Immunochemical detection of sequence-specific modifications to DNA induced by UV light

Sequence specificity of antibodies to UV-damaged DNA has not been described previously. The antisera investigated here were specific for UV-modified DNA and were absolutely dependent upon the presence of thymine residues. Using a series of oligonucleotides in competition ELISA, increased inhibition was observed with increasing chain length of UV-polythymidylate. A minimum of three adjacent thymines was required for effective inhibition; alone, dimers of thymine were poor antigens. Although UV-irradiated poly(dC) was not antigenic, cytosines could partially replace thymines within the smallest effective epitope (T-T-T) with a high degree of sequence specificity, not previously described. The main epitope induced by UV was formed from adjacent thymines and either a 3' or a 5' pyrimidine.

Photoinduced modifications in fiber gratings inscribed directly by infrared femtosecond irradiation

The structure of fiber Bragg gratings inscribed pointby-point by an infrared femtosecond laser is studied by quantitative phase microscopy. Results show that these gratings present a central region with a depressed refractive index surrounded by an outer corona with increased refractive index. The refractive index profile suggests the presence of microvoids embedded in a region of the core. © 2006 IEEE.

Strain distributionand electronic property modifications in Si/Ge axial nanowires hetrostructures

Molecular dynamics simulations were carried out for Si/Ge axial nanowire heterostructures using modified effective atom method (MEAM) potentials. A Si–Ge MEAM interatomic cross potential was developed based on available experimental data and was used for these studies. The atomic distortions and strain distributions near the Si/Ge interfaces are predicted for nanowires with their axes oriented along the [111] direction. The cases of 10 and 25 nm diameter Si/Ge biwires and of 25 nm diameter Si/Ge/Si axial heterostructures with the Ge disk 1 nm thick were studied. Substantial distortions in the height of the atoms adjacent to the interface were found for the biwires but not for the Ge disks. Strains as high as 3.5% were found for the Ge disk and values of 2%–2.5% were found at the Si and Ge interfacial layers in the biwires. Deformation potential theory was used to estimate the influence of the strains on the band gap, and reductions in band gap to as small as 40% of bulk values are predicted for the Ge disks. The localized regions of increased strain and resulting energy minima were also found within the Si/Ge biwire interfaces with the larger effects on the Ge side of the interface. The regions of strain maxima near and within the interfaces are anticipated to be useful for tailoring band gaps and producing quantum confinement of carriers. These results suggest that nanowire heterostructures provide greater design flexibility in band structure modification than is possible with planar layer growth.

Chemical synthesis of DNA and RNA containing thio-substituted bases and their post-synthetic modifications

Modified oligonucleotides containing sulphur group have been useful tools for studies of carcinogenesis, protein or nucleic acid structures and functions, protein-nucleic acid interactions, and for antisense modulation of gene expression. One successful example has been the synthesis and study of oligodeoxynucleotides containing 6-thio-2'-deoxyguanine. 6-Thio-2-deoxyguanosine was first discovered as metabolic compound of 6- mercaptopurine (6-MP). Later, it was applied as drug to cure leukaemia. During the research of its toxicity, a method was developed to use the sulphur group as a versatile position for post-synthetic modification. The advantage of application of post-synthetic modification lies in its convenience. Synthesis of oligomers with normal sequences has become routine work in most laboratories. However, design and synthesis of a proper phosphoramidite monomer for a new modified nucleoside are always difficult tasks even for a skilful chemist. Thus an alternative method (post-synthetic method) has been invented to overcome the difficulties. This was achieved by incorporation of versatile nucleotides into oligomers which contain a leaving group, that is sufficiently stable to withstand the conditions of synthesis but can be substituted by nucleophiles after synthesis, to produce, a series of oligomers each containing a different modified base. In the current project, a phosphoramidite monomer with 6-thioguanine has been successfully synthesised and incorporated into RNA. A deprotection procedure, which is specific for RNA was designed for oligomers containing 6-thioguanosine. The results were validated by various methods (UV, HPLC, enzymatic digestion). Pioneer work in utilization of the versatile sulphur group for post-synthetic modification was also tested. Post-synthetic modification was also carried out on DNA with 6- deoxythioguanosine. Electrophilic reagents with various functional groups (alphatic, aromatic, fluorescent) and bi-functional groups have been attached with the oligomers.

Cyclic and acyclic modifications of 5-aminoimidazole-4-carboxamide

The Introduction gives a brief resume' of the biologically important aspects of 5 -aminoimidazole -4 -carbozamide (1) and explores., in-depth, the synthetic routes to this imidazole. All documented reactions of 5 -aninoimidanole-4 -carboxamide are reviewed in detail, with particular emphasis on the preparation and subsequent coupling reactions of 5 –diazo-imidazole-4 -carboxamide (6). A series of thirteen novel amide 5-amino-2-arylazoimidazole-4-carboxamide derivatives (117-129) were prepared by the coupling of aryldiazonium salts with 5-aminoimidazole-4-carboxamide. Chemical modification of these azo-dyes resulted in the preparation of eight previously unknown acyl derivatives (136-143) Interaction of 5-amino-2-arylazoimidazole-4-carboxides with ethyl formate in sodium ethoxide effected pyrimidine ring closure to the novel 8-arylazohypoxanthines (144 and 145). Several reductive techniques were employed in an effort to obtain the elusive 2,5-diaminoimidazole-4-carboxamide (71),a candidate chemotherapeutic agent, from the arylazoiridazoles. No success can be reported although 5-amino-2-(3-aminoindazol-2-yl) imidazole-4-carboxamide (151) was isolated due to a partial reduction and intramolecular cyclisation of 5-amino72-(2-cyanaphenylazo)imidazole-4-carboxamide (122) .Further possible synthetic approaches to the diaminoimidazole are discussed in Chapter 4. An interesting degradation of a known unstable nitrohydrazone is described in Chapter 5.This resulted in formation of 1, 1-bis(pyrazol--3-ylazo)-1-nitroethane (164) instead of the expected cyclisation to a bicyclic tetrazine N-oxide. An improved preparation of 5-diazoinidazole-4-carboxamide has been achieved, and the diazo-azole formed cycloadducts with isocyanates to yield the hitherto unknown imidazo[5,1-d][1,2,3,5]tetrazin-7(6H)-ones. Eleven derivatives (167-177) of this new ring-system were prepared and characterised. Chemical and spectroscopic investigation showed this ring-system to be unstable under certain conditions, and a comparative study of stability within the group has been made. "Retro-cycloaddition" under protic and photolytic conditions was an unexpected property of 6-substituted imidazo[5,1-d][1,2,3,5]tetrazin--7(0)-ones.Selected examples of the imidazotetrazinone ring-system were tested for antitumour activity. The results of biological evaluation are given in Chapter 7, and have culminated in a Patent application by the collaborating body, May and Baker Ltd. One compound,3-carbamoyl-6-(2-chloro-ethyl)imidazo[5,1-d][1,2,3,5jtetrazin-7(6H)-one (175),shows striking anti-tumour activity in rodent test systems.

Development of novel mass spectrometric methods for identifying HOCl-induced modifications to proteins

Protein oxidation is thought to contribute to a number of inflammatory diseases, hence the development of sensitive and specific analytical techniques to detect oxidative PTMs (oxPTMs) in biological samples is highly desirable. Precursor ion scanning for fragment ions of oxidized amino acid residues was investigated as a label-free MS approach to mapping specific oxPTMs in a complex mixture of proteins. Using HOCl-oxidized lysozyme as a model system, it was found that the immonium ions of oxidized tyrosine and tryptophan formed in MS(2) analysis could not be used as diagnostic ions, owing to the occurrence of isobaric fragment ions from unmodified peptides. Using a double quadrupole linear ion trap mass spectrometer, precursor ion scanning was combined with detection of MS(3) fragment ions from the immonium ions and collisionally-activated decomposition peptide sequencing to achieve selectivity for the oxPTMs. For chlorotyrosine, the immonium ion at 170.1 m/z fragmented to yield diagnostic ions at 153.1, 134.1, and 125.1 m/z, and the hydroxytyrosine immonium ion at 152.1 m/z gave diagnostic ions at 135.1 and 107.1 m/z. Selective MS(3) fragment ions were also identified for 2-hydroxytryptophan and 5-hydroxytryptophan. The method was used successfully to map these oxPTMs in a mixture of nine proteins that had been treated with HOCl, thereby demonstrating its potential for application to complex biological samples.

Protein oxidative modifications:new mass spectrometry approaches to detection in biological samples

In inflammatory diseases, release of oxidants leads to oxidative damage to proteins. The precise nature of oxidative damage to individual proteins depends on the oxidant involved. Chlorination and nitration are markers of modification by the myeloperoxidase-H2O2-Cl- system and nitric oxide-derived oxidants, respectively. Although these modifications can be detected by western blotting, currently no reliable method exists to identify the specific sites damage to individual proteins in complex mixtures such as clinical samples. We are developing novel LCMS2 and precursor ion scanning methods to address this. LC-MS2 allows separation of peptides and detection of mass changes in oxidized residues on fragmentation of the peptides. We have identified indicative fragment ions for chlorotyrosine, nitrotyrosine, hydroxytyrosine and hydroxytryptophan. A nano-LC/MS3 method involving the dissociation of immonium ions to give specific fragments for the oxidized residues has been developed to overcome the problem of false positives from ions isobaric to these immonium ions that exist in unmodified peptides. The approach has proved able to identify precise protein modifications in individual proteins and mixtures of proteins. An alternative methodology involves multiple reaction monitoring for precursors and fragment ions are specific to oxidized and chlorinated proteins, and this has been tested with human serum albumin. Our ultimate aim is to apply this methodology to the detection of oxidative post-translational modifications in clinical samples for disease diagnosis, monitoring the outcomes of therapy, and improved understanding of disease biochemistry.

Reporter ion-based mass spectrometry approaches for the detection of non-enzymatic protein modifications in biological samples

Development of mass spectrometry techniques to detect protein oxidation, which contributes to signalling and inflammation, is important. Label-free approaches have the advantage of reduced sample manipulation, but are challenging in complex samples owing to undirected analysis of large data sets using statistical search engines. To identify oxidised proteins in biological samples, we previously developed a targeted approach involving precursor ion scanning for diagnostic MS3 ions from oxidised residues. Here, we tested this approach for other oxidations, and compared it with an alternative approach involving the use of extracted ion chromatograms (XICs) generated from high-resolution MSMS data using very narrow mass windows. This accurate mass XIC data methodology was effective at identifying nitrotyrosine, chlorotyrosine, and oxidative deamination of lysine, and for tyrosine oxidations highlighted more modified peptide species than precursor ion scanning or statistical database searches. Although some false positive peaks still occurred in the XICs, these could be identified by comparative assessment of the peak intensities. The method has the advantage that a number of different modifications can be analysed simultaneously in a single LC-MSMS run. This article is part of a Special Issue entitled: Posttranslational Protein modifications in biology and Medicine. Biological significance: The use of accurate mass extracted product ion chromatograms to detect oxidised peptides could improve the identification of oxidatively damaged proteins in inflammatory conditions. © 2013 Elsevier B.V.

Use of narrow mass-window, high-resolution extracted product ion chromatograms for the sensitive and selective identification of protein modifications

Protein modifications, including oxidative modifications, glycosylations, and oxidized lipid-protein adducts, are becoming increasingly important as biomarkers and in understanding disease etiology. There has been a great deal of interest in mapping these on Apo B100 from low density lipoprotein (LDL). We have used extracted ion chromatograms of product ions generated using a very narrow mass window from high-resolution tandem mass spectrometric data collected on a rapid scanning quadrupole time-of-flight (QTOF) instrument, to selectively and sensitively detect modified peptides and identify the site and nature of a number of protein modifications in parallel. We have demonstrated the utility of this method by characterizing for the first time oxidized phospholipid adducts to LDL and human serum albumin and for the detection of glycosylation and kynurenin formation from the oxidation of tryptophan residues in LDL. © 2013 American Chemical Society.

Lipoxidation adducts with peptides and proteins:deleterious modifications or signalling mechanisms?

Protein lipoxidation refers to the modification by electrophilic lipid oxidation products to form covalent adducts, which for many years has been considered as a deleterious consequence of oxidative stress. Oxidized lipids or phospholipids containing carbonyl moieties react readily with lysine to form Schiff bases; alternatively, oxidation products containing α,β-unsaturated moieties are susceptible to nucleophilic attack by cysteine, histidine or lysine residues to yield Michael adducts, overall corresponding to a large number of possible protein adducts. The most common detection methods for lipoxidized proteins take advantage of the presence of reactive carbonyl groups to add labels, or use antibodies. These methods have limitations in terms of specificity and identification of the modification site. The latter question is satisfactorily addressed by mass spectrometry, which enables the characterization of the adduct structure. This has allowed the identification of lipoxidized proteins in physiological and pathological situations. While in many cases lipoxidation interferes with protein function, causing inhibition of enzymatic activity and increased immunogenicity, there are a small number of cases where lipoxidation results in gain of function or activity. For certain proteins lipoxidation may represent a form of redox signaling, although more work is required to confirm the physiological relevance and mechanisms of such processes. This article is part of a Special Issue entitled: Posttranslational Protein modifications in biology and Medicine. © 2013 Elsevier B.V.

Post-translational modifications and mass spectrometry detection

In this review, we provide a comprehensive bibliographic overview of the role of mass spectrometry and the recent technical developments in the detection of post-translational modifications (PTMs). We briefly describe the principles of mass spectrometry for detecting PTMs and the protein and peptide enrichment strategies for PTM analysis, including phosphorylation, acetylation and oxidation. This review presents a bibliographic overview of the scientific achievements and the recent technical development in the detection of PTMs is provided. In order to ascertain the state of the art in mass spectrometry and proteomics methodologies for the study of PTMs, we analyzed all the PTM data introduced in the Universal Protein Resource (UniProt) and the literature published in the last three years. The evolution of curated data in UniProt for proteins annotated as being post-translationally modified is also analyzed. Additionally, we have undertaken a careful analysis of the research articles published in the years 2010 to 2012 reporting the detection of PTMs in biological samples by mass spectrometry. © 2013 Elsevier Inc.

Development of novel mass spectrometry methodology to detect post-translational modifications in oxidative stress and disease

This paper presented at the European Meeting of the Society-for-Free-Radical-Research-Europe 2007, discusses the development of novel mass spectrometry methodology to detect post-translational modifications in oxidative stress and disease.

Structural and chemical modifications of typical South African biomasses during torrefaction

Torrefaction experiments were carried out for three typical South African biomass samples (softwood chips, hardwood chips and sweet sorghum bagasse) to a weight loss of 30wt.%. During torrefaction, moisture, non-structural carbohydrates and hemicelluloses were reduced, resulting in a structurally modified torrefaction product. There was a reduction in the average crystalline diameter (La) (XRD), an increase in the aromatic fraction and a reduction in aliphatics (substituted and unsubstituted) (CPMAS 13C NMR). The decrease in the aliphatic components of the lignocellulosic material under the torrefaction conditions also resulted in a slight ordering of the carbon lattice. The degradation of hemicelluloses and non-structural carbohydrates increased the inclusive surface area of sweet sorghum bagasse, while it did not change significantly for the woody biomasses.