A new approach to the statistical treatment of 2D-maps in proteomics using fuzzy logic

A new approach to the statistical treatment of 2D-maps has been developed. This method is based on the use of fuzzy logic and allows to take into consideration the typical low reproducibility of 2D-maps. In this approach the signal corresponding to the presence of proteins on the 2D-maps is substituted with probability functions, centred on the signal itself. The standard deviation of the bidimensional gaussian probability function employed to blur the signal allows to assign different uncertainties to the two electrophoretic dimensions. The effect of changing the standard deviation and the digitalisation resolution are investigated.     

Monitoring dediazoniation product formation by high-performance liquid chromatography after derivatization

A derivatization protocol that exploits the rapid reaction between arenediazonium ions and a suitable coupling agent followed by high-performance liquid chromatography analyses of the reaction mixture was employed to determine the product distribution, the rate constants for product formation and the association constant of 4-nitrobenzenediazonium, PNBD, ion with beta-cyclodextrin, beta-CD. The derivatization of PNBD with the coupling agent leads to the formation of a stable azo dye that prevents by-side reactions of PNBD with the solvents of the mobile phase, including water, or the metallic parts of the chromatographic system that would eventually lead to erroneous identification and quantification of dediazoniation products. The results show that in the presence of beta-CD, nitrobenzene is formed at the expense of 4-nitrophenol, which is the major product in its absence. The observed rate constants for the interaction between PNBD and beta-CD increase upon increasing [beta-CD] showing a saturation profile indicative of the formation of an inclusion complex between PNBD and beta-CD. By fitting the experimental data to a simplified Lineaweaver-Burk equation, the corresponding association constant and the maximum acceleration rate of beta-CD towards PNBD were estimated. The protocol is applicable under a variety of experimental conditions provided that the rate of the coupling reaction is much faster than that of dediazoniation.     

A new class of flexible energetic salts, part 6: the structures of the hydrazinium and hydroxylammonium salts of dinitramide

The crystal structures of two amine base salts, the hydrazinium, 1, and the hydroxylammonium, 2, of dinitramide have been determined. 1 crystallizes in the monoclinic space group P 2₁/c with cell dimensions a = 8.312(3), b = 5.654(1), c = 10.659(3) Å, = 93.73(3)°, while 2 crystallizes in the orthorhombic space group Pcab (nonstandard setting of Pbca) with cell dimensions a = 6.439(2), b = 12.470(4), c = 30.816(14) Å. The structures of 1 and 2 contain protonated amine cations and dinitramide anions linked by hydrogen bonding. In addition, in 2 there are both neutral and zwitterionic hydroxylamine moieties involved in the hydrogen bonding scheme. Thus in 2 the complete formula unit is (NH₃ ⁺OH)₂[N₃O₄ ^–]₂ · (NH₂OH) middot; (NH₃ ⁺O^–), and in this structure the hydroxylamine exists in its three possible forms: protonated, neutral, and zwitterionic. In both structures the conformations adopted by the dinitramine anions can be related to the types of hydrogen bonds it forms with the surrounding amine cations.     

Production study of high specific activity NCA Re-186g by proton and deuteron cyclotron irradiation

Very high specific activity (A_S) ^186gRe could be produced by either proton or deuteron cyclotron irradiation on highly enriched ¹⁸⁶W target in no-carrier-added (NCA) form, leading to a A_S very close to the theoretical carrier free (CF) value of 6.88 GBq μg⁻¹. Thick Target Yields (TTYs), obtained irradiating both thick metal W targets of natural isotopic composition and highly enriched powdered ¹⁸⁶W targets, were measured at different particles energies taking into account high accuracy and precision. The evaluation of radionuclidic purities of ^186gRe obtained activating highly enriched ¹⁸⁶W by both p and d were also carried out and accurately compared. The thin-target excitation functions for all Re (A = 181, 182, 183, 184, 186 and their metastable levels), and W and Ta coproduced radionuclides will be presented elsewhere in deep details.     

Sequence and phosphorylation level determination of two donkey β‐caseins by mass spectrometry

Two coeluting components, with experimentally measured M_r values of 25529 and 24606 Da, were identified by reversed‐phase high‐performance liquid chromatography (RP‐HPLC) and mass spectrometric analysis in the dephosphorylated casein fraction of a milk sample collected from an individual donkey belonging to the Ragusano breed of the east of Sicily. By coupling enzymatic digestions, matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) and RP‐HPLC/nano‐electrospray ionization tandem mass spectrometry (nESI‐MS/MS) analysis, the two proteins were identified as donkey β‐CNs and their sequences characterized completely, using the two known β‐CNs from mare as references. The two donkey β‐CNs, showing a mass difference of 923 Da, differ by the presence of the domain E²⁷SITHINK³⁴ in the full‐length component (M_r 25529 Da). In comparison with the mare's β‐CNs used as reference, they present nine amino acid substitutions: L→S³⁷, R→H⁵², S→N⁸¹, P→V⁸⁴, L→V⁹¹, R→Q²⁰³, P→L/I²⁰⁶, L→F²¹⁰ and A→P²¹⁹. Together, these substitutions account for the increase of 18 Da in the M_r of the donkey β‐CNs with respect to the counterparts from the mare. The molecular mass determination by ESI‐MS for the phosphorylated proteins showed that the full‐length component was composed of highly multi‐phosphorylated isoforms with five to seven phosphate groups. By analogy with the homologous mare's β‐CNs, the full‐length (226 amino acids) β‐CN was termed variant A, whereas the shorter (218 amino acids) β‐CN was termed variant A^Δ5. Copyright © 2009 John Wiley & Sons, Ltd.     

Development and validation of a gas chromatography/mass spectrometry method for the assessment of genomic DNA methylation

A method for the determination of DNA global methylation, taken as the ratio (%) of 5‐methylcytosine (5mCyt) versus the sum of cytosine (Cyt) and 5mCyt, via gas chromatography/mass spectrometry (GC/MS), was developed and validated. DNA (2.5 µg) was hydrolyzed with aqueous formic acid 88%, spiked with cytosine‐2,4‐¹³C₂,¹⁵N₃ and 5‐methyl‐²H₃‐cytosine‐6‐²H₁ as internal standards, and derivatized with N‐methyl‐N‐(tert‐butyldimethylsilyl)trifluoroacetamide and 1% tert‐butyldimethylchlorosilane, in the presence of acetonitrile and pyridine. GC/MS, operating in single ion monitoring mode, separated and specifically detected all nucleobases as tert‐butyldimethylsilyl derivatives, without interferences, with the exception of guanosine. The method was linear throughout the range of clinical interest and had good sensitivity, with a limit of quantification of 3.2 pmol for Cyt and 0.056 pmol for 5mCyt, the latter corresponding to a methylation level of 0.41%. Intra‐ and inter‐day precision and accuracy were below 4.0% for both analytes and methylation. The matrix absolute effect, process efficiency and coefficient of variation ranged from 96.5 to 101.2%. The matrix relative effect was below 1%. The method was applied to the analysis of different human DNAs, including: nonmethylated DNA from PCR (methylation 0.00%), hypermethylated DNA prepared using M.SssI CpG methyltransferase (methylation 18.05%), DNA from peripheral blood leukocytes of healthy subjects (N = 6, median methylation 5.45%), DNA from bone marrow of leukemia patients (N = 5, 3.58%) and DNA from myeloma cell lines (N = 4, 2.74%). Copyright © 2009 John Wiley & Sons, Ltd.