Comparison of DNA sizes in a group of giant <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> phages by the PFGE method

Genome sizes of Pseudomonas aeruginosa phages ?KZ and EL earlier determined by sequence analysis were shown to correspond to sizes of their DNAs assessed by pulse-electrophoresis (PFGE). Putative “redundant” genes in ?KZ phage genome are supposed to control functions promoting vigorous growth of the phage belonging to this species, compared to phages of EL species.     

The secreted salivary proteome of the pea aphid Acyrthosiphon pisum characterised by mass spectrometry

Nine proteins secreted in the saliva of the pea aphid Acyrthosiphon pisum were identified by a proteomics approach using GE‐LC‐MS/MS and LC‐MS/MS, with reference to EST and genomic sequence data for A. pisum. Four proteins were identified by their sequences: a homolog of angiotensin‐converting enzyme (an M2 metalloprotease), an M1 zinc‐dependant metalloprotease, a glucose‐methanol‐choline (GMC)‐oxidoreductase and a homolog to regucalcin (also known as senescence marker protein 30). The other five proteins are not homologous to any previously described sequence and included an abundant salivary protein (represented by ACYPI009881), with a predicted length of 1161 amino acids and high serine, tyrosine and cysteine content. A. pisum feeds on plant phloem sap and the metalloproteases and regucalcin (a putative calcium‐binding protein) are predicted determinants of sustained feeding, by inactivation of plant protein defences and inhibition of calcium‐mediated occlusion of phloem sieve elements, respectively. The amino acid composition of ACYPI009881 suggests a role in the aphid salivary sheath that protects the aphid mouthparts from plant defences, and the oxidoreductase may promote gelling of the sheath protein or mediate oxidative detoxification of plant allelochemicals. Further salivary proteins are expected to be identified as more sensitive MS technologies are developed.     

Phenogenetic characterization of a group of giant Phi KZ-like bacteriophages of Pseudomonas aeruginosa

A comparative study was made of a group of Pseudomonas aeruginosa virulent giant DNA bacteriophages similar to phage phi KZ in several genetic and phenotypic properties (particle size, particle morphology, genome size, appearance of negative colonies, high productivity, broad spectrum of lytic activity, ability to overcome the suppressing effect of plasmids, absence of several DNA restriction sites, capability of general transduction, pseudolysogeny). We have recently sequenced the phage phi KZ genome (288,334 bp) [J. Mol. Biol., 2002, vol. 317, pp. 1-19]. By DNA homology, the phages were assigned to three species (represented by phage phi KZ, Lin68, and EL, respectively) and two new genera (phi KZ and EL). Restriction enzyme analysis revealed the mosaic genome structure in four phages of the phi KZ species (phi KZ, Lin21, NN, and PTB80) and two phages of the EL species (EL and RU). Comparisons with respect to phage particle size, number of structural proteins, and the N-terminal sequences of the major capsid protein confirmed the phylogenetic relatedness of the phages belonging to the phi KZ genus. The origin and evolution of the phi KZ-like phages are discussed. Analysis of protein sequences encoded by the phage phi KZ genome made it possible to assume wide migration of the phi KZ-like phages (wandering phages) among various prokaryotes and possibly eukaryotes. Since the phage phi KZ genome codes for potentially toxic proteins, caution must be exercised in the employment of large bacteriophages in phage therapy.     

Phenogenetic Characterization of a Group of Giant φKZ-like Bacteriophages of Pseudomonas aeruginosa

A comparative study was made of a group ofPseudomonas aeruginosa virulent giant DNA bacteriophages similar to phage KZ in several genetic and phenotypic properties (particle size, particle morphology, genome size, appearance of negative colonies, high productivity, broad spectrum of lytic activity, ability to overcome the suppressing effect of plasmids, absence of several DNA restriction sites, capability of general transduction, pseudolysogeny). We have recently sequenced the phage KZ genome (288 334 bp) [J. Mol. Biol., 2002, vol. 317, pp. 1–19]. By DNA homology, the phages were assigned to three species (represented by phages KZ, Lin68, and EL, respectively) and two new genera (KZ and EL). Restriction enzyme analysis revealed the mosaic genome structure in four phages of the KZ species (KZ, Lin21, NN, and PTB80) and two phages of the EL species (EL and RU). Comparisons with respect to phage particle size, number of structural proteins, and the N-terminal sequences of the major capsid protein confirmed the phylogenetic relatedness of the phages belonging to the KZ genus. The origin and evolution of the KZ-like phages are discussed. Analysis of protein sequences encoded by the phage KZ genome made it possible to assume wide migration of the KZ-like phages (wandering phages) among various prokaryotes and possibly eukaryotes. Since the phage KZ genome codes for potentially toxic proteins, caution must be exercised in the employment of large bacteriophages in phage therapy.     

Proteomic analysis of parthenogenetic and in vitro fertilized porcine embryos

Proteomic data from embryos are essential for the completion of whole proteome catalog due to embryo‐specific expression of certain proteins. In this study, using reverse phase LC‐MS/MS combined with 1‐D SDS‐PAGE, we identified 1625 mammalian and 735 Sus scrofa proteins from porcine zygotes that included both cytosolic and membranous proteins. We also found that the global protein profiles of parthenogenetically activated (PA) and in vitro fertilized (IVF) zygotes were similar but differences in expression of individual proteins were also evident. These differences were not due to culture conditions, polyspermy or non‐activation of oocytes, as the same culture method was used in both groups, the frequency of polyspermy was 24.3±3.0% and the rates of oocyte activation did not differ (p>0.05) between PA and IVF embryos. Consistent with proteomic data, fluorescent Hoechst 33 342 staining and terminal deoxynucleotidyl transferase dUTP nick end labeling assay also revealed that PA embryos were of poor quality as they contained less cells per blastocyst and were more predisposed to apoptosis (p<0.05), although their in vitro development rates were similar. To our knowledge, this is the first report on global peptide sequencing and quantification of protein in PA and IVF embryos by LC‐MS/MS that may be useful as a reference map for future studies.     

Comparative proteomic analysis of human lung telocytes with fibroblasts

Telocytes (TCs) were recently described as interstitial cells with very long prolongations named telopodes (Tps; www.telocytes.com ). Establishing the TC proteome is a priority to show that TCs are a distinct type of cells. Therefore, we examined the molecular aspects of lung TCs by comparison with fibroblasts (FBs). Proteins extracted from primary cultures of these cells were analysed by automated 2‐dimensional nano‐electrospray ionization liquid chromatography tandem mass spectrometry (2D Nano‐ESI LC‐MS/MS). Differentially expressed proteins were screened by two‐sample t‐test (P < 0.05) and fold change (>2), based on the bioinformatics analysis. We identified hundreds of proteins up‐ or down‐regulated, respectively, in TCs as compared with FBs. TC proteins with known identities are localized in the cytoskeleton (87%) and plasma membrane (13%), while FB up‐regulated proteins are in the cytoskeleton (75%) and destined to extracellular matrix (25%). These identified proteins were classified into different categories based on their molecular functions and biological processes. While the proteins identified in TCs are mainly involved in catalytic activity (43%) and as structural molecular activity (25%), the proteins in FBs are involved in catalytic activity (24%) and in structural molecular activity, particularly synthesis of collagen and other extracellular matrix components (25%). Anyway, our data show that TCs are completely different from FBs. In conclusion, we report here the first extensive identification of proteins from TCs using a quantitative proteomics approach. Protein expression profile shows many up‐regulated proteins e.g. myosin‐14, periplakin, suggesting that TCs might play specific roles in mechanical sensing and mechanochemical conversion task, tissue homoeostasis and remodelling/renewal. Furthermore, up‐regulated proteins matching those found in extracellular vesicles emphasize TCs roles in intercellular signalling and stem cell niche modulation. The novel proteins identified in TCs will be an important resource for further proteomic research and it will possibly allow biomarker identification for TCs. It also creates the premises for understanding the pathogenesis of some lung diseases involving TCs.     

Protein recovery and identification from the gulf killifish, Fundulus grandis: comparing snap-frozen and RNAlater® preserved tissues

Reliable proteomic analysis of biological tissues requires sampling approaches that preserve proteins as close to their in vivo state as possible. In the current study, the patterns of protein abundance in one‐dimensional (1‐D) gels were assessed for five tissues of the gulf killifish, Fundulus grandis, following snap‐freezing tissues in liquid nitrogen or immersion of fresh tissues in RNAlater^®. In liver and heart, the protein profiles in 1‐D gels were better preserved by snap‐freezing, while in gill, the 1‐D protein profile was better preserved by immersion in RNAlater^®. In skeletal muscle and brain, the two approaches yielded similar patterns of protein abundance. LC‐MS/MS analyses and database searching resulted in the identification of 17 proteins in liver and 12 proteins in gill. Identified proteins include enzymes of energy metabolism, structural proteins, and proteins serving other biological functions. These protein identifications for a species without a sequenced genome demonstrate the utility of F. grandis as a model organism for environmental proteomic studies in vertebrates.     

Comparison of the genome for phylogenetically related bacteriophages ϕKZ and EL of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>: Evolutionary aspects and minimal genome size

Bacteriophages of the family Myoviridae represent one of the most widespread domains of the biosphere substantially affecting the ecological balance of microorganisms. Interestingly, sequence analysis of genomic DNAs of large bacteriophages revealed many genes coding for proteins with unknown functions. A new approach is proposed to improve the functional identification of genes. This approach is based on comparing the genome sequence for phylogenetically and morphologically related phages showing no considerable homology at the level of genomic DNA. It is assumed that gene functions essential for the development of phages of a given family are conserved and that the corresponding genes code for similar orthologous proteins even when lacking sequence homology. The genome was sequenced and compared for two Pseudomonas aeruginosa giant bacteriophages, KZ and EL, which belong to a group of KZ-related phages. A substantial difference in genome organization was observed, suggesting specific features of phage evolution. In addition, the problem of the minimal genome of the superfamily is discussed on the basis of the difference in size and structure between the KZ and EL genomes.__________Translated from Genetika, Vol. 41, No. 4, 2005, pp. 455–465.Original Russian Text Copyright © 2005 by Krylov, Pleteneva, Lavigne, Hertveldt, Volckaert, Sernova, Georgopoulos, Korchevskii, Kurochkina, Mesyanzhinov.     

Ser/Thr/Tyr phosphoproteome analysis of pathogenic and non‐pathogenic Pseudomonas species

Protein phosphorylation on serine, threonine, and tyrosine is well established as a crucial regulatory posttranslational modification in eukaryotes. With the recent whole‐genome sequencing projects reporting the presence of serine/threonine kinases and two‐component proteins both in prokaryotes and eukaryotes, the importance of protein phosphorylation in archaea and bacteria is gaining acceptance. While conventional biochemical methods failed to obtain a snapshot of the bacterial phosphoproteomes, advances in MS methods have paved the way for in‐depth mapping of phosphorylation sites. Here, we present phosphoproteomes of two ecologically diverse non‐enteric Gram‐negative bacteria captured by a nanoLC‐MS‐based approach combined with a novel phosphoenrichment method. While the phosphoproteome data from the two species are not very similar, the results reflect high similarity to the previously published dataset in terms of the pathways the phosphoproteins belong to. This study additionally provides evidence to prior observations that protein phosphorylation is common in bacteria. Notably, phosphoproteins identified in Pseudomonas aeruginosa belong to motility, transport, and pathogenicity pathways that are critical for survival and virulence. We report, for the first time, that motility regulator A, probably acting via the novel secondary messenger cyclic diguanylate monophosphate, significantly affects protein phosphorylation in Pseudomonas putida.     

Improving basic and membrane protein MS detection of the culture filtrate proteins from Mycobacterium tuberculosis H37Rv by biomimetic affinity prefractionation

The culture filtrate proteins (CFPs) from Mycobacterium tuberculosis have been shown to induce protective immune responses in human and animal models, making them a promising source of candidate targets for tuberculosis drugs, vaccines, and diagnostics. The constituents of the M. tuberculosis CFP proteome are complex and vary with growth conditions. To effectively profile CFPs, gel‐based prefractionation is usually performed before MS analysis. In this study, we describe a novel prefractionation approach by which the proteome is divided into seven partially overlapping fractions by biomimetic affinity chromatography (BiAC) using a six‐column cascade. The LC‐MS/MS analysis of individual fractions identified a total of 541 CFPs, including 61 first‐time identifications. Notably, ～1/3 (20/61) of these novel CFPs are membrane proteins, among which nine proteins have 2–14 transmembrane domains. In addition, ～1/4 (14/61) of the CFPs are basic proteins with pI values greater than 9.0. Our data demonstrate that biomimetic affinity chromatography prefractionation markedly improves protein detection by LC‐MS/MS, and the coverage of basic and hydrophobic proteins in particular is remarkably increased.     

Plant defense in response to chewing insects: proteome analysis of Arabidopsis thaliana damaged by Plutella xylostella

The interactions between Arabidopsis thaliana and Plutella xylostella have been considered as a model system to unravel the responses of plants to herbivorous insects. Here, we use a 2-DE proteome approach to detect protein expression changes in the leaves of Arabidopsis plants exposed to P. xylostella larval infestation at 27°C within 8?h. Approximately 450 protein spots were reproducibly detected on gels. Of these, comparing healthy and infested leaves, we identified 18 differentially expressed protein spots. Thirteen proteins were successfully identified by MALDI-TOF/MS and LC-ESI-MS/MS. Functional classification analysis indicated that the differentially identified proteins were associated with amino acid, carbohydrate, energy, lipid metabolism, and photosynthesis. In addition, their relative abundances were assessed according to larval pest feeding on Arabidopsis leaves. These data provide valuable new insights for further works in plant-biotic and environmental stress interaction.     

Proteomics of the milk fat globule membrane from Camelus dromedarius

Camel milk has been widely characterized with regards to casein and whey proteins. However, in camelids, almost nothing is known about the milk fat globule membrane (MFGM), the membrane surrounding fat globules in milk. The purpose of this study was thus to identify MFGM proteins from Camelus dromedarius milk. Major MFGM proteins (namely, fatty acid synthase, xanthine oxidase, butyrophilin, lactadherin, and adipophilin) already evidenced in cow milk were identified in camel milk using MS. In addition, a 1D‐LC‐MS/MS approach led us to identify 322 functional groups of proteins associated with the camel MFGM. Dromedary MFGM proteins were then classified into functional categories using DAVID (the Database for Annotation, Visualization, and Integrated Discovery) bioinformatics resources. More than 50% of MFGM proteins from camel milk were found to be integral membrane proteins (mostly belonging to the plasma membrane), or proteins associated to the membrane. Enriched GO terms associated with MFGM proteins from camel milk were protein transport (p‐value = 1.73 × 10^?14), translation (p‐value = 1.08 × 10^?11), lipid biosynthetic process (p‐value = 6.72 × 10^?10), hexose metabolic process (p‐value = 1.89 × 10^?04), and actin cytoskeleton organization (p‐value = 2.72 × 10^?04). These findings will help to contribute to a better characterization of camel milk. Identified MFGM proteins from camel milk may also provide new insight into lipid droplet formation in the mammary epithelial cell.     

Analysis of sesterterpenoids from Aspergillus terreus using ESI‐QTOF and ESI‐IT

Introduction – Biosynthesis of terretonin was studied due to the interesting skeleton of this series of sesterterpenoids. Very recently, López‐Gresa reported two new sesterterpenoids (terretonins E and F) which are inhibitors of the mammalian mitochondrial respiratory chain. Mass spectrometry (MS), especially tandem mass spectrometry, has been one of the most important physicochemical methods for the identification of trace natural products due to it rapidity, sensitivity and low levels of sample consumption. The potential application prospect and unique skeleton prompted us to study structural characterisation using MS. Objective – To obtain sufficient information for rapid structural elucidation of this class of compounds using MS. Methodology – The elemental composition of the product ions was confirmed by low‐energy ESI‐CID‐QTOF‐MS/MS analyses. The fragmentation pathways were postulated on the basis of ESI‐QTOF‐MS/MS/MS and ESI‐IT‐MSⁿ spectra. Common features and major differences between ESI‐QTOF‐MS/MS and IT‐MSⁿ spectra were compared. For ESI‐QTOF‐MS/MS/MS experiments, capillary exit voltage was raised to induce in‐source dissociation. Ammonium acetate or acetic acid were added into solutions to improve the intensity of [M + H]⁺. The collision energy was optimised to achieve sufficient fragmentation. Some fragmentation pathways were unambiguously proposed by the variety of abundance of fragment ions at different collision energies even without MSⁿ spectra. Results – Fragmentation pathways of five representative sesterterpenoids were elucidated using ESI‐QTOF‐MS/MS/MS and ESI‐IT‐MSⁿ in both positive‐ and negative‐ion mode. The key group of characterising fragmentation profiles was ring B, and these fragmentation patterns are helpful to identify different types of sestertepenoids. Conclusion – Complementary information obtained from fragmentation experiments of [M + H]⁺ (or [M + NH₄]⁺) and [M ? H]^? precursor ions is especially valuable for rapid identification of this kind of sesterterpenoid.     

Protein interactomics based on direct molecular fishing on paramagnetic particles: Practical realization and further SPR validation

There is increasing evidence that proteins function in the cell as integrated stable or temporally formed protein complexes, interactomes. Previously, using model systems we demonstrated applicability of direct molecular fishing on paramagnetic particles for protein interactomics (Ershov et al. Proteomics, 2012, 12, 3295). In the present study, we have used a combination of affinity‐based molecular fishing and subsequent MS for investigation of human liver proteins involved in interactions with immobilized microsomal cytochrome b5 (CYB5A), and also transthyretin and BSA as alternative affinity ligands (baits). The LC?MS/MS identification of prey proteins fished on these baits revealed three sets of proteins: 98, 120, and 220, respectively. Comparison analysis of these sets revealed only three proteins common for all the baits. In the case of paired analysis, the number of common proteins varied from 2 to 9. The binding capacity of some identified proteins has been validated by a SPR‐based biosensor. All the investigated proteins effectively interacted with the immobilized CYB5A (Kd values ranged from 0.07 to 1.1 μM). Results of this study suggest that direct molecular fishing is applicable for analysis of protein–protein interactions (PPI) under normal and pathological conditions, in which altered PPIs are especially important.     

Proteomic characterization of EL4 lymphoma‐derived tumors upon chemotherapy treatment reveals potential roles for lysosomes and caspase‐6 during tumor cell death in vivo

The murine mouse lymphoblastic lymphoma cell line (EL4) tumor model is an established in vivo apoptosis model for the investigation of novel cancer imaging agents and immunological treatments due to the rapid and significant response of the EL4 tumors to cyclophosphamide and etoposide combination chemotherapy. Despite the utility of this model system in cancer research, little is known regarding the molecular details of in vivo tumor cell death. Here, we report the first in‐depth quantitative proteomic analysis of the changes that occur in these tumors upon cyclophosphamide and etoposide treatment in vivo. Using a label‐free quantitative proteomic approach a total of 5838 proteins were identified in the treated and untreated tumors, of which 875 were determined to change in abundance with statistical significance. Initial analysis of the data reveals changes that may have been predicted, such as the downregulation of ribosomes, but demonstrates the robustness of the dataset. Analysis of the dataset also reveals the unexpected downregulation of caspase‐3 and an upregulation of caspase‐6 in addition to a global upregulation of lysosomal proteins in the bulk of the tumor.     

Growth condition‐dependent cell surface proteome analysis of Enterococcus faecium

The last 30 years Enterococcus faecium has become an important nosocomial pathogen in hospitals worldwide. The aim of this study was to obtain insight in the cell surface proteome of E. faecium when grown in laboratory and clinically relevant conditions. Enterococcus faecium E1162, a clinical blood stream isolate, was grown until mid‐log phase in brain heart infusion medium (BHI) with, or without 0.02% bile salts, Tryptic Soy Broth with 1% glucose (TSBg) and urine, and its cell surface was “shaved” using immobilized trypsin. Peptides were identified using MS/MS. Mapping against the translated E1162 whole genome sequence identified 67 proteins that were differentially detected in different conditions. In urine, 14 proteins were significantly more and nine proteins less abundant relative to the other conditions. Growth in BHI‐bile and TSBg, revealed four and six proteins, respectively, which were uniquely present in these conditions while two proteins were uniquely present in both conditions. Thus, proteolytic shaving of E. faecium cells identified differentially surface exposed proteins in different growth conditions. These proteins are of special interest as they provide more insight in the adaptive mechanisms and may serve as targets for the development of novel therapeutics against this multi‐resistant emerging pathogen. All MS data have been deposited in the ProteomeXchange with identifier PXD002497 ( http://proteomecentral.proteomexchange.org/dataset/PXD002497 ).     

Proteomic profiling of Cronobacter turicensis 3032, a food‐borne opportunistic pathogen

Members of the genus Cronobacter are opportunistic pathogens for neonates and are often associated with contaminated milk powder formulas. At present little is known about the virulence mechanisms or the natural reservoir of these organisms. The proteome of Cronobacter turicensis 3032, which has recently caused two deaths, was mapped aiming at a better understanding of physiology and putative pathogenic traits of this clinical isolate. Our analyses of extracellular, surface‐associated and whole‐cell proteins by two complementary proteomics approaches, 1D‐SDS‐PAGE combined with LC‐ESI‐MS/MS and 2D‐LC‐MALDI‐TOF/TOF MS, lead to the identification of 832 proteins corresponding to a remarkable 19% of the theoretically expressed protein complement of C. turicensis. The majority of the identified proteins are involved in central metabolic pathways, translation, protein folding and stability. Several putative virulence factors, whose expressions were confirmed by phenotypic assays, could be identified: a macrophage infectivity potentiator involved in C. turicensis persistence in host cells, a superoxide dismutase protecting the pathogen against reactive oxygen species and an enterobactin‐receptor protein for the uptake of siderophore‐bound iron. Most interestingly, a chitinase and a metalloprotease that might act against insects and fungi but no casein hydrolysing enzymes were found, suggesting that there is an environmental natural habitat of C. turicensis 3032.     

Proteomic characterization of seeds from yellow lupin (Lupinus luteus L.)

Yellow lupin (Lupinus luteus L.) is a legume crop containing a large amount of protein in its seeds. In this study, we constructed a seed‐protein catalog to provide a foundation for further study of the seeds. A total of 736 proteins were identified in 341 2DE spots by nano‐LC‐MS/MS. Eight storage proteins were found as multiple spots in the 2DE gels. The 736 proteins correspond to 152 unique proteins as shown by UniRef50 clustering. Sixty‐seven of the 152 proteins were associated with KEGG‐defined pathways. Of the remaining proteins, 57 were classified according to a GO term. The functions of the remaining 28 proteins have yet to be determined. This is the first yellow lupin seed–protein catalog, and it contains considerably more data than previously reported for white lupin (L. albus L.).