Utility of the gas-phase sequencer for both liquid- and solid-phase degradation of proteins and peptides at low picomole levels

The utility of the commercially available gas-phase sequencer for complete analysis of peptide samples was investigated. Using the program supplied with the instrument, significant extractive loss of samples in Polybrene was observed, even at input levels up to 500 pmol. In order to reduce this loss, the sequencer program was modified by increasing the phenylisothiocyanate (PITC)-coupling steps from two to three and lengthening the duration of ethyl acetate (S2) delivery while reducing the delivery rate. These changes gave improved results with peptides, e.g., all eight residues of angiotensin II were identified at the 25-pmol level. In addition, background contamination was decreased and repetitive yields were increased. The instrument was also found to function well with samples coupled to solid supports; however, some of the methodologies that work adequately for covalent attachment of peptides to solid supports at the level 1-10 nmol were found to give unacceptable coupling/sequenceable yields at or below the 100-pmol level. The coupling methods tried were (1) reaction of homoserine lactone with aminopropyl (AP)-glass, (2) reaction of alpha- and epsilon-NH2 groups with p-phenylenediisothiocyanate (DITC)-glass, and (3) reaction of alpha-COOH groups with aminoaryl (AA)-glass via EDAC (1-ethyl-3,3'-dimethylaminopropyl-carbodiimide). Of these, the first method gave combined yields of 42-94% while the latter two were only 9-35% efficient. The covalently bound samples provided sequence information even at the resulting low levels, e.g., 9/13 residues of dynorphin including Lys-13 at 11 pmol. In general, sequencer runs on solid-phase samples gave "cleaner" analyses and slightly higher repetitive yields (1-2%). Sequence information has also been obtained on peptides made by solid-phase synthesis prior to cleavage from the polystyrene support. With improved coupling efficiencies, solid-phase techniques would provide an alternative to immobilization of peptides in Polybrene films for low picomole level gas-phase sequencing.     

Solution versus solid-phase cyclization strategies for large sidechain lactam-bridged peptides: A comparative study

A 22-residue peptide with a sidechain lactam bridge involving 18 residues (60-atom cycle) has been synthesized. Three different protection schemes using Fmoc/^tBu/cyclohexyl, Fmoc/^tBu/allyl or Boc/Bzl/ fluorenylmethyl protecting group combinations have been explored for the solid phase of the linear precursors, which have been subsequently cyclized in solution or in the solid phase. Cyclization yields in solution have been consistently better than on solid phase; however, the solid-phase strategy requires fewer purification steps and therefore global yields are comparable.     

收集挥发性样品的吸附剂法和固相微萃取法

本文介绍挥发性信息化学物质的两种收集方法——吸附剂收集法和固相微萃取收集法,以植物挥发性化学物质收集为例为例,详述了两种方法的原理、步骤、注意事项和实例,最后比较了两种收集方法的优缺点,供相关工作者使用时参考。     

A versatile modelling approach to determine the hydrophobicity of peptides at the atomic level

This study describes a versatile computational method to determine the hydrophobicity of small peptides at the atomic level. Free energies of transfer for individual atoms in peptide structures were derived, utilising two specifically defined parameters: (i) the water-excluding distance to define the dynamic interface between a peptide solute and its surrounding solvent and (ii) the corresponding hydrophobicity index as a relative measure for water occlusion/repulsion. The method was tested on a range of small peptide models (Ac-X-NH₂, G-X-G, Ac-WL-X-LL and Ac-GG-X-GG-NH₂) and several derivatives of these structures, whereby X was any of the 20 most common amino acids that naturally occur in polypeptides or proteins. The advantage of this new method lies in its versatility, ease to implement and capability to provide information on the hydrophobicity characteristics at the atomic level. The approach also encapsulates the impact of factors that influence these properties, but which have hitherto been difficult to accurately quantify, e.g. steric hindrance or proximity effects due to nearby polarised atoms. The method is not conditional on the knowledge of hydrophobicity parameters from the literature and does not require a sophisticated computer software/hardware to enable the atomic solvent-accessible surface areas or other hydrophobicity parameters to be de novo obtained.     

Methionine anchoring applied to the solid-phase synthesis of lysine-containing 'head-to-tail' cyclic peptides

Lysine-containing 'head-to-tail' cyclic peptides can be prepared via a side-chain anchoring solid-phase synthesis strategy. A handle is prepared using a methionine residue, the C -carboxylof which forms an amide with the N -amine of lysine. Subsequently, the linear peptide sequence is assembled, appropriatedeblocking steps are carried out, and on-resin head-to-tail cyclizationfollows. Optionally, acid-labile protecting groups may be removed while the peptide remains resin-bound. The final cleavage step uses CNBr, and releases the free or protected cyclic peptide into solution.     

Methionine anchoring applied to the solid-phase synthesis of lysine-containing 'head-to-tail' cyclic peptides

Summary Lysine-containing ‘head-to-tail’ cyclic peptides can be prepared via a side-chain anchoring solid-phase synthesis strategy. A handle is prepared using a methionine residue, theC ^α-carboxyl of which forms an, amide with theN ^ε-amine of lysine. Subsequently, the linear peptide sequence is assembled, appropriate deblocking steps are carried out, and on-resin head-to-tail cyclization follows. Optionally, acid-labile protecting groups may be removed while the peptide remains resin-bound. The final cleavage step uses CNBr, and releases the free or protected cyclic peptide into solution. Taken in part from the Ph.D. Thesis of J. C. Kappel, University of Minnesota, November 2003. Portions of this work were reported in preliminary form at the Eighteenth American Peptide Symposium, Boston, MA, U.S.A., 19–23 July 2003, and at the Eighth International Symposium on Solid Phase Synthesis and Combinatorial Chemical Libraries, London, England, U.K., 2–5 September 2003.     

A solid-phase assay for the quantitative analysis of hyaluronic acid at the nanogram level

A sensitive and accurate solid-phase assay for the quantitative determination of hyaluronic acid (HA) is described. The wells of the polystyrene microplates used were coated with glutaraldehyde followed, via a Schiff's base bond, with spermine to introduce amino groups. HA was added to the activated microwells in the presence of carbodiimide and left to bind via a peptide bond to the amino groups. Then aggrecan solution was added to the wells of the microtiter plates to interact with its G1 domain with hyaluronic acid, and the amounts of aggrecan bound were measured immunochemically. The inhibition of the binding between aggrecan and immobilized HA due to soluble HA present in reference solutions showed linearity in the range of concentrations 0.1 to 0.7 microg/ml. The reaction is specific and rapid and can be widely used for the calculation of HA in body fluids directly and in tissue samples after a brief digestion with a proteolytic enzyme.     

A solid-phase immunoassay for the binding of cartilage proteoglycan to hyaluronic acid

A solid-phase assay for detecting the binding of cartilage proteoglycan (PG) to hyaluronic acid (HA) is described. In the assay, HA is immobilized on protamine-treated microtiter wells, the wells are incubated with PG monomer and antibody to PG monomer, and then an ELISA system is used to detect binding of the PG to HA. The specificity of the assay is indicated by the failure to detect PG binding to chondroitin sulfate or albumin-coated microtiter wells, the absence of binding with tryptic fragments of PG monomer other than the HA-binding segment, the loss of binding after reduction and alkylation of PG monomer, and the inhibition of binding by preincubation of PG monomer with small amounts of HA. In contrast to the HA-PG interaction in solution, hyaluronidase digestion of HA does not affect its ability to inhibit the reaction of PG monomer with immobilized HA. The microtiter well-based assay appears to be a rapid, simple, and potentially versatile method for studying interactions with HA.     

A comparison of fluorescamine and o-phthaldialdehyde as effective blocking reagents in protein sequence analyses by the Beckman sequencer

Use of o-phthaldialdehyde to chemically reduce the newly generated amino termini responsible for the progressively increasing background during an extended amino acid sequence analysis in a liquid phase sequencer has been described. The results have been compared with Fluram blocking using apomyoglobin and rabbit C-reactive protein as standard and unknown samples, respectively.     

一种快速有效纯化DNA序列分析模板的方法

介绍一种ＤＮＡ序列分析模板的快速、有效的纯化方法。该法对ＤＮＡ模板的回收率可达９５％以上。多次测序结果表明,此法与其他常规纯化方法相比,具有简便、快速、有效、可靠等优点,其测序结果电泳带清晰,无模糊带及“鬼带”出现,重复性及稳定性较好。     

The cancellation effect at the group level

Group selection models combine selection pressure at the individual level with selection pressure at the group level. Cooperation can be costly for individuals, but beneficial for the group, and therefore, if individuals are sufficiently much assorted, and cooperators find themselves in groups with disproportionately many other cooperators, cooperation can evolve. The existing literature on group selection generally assumes that competition between groups takes place in a well-mixed population of groups, where any group competes with any other group equally intensely. Competition between groups however might very well occur locally; groups may compete more intensely with nearby than with far-away groups. We show that if competition between groups is indeed local, then the evolution of cooperation can be hindered significantly by the fact that groups with many cooperators will mostly compete against neighboring groups that are also highly cooperative, and therefore harder to outcompete. The existing empirical method for determining how conducive a group structured population is to the evolution of cooperation also implicitly assumes global between-group competition, and therefore gives (possibly very) biased estimates.     

A direct approach to the measurement of genetic variation in fish populations: applications of the polymerase chain reaction to studies of Atlantic cod, Gadus morhua L.

We used the polymerase chain reaction (PCR) and direct DNA sequencing to study genetic variation within and among populations of Atlantic cod, Gadus morhua , in the western North Atlantic. In a 307 bp region of the mitochondrial cytochrome b gene, 24 variable nucleotide positions define 24 genotypes, which differ by from one to six nucleotide substitutions. Greenland cod ( G. ogac ) differs from the most similar G. morhua genotype by an additional 12 nucleotide substitutions. Silent transitions dominate both intra- and interspecific comparisons, however four nucleotide substitutions within morhua result in amino acid replacements. Direct sequencing of DNA reveals substantially more of the genetic variation that exists within and between species than do previous indirect methods based on restriction fragment length polymorphisms, and thus has far greater potential to quantify such differences as may exist among fish stocks. Preliminary experiments also indicate that automation of DNA sequencing provides an efficient, rapid, and accurate means for detection of genetic variation in natural populations offish.     

A comparison of the nucleotide sequence of the A subunit of heat-labile enterotoxin and cholera toxin

Abstract The heat-labile enterotoxin genes from plasmid P307 have been cloned into pAT153. A comparison of the sequence of the LT-A gene with that of the A subunit of cholera toxin shows extensive homology. There are a number of significant differences between the sequence of the LT-A gene reported here and that published previously.     

Adaptation to salinity at the plant cell level

Summary Various mechanisms of adaptation of plant cells to salinity are reviewed: (1) protection of enzymes and maintenance of turgor by organic solutes; (2) prevention of ion toxicity by compartmentation; and (3) energization of solute transport by the proton pump. All these mechanisms seem to play a role in adaptation. The particular advantages of using salt-adapted cells in suspension culture to identify mechanisms of adaptation are pointed out.     

A microbial fuel cell operating at low pH using the acidophile Acidiphilium cryptum

For the first time, a microbial fuel cell has been developed using an acidophile, Acidiphilium cryptum, as the anode biocatalyst. Electricity production using its natural electron acceptor, iron, as the electron mediating agent at pH values < or =4.0 was demonstrated. Accumulation of Fe(III) at the electrode, however, restricted current output. The combination of nitrilotriacetic acid and Phenosafranin as electron mediators increased the power output to 12.7 mW/m(2) in a two-chamber air-sparged fuel cell. Direct electron transfer from the microorganisms to the anode was also investigated but was not detected under the conditions studied.     

A universal protocol to generate consensus level genome sequences for foot-and-mouth disease virus and other positive-sense polyadenylated RNA viruses using the Illumina MiSeq

Background

Next-Generation Sequencing (NGS) is revolutionizing molecular epidemiology by providing new approaches to undertake whole genome sequencing (WGS) in diagnostic settings for a variety of human and veterinary pathogens. Previous sequencing protocols have been subject to biases such as those encountered during PCR amplification and cell culture, or are restricted by the need for large quantities of starting material. We describe here a simple and robust methodology for the generation of whole genome sequences on the Illumina MiSeq. This protocol is specific for foot-and-mouth disease virus (FMDV) or other polyadenylated RNA viruses and circumvents both the use of PCR and the requirement for large amounts of initial template.

Results

The protocol was successfully validated using five FMDV positive clinical samples from the 2001 epidemic in the United Kingdom, as well as a panel of representative viruses from all seven serotypes. In addition, this protocol was successfully used to recover 94% of an FMDV genome that had previously been identified as cell culture negative. Genome sequences from three other non-FMDV polyadenylated RNA viruses (EMCV, ERAV, VESV) were also obtained with minor protocol amendments. We calculated that a minimum coverage depth of 22 reads was required to produce an accurate consensus sequence for FMDV O. This was achieved in 5 FMDV/O/UKG isolates and the type O FMDV from the serotype panel with the exception of the 5′ genomic termini and area immediately flanking the poly(C) region.

Conclusions

We have developed a universal WGS method for FMDV and other polyadenylated RNA viruses. This method works successfully from a limited quantity of starting material and eliminates the requirement for genome-specific PCR amplification. This protocol has the potential to generate consensus-level sequences within a routine high-throughput diagnostic environment.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-828) contains supplementary material, which is available to authorized users.     

A computational approach to studying ageing at the individual level

The ageing process is actively regulated throughout an organism''s life, but studying the rate of ageing in individuals is difficult with conventional methods. Consequently, ageing studies typically make biological inference based on population mortality rates, which often do not accurately reflect the probabilities of death at the individual level. To study the relationship between individual and population mortality rates, we integrated in vivo switch experiments with in silico stochastic simulations to elucidate how carefully designed experiments allow key aspects of individual ageing to be deduced from group mortality measurements. As our case study, we used the recent report demonstrating that pheromones of the opposite sex decrease lifespan in Drosophila melanogaster by reversibly increasing population mortality rates. We showed that the population mortality reversal following pheromone removal was almost surely occurring in individuals, albeit more slowly than suggested by population measures. Furthermore, heterogeneity among individuals due to the inherent stochasticity of behavioural interactions skewed population mortality rates in middle-age away from the individual-level trajectories of which they are comprised. This article exemplifies how computational models function as important predictive tools for designing wet-laboratory experiments to use population mortality rates to understand how genetic and environmental manipulations affect ageing in the individual.     

Data aggregation at the level of molecular pathways improves stability of experimental transcriptomic and proteomic data

High throughput technologies opened a new era in biomedicine by enabling massive analysis of gene expression at both RNA and protein levels. Unfortunately, expression data obtained in different experiments are often poorly compatible, even for the same biologic samples. Here, using experimental and bioinformatic investigation of major experimental platforms, we show that aggregation of gene expression data at the level of molecular pathways helps to diminish cross- and intra-platform bias otherwise clearly seen at the level of individual genes. We created a mathematical model of cumulative suppression of data variation that predicts the ideal parameters and the optimal size of a molecular pathway. We compared the abilities to aggregate experimental molecular data for the 5 alternative methods, also evaluated by their capacity to retain meaningful features of biologic samples. The bioinformatic method OncoFinder showed optimal performance in both tests and should be very useful for future cross-platform data analyses.