Assessment of protein spot components applying correspondence analysis for peptide mass fingerprint data

Proteins separated by two-dimensional gel electrophoresis (2-DE) may be distributed over several spots. Otherwise, one spot may contain more than one component. The same protein occurring in several spots supposedly represents differently modified protein species that might be of biological relevance. Identification of spots with peptide mass fingerprinting and database searching leads only to the detection of the major spot components. If a spot also contains additional minor protein components, quantitation of spots with protein staining techniques or antibody detection becomes misleading. In order to find spots containing minor components we applied correspondence analysis, a multivariate data exploration method, to peptide mass fingerprint data. Correspondence analysis using peak lists revealed groups of spots containing the same protein with their characteristic mass-to-charge ratio (m/z) values. In order to detect different protein spot components an interactive threshold setting and removal of m/z values with subsequent recalculation of the correspondence analysis using our software tool CorrAn are performed. The usefulness of this methodical approach was shown by a data set of peptide mass fingerprints of 284 spots of Helicobacter pylori 26695 separated by 2-DE.     

Rapid and automatic on-plate desalting protocol for MALDI-MS: using imprinted hydrophobic polymer template

A novel protocol of rapid and automatic on-plate desalting (OPD) and peptide concentration for 2-DE-MALDI-MS has been developed by the approach of templating the hydrophobic polymer solution over Kapton-etched mask. For the template technique, small hydrophobic polymer [linear poly(methyl methacrylate) (PMMA), PMMA derivatized with fullerene-C60 (PMMA-C60), linear polystyrene (PSt), or PSt derivatized with fullerene-C60 (PSt-C60)] spots (990 microm od) are patterned at the centers of stainless MALDI plate wells (1400 microm id). Tryptic-peptide solution with no predesalting was dropped onto the central hydrophobic spots, resulting in a concentration of proteolytic peptides on the hydrophobic polymer surface with a reduced spot size. The dried peptide layer was then covered subsequently with over-volume matrix solution, causing the removal of redissolved salts from the spot center to the spot edge by means of a natural "outward flow." The proposed OPD protocol exhibited a dramatic enhancement in S/N up to 850 for 14 fmol BSA digests in the coexistence of 100 mM salts, compared with barely detectable peaks in ordinary way. This analysis has shown that the success rate of identification was increased by two-fold for low abundance proteins in the human liver tissue with no need for the conventional ZipPlate desalting strategy.     

Deposition of Bacillus subtilis spores using an airbrush-spray or spots to study surface decontamination by pulsed light

Microbial contamination on surfaces of food processing equipment is a major concern in industries. A new method to inoculate a single-cell layer (monolayer) of microorganisms onto polystyrene was developed, using a deposition with an airbrush. A homogeneous dispersion of Bacillus subtilis DSM 402 spores sprayed on the surface was observed using both plate count and scanning electron microscopy. No clusters were found, even with high spore concentrations (10⁷ spores/inoculated surface). A monolayer of microorganisms was also obtained after deposition of 10 μL droplets containing 3 × 10⁴ spores/spot on polystyrene disks, but not with a higher spore concentration. Pulsed light (PL) applied to monolayers of B. subtilis spores allowed log reductions higher than 6. As a consequence of clusters formation in spots of 10 μL containing more than 3 × 10⁵ spores, log reductions obtained by PL were significantly lower. The comparative advantages of spot and spray depositions were discussed.     

Natural meiotic recombination hot spots in the Schizosaccharomyces pombe genome successfully predicted from the simple sequence motif M26

The M26 hot spot of meiotic recombination in Schizosaccharomyces pombe is the eukaryotic hot spot most thoroughly investigated at the nucleotide level. The minimum sequence required for M26 activity was previously determined to be 5'-ATGACGT-3'. Originally identified by a mutant allele, ade6-M26, the M26 heptamer sequence occurs in the wild-type S. pombe genome approximately 300 times, but it has been unclear whether any of these are active hot spots. Recently, we showed that the M26 heptamer forms part of a larger consensus sequence, which is significantly more active than the heptamer alone. We used this expanded sequence as a guide to identify a smaller number of sites most likely to be active hot spots. Ten of the 15 sites tested showed meiotic DNA breaks, a hallmark of recombination hot spots, within 1 kb of the M26 sequence. Among those 10 sites, one occurred within a gene, cds1(+), and hot spot activity of this site was confirmed genetically. These results are, to our knowledge, the first demonstration in any organism of a simple, defined nucleotide sequence accurately predicting the locations of natural meiotic recombination hot spots. M26 may be the first example among a diverse group of simple sequences that determine the distribution, and hence predictability, of meiotic recombination hot spots in eukaryotic genomes.     

Analysis of cyclosporin A in dried blood spots using liquid chromatography tandem mass spectrometry

Dried blood spot sampling is a promising and patient friendly alternative for venous sampling. A liquid chromatography tandem mass spectrometer assay was developed for analyzing cyclosporin A in dried blood spots. Linearity ranged from 25 to 1440 μg/L. Within and between run accuracy and precision were within limits. The developed assay has a negligible matrix-effect and a recovery of 97%. The dried blood spots were stable during a period of at least 17 days in the refrigerator. The developed assay is suitable for analyzing cyclosporin A in dried blood spots.     

Molecular dynamic simulations study of the effect of salt valency on structure and thermodynamic solvation behaviour of anionic polyacrylate PAA in aqueous solutions

The effect of salt concentration and valency on intermolecular structure and solvation thermodynamic properties of aqueous solution containing polyacrylicacid (PAA) chains and multi-valent salts calcium chloride (CaCl₂) and aluminium chloride (AlCl₃) as a function of charge density was investigated using atomistic molecular dynamic simulations with explicit solvent. Salt-free solution favours the self-association of uncharged (acidic form) PAA chains facilitated by inter-chain hydrogen bonds. The ionised (charged) PAA chains are not associated in salt-free aqueous solutions and undergo self-association in the salt solutions due to bridging effect induced by condensed salt ions in agreement with scattering investigations available in literature. The collapse behaviour of PAA in presence of CaCl₂ and re-expansion behaviour of PAA chains in case of AlCl₃ salt solutions are observed. The rigidity of PAA chains decrease with increase in salt concentration, in agreement with experimental results available in literature. The trivalent salt favours relatively the greater extent of shrinking of PAA chains as well as inter-chain interactions as compared to divalent salts as evident from radius-of-gyration, H-bond and pair-wise solvation enthalpy data. The conformation and hydration behaviour of the acid form of PAA chains are not significantly altered by added salt ions. The hydration behaviour of ionised PAA chains is significantly reduced by added salts due to screening effect of the condensed salt ions. The pair correlation functions of solutions species such as Ca²⁺, Al³⁺, Na⁺ and Cl^? with respect to PAA oxygen show the greater affinity of PAA units with the higher valency Al³⁺ ions over Ca²⁺ and Na⁺ in solution. With increase in concentration of AlCl₃ and CaCl₂ salts, a decrease in effective charge density of ionised PAA chains is observed from the existence of unfavourable PAA–water, PAA–Ca²⁺ and PAA–Al³⁺ interactions.     

Mutagenic activity of 2-amino-N6-hydroxyadenine in the mouse spot test.

The base analogue 2-amino-N6-hydroxyadenine (AHA) was mutagenic in the spot test in (T x HT)F1 mouse embryos. Females were injected with single doses of 20 or 40 mg AHA per kg body weight on the 9th day of pregnancy. To rank the mutagenic potency of different compounds, the frequencies of genetically relevant spots induced by 1 mg/kg body weight were calculated. The observed somatic mutation frequency for 1 mg/kg AHA was lower (1.95 x 10(-3)) spots of genetic relevance) than that of mitomycin C (16 x 10(-3)), ethylnitrosourea (6.8 x 10(-3)) and cyclophosphamide (6.4 x 10(-3)) and therefore AHA was not classified as a very potent mutagen in this test system. The doubling dose to induce genetically relevant spots was calculated to be 20 mg/kg b.w. Based on these data, AHA is suggested to be a candidate to induce recessive specific-locus mutations in germ cells of mice.     

Genetic dimension of the coevolution of virulence-resistance in Drosophila -- parasitoid wasp relationships

Variations observed in parasite virulence and host resistance may be the outcome of coevolutionary processes. Recent theoretical developments have led to a 'geographic mosaic theory' of coevolution according to which there are some localities where reciprocal selection occurs (hot spots) and others where it is strongly reduced (cold spots). Studies of host-parasitoid systems back this up, revealing a geographical variation of traits subjected to antagonistic selection governed by variations in the strength of the ecological interactions. A more detailed analysis of the genetic basis of these geographic variations in a model system -- the interaction between Drosophila melanogaster and its specific parasitoid Leptopilina boulardi -- suggests that cold spots and hot spots are also driven by the amount of genetic variation available for the trait considered. Our approach, based on isolating reference strains, has been found to predict the result of sympatric interactions and it will be helpful in identifying the selective forces responsible for the coevolution. In this model, host resistance to a standardised reference strain is a weak predictor of the outcome of interactions in the field, and the main parameter accounting for the geographic variations is the number of host species available, with less parasitoid virulence towards D. melanogaster being found in areas displaying a more diversified host community.     

Effect of ATP on protein synthesis in cultured nerve tissue of the edible snail]

External ATP in concentrations of 10(-6)--10(-3) M is shown to stimulate the label incorporation from intracellular labeled pool of 14C-leucine into proteins of mollusc nervous tissue. The maximum effect (by 45% higher than in control) is observed at the 10(-5) ATP concentration. In solutions with high concentration of bivalent ions, ATP action increases by 10--15%. Being incubated for an hour in physiological solutions without energic substrates nervous tissue loses 30--50% of labeled amino acids. Outwashing of 14C-leucine depends only a little on the bivalent ion concentration in the external solution and on the presence of helating agents. Addition of 10(-4) M ATP into the solution, completely inhibits the washing of amino acids out of tissue. At low bivalent ion concentrations 14C-leucine incorporation into nervous tissue in the presence of ATP changes inversely to the ATP concentration: low ATP concentrations (10(-5)--10(-6) M) activate label incorporation by 60--40%, whereas high concentrations lead to the corresponding inhibition. This inhibition is due to helating action of ATP.     

High-throughput array production using precision glass syringes

The advantages of using 1, 96, or 384 precision glass syringes in automated high-throughput microdispensers in creating highly uniform and reproducible DNA, protein, and organic compound array filters and slides are described. Using the Hydra Microdispenser and Tango Liquid Handling system, 0.1-5 ng (in 50-300 nL) PCR-amplified, human cancer-related genes and housekeeping genes were spotted onto nylon membranes and coated slides. Protein solutions of 50 microg/mL to 1 mg/mL were spotted onto coated slides or onto MaxiSorp 96-well plates. Up to 6144 spots/membrane and up to 1000 spots/slide were printed. The size of the spots created by glass syringes was uniform and reproducible (precision variation of less than 5%) from spot to spot and membrane to membrane. Using a Tango 384 system, a total of ten 6144-spot filters can be produced in approximately 25 min, translating into a spotting speed of 2.5 min/membrane.     

Mutagenicities of N-nitrosodimethylamine and N-nitrosodiethylamine in Drosophila and their relationship to the levels of O-alkyl adducts in DNA

N-Nitrosodialkylamines are potent carcinogens in experimental animals. Previously, we reported that the mutagenicity of N-nitrosodimethylamine (NDMA) was 10 times higher than that of N-nitrosodiethylamine (NDEA) in the Drosophila wing spot test. To find out how to explain this difference, we have measured the levels of O-alkylated bases in the DNA of exposed Drosophila larvae. Third instar larvae were fed for 3 or 6 h with NDMA or NDEA. Part of the treated larvae were grown to adult flies to score their wings for the presence of mutant spots. From the remaining larvae, DNA was isolated and digested to deoxyribonucleosides, and the digest fractionated by high-performance liquid chromatography (HPLC). The amounts of specific alkyldeoxyribonucleosides present in the fractions were quantified by a radioimmunoassay (RIA) using monoclonal antibodies. Dose-dependent O6-methylguanine, O6-ethylguanine and O4-ethylthymine formations were found to be correlated with the induction frequencies of mutant wing spots. At the same exposure dose, the values of O6-alkylde- oxyguanosine/106 deoxyguanosine were similar for NDMA and NDEA: on feeding 20 micromol/1.5 ml feeding solution, the values for NDMA were 4.0 with 3 h and 18.5 with 6 h of exposure; with 20 micromol NDEA, the corresponding values were 5.4 with 3 h and 14.6 with 6 h of exposure. The wing spot frequencies were very different; however, with NDMA, the total numbers of spots/wing were 3.5 (3 h) and 15 (6 h), and with NDEA 0.8 (3 h) and 0.9 (6 h). Similar discrepancies exist as well between the mutagenicities and the alkylation rates observed for O4-alkylthymidines. These results suggest that the difference between the mutagenic potencies of NDMA and NDEA cannot be explained by the amounts of O-alkyl adducts formed. Different mechanisms are considered by which NDMA and NDEA may produce the genetic effects observed.     

An automatic image analysis system for RLGS films

The RLGS (Restriction Landmark Genome Scanning) method was originally developed as a powerful method for enabling viewing of thousands of restriction landmarks. It offers a tool for obtaining information about genetic loci, with a single RLGS profile displaying approximately 2000 restriction landmarks as spots. One of the most useful applications is RLGS spot mapping, which allows the efficient, low-cost construction of the genetic map of any organism. However, analyses of the profiles depend mainly on human visual observation and are tedious and laborious. Although several commercially available image analyzing systems for profile comparison have been examined, they cannot be used for the RLGS spot mapping system owing to the background characteristics of the RLGS profiles, unsatisfactory rates of correspondence, and inefficient correction of informative genetic data. We therefore developed a novel automatic image analysis system for RLGS spot mapping, using an original algorithm based on the binary image transferred from the original RLGS profile. This system was employed for identifying non-polymorphic and parental strain-specific polymorphic spots of the F₁ mouse profile and yielded efficient initial screening of RLGS profiles. Received: 5 December 1997 / Accepted: 6 April 1998     

Investigation of the potential for mitotic recombination in the mouse

A variation of the mouse spot test is described that is designed to distinguish between spots of altered coat colour that arise by reciprocal mitotic recombination and those caused by somatic mutation or non-disjunction. Mouse fetuses that were heterozygous for two, linked coat colour genes were irradiated (1.5 Gy X-rays) in utero at 10.25-10.50 days post coitum (p.c.) or left untreated. Subsequently, the coats were classified for the presence of spots of altered colour. The irradiated embryos were heterozygous for the linked genes pink-eyed dilution (p) and albino (c) and were produced by both the repulsion and coupling crosses. Half of the reciprocal recombination events between the centromere and the proximal marker (p), in heterozygotes with p and c in repulsion, should produce twin spots. No such twin spots would be expected from a similar event in the coupling heterozygotes. The coats of 238 irradiated and 208 untreated repulsion heterozygotes plus 107 irradiated and 314 untreated coupling heterozygotes were classified for spots. One irradiated, repulsion heterozygote had a diffuse twin spot that was only recognisable by microscopic examination of the hairs. We conclude that if the treatment described induces mitotic recombination in the mouse, it does so with low efficiency.     

Detection of mercaptopyridines and mercaptopyrimidines in planar chromatography with iodine-azide reaction as a detection system

Reaction between iodine and azide ion induced by mercaptopyridines and mercaptopyrimidines was utilized as a detection system in TLC and HPTLC. The developed plates were sprayed with a freshly prepared mixtures of sodium azide and starch solution adjusted to pH 5.5, and exposed to iodine vapour. The spots became visible as white spots on violet-grey background. The iodine-azide detection system has been proved to be the most favourable and enabled to detect quantities per spot in the range of 1-20 pmol (HPTLC) and 1-60 pmol (TLC). The iodine-azide tests were compared with other visualizing techniques commonly used in planar chromatography (iodine vapour and UV254). The developed method was applied to detection of thiopental in biological samples.     

Francisella tularensis proteome: low levels of ASB-14 facilitate the visualization of membrane proteins in total protein extracts

Proteomic analysis of bacterial pathogens isolated from in vivo sources, such as infected tissues, provides many challenges not the least of which is the limited quantity of sample available for analysis. It is, therefore, highly desirable to develop a one-step cellular lysis and protein solubilization method that minimizes protein losses and allows the maximum possible coverage of the proteome. Here, we have used standard sample buffer constituents including urea, thiourea and DTT, but varied the detergent composition of the buffers in order to achieve the best quality of gels and the greatest spot resolution. We found that the most efficient solubilizing solution in this case consisted of 7 M urea, 2 M thiourea, 1% DTT, 0.5% amidosulfobetaine-14 (ASB-14) and 4% 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). Inclusion of low levels of ASB-14 in solutions allowed visualization of a subset of 24 new protein spots in the Live Vaccine Strain (LVS) of Francisella tularensis and 21 spots in a virulent A-strain of the pathogen. Further investigation showed that 15 of the 24 enriched LVS spots were membrane or membrane-associated proteins suggesting that the optimized lysis and solubilization solution aids in the detection of more hydrophobic proteins. This methodology is now being applied to the analysis of Francisella obtained from in vivo sources.     

Spot overlapping in two-dimensional maps: a serious problem ignored for much too long

In the analysis of a neuroblastoma xenograft implanted in mice using two-dimensional maps, some 85 proteins were found to be up- or down-regulated (out of a total of 264 detected by a medium-sensitivity colloidal Coomassie stain). When these spots were eluted and analysed by mass spectrometry in a quadrupole time of flight mass spectrometer, a number of spots were found to be envelopes of different polypeptide chains. Out of a total of 74 proteins identified, 52 (71%) were found to be singlets, 14 (19%) were doublets, 6 (8%) were triplets, 1 was a quadruplet and 1 a quintuplet. Analysis of the DeltapI and DeltaMr of all species contained in a single gel segment eluted helped point out potential errors in protein identification. This was a unique case, in that very minute bioptic sample loads were applied to the gel. In normal cases, where sample loads of ca. 1 mg of total protein are applied and typically at least 1000 spots are visualised, the singlets will be the minority, rarely exceeding 30% of all spots analysed. The experimental data on the abundance of overlapping spots were in excellent agreement with theoretical data calculated on the basis of the statistical theory of spot overlapping, originally proposed by Davis and further developed by some of the authors. Ways and means for minimizing spot overlap and visualising a greater number of spots in a two-dimensional map are discussed.     

Dose and effect of methyl-2-benzimidazolylcarbamate in the "mammalian spot test", an in vivo method for the detection of genetic alterations in somatic cells of mice.

In the spot test, mouse embryos which are heterozygous for four different recessive coat-colour genes are treated in utero by injection of a mutagen into the peritoneal cavity of the mother or by other appropriate routes of administration. If this treatment leads in a pigment precursor cell to an alteration of the wild type allele of one of the genes under study or to its loss, a colour spot in the adult coat may be seen. Peroral application of 100-300 mg methyl-2-benzimidazolylcarbamate (MBC)/kg to the mother during the tenth day postconception led to an increase in the frequency of colour spots in the coats of offspring. The data are consistent with the hypothesis that MBC is a point mutagen.     

Abscisic acid and sucrose increase the protein content in date palm somatic embryos,causing changes in 2-DE profile

Various supplements (abscisic acid (ABA) or sucrose) were added to the initial embryo culture medium (M3) with the aim of improving the vigour of vitroplants deriving from date palm somatic embryogenesis. ABA (20 and 40 μM) and sucrose (90 g/l) applied for 4 and 2 weeks respectively increased embryo thickness, with no apparent difference in length. ABA (5–40 μM) increased embryo proliferation rate. Somatic embryos maintained in modified M3 (M3 supplemented with ABA and an increased sucrose concentration) contained a higher amount of protein than those maintained in initial M3 (no ABA, 30 g/l of sucrose), with a 1.5–1.7-fold increase depending on the compound and concentration assayed. The 1-D and 2-DE protein profiles showed qualitative and quantitative differences between the somatic embryos cultured in initial M3 (control) and in modified M3. Statistical analysis of spot intensity was performed by principal component analysis, yielding two accurate groups of samples and determining the most discriminating spots. Samples were also clustered using Euclidean distance with an average linkage algorithm. Thirty-four variable spots were identified using mass spectrometry analysis. Identified proteins were classified into the following functional categories: energy metabolism (five proteins); protein translation, folding and degradation (9); redox maintenance (5); cytoskeleton (3); storage protein (2); and with no assigned function as (10). While “up-regulation” of stress-related proteins and “down-regulation” of energy metabolism proteins were observed in somatic embryos matured in M3 supplemented with ABA, storage proteins (legumin) were “up-regulated” in somatic embryos matured in M3 supplemented with increased sucrose.     

Involvement of eddy currents in the mutagenicity of ELF magnetic fields

Possible carcinogenic and/or mutagenic activity of extremely low frequency magnetic fields was examined using somatic mutation and recombination test system of Drosophila melanogaster. An X-linked semi-dominant DNA repair defective mutation mei-41(D5) was introduced into the conventional mwh/flr test system to enhance mutant spot frequency. Virgin females of w mei-41(D5)/FM6; flr/TM6 were crossed with w mei-41(D5)/Y; mwh jv; spa(pol) males. The F(1) third instar larvae were exposed to a 50Hz, 20mT sinusoidal AC magnetic field for 24h. After moulting from pupal cases, their wings were examined under a bright field microscope to detect hair spots with mwh or flr mutant morphology. The exposure caused a statistically significant enhancement in somatic recombination spot frequency. Mutant spots arising due to chromosomal non-disjunction or terminal deletion also increased but the frequency of spots resulting from point mutation was not altered. The enhancement in the recombination spot frequency was suppressed to the control level when a culture medium without electrolytes was used during exposure. When larvae were exposed to a magnetic field in an annular dish, flies from the outer ring showed more mutant spots compared to those from the inner ring. These results suggest that the detected mutagenic activity was that of the induced eddy current, rather than that of the magnetic field itself.     

Two-dimensional reference map of Agrobacterium tumefaciens proteins

Proteomics based on two-dimensional (2-D) gel electrophoresis of proteins followed by spot identification with mass spectrometry is a commonly used method for physiological studies. Physiological proteomics requires 2-D reference maps, on which most of the main proteins are identified. We present a reference map for the bacterial plant pathogen Agrobacterium tumefaciens proteins, which contains more than 300 entries with an isoelectric point (pI) between 4 and 7. The quantitative study of the proteins in the analytical window of the master gel demonstrated unique features, in comparison with other bacteria. In addition, a theoretical analysis of several protein parameters was performed and compared with the experimental results. A comparison of the theoretical molecular weight (MW) of the proteins and their theoretical pI with their vertical and horizontal migration distances, respectively, pointed out the existence of several proteins that strongly diverted from the graph trend-line. These proteins were clearly subjected to post-translational modifications, which changed their pI and/or MW. Additional support for post-translational modifications comes from the identification of multiple spots of the same gene products. Post-translational modifications appear to be more common than expected, at least for soluble proteins, as more than 10% of the proteins were associated with multiple spots.