Electrospray ionization linear trap quadrupole Orbitrap in analysis of old tempera paintings: application to nineteenth-century Orthodox icons.

Proteomic approach in combination with mass spectrometry demonstrates a great potential for identification of proteinaceous materials in works of art. In this study we used a linear trap quadrupole Orbitrap (LTQ-Orbitrap), a state-of-the-art mass spectrometer for parts per million accuracy analyses of peptides behind tryptic hydrolysis. After the efficiency of the proteomic method was confirmed for reference and model samples, micro-samples from historical paintings were for the first time analysed using this technique. Superior performances of the liquid chromatography-mass spectrometry approach using a LTQ-Orbitrap mass spectrometer allowed identification of egg yolk peptides in two samples from nineteenth-century Orthodox icons, indicating egg tempera as the painting technique. Accurate precursor ion masses, in the range of ±2 ppm, and retention times of tryptic peptides strengthen protein identification. Additionally, in all historical samples the presence of animal glues suggested that the ground layer was likely bound using bovine collagen. Comparing to results acquired using matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry in our previous study, here we achieved higher ion scores and protein scores, better sequence coverage and more identified proteins. In fact, a combination of the two mass spectrometric techniques provided overlapping and complementary data, related to the detection of peptides with different physicochemical properties.

Secondary metabolites of Seseli rigidum: Chemical composition plus antioxidant, antimicrobial and cholinesterase inhibition activity.

Extracts of different polarity obtained from various plant parts (root, leaf, flower and fruit) of Seseli rigidum were studied by different antioxidant assays: DPPH and ABTS radical scavenging activity, by total reducing power method as well as via total content of flavonoids and polyphenols. Essential oils of all plant parts showed weak antioxidant characteristics. The inhibitory concentration range of the tested extracts, against bacteria Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus cereus, and fungi Candida albicans and Aspergillus niger was 0.01-1.50 mg/mL and of a microbicidal 0.02-3.00 mg/mL. In the interaction with cholinesterase, all essential oils proved effective as inhibitors. The highest percentage of inhibition versus human and horse cholinesterase was shown by root essential oil (38.20% and 48.30%, respectively) among oils, and root hexane extract (40.56% and 50.65% respectively). Essential oils and volatile components of all plant parts were identified by GC, GC-MS and headspace/GC-MS. Statistical analysis of the ensemble of results showed that the root essential oil composition differed significantly from essential oils of other parts of the plant. Taking into account all of the studied activities, the root hexane extract showed the best overall properties. By means of high performance liquid chromatography coupled to high resolution mass spectrometry, the 30 most abundant constituents were identified in extracts of different polarity. The presence of identified constituents was linked to observed specific biological activities, thus designating compounds potentially responsible for each exhibited activity.

Identification of protein binders in artworks by MALDI-TOF/TOF tandem mass spectrometry.

Aim of this work is to propose an analytical protocol for proteinaceous binder identification in paintings using tryptic peptide analysis and MALDI-TOF mass spectrometry strengthened with MALDI-TOF/TOF tandem mass spectrometry (LIFT method). Proteinaceous binders are enzymatically digested with trypsin. From each individual protein frequently occurring in binders, a specific set of peptides is releasing during enzymatic digestion giving a peptide mass fingerprint (PMF) of that particular protein. The most intensive peptide peaks in PMF were determined and annotated with their corresponding amino acid sequence by MALDI-TOF/TOF analysis and subsequent database search. Before analyzing historical painting samples, procedure was tested and optimized on several painting model samples for a reliable and efficient identification of proteinaceous materials. The method is avoiding sample manipulation as much as possible in order to reduce sample loss. Since the applied procedures led to protein identification of binding media in model samples, MALDI-TOF/TOF was for the first time applied for analysis of proteinaceous binders in old painting samples.

Wild fire impact on copper, zinc, lead and cadmium distribution in soil and relation with abundance in selected plants of Lamiaceae family from Vidlic Mountain (Serbia).

Fire has been considered as an improving factor in soil quality, but only if it is controlled. Severe wild fire occurred in the summer 2007 on the Vidlic Mountain (Serbia) overspreading a huge area of meadows and forests. Main soil characteristics and content of heavy metals (Cu, Pb, Cd, Zn) in different fractions obtained after sequential extraction of soil from post-fire areas and from fire non disturbed areas were studied. In four plant species of Lamiaceae family (Ajuga genevensis L., Lamium galeobdolon (L.) L., Teucrium chamaedrys L., Acinos alpinus (L.) Moench.), that grow in typical habitats of the mountain, distribution of heavy metals in aerial parts and roots was investigated too. For all samples from post-fire area cation exchange capacity and soil organic matter content are increased while rH is decreased. Fire caused slightly increased bioavailability of the observed metals but more significant rise happened in metal amounts bound to oxides and organics. The plants showed variable behavior. T. chamaedrys collected on the post-fire area contained elevated concentrations of all analyzed metals. A. alpinus showed higher phytoaccumulation for Zn and Cd, while the other two plant species for Pb and Cd in the post-fire areas.

A modification of the kinetic determination of pancuronium bromide based on its inhibitory effect on cholinesterase.

A modification of the existing spectrophotometric kinetic method for the determination of pancuronium bromide (PCBr), based on pooled human serum cholinesterase (ChE, EC 3.1.1.8 acylcholine acylhydrolase) inhibition, was developed. Butyrylthiocholine iodide (concentration 1.667 mmol/L) was used as substrate and determination was performed at pH 7.6. Essential basic kinetic parameters were also determined: Michaelis-Menten's constant KM=0.33 mmol/L, maximal reaction rate Vmax=42.29 micromol/L min, inhibition constant KI=0.34 micromol/L, and IC50=0.235 micromol/L. Linear dependence between the reaction rate and the inhibitor concentration exists in PCBr concentration range 8.29-265.28 nmol/L, which corresponds to the real sample concentrations from 0.166 to 5.306 micromol/L. The method detection limit was established to be 1.86 nmol/L and the quantification limit was 6.18 nmol/L. Precision of the method was tested for three pancuronium concentrations (16.58, 99.48, and 198.96 nmol/L). The relative standard deviation (RSD) was in the range 0.78-5.13%. Accuracy was examined by the standard addition method. The influence of substances usually present in serum and urine on the reaction rate was determined. The method developed was applied for PCBr determination in spiked serum and urine samples and in the urine taken during surgery. The method was proven to have good sensitivity, accuracy, and precision and can be considered suitable for clinical practice.