein, based on the effectiveness of sodium deoxycholate and a detergent removal spin column, we developed a modified FASP (mFASP) method and compared its overall performance, total number of peptides and proteins identified for shotgun proteomic experiments with that of the FASP method. Identification of 4570±392 and 9139±317 peptides and description of 862±46 and 1377±33 protein groups with two or more peptides from the ovarian cancer cell line A2780 was accomplished by FASP and mFASP methods, respectively. The mFASP method (21.2±0.2%) had higher average peptide to protein coverage than FASP method (13.2±0.5%). More hydrophobic peptides were identified by mFASP than by FASP, as indicated by the GRAVY score distribution. The reported method enables reliable and efficient identification of proteins and peptides in whole-cell extracts containing SDS. The new approach allows for higher throughput (the simultaneous identification of more proteins), a more comprehensive investigation of proteins, and potentially the discovery of new biomarkers. Copyright © 2016 John Wiley & Sons, Ltd.

Surface metabolites of the brown alga Taonia atomaria have the ability to regulate epibiosis

This study aimed to improve understanding of the strategies developed by the Mediterranean seaweed Taonia atomaria to chemically control bacterial epibiosis. An experimental protocol was optimized to specifically extract algal surface-associated metabolites by a technique involving dipping in organic solvents whilst the integrity of algal cell membranes was assessed by fluorescent microscopy. This methodology was validated using mass spectrometry-based profiles of algal extracts and analysis of their principal components, which led to the selection of methanol as the extraction solvent with a maximum exposure time of 15 s. Six compounds (A–F) were identified in the resulting surface extracts. Two of these surface-associated compounds (B and C) showed selective anti-adhesion properties against reference bacterial strains isolated from artificial surfaces while remaining inactive against epibiotic bacteria of T. atomaria. Such specificity was not observed for commercial antifouling biocides and other molecules identified in the surface or whole-cell extracts of T. atomaria.