GenoMass software: a tool based on electrospray ionization tandem mass spectrometry for characterization and sequencing of oligonucleotide adducts

The analysis of DNA adducts is of importance in understanding DNA damage, and in the last few years mass spectrometry (MS) has emerged as the most comprehensive and versatile tool for routine characterization of modified oligonucleotides. The structural analysis of modified oligonucleotides, although routinely analyzed using mass spectrometry, is followed by a large amount of data, and a significant challenge is to locate the exact position of the adduct by computational spectral interpretation, which still is a bottleneck. In this report, we present an additional feature of the in‐house developed GenoMass software, which determines the exact location of an adduct in modified oligonucleotides by connecting tandem mass spectrometry (MS/MS) to a combinatorial isomer library generated in silico for nucleic acids. The performance of this MS/MS approach using GenoMass software was evaluated by MS/MS data interpretation for an unadducted and its corresponding N‐acetylaminofluorene (AAF) adducted 17‐mer (5'OH‐CCT ACC CCT TCC TTG TA‐3′OH) oligonucleotide. Further computational screening of this AAF adducted 17‐mer oligonucleotide (5′OH‐CCT ACC CCT TCC TTG TA‐3′OH) from a complex oligonucleotide mixture was performed using GenoMass. Finally, GenoMass was also used to identify the positional isomers of the AAF adducted 15‐mer oligonucleotide (5′OH‐ATGAACCGGAGGCCC‐3′OH). GenoMass is a simple, fast, data interpretation software that uses an in silico constructed library to relate the MS/MS sequencing approach to identify the exact location of adduct on oligonucleotides. Copyright © 2012 John Wiley & Sons, Ltd.     

两种非对称芳基二噻吩并[2,3-b:3',2'-d]噻吩的合成与光谱特性

合成了两种非对称芳基取代的并三噻吩化合物.以2-溴-5-三甲基硅-二噻吩并[2,3-b:3',2'-d]噻吩为原料,经脱除四甲基硅烷(TMS)和Suzuki偶联两步反应制备了2-噻吩基二噻吩并[2,3-b:3',2'-d]噻吩,总产率为67.4%;以2-苯基二噻吩并[2,3-b:3',2'-d]噻吩为原料,经N-溴代琥...     

Anomeric 5-Aza-7-deaza-2′-deoxyguanosines in Silver-Ion-Mediated Homo and Hybrid Base Pairs: Impact of Mismatch Structure,Helical Environment,and Nucleobase Substituents on DNA Stability

Nucleoside configuration (α-d vs. β-d ), nucleobase substituents, and the helical DNA environment of silver-mediated 5-aza-7-deazaguanine-cytosine base pairs have a strong impact on DNA stability. This has been demonstrated by investigations on oligonucleotide duplexes with silver-mediated base pairs of α-d and β-d anomeric 5-aza-7-deaza-2′-deoxyguanosines and anomeric 2′-deoxycytidines incorporated in 12-mer duplexes. To this end, a new synthetic protocol has been developed to access the pure anomers of 5-aza-7-deaza-2′-deoxyguanosine by glycosylation of either the protected nucleobase or its salt followed by separation of the glycosylation products by crystallization and chromatography. Thermal stability measurements were performed on duplexes with α-d /α-d and β-d /β-d homo base pairs or α-d /β-d and β-d /α-d hybrid pairs within two sequence environments, positions 6 or 7, of oligonucleotide duplexes. The respective T_m stability increases observed after silver ion addition differ significantly. Homo base pairs with β-d /β-d or α-d /α-d nucleoside combinations are more stable than α-d /β-d hybrid base pairs. The positional switch of silver-ion-mediated base pairs has a significant impact on stability. Nucleobase substituents introduced at the 5-position of the dC site of silver-mediated base pairs affect base pair stability to a minor extent. Our investigation might lead to applications in the construction of bioinspired nanodevices, in DNA diagnostics, or metal-DNA hybrid materials.     

Mass spectrometric investigation of the sequence selectivity for adduction of heterocyclic aromatic amines on single-strand oligonucleotides

Heterocyclic aromatic amines (HAAs) generated during the cooking of meats are known to be genotoxic substances able to form covalent bonds with DNA bases after metabolic activation. This work aimed at the investigation of the influence of the local environment of nucleobases along the nucleotidic sequence on its modification induced by two different HAAs, namely 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), in order to identify possible sequences more susceptible to modification. A systematic study of the neighbouring base effect on the adduction was emphasized. Thus, PhIP and IQ adducts have been synthesized with various T-rich model single-strand oligonucleotides displaying different flanking bases (A, G, C or T) at the 3' or the 5' side of the targeted guanine, which allowed a comparison of the flanking base effects on adduction. Modified oligonucleotides were then analyzed by high-performance liquid chromatography (HPLC) coupled to electrospray ionization mass spectrometry. The localization of the modifications induced by PhIP or IQ along the oligonucleotide sequence was achieved by tandem mass spectrometry, and modification yields of the various model sequences were compared. Results indicate a favouring sequence context effect on the G-C8-IQ adduct formation with the sequence 5'GGG3'. Although higher than IQ, modification yields observed with PhIP showed a less obvious effect of the neighbouring base on the G-C8-PhIP adduct formation, with a preferential sequence 5'GGA/G/T3'.     

Ion trap collisional activation of the deprotonated deoxymononucleoside and deoxydinucleoside monophosphates

Deoxymononucleoside and deoxydinucleoside monophosphate anions formed by electrospray have been subjected to ion trap collisional activation. The threshold for decomposition via loss of base is significantly lower for the deoxymononucleoside 3′-monophosphates than for the corresponding 5′-monophosphates, which indicates that the presence of a charged 3′ phosphate group facilitates base loss. The behavior of the bases among each class of isomers shows slight variation in threshold and tandem mass spectrometry efficiency with tile notable exception of 2′-deoxyguanosine 5′-monophosphate. This ion is exceptionally stable toward decomposition via base loss, which reflects a strong hydrogen bonding interaction between the base and the phosphate group. All dinucleotides fragment via similar mechanisms, but the propensity for neutral base loss relative to loss of a charged base is highly dependent on the identities of both the 5′ and 3′ bases. The behavior of the dinucleotides under collisional activation conditions supports the proposal that base loss proceeds via a proton-bound dimer intermediate in which loss of the charged base directly competes with loss of the neutral base. Application of the kinetic method allows for quantitative predictions of the differences of the gas-phase acidities of the dimer components.     

Characterization of the DNA adducts induced by aristolochic acids in oligonucleotides by electrospray ionization tandem mass spectrometry

Metabolic activation of carcinogenic aristolochic acids (AA) produces reactive aristolactam-nitrenium ion intermediates. Electrophilic attack of the aristolactam-nitrenium ion via its C7 position to the exocyclic amino group in the purine bases leads to the formation of DNA adducts. DNA-binding assays have demonstrated that carcinogens show site- and sequence-specificity and the biological consequence is defined by the nature of binding as well as their position in the genome. In this study, electrospray ionization tandem mass spectrometry was applied for the identification and position mapping of DNA adducts in oligonucleotides (ODNs). The developed method was successfully applied for the analysis of unmodified and AA-modified ODNs (5'-TTTATT-3', 5'-TTTGTT-3' and 5'-TACATGTGT-3'). The observation of the modified bases (modified adenine and guanine) together with the complementary product ions ([a(n)-B*(n)](-), w(-)) from the cleavage of the 3' C--O bond adjacent to the modified base in MS/MS analyses readily enabled the identification of the AA-binding site in ODNs.     

Functionalized Silver-Ion-Mediated α-dC/β-dC Hybrid Base Pairs with Exceptional Stability: α-d-5-Iodo-2′-Deoxycytidine and Its Octadiynyl Derivative in Metal DNA

Silver-mediated α-dC–Ag⁺–β-dC hybrid base pairs decorated with 5-iodo- or 5-octadiynyl residues are well accommodated in duplex DNA. A strong T_m increase and favorable thermodynamic data for duplex DNA were observed after addition of silver ions. The phenomenon is particularly obvious when both nucleobases of the base pairs are functionalized. Neither the position of the base pair, nor the type of 5-substituent had a negative influence. On the contrary, functionalization of conventional silver-mediated β-dC–Ag⁺–β-dC homo base pairs showed a negative impact induced by the bulky substituents. To this end, cytosine modified 12-mer oligodeoxynucleotides were prepared by solid-phase synthesis employing new α-anomeric 2′-deoxycytidine phosphoramidites. A multigram scale synthesis was developed for 5-iodo-α-d -2′-deoxycytidine ( 1 ) employing the direct glycosylation of cytosine with Hoffer's α-d -halogenose followed by separation of anomeric DMT nucleosides. Regarding base-pair stability and functionalization silver-mediated α/β-dC hybrid base pairs were found to be superior to β/β-dC homo pairs. According to their extraordinary properties, they might find applications in DNA diagnostics, material science, or nanotechnology.     

Location of abasic sites in oligodeoxynucleotides by tandem mass spectrometry and by a chemical cleavage initiated by an unusual reaction of the ODN with MALDI matrix

We describe two approaches employing electrospray ionization (ESI) tandem mass spectrometry (MS/MS) and matrix assisted laser desorption/ionization (MALDI) post-source decay (PSD) for determining the location of an abasic site in modified oligodeoxynucleotides (ODNs). With MS/MS, we found both complementary fragment ions (a_n′ and w_n′) produced at the abasic site were predominant in the mass spectra and allowed the location to be determined. Under MALDI conditions, most ODNs carrying an abasic site are singly charged, and PSD gives predominately w_n′ ions at the abasic sites, revealing their location. We also describe another approach for identifying and locating abasic sites in model ODNs; namely, an “in situ” derivatization coupled with MALDI mass spectrometry (MS). In general, an ODN n-mer containing an abasic site at the m-th position from the 5′-terminus can react with the matrix component, anthranilic acid, to form a Schiff base. The adduct upon MALDI breaks into 3′ and 5′ fragments (w_n−m, b_m, a_m, d_m−1) at the abasic site, revealing its location. ESI MS methods are also applicable for detecting the hydrazone derivatives of abasic sites, and the fragmentation of hydrazones shows the location of the abasic site.     

Liquid chromatography-thermospray mass spectrometry of DNA adducts formed with mitomycin C, porfiromycin and thiotepa

High-performance liquid chromatography (HPLC) and thermospray mass spectrometry were combined for the analysis of DNA adducts formed from the interaction of the anticancer drugs mitomycin C, porfiromycin and thiotepa with calf thymus DNA. The adducts formed from reaction of mitomycin C and porfiromycin with DNA were separated from unmodified nucleosides by HPLC on a C18 column and identified by thermospray mass spectrometry. Thiotepa DNA adducts readily depurinated from DNA and were chromatographed and identified by thermospray liquid chromatography-mass spectrometry as the modified bases without the ribose moiety attached. The utility of thermospray mass spectrometry for the identification of microgram quantities of nucleoside adducts and depurinated base adducts of these anticancer drugs was demonstrated.     

UV Light-induced Cross-linking of Nucleosides, Nucleotides and a Dinucleotide to the Carboxy-terminal Heptad Repeat Peptide of RNA Polymerase II as Studied by Mass Spectrometry

Abstract— We report here the results of a study to assess the usefulness of mass spectrometry as a method for rapidly locating cross-linking sites in peptides modified by UV irradiation in the presence of nucleic acid components. For this study, we selected two nucleosides (thymidine and 5-bromo-2'-deoxyuridine), two nucleotides (thymi-dine-5'-monophosphate and 5-bromo-2'-deoxyuridine-5-monophosphate) and a dinucleotide (thymidylyl-[3'→5']-2'-deoxyadenosine). The peptide picked was SPSYSPT ( l -seryl- l -prolyl- l -seryl- l -tyrosyl- l -seryl- l -prolyl- l -threonine), the heptad repeat unit found in the largest subunit of the RNA polymerase II multiprotein complex. Modified peptides were isolated by reversed-phase HPLC. Molecular mass measurements confirmed that covalent adducts had been formed. High-energy tandem collision-induced dissociation mass spectrometry pinpointed the location of cross-linking in each modified peptide as being at the tyrosine residue. These results indicate that mass spectrometry is a potentially applicable technique for location of cross-linking sites in peptides, modified by attachment of nucleosides, nucleotides and dinucleotides. Such modified peptides would be among the products expected after application of standard proteolytic and nucleolytic digestion protocols to digestion of cross-linked DNA-protein complexes.     

Permethylation Linkage Analysis Techniques for Residual Carbohydrates

Permethylation analysis is the classic approach to establishing the position of glycosidic linkages between sugar residues. Typically, the carbohydrate is derivatized to form acid-stable methyl ethers, hydrolyzed, peracetylated, and analyzed by gas chromatography-mass spectrometry. The position of glycosidic linkages in the starting carbohydrate are apparent from the mass spectra as determined by the location of acetyl residues. The completeness of permethylation is dependent upon the choice of base catalyst and is readily confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry mass spectrometry. For the permethylation of β-cyclodextrin, Hakomori dimsyl base is shown to be superior to the NaOH–dimethyl sulfoxide system, and the use of the latter resulted in selective under-methylation of the 3-hydroxy groups. These techniques are highly applicable to residual carbohydrates from biofuel processes. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture.     

Traveling-wave ion mobility mass spectrometry analysis of isomeric modified peptides arising from chemical cross-linking

Traveling-wave ion mobility (TWIM) coupled to mass spectrometry (MS) has emerged as a powerful tool for structural and conformational analysis of proteins and peptides, allowing the analysis of isomeric peptides (or proteins) with the same sequence but modified at different residues. This work demonstrates the use of the novel TWIM-MS technique to separate isomeric peptide ions derived from chemical cross-linking experiments, which enables the acquisition of distinct product ion spectra for each isomer, clearly indicating modification on different sites. Experiments were performed with four synthetic peptides, for which variable degrees of mobility separation were achieved. In cases of partially overlapping mobility arrival time distributions (ATDs), extracting the ATDs of fragment ions belonging to each individual isomer allowed their separation into two distinct ATDs. Accumulation over regions from the specific ATDs generates the product ion spectrum of each isomer, or a spectrum highly enriched in their fragments. The population of both modified peptide isomers was correlated with the intrinsic reactivities of different Lys residues from reactions conducted at different pH conditions.     

SYNTHESIS AND DETERMINATION OF THE BIOLOGICAL ACTIVITIES OF YEAST ALANINE tRNA ANALOGUES

The role of base modification in yeast tRNAAl(?) function in protein synthesis was examined by the use of unmodified tRNA analogues. Unmodified full length tRNAs, 5'-half tRNAs (nucleotides 1-35) and 3'-half tRNAs (nucleotides 37-75) were transcribed in vitro using T7-RNA polymerase. Unmodified tRNA half molecules were joined to normally modified half molecules (5'-half, nucleotides 1-36; 3'-half, nucleotides 36-75) by T4-RNA ligase. Using this method, we synthesized three analogues of yeast tRNAAl(?): (i) tRNAAl(?) which lacks base modifications in the 5'-half of the molecule; (ii) tRNAAl(?) which lacks base modifications in the 3'-half of the molecule; and (iii) tRNAAla completely lacking base modifications. We determined the biological activities of these analogues. In rat aminoacyl-tRNA synthetase reactions, the alanine acceptance activity decreased by 52%, 79% and 85% when modified bases were absent from the 5'-half molecule, the 3'-half molecule or the total molecule, respectively. In rabbit retic     

Liquid injection field desorption/ionization-mass spectrometry of ionic liquids

Ten ionic liquids based on four types of organic cations, C(+) (imidazolium, pyrrolidinium, pyridinium, and phosphonium), combined with various types of anions, A-, were analyzed by liquid injection field desorption/ionization- (LIFDI) mass spectrometry. For the purpose of LIFDI analysis the ionic liquids were dissolved in methanol, acetonitrile or tetrahydrofuran at concentrations of 0.01-0.1 microl mL(-1). The measurements were performed on a double-focusing magnetic sector instrument. In all ionic liquid LIFDI spectra, the intact cation of the compound yielded the base peak accompanied by cluster ions of the general formula [C(2)A](+) and occasionally [C(3)A(2)](+). Tandem mass spectrometry and reconstructed ion chromatograms were employed to reveal the identity of the observed ions. Although limited to positive-ion mode, LIFDI also provided analytical information on the anions due to cluster ion formation. Depending on actual emitter condition and ionic liquid the limit of detection in survey scans was determined to 5-50 pg of ionic liquid.     

Thermal characterization of natural and modified cyclodextrins using TG-MS combined technique

Summary Thermoanalytical techniques (TG, DSC) are frequently used in the investigation of the thermal properties of cyclodextrins and their inclusion complexes. However, the above techniques do not provide information on the chemical composition of the evolved fragments upon the thermal decomposition. In this study &agr;-, &bgr;- and &ggr;-cyclodextrins and 4 methylated and 3 ethylated &bgr;-CD derivatives were investigated with a TG-MS combined thermoanalytical technique in order to get information about their fragmentation behaviour. By comparison of the TG/DTA curves, a different thermal behaviour was found for each of the native and the chemically modified cyclodextrins. Except for the water loss profiles and the solid-solid phase transformations, the thermal behaviour of the (investigated) native CDs do not show remarkable differences. However, the chemical modification of the native &bgr;-CD resulting in a new compound may change the strength of interactions between host and guest causing differences in the thermal stabilities of the derivatives. The mass spectrometry results supported the observed thermal differences and showed significant alterations in the fragmentation of ethylated and methylated compounds. The investigated natural CDs possess a very similar fragmentation profile, due to the common &agr;-D-glucopyranose building units. In the case of modified CDs characteristic signals of the substituents are present.     

Characterization of blocked nucleosides by mass spectrometry. Benzyl derivatives

The mass spectra of eleven model monobenzylated nucleosides were studied using low and high resolution mass spectrometry. Structural assignments to the major ions were made and several decomposition mechanisms proposed, with the goal of establishing the uses and limitations of mass spectrometry for the characterization of benzylaled nucleosides. Mass spectra generally permit determination of the extent and site of benzylation, with particular regard to base vs. sugar substitution, 0–2′ vs. 0–3′ or 0–5′, and in some cases 0–5′ vs. other isomers.     

Sequence, structure and energy transfer in DNA

Excitation energy transfer in DNA has similarities to charge transfer, but the transport is of an excited state, not of mass or charge. Use of the fluorescent, modified adenine base 2‐aminopurine (2AP) as an energy trap in short (3‐ to 20‐base) single‐ and double‐stranded DNA oligomers is reviewed. Variation of 2AP’s neighboring sequence shows (1) relatively efficient transfer from adenine compared to that from cytosine and thymine, (2) efficient transfer from guanine, but only when 2AP is at the 3′ end, (3) approximate equality of efficiencies for 3′ to 5′ and 5′ to 3′ directional transfer in adenine tracks. The overall, average transfer distance at room temperature is about four adenine bases or less before de‐excitation. The transfer fluorescence excitation spectral shape is similar to that of the absorption spectrum of the neighboring normal bases, confirming that initial excitation of the normal bases, followed by emission from 2AP (i.e. energy transfer), is occurring. Transfer apparently may take place both along one strand and cross‐strand, depending on the oligomer sequence. Efficiency increases when the temperature is decreased, rising above 50% (overall efficiency) in decamers of adenine below ?60°C (frozen media). Modeling of the efficiencies of transfer from the nearest several adenine neighbors of 2AP in these oligomers suggests that the nearest two neighbors transfer with near 100% efficiency. As bases in B DNA, as well as in single‐stranded DNA, are separated by less than 5 Å (less than the size of a base), standard Förster transfer theory should not apply. Indeed, while both theory and experiment show efficiency decreasing with donor–acceptor distance, the experimental dependence clearly disagrees with Förster 1/r⁶ dependence. It is not yet clear what the best theoretical approach is, but any calculation must deal accurately with the excited states of bases, including strong base–base interactions and structural fluctuations, and should reflect the increase of efficiency with temperature decrease and the relative insensitivity to strandedness (single, double). Attempts to use DNA as a molecular “fiber optic” face three primary challenges. First, reasonable efficiency over more than a base or two occurs only in adenine stretches at temperatures well below freezing. Second, transfer in these adenine tracks is efficient in both directions. Third, absorption of UV light occurs randomly, making excitation at a specific site on this “fiber optic” a challenge.     

Mass spectrometric identification of in vivo carbamylation of the amino terminus of Ectothiorhodospira mobilis high-potential iron-sulfur protein,isozyme 1

The complete amino acid sequence of a novel high-potential iron-sulfur protein (HiPIP) isozyme 1 from the moderately halophilic phototrophic bacterium Ectothiorhodospira mobilis was determined by a combined approach of chemical and mass spectrometric sequencing techniques. By mass analysis of the apo- and holo-protein in the positive electrospray ionization mode using different electrospray solvents, the protein was found to be post-translationally modified by a moiety of 43 Da. Further analysis showed the nature and location of this modification to be a carbamyl group at the N-terminus of the HiPIP. This rare type of modification has previously been reported to occur in the water-soluble human lens alphaB-crystallin, class D beta-lactamases and some prokaryotic ureases, albeit at an internal lysine residue. In this paper, we discuss the mass spectrometric features of a carbamylated residue at the N-terminus of a peptide or a lysine side-chain during sequence analysis by collision-induced dissociation tandem mass spectrometry. Our data provide evidence for the first case of a prokaryotic carbamylated electron transport protein occurring in vivo.     

One-Pot Synthesis of Novel Spiro Pyrano[2,3-d][1,3]thiazolo[3,2-a]pyrimidine Derivatives

In a one-pot synthesis, 1′-methyl-2,3″-dioxo-5″-aryl-1,2,5a″,7″,8″,9a″-hexahydro-5″H,6″H-dispiro[indole-3,2′-pyrrolidine-3′,2″-pyrano[2,3-d][1,3]thiazolo[3,2-a]pyrimidine]-4′-carboxylic acid methyl ester was prepared via the sequential reaction of 4-aryl-octahydro-pyrano[2,3-d]pyrimidine-2-thione, dimethyl acetylenedicarboxylate (DMAD), and a mixture of isatin and sarcosine. All the novel spiro compounds, in moderate yields, were characterized thoroughly by infrared, NMR, mass spectromentry, and elemental analysis together with x-ray crystallographic analysis.