Latest developments in sample treatment for O-isotopic labeling for proteomics mass spectrometry-based approaches: A critical review

Nowadays isotopic ¹⁸O-labeling of peptides has recalled the attention of researchers due to its simplicity of application and high versatility for proteomics studies. Protein quantification, differential peptide mass mapping, studies regarding proteins overexpressed or underexpressed, or the searching of biomarkers can be accomplished by using ¹⁸O-labeling. In this critical review we comment on the different ways in which ¹⁸O-labeling can be done, highlighting the key parameters of the different sample treatments to obtain a reliable and reproducible labeling. In addition we describe and compare the latest improvement in terms of sample treatment that allows to reduce the handling and to increase the throughput for this sample treatment. Finally, we hypothesize on the future trends of these methods under the light of the new technological advances to speed protein cleavage.     

Determination of phytoextraction potential of plant species for toxic elements in soils of abandoned sulphide-mining areas

This study has determined contamination levels in soils and plants from the São Domingos mining area, Portugal, by k ₀-INAA. Total concentrations of As, Sb, Cr, Hg, Cu, Zn and Fe in soils were very high, exceeding the maximum limits in Portuguese legislation. Concentrations of toxic elements like As, Sb and Zn were highest in roots of Erica andevalensis, Juncus acutus, Agrostis castellana and Nicotiana glauca. Additionally, As, Br, Cr, Fe, Sb and Zn in all organs of most plants were above toxicity levels. Those species that accumulated relatively high concentrations of toxic elements in roots (and tops) may be cultivated for phytostabilisation of similar areas.     

Direct sample fraction deposition using electrospray in narrow-bore size-exclusion chromatography/matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for polymer characterization

A direct sample fraction deposition method was developed for off-line size-exclusion chromatography (SEC)/matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry. By using electrospray, the SEC eluent, together with a suitable matrix solution added coaxially, was directly deposited on the MALDI plate. Owing to the formation of very small droplets in electrospray, solvent evaporation is much faster. The fractionation volume in narrow-bore SEC, which can directly be collected in one MALDI spot, can easily be optimized in the range of a few microlitres. In addition, fairly homogeneous sample spots were obtained. The possible influence of composition variation of the SEC effluent on the analytical results using direct fraction deposition was investigated; no substantial effects were observed. The applicability of the method was demonstrated by characterizing a broad poly(methyl methacrylate) sample. Copyright 2000 John Wiley & Sons, Ltd.     

Comparison of ultrasound-assisted extraction and microwave-assisted digestion for determination of magnesium, manganese and zinc in plant samples by flame atomic absorption spectrometry

In this paper, a sample preparation method based on acid extraction of magnesium, manganese and zinc from plant tissue by means of high intensity probe ultrasonication is described. Acid extracts obtained upon sonication were directly nebulised into an air-acetylene flame for fast metal determination by atomic absorption spectrometry. Parameters influencing extraction such as sonication time, ultrasound amplitude, sample mass, particle size, extractant composition and volume were fully optimised. Optimum conditions for metal extraction were as follows: a 3-min sonication time, a 30% ultrasonic amplitude, a 0.1-g sample mass, a particle size less than 50 mum, a 0.3% m/v HCl concentration in the extractant solution and a 5-ml extractant volume. Six plant samples used in the human diet were analysed, the concentration range of the three metals approximately being in the range of 1500-3000 mug g(-1) for Mg, 30-735 mug g(-1) for Mn and 20-45 mug g(-1) for Zn. Limits of detection corresponding to the ultrasound-assisted extraction method were 0.10, 1.26 and 0.65 mug g(-1) for Mg, Mn and Zn, respectively. Between-batch precision, expressed as R.S.D., was about 0.5, 1.5 and 1% for Mg, Mn and Zn, respectively. Analytical results for the three metals by ultrasound-assisted extraction and microwave-assisted digestion showed a good agreement, thus indicating the possibility of using mild conditions for sample preparation instead of intensive treatments inherent with the digestion method. The advantages and drawbacks of ultrasound-assisted extraction in respect to the microwave-assisted digestion are discussed.     

Ultrasonic energy as a new tool for fast isotopic 18O labeling of proteins for mass spectrometry-based techniques: preliminary results

Preliminary results regarding fast isotopic labeling of proteins with (18)O in conjunction with matrix assisted laser desorption ionization time of flight mass spectrometry technique are presented. Similar (16)O/(18)O isotopic labeling ratios were found for the overnight procedure (12h) and the new fast ultrasonic one (30 min) for the BSA, ovalbumin and alpha-lactalbumin proteins. The procedure, however, failed to promote double (18)O isotopic labeling for the proteins, ovalbumin and alpha-lactalbumin. Two different sonication frequencies, 35 and 130 kHz, were studied at two different sonication times of 15 and 30 min, being best results obtained with the procedure at 130 kHz of sonication frequency and 30 min of sonication time. For comparative purposes the overnight isotopic (18)O labeling procedure was done. In addition, the new fast isotopic labeling procedure was also studied without ultrasonication, in a water bath at 60 degrees C.     

Dating Archaeological Copper/Bronze Artifacts by Using the Voltammetry of Microparticles

A method for dating copper/bronze archaeological objects aged in atmospheric environments is proposed based on the specific signals for cuprite and tenorite corrosion products measured through the voltammtry of microparticles method. The tenorite/cuprite ratio increased with the corrosion time and fitted to a potential law that yielded a calibration curve usable for dating purposes.     

Decision peptide-driven: A free software tool for accurate protein quantification using gel electrophoresis and matrix assisted laser desorption ionization time of flight mass spectrometry

The decision peptide-driven tool implements a software application for assisting the user in a protocol for accurate protein quantification based on the following steps: (1) protein separation through gel electrophoresis; (2) in-gel protein digestion; (3) direct and inverse ¹⁸O-labeling and (4) matrix assisted laser desorption ionization time of flight mass spectrometry, MALDI analysis. The DPD software compares the MALDI results of the direct and inverse ¹⁸O-labeling experiments and quickly identifies those peptides with paralleled loses in different sets of a typical proteomic workflow. Those peptides are used for subsequent accurate protein quantification. The interpretation of the MALDI data from direct and inverse labeling experiments is time-consuming requiring a significant amount of time to do all comparisons manually. The DPD software shortens and simplifies the searching of the peptides that must be used for quantification from a week to just some minutes. To do so, it takes as input several MALDI spectra and aids the researcher in an automatic mode (i) to compare data from direct and inverse ¹⁸O-labeling experiments, calculating the corresponding ratios to determine those peptides with paralleled losses throughout different sets of experiments; and (ii) allow to use those peptides as internal standards for subsequent accurate protein quantification using ¹⁸O-labeling. In this work the DPD software is presented and explained with the quantification of protein carbonic anhydrase.     

Simplifying sample handling for protein identification by peptide mass fingerprint using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

An ultrasonic bath, an ultrasonic probe and a sonoreactor were used to speed up the kinetics of the reactions involved in each step of the sample handling for in-gel protein identification by peptide mass fingerprint, PMF, using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The following steps were successfully accelerated using ultrasonic energy: gel washing, protein reduction, and protein alkylation. As a result, a reduction comprising 80% to 90% of the total time involved in the classic approach was achieved. In addition the sample handling was also drastically simplified. The number of peptides identified and the protein sequence coverage obtained for the new procedure were comparable to those obtained with the traditional sample treatment for the following protein standards: glycogen phosphorylase b, BSA, ovalbumin, carbonic anhydrase, trypsin inhibitor and alpha-lactalbumin. Finally, as a proof of the procedure, specific proteins were identified from complex protein mixtures obtained from three different sulphate-reducing bacteria: Desulfovibrio desulfuricans G20, Desulfuvibrio gigas NCIB 9332, and Desulfuvibrio desulfuricans ATCC 27774.     

Diels-alder reactions with 2-(Arylsulfinyl)-1,4-benzoquinones: effect of aryl substitution on reactivity, chemoselectivity, and pi-facial diastereoselectivity

Diels-Alder reactions of (SS)-2-(2'-methoxynaphthylsulfinyl)-1, 4-benzoquinone (1b), 2-(p-methoxyphenylsulfinyl)-1,4-benzoquinone (1c), and 2-(p-nitrophenylsulfinyl)-1,4-benzoquinone (1d) with cyclopentadiene are reported. These cycloadditions allowed the highly chemo- and stereoselective formation of both diastereoisomeric endo-adducts resulting from reaction on the unsubstituted double bond C(5)-C(6) of quinones working under thermal and Eu(fod)(3)- or BF(3).OEt(2)-catalyzed conditions. The synthesis of endo-adduct [4aS,5S,8R,8aR,SS]-9d resulting from cycloaddition on the substituted C(2)-C(3) double bond was achieved in a chemo- and diastereoselective way from quinone 1d in the presence of ZnBr(2). The reactivity and selectivity of the process proved to be dependent on the electron density of the arylsulfinyl group.     

Exploring the interaction of mercury(II) by N(2)S(2) and NS(3) anthracene-containing macrocyclic ligands: photophysical, analytical, and structural studies

The complexation properties toward Hg(II) of six macrocyclic ligands, 3,11-dithia-7,17-diazabicyclo[11.3.1]heptadeca-1(17),13,15-triene (L1), 7-(9-anthracenylmethyl)-3,11-dithia-7,17-diazabicyclo[11.3.1]heptadeca-1(17),13,15-triene (L2), 7-(10-methyl-9-anthracenylmethyl)-3,11-dithia-7,17-diazabicyclo[11.3.1]heptadeca-1(17),13,15-triene (L3), 7,7'-[9,10-anthracenediylbis(methylene)]bis-3,11-dithia-7,17-diazabicyclo[11.3.1]heptadeca-1(17),13,15-triene (L4), 1,4,7-trithia-11-azacyclotetradecane (L5), and 11,-(anthracen-9-ylmethyl)-1,4,7-trithia-11-azacyclotetradecane (L6), were studied. The stoichiometries of the formed species were determined from absorption and fluorescence titrations. In these anthracene-containing macrocycles, a fluorescent quenching of the emission was found upon Hg(II) addition. The X-ray crystal structure of [HgCl2(L2)] x 1/2CH2Cl2 was determined. The asymmetric unit contains two independent [HgCl2(L2)] molecules and one dichloromethane molecule. Each Hg(II) ion is coordinated by the pyridine nitrogen, the two sulfur atoms of one L2 molecule, and two chloride ions. Analytical studies using solvent extraction separation of Hg(II) from aqueous solutions were performed to determine the Hg(II) extraction capability of ligands L1, L2, and L5.