Stable isotope-free quantitative shotgun proteomics combined with sample pattern recognition for rapid diagnostics

Mass spectrometry (MS) has become a powerful tool for the quantitative analysis of complex protein samples. A high-throughput strategy for the comparative analysis of multiple protein samples with high complexity becomes more and more important. Two strategies, spectral count and peak intensity, for label-free MS analysis of prefractionated complex mixtures have been described recently to be useful for quantitation. Here we compare both strategies for rapid and quantitative 1-D shotgun LC/MS/MS analyses of highly complex protein mixtures using silica-based monolithic columns. First, we validated linearity and sensitivity of these methods by spiking varying amounts of an internal standard protein in a complex plant protein extract. Secondly, quantitative data of proteins of Medicago truncatula nodules were visualized with independent components analysis using data either obtained from spectral count or peak integration performed with commercial software. Spectral count showed apparent advantages over peak integration because several peptides per protein are automatically averaged, the linear dynamic range of quantitation increases in complex matrices and the number of quantified proteins surpasses the number of proteins using peak integration. Thus, for the need of rapid comparative analysis of highly complex protein samples, spectral count enables sample pattern recognition and identification of biomarkers in nongel based proteomic studies.     

Comparison of Benzyl Celluloses Synthesized in Aqueous NaOH and Dimethyl Sulfoxide/Tetrabutylammonium Fluoride

Cellulose was benzylated starting from the dissolved polymer in dimethyl sulfoxide (DMSO)/tetrabutylammonium fluoride trihydrate (TBAF) and heterogeneously in aqueous NaOH as reaction media. Differences in the distribution of benzyl moieties within the anhydroglucose unit in dependence on the synthesis pathway couldn't be found. However, as revealed by means of ¹H-NMR spectroscopy, significant differences in the distribution of substituents along the cellulose chains occurred. Conventionally synthesized samples (in aqueous NaOH), even in the presence of a phase transfer catalyst, possess a block-wise distribution of substituents, while a dissolution of cellulose in DMSO/TBAF prior to the reaction gives products that are uniformly substituted along the cellulose chains.     

Chemical and photochemical reactivity of 6‐hydroxymethyl‐7,8‐dihydropterin in aqueous solutions

6‐Hydroxymethyl‐7,8‐dihydropterin (H₂Hmp) is an intermediate in the biosynthesis of folate, a precursor of coenzymes involved in the metabolism of nucleotides and amino acids. In this work, we have investigated the reactions undergone by H₂Hmp in aqueous solutions at physiological pH, in the absence and in the presence of UV‐A radiation (320–400 nm). In air‐equilibrated solutions, H₂Hmp undergoes slow thermal oxidation (half‐life 37 h) to yield 7,8‐dihydroxanthopterin (H₂Xap) as the main product. The reaction of H₂Hmp with hydrogen peroxide also yields H₂Xap as a main product. In contrast, UV‐A excitation of H₂Hmp leads to the formation of a dimer identified by electrospray ionization mass spectrometry. The corresponding quantum yield of H₂Hmp consumption (Φ_?R) was independent of O₂ and reactant concentration and has a value of 0.10 (±0.02), more than twice higher than that measured for other 6‐subtituted 7,8‐dihydropterins. Copyright © 2012 John Wiley & Sons, Ltd.     

Halichonic acid,a new rearranged bisabolene-type sesquiterpene from a marine sponge Halichondria sp.

A new compound, halichonic acid (1), was isolated from a marine sponge Halichondria sp., together with (6R,7S)-7-amino-7,8-dihydro-α-bisabolene (2). The structure of 1 was elucidated by spectroscopic analysis and ECD spectrum calculation to be a rearranged bisabolene-type sesquiterpene having a 3-azabicyclo[3.3.1]nonene moiety. Compound 2 was cytotoxic against HeLa cells with an IC₅₀ value of 50?μM, whereas 1 did not show cytotoxicity even at 50?μM. It is possible that 1 is biosynthesized from farnesyl pyrophosphate and glycine, with rearrangement.     

Oxidation of 2'-deoxyguanosine 5'-monophosphate photoinduced by pterin: type I versus type II mechanism

UV-A radiation (320-400 nm) induces damage to the DNA molecule and its components through different photosensitized reactions. Among these processes, photosensitized oxidations may occur through electron transfer or hydrogen abstraction (type I) and/or the production of singlet molecular oxygen ((1)O2) (type II). Pterins, heterocyclic compounds widespread in biological systems, participate in relevant biological processes and are able to act as photosensitizers. We have investigated the photosensitized oxidation of 2'-deoxyguanosine 5'-monophosphate (dGMP) by pterin (PT) in aqueous solution under UV-A irrradiation. Kinetic analysis was employed to evaluate the participation of both types of mechanism under different pH conditions. The rate constant of (1)O2 total quenching (k(t)) by dGMP was determined by steady-state analysis of the (1)O2 NIR luminescence, whereas the rate constant of the chemical reaction between (1)O2 and dGMP (k(r)) was evaluated from kinetic analysis of concentration profiles obtained by HPLC. The results show that the oxidation of dGMP photosensitized by PT occurs through two competing mechanisms that contribute in different proportions depending on the pH. The dominant mechanism in alkaline media involves the reaction of dGMP with (1)O2 produced by energy transfer from the PT triplet state to molecular oxygen (type II). In contrast, under acidic pH conditions, where PT and the guanine moiety of dGMP are not ionized, the main pathway for dGMP oxidation involves an initial electron transfer between dGMP and the PT triplet state (type I mechanism). The biological implications of the results obtained are also discussed.     

New Copper,Palladium and Nickel Catalytic Systems: An Evolution towards More Efficient Procedures

Metal‐catalysed reactions are a fundamental tool in synthetic chemistry. Increasingly challenging transformations can be accomplished only by means of certain metal catalysts. However, there still remains the need for a substantial decrease of the amount of catalyst, for better reuse or recycling of such active species, and for the avoidance of relatively toxic solvents in favour of environmentally friendly media. These facts apply to copper‐, palladium‐, and nickel‐catalysed cross‐coupling reactions, direct arylations, and oxidative processes. This account summarises our research on the last reactions, featuring an evolution towards more sustainable procedures in this field.     

The multichoice consistent value

We consider multichoice NTU games, i.e., cooperative NTU games in which players can participate in the game with several levels of activity. For these games, we define and characterize axiomatically the multichoice consistent value, which is a generalization of the consistent NTU value for NTU games and of the multichoice value for multichoice TU games. Moreover, we show that this value coincides with the consistent NTU value of a replicated NTU game and we provide a probabilistic interpretation. Received: May 1998/Final version: January 2000