Short monolithic columns for purification and fractionation of peptide samples for matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry analysis in proteomics

This study records a novel application of methacrylate-based monolithic columns for MALDI-TOF/TOF MS analyses in proteomics for pre-concentration and separation of peptides derived from protein digestion. Reversed-phase monolithic capillary columns (30 mm × 0.32 mm i.d.) were created inside the fused silica capillary via thermal-initiated free-radical polymerization of ethylene glycol dimethacrylate and lauryl methacrylate monomers in the presence of 1-propanol and 1,4-butandiol as a porogen system. The elution of peptides was achieved using a linear gradient of acetonitrile from 0 to 60% in water with 0.1% trifluoroacetic acid formed in a microsyringe. Individual fractions of separated peptides were collected on the MALDI target spots covered with alpha-cyano-4-hydroxycinnamic acid used as a matrix and then they were analyzed using MALDI-TOF/TOF mass spectrometry. The developed method was tested with a mixture of tryptic peptides from bovine serum albumin and its applicability was also tested for tryptic in-gel digests from barley grain extracts of water soluble proteins separated using SDS gel electrophoresis. The number of detected peptides was approximately three to four times higher compared to the analysis without previous separation. These results show an improved quality of sample information with the higher amount of identified peptides which increased protein sequence coverage and improved sensitivity of mass spectrometry measurements.     

Retention studies of 2′-2′-difluorodeoxycytidine and 2′-2′-difluorodeoxyuridine nucleosides and nucleotides on porous graphitic carbon: Development of a liquid chromatography–tandem mass spectrometry method

The development of a method for the separation of 2′-2′-difluorodeoxycytidine (gemcitabine, dFdC), 2′-2′-difluorodeoxyuridine (dFdU) and their mono-, di- and triphosphates using a porous graphitic carbon column (Hypercarb), without ion-pairing agent, is described. The retention of dFdC and dFdU could be controlled with an organic modifier (acetonitrile, CH₃CN) and the retention of the anionic nucleotides with an eluting ion (bicarbonate). Separation of all analytes was achieved using a 0–25 mM ammonium bicarbonate gradient in CH₃CN–H₂O (15:85, v/v). Under these conditions, however, very long re-equilibration times were required. Injection of an acidic solution (100 μL 10% formic acid in H₂O, v/v; 2.65 M) after running a gradient directly restored the separation capabilities of the column. Still, separation between the analytes slowly deteriorated over a period of months. These problems were solved by preconditioning the column with a pH buffered hydrogen peroxide (H₂O₂) solution (0.05% H₂O₂ in CH₃CN–H₂O (15:85, v/v), pH 4) before starting an analytical run. The oxidation of the stationary phase with H₂O₂ prevented its slow reduction, which most likely caused the decreasing retention times. The analytes were detected using tandem mass spectrometry.     

Sodium and potassium benzeneazophosphonate complexes with crown ethers: Solid-state microwave synthesis, characterization and biological activity

The eco-friendly synthesis, spectroscopic (IR, MS, ¹H and ¹³C NMR) study and biological (cytostatic, antiviral) activity of sodium and potassium benzeneazophosphonate complexes, obtained by reaction in the solid state under microwave irradiation of the alkali salts of ethyl [α-(4-benzeneazoanilino)-N-benzyl]phosphonic acid and [α-(4-benzeneazoanilino)-N-4-methoxybenzyl]phosphonic acid with crown ethers containing 18-membered (dibenzo-18-crown-6 and bis(4′-di-tert-butylbenzo)-18-crown-6), 24-membered (dibenzo-24-crown-8) and 30-membered (dibenzo-30-crown-10) macrocyclic rings, have been described. The simple work-up solvent free reaction is an efficient green procedure for the formation of mononuclear crown ether complexes in which the sodium/potassium ion is bound to oxygen atoms of the macrocycle and the phosphonic acid oxygen. The free crown ethers, alkali benzeneazophosphonate salts and their complexes were evaluated for their cytostatic activity in vitro against murine leukemia L1210, murine mammary carcinoma FM3A and human T-lymphocyte CEM and MT-4 cell lines, as well as for their antiviral activity against a wide variety of DNA and RNA viruses. The investigated compounds showed no specific antiviral activity, whereas all the free crown ethers and their complexes demonstrated cytostatic activity, which was especially pronounced in the case of bis(4′-di-tert-butylbenzo)-18-crown-6 and its complexes.     

Environmental biodegradation of synthetic polymers I. Test methodologies and procedures

Biodegradation of synthetic polymers is an important property that is used in many applications. Evaluation of the extent of biodegradation has used different methods in recent years. For each environmental compartment, different approaches have to be made in order to obtain valuable data on biodegradability.This review describes validated and accepted methods based on standardized biodegradation tests, analytical tests, enzymatic tests or tests of physical properties to evaluate the biodegradability of synthetic polymers for different types of environmental compartments (e.g., soil, compost or aqueous media).Part II of this review will subsequently report on the environmental biodegradation of different groups of synthetic polymers.     

One step synthesis of Diflunisal using a Pd-diamine complex

Diflunisal and Felbinac, two FDA-approved NSAIDs and other biphenyl carboxylic acids were prepared in one step by a simple and clean Suzuki cross-coupling reaction using an easily synthesized, air and moisture stable, palladium-diamine complex. The yield (93%) for the one-step preparation of Diflunisal is the best reported without using a glovebox and a phoshine-based catalyst.      

Surface-induced dissociation and chemical reactions of C2D 4 + on stainless steel, carbon (HOPG), and two different diamond surfaces

Surface-induced interactions of the projectile ion C₂D₄⁺ with room-temperature (hydrocarbon covered) stainless steel, carbon highly oriented pyrolytic graphite (HOPG), and two different types of diamond surfaces (O-terminated and H-terminated) were investigated over the range of incident energies from a few eV up to 50 eV. The relative abundance of the product ions in dependence on the incident energy of the projectile ion [collision-energy resolved mass spectra, (CERMS) curves] was determined. The product ion mass spectra contained ions resulting from direct dissociation of the projectile ions, from chemical reactions with the hydrocarbons on the surface, and (to a small extent) from sputtering of the surface material. Sputtering of the surface layer by low-energy Ar⁺ ions (5–400 eV) indicated the presence of hydrocarbons on all studied surfaces. The CERMS curves of the product ions were analyzed to obtain both CERMS curves for the products of direct surface-induced dissociation of the projectile ion and CERMS curves of products of surface reactions. From the former, the fraction of energy converted in the surface collision into the internal excitation of the projectile ion was estimated as 10% of the incident energy. The internal energy of the surface-excited projectile ions was very similar for all studied surfaces. The H-terminated room-temperature diamond surface differed from the other surfaces only in the fraction of product ions formed in H-atom transfer surface reactions (45% of all product ions formed versus 70% on the other surfaces).     

Studies on the mechanism of decomposition of 1-methyl-1-(1-naphthyl)ethyl hydroperoxides to 2-(1-naphthyloxy)propenes

Abstract  

Thermal decomposition of 1-methyl-1-(4-methyl-1-naphthyl)ethyl hydroperoxide under gas chromatography-mass spectroscopy (GC–MS) conditions gives 2-((4-methyl-1-naphthyl)oxy)propene as the main product (50.5%), without any detectable traces of the isomeric 2-((5-methyl-1-naphthyl)oxy)propene. This finding excludes the rearrangement pathway of 1-methyl-1-(1-naphthyl)ethyl hydroperoxides to the corresponding 2-(1-naphthyloxy)propenes, which involves formation of a naphthofuran derivative as an intermediate and transfer of the isopropenyloxy group to the 8 position. This result, as well as our previous density functional theory (DFT) calculations, points to the rearrangement pathway involving an oxirane-type intermediate as the most plausible pathway to 2-(1-naphthyloxy)propenes. This rearrangement is responsible for the unusual inhibition effects of 1-methyl-1-(1-naphthyl)ethyl hydroperoxide on the liquid-phase oxidation of isopropylarenes with oxygen.     

Comparison of photodynamic efficacy of tetraarylporphyrin pegylated or encapsulated in liposomes: In vitro studies

Two photosensitizing systems: (1) tetrakis(4-hydroxyphenyl)porphyrin (p-THPP) encapsulated in sterically stabilized liposomes (SSL) and (2) p-THPP functionalized by covalent attachment of poly(ethylene glycol) (p-THPP–PEG₂₀₀₀) were studied in vitro. The dark and photo cytotoxicity of these systems were evaluated on two cell lines: HCT 116, a human colorectal carcinoma cell line, and DU 145, a prostate cancer cell line and compared with these determined for free p-THPP. It was demonstrated that both encapsulation in liposomes as well as attachment of PEG chain result in pronounced reduction of the dark cytotoxicity of the parent porphyrin. The liposomal formulation showed higher than p-THPP–PEG₂₀₀₀ photocytotoxicity towards both cell lines used in the studies.     

Coordination chemistry of 5,6,7-trimethyl-[1,2,4]triazolo[1,5-a]pyrimidine with first-row transition-metal salts: Synthesis, spectroscopy and single-crystal structures, with counter-anion dependence of the structures

A variety of new coordination compounds with transition-metal salts and the ligand trimethyl[1,2,4]triazolo[1,5-a]pyrimidine (abbreviated as tmtp) is described, together with several of their 3D crystal structures and spectroscopic and magnetic properties. The compounds were selected based on the coordination ability of the counterion, halide, nitrate, sulfate, thiocyanate and perchlorate. The formed coordination compounds and their coordination numbers were found to be strongly dependent on both the cation and the used counter-anion. The several compounds studied have the following structural formulae: [CuCl₂(tmtp)₂], [CuBr₂(tmtp)₂], [ZnBr₂(tmtp)₂], [Cu(NO₃)₂(tmtp)₂], [CuSO₄(tmtp)₂]₂(H₂O)(MeOH), [Cu(H₂O)(NCS)₂(tmtp)₂], [Zn(NCS)₂(tmtp)₂], [Cd(NCS)₂(tmtp)₂] and [M(H₂O)₂(tmtp)₄](BF₄)₂, in which M = Co, Ni, Zn.The new coordination compounds have been further characterized by NMR, (far-)IR and LF spectra, as well as by C, H, N element analyses, and EPR spectra for the Cu(II) compounds. The coordination around the metal varies from 4 (Zn, Cu), via 5 (Cu) to 6 (for Co, Cu and Cd). The anions usually complete the coordination sphere; only the Co and Zn compounds with the tetrafluoridoborate anions have no coordinated anions, but water ligands complete the octahedral coordination sphere. In the 5-coordinated [Cu(H₂O)(NCS)₂(tmtp)₂] water completes the square pyramid geometry.     

Current applications and future trends of molecular diagnostics in clinical bacteriology

Molecular diagnostics of infectious diseases, in particular, nucleic-acid-based methods, are the fastest growing field in clinical laboratory diagnostics. These applications are stepwise replacing or complementing culture-based, biochemical, and immunological assays in microbiology laboratories. The first-generation nucleic acid assays were monoparametric such as conventional tests, determining only a single parameter. Improvements and new approaches in technology now open the possibility for the development of multiparameter assays using microarrays, multiplex nucleic acid amplification techniques, or mass spectrometry, while the introduction of closed-tube systems has resulted in rapid microbial diagnostics with a subsequently reduced contamination risk. Whereas the first assays were focused on the detection and identification of microbial pathogens, these new technologies paved the way for the parallel determination of multiple antibiotic resistance determinants or to perform microbial epidemiology and surveillance on a genetic level.