Extrachromosomal circular DNAs in Drosophila melanogaster: Comparison between embryos and Kc0% cells

We established the size distribution of extrachromosomal covalently closed circular DNA molecules from embryos of various Drosophila melanogaster strains and from Kc0% tissue culture cells. In embryos, more than 80% of the circular DNA molecules are smaller than 2.5 kb and all the distributions show a peak of molecules of between 200 and 400 bp. The Kc0% cell distribution differs mainly from that of embryos in that 48% of the molecules have a size between 4 and 8 kb. Correlating with this, circular molecules homologous to copia, 412 and 297 were detected only in Kc0% cells. The three tandemly repeated families containing the 5S genes, the histone genes and the 240 bp repeat of the ribosomal DNA intergenic spacer, which had previously been identified in circular DNAs from embryos, were also found in cultured cells. A fourth tandemly repeated family corresponding to the 1.688 g/cm³ satellite DNA was detected, both in embryos and Kc0% cells. It consists of circular multimeric molecules containing multiple copies of the 359 bp repeated unit. No circular DNA molecules homologous to the actin genes, the type I ribosomal DNA insertion, or the F and I transposable elements were found in embryos or Kc0% cells. Thus it appears that the extrachromosomal circular DNA molecules from embryos and from tissue culture cells differ mainly in the presence of circular copies of the copia-like transposable elements.     

cDNA clones for the ecdysone-inducible polypeptide (EIP) mRNAs of Drosophila Kc cells

The ecdysone-inducible polypeptides (EIPs) 28, 29 and 40 were identified previously as polypeptides whose synthesis is stimulated early in the ecdysone response of Drosophila Kc cells. We have now shown, using two-dimensional gels, that each of these EIPs consists of three species differing in pI, and all stimulated by ecdysone. Translations and hybrid-arrested translations indicated that the poly(A)⁺ EIP mRNAs increase ˜10-fold in abundance during the first 4 h of ecdysone treatment. By a differential screen of a cDNA library we have identified cDNA clones corresponding to all three EIPs. Two kinds of clones were isolated: one hybridizes to the EIP 40 mRNA(s); the second hybridizes to the mRNA(s) encoding all the EIPs 28 and 29. The EIP 28/29 and EIP 40 loci detected by these clones are each present at single sites on the polytene chromosomes and each is at or in the vicinity of an ecdysone-regulated puff.     

An integrated computational workflow for efficient and quantitative modeling of renin inhibitors

A new integrated computational workflow that couples the strength of the molecular overlay methods to achieve rapid and automated alignments along with 3D-QSAR techniques like CoMFA and CoMSIA for quantitative binding affinity prediction is presented. The results obtained from such techniques are compared with rule-based Topomer CoMFA method, where possible. The developed 3D-QSAR models were prospectively used to predict the affinities of new compounds designed through R-group deconvolution starting from the core chemical scaffold and subsequent virtual combinatorial library enumeration. The general applicability of the seamless in silico modeling workflow is demonstrated using several datasets reported for small molecule inhibitors of renin.     

An integrated, directed mass spectrometric approach for in-depth characterization of complex peptide mixtures

LC-MS/MS has emerged as the method of choice for the identification and quantification of protein sample mixtures. For very complex samples such as complete proteomes, the most commonly used LC-MS/MS method, data-dependent acquisition (DDA) precursor selection, is of limited utility. The limited scan speed of current mass spectrometers along with the highly redundant selection of the most intense precursor ions generates a bias in the pool of identified proteins toward those of higher abundance. A directed LC-MS/MS approach that alleviates the limitations of DDA precursor ion selection by decoupling peak detection and sequencing of selected precursor ions is presented. In the first stage of the strategy, all detectable peptide ion signals are extracted from high resolution LC-MS feature maps or aligned sets of feature maps. The selected features or a subset thereof are subsequently sequenced in sequential, non-redundant directed LC-MS/MS experiments, and the MS/MS data are mapped back to the original LC-MS feature map in a fully automated manner. The strategy, implemented on an LTQ-FT MS platform, allowed the specific sequencing of 2,000 features per analysis and enabled the identification of more than 1,600 phosphorylation sites using a single reversed phase separation dimension without the need for time-consuming prefractionation steps. Compared with conventional DDA LC-MS/MS experiments, a substantially higher number of peptides could be identified from a sample, and this increase was more pronounced for low intensity precursor ions.     

Highly robust, automated, and sensitive online TiO2-based phosphoproteomics applied to study endogenous phosphorylation in Drosophila melanogaster

Reversible protein phosphorylation ranks among the most important post-translational modifications, and elucidation of phosphorylation sites is essential to understand the regulation of key cellular processes such as signal transduction. Enrichment of phosphorylated peptides is a prerequisite for successful analysis due to their low stoichiometry, heterogeneity, and low abundance. Enrichment is often performed manually, which is inherently labor-intensive and a major hindrance in large-scale analyses. Automation of the enrichment method would vastly improve reproducibility and thereby facilitate 'high-throughput' phosphoproteomics research. Here, we describe a robust and automated online TiO 2-based two-dimensional chromatographic approach to selectively enrich phosphorylated peptides from digests of complete cellular lysates. We demonstrate method enhancement for both adsorption and desorption of phosphorylated peptides resulting in lower limits of detection. Phosphorylated peptides from a mere 500 attomole tryptic digest of a protein mixture were easily detected. With the combination of strong cation exchange chromatography with the online TiO 2 enrichment, 2152 phosphopeptides were enriched from 250 microg of protein originating for the cell lysate of Drosophila melanogaster S2 cells. This is a 4-fold improvement when compared to an enrichment strategy based solely on strong cation exchange/LC-MS. Phosphopeptide enrichment methods are intrinsically biased against relatively basic phosphopeptides. Analysis of the p I distributions of the enriched/detected phosphopeptides showed that the p I profile resembles that of a total Drosophila protein digest, revealing that the current described online procedure does not discriminate against either more acidic or basic phosphopeptides. However, careful comparison of our new and existing phosphopeptide enrichment techniques also reveal that, like many enrichment techniques, we are still far from comprehensive phosphoproteomics analyses, and we describe several factors that still require to be addressed. Still, as the online approach allows the complementary measurements of phosphopeptides and their nonphosphorylated counterparts in subsequent analyses, this method is well-suited for automated quantitative phosphoproteomics.     

An accurate mass tag strategy for quantitative and high-throughput proteome measurements

We describe and demonstrate a global strategy that extends the sensitivity, dynamic range, comprehensiveness, and throughput of proteomic measurements based upon the use of peptide "accurate mass tags" (AMTs) produced by global protein enzymatic digestion. The two-stage strategy exploits Fourier transform-ion cyclotron resonance (FT-ICR) mass spectrometry to validate peptide AMTs for a specific organism, tissue or cell type from "potential mass tags" identified using conventional tandem mass spectrometry (MS/MS) methods, providing greater confidence in identifications as well as the basis for subsequent measurements without the need for MS/MS, and thus with greater sensitivity and increased throughput. A single high resolution capillary liquid chromatography separation combined with high sensitivity, high resolution and accurate FT-ICR measurements has been shown capable of characterizing peptide mixtures of significantly more than 10(5) components with mass accuracies of < 1 ppm, sufficient for broad protein identification using AMTs. Other attractions of the approach include the broad and relatively unbiased proteome coverage, the capability for exploiting stable isotope labeling methods to realize high precision for relative protein abundance measurements, and the projected potential for study of mammalian proteomes when combined with additional sample fractionation. Using this strategy, in our first application we have been able to identify AMTs for >60% of the potentially expressed proteins in the organism Deinococcus radiodurans.     

Tyrosine phosphoproteomics and identification of substrates of protein tyrosine phosphatase dPTP61F in Drosophila S2 cells by mass spectrometry-based substrate trapping strategy

Recent biochemical and genetic approaches have clearly defined the functional role of critical components in tyrosine phosphorylation-dependent signal transduction. These signaling modulators often exhibit evolutionarily conserved functions across various species. It has been proposed that if protein tyrosine kinases (PTKs), protein tyrosine phosphatases (PTPs), and thousands of their substrates could be identified and characterized, it would significantly advance our understanding of the underlying mechanisms that control animal development and physiological homeostasis. The fruit fly Drosophila melanogester has been used extensively as a model organism for investigating the developmental processes, but the state of its tyrosine phosphorylation is poorly characterized. In the current study, we used advanced mass spectrometry (MS)-based shotgun analyses to profile the tyrosine phosphoproteome of Drosophila S2 cells. Using immunoaffinity isolation of the phosphotyrosine (pTyr) subproteome from cells treated with pervanadate followed by enrichment of phosphopeptides, we identified 562 nonredundant pTyr sites in 245 proteins. Both this predefined pTyr proteome subset and the total cell lysates were then used as sample sources to identify potential substrates of dPTP61F, the smallest member in terms of amino acid number and molecular weight in the Drosophila PTP family and the ortholog of human PTP1B and T Cell-PTP, by substrate trapping. In total, 20 unique proteins were found to be specifically associated with the trapping mutant form of dPTP61F, eluted by vanadate (VO4(3-)), and identified by MS analyses. Among them, 16 potential substrates were confirmed as tyrosine phosphorylated proteins, including a receptor PTK PDGF/VEGF receptor, a cytosolic PTK Abl, and several components of SCAR/WAVE complex, which may work in coordination to control actin dynamics. Thus, our data suggest that dPTP61F plays a central role in counteracting PTK-mediated signaling pathways as well as in regulating actin reorganization and remodeling through tyrosine dephosphorylation of critical substrates in Drosophila cells.     

A new trap and lure for Drosophila melanogaster (Diptera: Drosophilidae)

We conducted a series of nine laboratory experiments testing the response of "vinegar flies," Drosophila melanogaster Meigen (Diptera: Drosophilidae), released in bioassay chambers to experimental traps and lures. These experiments showed that an effective trap could be constructed from a clear 225-ml screw-cap jar fitted with a hollow 8-mm-diameter cylindrical cross bridge. Flies could enter the trap from either end of the cylindrical "gate" and in turn could enter the interior chamber of the trap through a cut out portion at mid-span of the cylinder. The experiments also showed that a natural-component lure could be made using a teabag containing freeze-dried banana powder, yeast, and carrageenan gum powder as a humectant. When dipped in water for 10-15 s and then placed in the bottom of a trap, the teabag provided effective attraction for at least 7 d. Captured flies were immobilized on a sticky card placed in the trap, allowing them to be easily seen. Unlike other traps that cannot be opened and have liquid lures, the cylindrical-gate trap can be reused repeatedly if the teabag and sticky card are replaced. A final two experiments showed that the prototype operational cylindrical-gate trap with a teabag lure captured 3.3 and 2.3 times more released flies, respectively, than the next best of three commercially available traps.     

Chromatographic and mass spectrometric methods for quantitative determination of 3-nitrotyrosine in biological samples and their application to human samples

The permanent modification of soluble and protein-associated tyrosine by nitration results in the formation of 3-nitrotyrosine, which can be used as a marker of "nitro-oxidative" damage to proteins. Based on the analysis of patient materials, over 40 different diseases and/or conditions have been linked to increased nitration of tyrosine. They include many cardiovascular diseases, conditions associated with immunological reactions and neurological diseases. In this article we review the existing chromatographic and mass spectrometric methods for quantitative measurements of 3-nitrotyrosine in different human biological samples including plasma, either from the free amino acid pool or from hydrolyzed proteins from different matrices.     

Selection on viability of individuals heterozygous for the temperature-sensitive lethal mutation l(2)M167(DTS) in experimental populations of Drosophila melanogaster

In experiments on introduction of mutation l(2)M167(DTS) in Drosophila melanogaster populations, larval and pupal viability and developmental rate are limiting factors determining the intensity of selection on the l(2)M167(DTS) mutation. Notwithstanding the rapid elimination of the mutation from the population, positive selection for viability was shown, which increased fitness of the mutation carriers in generations. The fitness component viability was estimated in individuals l(2)M167(DTS)/+; relative to that of wild-type individuals, it varied from 0.1 to 1. Factors affecting this trait in overcrowded populations were found.     

Synthetic sulfogalactosylceramide (sulfatide) and its use for the mass spectrometric quantitative urinary determination in metachromatic leukodystrophies

3-O-Sulfogalactosylceramides (sulfatides) accumulate in the genetic disease metachromatic leukodystrophy which is due to a defect in the catabolic enzyme, arylsulfatase A. Clinical diagnosis is usually confirmed by in vitro enzymatic deficiency of arylsulfatase A activity. The diagnosis may be complicated because of arylsulfatase A pseudo-deficiencies and another cause of MLD, sphingolipid activator B deficiency. As large quantities of sulfatides can be found in the urine in this disease, sulfatiduria appears as an extremely useful test. As recently enzyme replacement is underway, the quantitative determination, using an internal standard, appears particularly useful as a follow-up. Thus a non-physiological sulfatide was synthesized for this purpose, i.e. 3-O-sulfo-β-d-C17 galactosylceramide (3-O-Sulfo-d-Galactosyl-β1′→1-N-Heptadecanoyl-d-erythro-Sphingosine). It has been prepared through condensation of an azidosphingosine derivative with a protected d-galactopyranosyltrichloroacetimidate. Reduction of the azide was followed by acylation of a C-17 fatty acid. The key step was achieved by selective sulfation of the desired hydroxyl group on the sugar residue of the galactosylceramide using the stannylene methodology to give a 3′-sulfated beta-galactosyl C-17 ceramide. An erratum to this article can be found at     

RNAi screening of Drosophila (Sophophora) melanogaster S2 cells for ricin sensitivity and resistance

The ribosome-inhibiting toxin ricin binds exposed β1→4 linked galactosyls on multiple glycolipids and glycoproteins on the cell surface of most eukaryotic cells. After endocytosis, internal cell trafficking is promiscuous, with only a small proportion of ricin proceeding down a productive (cytotoxic) trafficking route to the endoplasmic reticulum (ER). Here, the catalytic ricin A chain traverses the membrane to inactivate the cytosolic ribosomes, which can be monitored by measuring reduction in protein biosynthetic capacity or cell viability. Although some markers have been discovered for the productive pathway, many molecular details are lacking. To identify a more comprehensive set of requirements for ricin intoxication, the authors have developed an RNAi screen in Drosophila S2 cells, screening in parallel the effects of individual RNAi treatments alone and when combined with a ricin challenge. Initial screening of 806 gene knockdowns has revealed a number of candidates for both productive and nonproductive ricin trafficking, including proteins required for transport to the Golgi, plus potential toxin interactors within the ER and cytosol.     

The application of robotics and mass spectrometry to the characterisation of the Drosophila melanogaster indirect flight muscle proteome

Summary The rapid accumulation of sequence data generated by the various genome sequencing projects and the generation of expressed sequence tag databases has resulted in the need for the development of fast and sensitive methods for the identification and characterisation of large numbers of gel electrophoretically separated proteins to translated the sequence data into biological function. To achieve this goal it has been necessary to devise new approaches to protein analysis: matrix-assisted laser desorption and electrospray mass spectrometry have become important protein analytical tools which are both fast and sensitive. When combined with a robotic system for the in-gel digestion of electrophoretically separated proteins, it becomes possible to rapidly identify many proteins by searching databases with MS data. The power of this combination of techniques is demonstrated by an analysis of the proteins present in the myofibrillar lattice of the indirect flight muscle ofDrosophila melanogaster. The proteins were separated by SDS-PAGE and in-gel proteolysis was performed both automatically and manually. All 16 major proteins could quickly be identified by mass spectrometry. Although most of the protein components were known to be present in the flight muscle, two new components were also identified. The combination of methods described offers a means for the rapid identification of large numbers of gel separated proteins.     

The application of robotics and mass spectrometry to the characterisation of the Drosophila melanogaster indirect flight muscle proteome

The rapid accumulation of sequence data generated by the various genome sequencingprojects and the generation of expressed sequence tag databases has resulted in the need forthe development of fast and sensitive methods for the identification and characterisation oflarge numbers of gel electrophoretically separated proteins to translate the sequence data intobiological function. To achieve this goal it has been necessary to devise new approaches toprotein analysis: matrix-assisted laser desorption and electrospray mass spectrometry havebecome important protein analytical tools which are both fast and sensitive. When combinedwith a robotic system for the in-gel digestion of electrophoretically separated proteins, itbecomes possible to rapidly identify many proteins by searching databases with MS data. Thepower of this combination of techniques is demonstrated by an analysis of the proteins presentin the myofibrillar lattice of the indirect flight muscle of Drosophila melanogaster. Theproteins were separated by SDS-PAGE and in-gel proteolysis was performed bothautomatically and manually. All 16 major proteins could quickly be identified by massspectrometry. Although most of the protein components were known to be present in theflight muscle, two new components were also identified. The combination of methodsdescribed offers a means for the rapid identification of large numbers of gel separatedproteins.     

Liquid chromatographic–tandem mass spectrometric method for the determination of the neuraminidase inhibitor zanamivir (GG167) in human serum

An LC–MS–MS method for the analysis of the neuraminidase inhibitor, zanamivir, in human serum is described. Zanamivir was extracted from protein precipitated human serum samples using Isolute SCX solid-phase extraction cartridges and analysed using reversed-phase chromatography with TurboIonSpray atmospheric pressure ionisation followed by mass spectrometric detection. The method uses a stable isotope internal standard, is highly specific and sensitive for a compound of this type and has been used for the analysis of human serum and urine samples from clinical studies. The method was extended to the analysis of serum and plasma samples from pre-clinical studies involving the rat, ferret and cell culture media. The method has been shown to be robust and valid over a concentration range of 10–5000 ng/ml using a 0.2-ml sample volume. The main advantages of this method compared to earlier procedures are primarily specificity, sensitivity, ease of sample preparation, small sample volume and short analysis time (ca. 5 min).