Structural analysis of drug-DNA adducts by tandem mass spectrometry

The utility of electrospray ionisation (ESI) tandem mass spectrometry (MS/MS) for the characterisation of ligand-oligonucleotide adducts is demonstrated with adducts formed between the oligonucleotide 5'-CACGTG-3' and both a platinating agent, cis-diamminedichloroplatinum(II) (cisplatin), and an alkylating ligand, n-bromohexylphenanthridinium bromide (phenC6Br). We have demonstrated previously that negative ion MS/MS spectra of alkylated oligonucleotides show a highly specific fragmentation pathway that enables the site of binding of the ligand to be readily identified. In comparison, the positive ion ESI-MS/MS spectra reported here also show a single major fragmentation pathway, but the dominant ion is the protonated ligand-base adduct. MS/MS of this ion confirms the site on binding of the ligand to the guanine base. MS/MS spectra of cisplatin adducts show much less specific fragmentation than alkylated adducts, particularly in the negative ion mode. This suggests that the ESI-MS/MS spectra of ligand-DNA adducts are strongly influenced by the extent to which the ligand weakens the glycosidic bond in the residue to which it is bound. For platinating agents, which do not labilise the glycosidic bond, additional experiments involving MS/MS of source-generated product ions were required to enable isomeric adducts to be distinguished.     

High resolution mass spectrometry for studying the interactions of cisplatin with oligonucleotides

Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) has been used to probe the interaction of the anticancer drug cisplatin with oligonucleotides. The binding kinetics, the nature of the adducts formed, and the location of the binding site within the specifically designed double-stranded DNA oligonucleotides, ds(GTATTGGCACGTA) and ds(GTACCGGTGTGTA), were determined by recording mass spectra over time and/or employing tandem mass spectrometry (MS/MS). The FT-ICR MS studies show that binding to DNA takes place via a [Pt(NH 3) 2Cl] (+) intermediate prior to formation of bifunctional [Pt(NH 3) 2] (2+) adducts. Tandem MS reveals that the major binding sites correspond to GG and GTG, the known preferred binding sites for cisplatin, and demonstrates the preference for binding to guanosine within the oligonucleotide. The obtained results are discussed and compared to published data obtained by other mass spectrometric techniques, NMR spectroscopy and X-ray crystallography.     

Tautomerization in gas-phase ion chemistry of isomeric C-8 deoxyguanosine adducts from phenol-induced DNA damage

Collision-induced dissociation (CID) of 8-(4'-hydroxyphenyl)-2'-deoxyguanosine and 8-(2'-hydroxyphenyl)-2'-deoxyguanosine was investigated using sequential tandem mass spectrometry. These adducts represent biomarkers of DNA damage linked to phenolic radicals and were investigated to gain insight into the effects of chemical structure of a C-8 modification on fragmentation pathways of modified 2'-deoxyguanosine (dG). CID in MS(2) of the deprotonated molecules of both the isomers generated the same product ion having the same m/z values. CID in MS(3) of the product ion at m/z 242 and CID in MS(4) experiments carried out on the selected product ions at m/z 225 and m/z 218 afford distinct fragmentation patterns. The conformational properties of isomeric product ions from CID showed that the ortho-isomers possess the unique ability to tautomerize through an intramolecular proton transfer between the phenolic OH group and the imine nitrogen (N7). Tautomerization of ortho-isomers to their keto-tautomers led to differences in their system of conjugated double bonds compared with either their enol-tautomer or the para-isomer. The charge redistribution through the N-7 site on the imidazole ring is a critical step in guanosine adduct fragmentation which is disrupted by the formation of the keto-tautomer. For this reason, different reaction pathways are observed for 8-(4'-hydroxyphenyl)-2'-deoxyguanosine and 8-(2'-hydroxyphenyl)-2'-deoxyguanosine. We present herein the dissociation and the gas-phase ion-molecule reactions for highly conjugated ions involved in the CID ion chemistry of the investigated adducts. These will be useful for those using tandem mass spectrometry for structural elucidation of C-8 modified dG adducts. This study demonstrates that the modification at the C-8 site of dG has the potential to significantly alter the reactivity of adducts. We also show the ability of tandem mass spectrometry to completely differentiate between the isomeric dG adducts investigated.     

Structural analysis of diacyl peroxides by electrospray tandem mass spectrometry with ammonium acetate: bond homolysis of peroxide-ammonium and peroxide-proton adducts

Organic peroxides have significant implications in organic chemistry and biological processes. The weak O-O bond makes them extremely difficult to characterize by conventional analytical methods. Diacyl peroxides are one of the major radical sources in polymerization and organic synthesis. It is well known that diacyl peroxides are thermal labile and thus are not amenable to study by gas chromatography/mass spectrometry (GC/MS). Electrospray tandem mass spectrometry (ESI-MS/MS) has been applied to the structural analysis of diacyl peroxides by formation of ammonium adducts. Collision induced dissociation (CID) studies of the ammonium adducts of the peroxide [M + NH(4)](+) give collision energy dependent fragments. For most diacyl peroxides, homolysis of the peroxy bond predominates the fragmentation pathways of the peroxide-ammonium adducts. Deuterated substrates have been employed to provide evidence for typical fragmentation pathways. The CID studies were also used to locate the O-18 in some O-18 specifically labeled diacyl peroxides. For branched alkyl or alkoxy substrates, McLafferty rearrangement and decarboxylation become a major pathway. By comparison with some anhydride analogues, ESI-MS/MS can also be used to study this class of compounds.     

Gas Phase Chemistry of Li+ with Amides: the Observation of LiOH Loss in Mass Spectrometry

Collision-induced dissociation (CID) of Li(+) adducts of three sets of compounds that contains an amide bond, including 2-(4, 6-dimethoxypyrimidin-2-ylsulfanyl)-N-phenylbenzamide, its derivatives and simpler structures was investigated by electrospray ionization tandem mass spectrometry (ESI-MS/MS). Observed fragment ions include those that reflect loss of LiOH. Other product ions result from the Smiles rearrangement and direct C-S bond cleavage. MS/MS of H/D exchange products demonstrated occurrence of a 1,3-H shift from the amide nitrogen atom to the phenyl ring of these compounds. The LiOH loss from Li(+) adducts of amides was further examined by CID of [M + Li](+) ions of N-phenylbenzamide and N-phenylcinnamide. Loss of LiOH was essentially the sole fragmentation reaction observed for the former. For the latter, both losses of LiOH and H(2)O were discovered. The presence of electron-donating substituents of the phenyl ring of these compounds was found to facilitate elimination of LiOH, while that loss was retarded by electron-withdrawing substituents. Proposed fragment ion structures were supported by elemental compositions deduced from ultrahigh resolution Fourier transform ion cyclotron resonance tandem mass spectrometry (FTICR-MS/MS) m/z value determinations. Density functional theory-based (DFT) calculations were performed to evaluate potential mechanisms for these reactions.     

Liquid secondary ionization,tandem and matrix-assisted laser desorption/ionization time-of-flight mass spectrometric characterization of glycosphingolipid derivatives

Permethylated, peracetylated and perbenzoylated derivatives of glycosphingolipids (GSLs) were prepared to compare their liquid secondary ionization mass spectrometric (LSIMS) and collision-induced dissociation tandem mass spectrometric (CID/MS/MS) fragmentation patterns and also to determine sensitivity improvement in LSIMS and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) relative to the native species. Permethylation was carried out in the liquid phase, whereas peracetylation and perbenzoylation could be effected using either liquid (bulk)-phase or gas-phase procedures. Lower amounts of starting material were required for the gas-phase derivatization (? 100 pmol) compared with the bulk phase (?1 nmol), because the former method permits more efficient sample handling. All three types of derivatives yielded sensitivity improvements of at least two orders of magnitude over the native species in both LSIMS and MALDI-TOFMS. The behavior of the permethylated compounds was used as the benchmark for GSL structural information content in normal and tandem mass spectra. Fragments present in spectra of the three types of derivatives generated complementary information. Permethylated GSLs favored the formation of ions related to the ceramide moieties, whereas peracetylation enhanced the production of carbohydrate-related ions. The LSI mass spectra of perbenzoylated GSLs contained information on both ceramide and sugar portions of the molecules. Each of the LSIMS, MS/MS and MALDI-TOFMS techniques proved to be complementary to the others in this study; the use of all three is recommended for the generation of complete structural information.     

Mass spectrometry of cis-diamminedichloroplatinum(II) adducts with the dinucleosidemonophosphates d(ApG), d(GpG) and d(TpC) in an ion trap

The detection and fragmentation behaviour of adducts of the chemotherapeutic cis-diamminedichloroplatinum(II) (cisplatin) with the dinucleosidemonophosphates d(ApG), d(GpG) and d(TpC) as model compounds for DNA adducts in an ion trap with electrospray ionization were studied. Mainly the monofunctional adduct, the bifunctional adduct and the bifunctional adduct with platinum bridging two dinucleosidemonophosphates were detected. In addition, several more complex adducts were seen resulting from reactions among these species. Adduct formation was low in the case of d(TpC). Fragmentation could be controlled strongly by varying the temperature of the transfer capillary; furthermore, tandem mass spectrometric (MS/MS) experiments on both the monofunctional and the bifunctional adducts were performed. For the adducts of d(ApG) and d(GpG) losses of NH(3) and HCl were the most dominant reactions, followed by the losses of one, then another two units of 98 amu from the sugar-phosphate backbone, whereas d(TpC)-Pt predominantly forms the dinucleosidemonophosphate. In the gas phase, the conversion of the monofunctional into the bifunctional adducts through binding to another site in the dinucleotide accompanied by loss of NH(3) or HCl could also be observed. The removal of a ligand from the coordination sphere of the square-planar platinum complexes appeared to be the crucial step for the induction of further fragmentation of the dinucleotide ligand. MS(n) experiments of the bifunctional adducts of d(ApG) and d(GpG) revealed different fragmentation pathways involving the loss of phosphoric acid, metaphosphoric acid, deoxyribose units (intact or dehydrated) and the nucleobases in different orders, leaving characteristic binding site-determining fragments. Fragmentation of these ions was also performed, mainly resulting in fragmentation of the bases. The study confirmed the remarkable stability of the platinum-guanine bond compared with other nucleobases.     

Mechanisms for the selective gas-phase fragmentation reactions of methionine side chain fixed charge sulfonium ion containing peptides

To enable the development of improved tandem mass spectrometry based methods for selective proteome analysis, the mechanisms, product ion structures, and other factors influencing the gas-phase fragmentation reactions of methionine side-chain derivatized "fixed-charge" phenacylsulfonium ion containing peptide ions have been examined. Dissociation of these peptide ions results in the exclusive characteristic loss of the derivatized side chain, thereby enabling their selective identification. The resultant product ion(s) are then subjected to further dissociation to obtain sequence information for subsequent protein identification. Molecular orbital calculations (at the B3LYP/6-31 + G (d,p) level of theory) performed on a simple peptide model, together with experimental evidence obtained by multistage dissociation of a regioselectively deuterated methionine derivatized sulfonium ion containing tryptic peptide, indicate that fragmentation of the fixed charge containing peptide ions occurs via SN2 reactions involving the N- and C-terminal amide bonds adjacent to the methionine side chain, resulting in the formation of stable cyclic five- and six-membered iminohydrofuran and oxazine product ions, respectively. These studies further indicate that the rings formed via these neighboring group reactions are stable to further dissociation by MS3. As a consequence, the formation of b- or y-type sequence ions are "skipped" at the site of cyclization. Despite this, complete sequence information is still obtained because of the presence of both cyclic products.     

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.     

Analysis of the binding sites with NL-101 to amino acids and peptides by HPLC/MS/MS

Collision-induced dissociation experiment demonstrated that under the same collision energy, different fragmentation pattern could help distinguish the accurate location of amino acids-NL adducts.     

Tandem mass spectrometry and hydrogen/deuterium exchange studies of protonated species of 1,1'-bis(diphenylphosphino)-ferrocene oxidative impurity generated during a Heck reaction

Oxidation of 1,1'-bis(diphenylphosphino)-ferrocene (DPPF) was found to occur when it served as the ligand for Pd(II)(CH3COO)2 in a Heck reaction. This oxidative impurity of DPPF, referred to as DPPF(O), was identified by high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) and exact mass measurements. Protonated DPPF(O) exhibited unique fragmentation pathways in the gas phase. Hydrogen/deuterium (H/D) exchange experiments provided important insights into the dissociation mechanisms of protonated DPPF(O), suggesting the existence of isomeric structures of the product ions by retaining or losing a proton (or deuteron) upon collision-induced dissociation (CID). The specific fate of the proton (or deuteron) upon CID is postulated to be dependent on the distance between the exchangeable proton (or deuteron) and the sites of bond cleavage. Density functional theory (DFT) calculations at the B3LYP/LANL2DZ level of theory showed that oxygen in DPPF(O) plays a pivotal role in invoking pi-cation interactions between the p-type lone pair electrons (n pi) in oxygen and the anti-bonding orbital of Fe(II), accounting for the major fragmentation pathways of protonated DPPF(O). Facile formation of organometallic distonic ions in dissociation of protonated DPPF(O), and especially of protonated DPPF, could be useful for further exploration of their chemical properties by gas-phase ion/molecule reactions.     

Gas-phase binding of non-covalent protein complexes between bovine pancreatic trypsin inhibitor and its target enzymes studied by electrospray ionization tandem mass spectrometry

The potential of electrospray ionization (ESI) mass spectrometry (MS) to detect non-covalent protein complexes has been demonstrated repeatedly. However, questions about correlation of the solution and gas-phase structures of these complexes still produce vigorous scientific discussion. Here, we demonstrate the evaluation of the gas-phase binding of non-covalent protein complexes formed between bovine pancreatic trypsin inhibitor (BPTI) and its target enzymes over a wide range of dissociation constants. Non-covalent protein complexes were detected by ESI-MS. The abundance of the complex ions in the mass spectra is less than expected from the values of the dissociation constants of the complexes in solution. Collisionally activated dissociation (CAD) tandem mass spectrometry (MS/MS) and a collision model for ion activation were used to evaluate the binding of non-covalent complexes in the gas phase. The internal energy required to induce dissociation was calculated for three collision gases (Ne, Ar, Kr) over a wide range of collision gas pressures and energies using an electrospray ionization source. The order of binding energies of the gas-phase ions for non-covalent protein complexes formed by the ESI source and assessed using CAD-MS/MS appears to differ from that of the solution complexes. The implication is that solution structure of these complexes was not preserved in the gas phase.     

Gas‐phase fragmentation of protonated piplartine and its fungal metabolites using tandem mass spectrometry and computational chemistry

Piplartine, an alkaloid produced by plants in the genus Piper , displays promising anticancer activity. Understanding the gas‐phase fragmentation of piplartine by electrospray ionization tandem mass spectrometry can be a useful tool to characterize biotransformed compounds produced by in vitro and in vivo metabolism studies. As part of our efforts to understand natural product fragmentation in electrospray ionization tandem mass spectrometry, the gas‐phase fragmentation of piplartine and its two metabolites 3,4‐dihydropiplartine and 8,9‐dihydropiplartine, produced by the endophytic fungus Penicillium crustosum VR4 biotransformation, were systematically investigated. Proposed fragmentation reactions were supported by ESI‐MS/MS data and computational thermochemistry. Cleavage of the C‐7 and N‐amide bond, followed by the formation of an acylium ion, were characteristic fragmentation reactions of piplartine and its analogs. The production of the acylium ion was followed by three consecutive and competitive reactions that involved methyl and methoxyl radical eliminations and neutral CO elimination, followed by the formation of a four‐member ring with a stabilized tertiary carbocation. The absence of a double bond between carbons C‐8 and C‐9 in 8,9‐dihydropiplartine destabilized the acylium ion and resulted in a fragmentation pathway not observed for piplartine and 3,4‐dihydropiplartine. These results contribute to the further understanding of alkaloid gas‐phase fragmentation and the future identification of piplartine metabolites and analogs using tandem mass spectrometry techniques. Copyright © 2017 John Wiley & Sons, Ltd.     

Conformational Isomers of Calcineurin Follow Distinct Dissociation Pathways

In the gas-phase, ions of protein complexes typically follow an asymmetric dissociation pathway upon collisional activation, whereby an expelled small monomer takes a disproportionately large amount of the charges from the precursor ion. This phenomenon has been rationalized by assuming that upon activation, a single monomer becomes unfolded, thereby attracting charges to its newly exposed basic residues. Here, we report on the atypical gas-phase dissociation of the therapeutically important, heterodimeric calcium/calmodulin-dependent serine/threonine phosphatase calcineurin, using a combination of tandem mass spectrometry, ion mobility mass spectrometry, and computational modeling. Therefore, a hetero-dimeric calcineurin construct (62?kDa), composed of CNa (44?kDa, a truncation mutant missing the calmodulin binding and auto-inhibitory domains), and CNb (18?kDa), was used. Upon collisional activation, this hetero-dimer follows the commonly observed dissociation behavior, whereby the smaller CNb becomes highly charged and is expelled. Surprisingly, in addition, a second atypical dissociation pathway, whereby the charge partitioning over the two entities is more symmetric is observed. The presence of two gas-phase conformational isomers of calcineurin as revealed by ion mobility mass spectrometry (IM-MS) may explain the co-occurrence of these two dissociation pathways. We reveal the direct relationship between the conformation of the calcineurin precursor ion and its concomitant dissociation pathway and provide insights into the mechanisms underlying this co-occurrence of the typical and atypical fragmentation mechanisms.     

Fragmentation of <Emphasis Type="Italic">α</Emphasis>-radical cations of arginine-containing peptides

Fragmentation pathways of peptide radical cations, M^+·, with well-defined initial location of the radical site were explored using collision-induced dissociation (CID) experiments. Peptide radical cations were produced by gas-phase fragmentation of Co^III(salen)-peptide complexes [salen=N,N′-ethylenebis (salicylideneiminato)]. Subsequent hydrogen abstraction from the β-carbon of the side-chain followed by C_α-C_β bond cleavage results in the loss of a neutral side chain and formation of an α-radical cation with the radical site localized on the α-carbon of the backbone. Similar CID spectra dominated by radical-driven dissociation products were obtained for a number of arginine-containing α-radicals, suggesting that for these systems radical migration precedes fragmentation. In contrast, proton-driven fragmentation dominates CID spectra of α-radicals produced via the loss of the arginine side chain. Radical-driven fragmentation of large M^+· peptide radical cations is dominated by side-chain losses, formation of even-electron a-ions and odd-electron x-ions resulting from C_α-C bond cleavages, formation of odd-electron z-ions, and loss of the N-terminal residue. In contrast, charge-driven fragmentation produces even-electron y-ions and odd-electron b-ions.     

Fragmentation methods on the balance: unambiguous top–down mass spectrometric characterization of oxaliplatin–ubiquitin binding sites

The interaction between oxaliplatin and the model protein ubiquitin (Ub) was investigated in a top–down approach by means of high-resolution electrospray ionization mass spectrometry (ESI-MS) using diverse tandem mass spectrometric (MS/MS) techniques, including collision-induced dissociation (CID), higher-energy C-trap dissociation (HCD), and electron transfer dissociation (ETD). To the best of our knowledge, this is the first time that metallodrug–protein adducts were analyzed for the metal-binding site by ETD-MS/MS, which outperformed both CID and HCD in terms of number of identified metallated peptide fragments in the mass spectra and the localization of the binding sites. Only ETD allowed the simultaneous and exact determination of Met1 and His68 residues as binding partners for oxaliplatin. CID-MS/MS experiments were carried out on orbitrap and ion cyclotron resonance (ICR)-FT mass spectrometers and both instruments yielded similar results with respect to number of metallated fragments and the localization of the binding sites. A comparison of the protein secondary structure with the intensities of peptide fragments generated by collisional activation of the [Ub + Pt-(chxn)] adduct [chxn = (1R,2R)-cyclohexanediamine] revealed a correlation with cleavages in solution phase random coil areas, indicating that the N-terminal β-hairpin and α-helix structures are retained in the gas phase.     

Direct determination of the primary binding site of cisplatin on cytochrome c by mass spectrometry

Protein—cisplatin interactions lie at the heart of both the effectiveness of cisplatin as a therapeutic agent and side effects associated with cisplatin treatment. Because a greater understanding of the protein—cisplatin interactions at the molecular level can inform the design of cisplatin-like agents for future use, mass spectrometric determination of the binding site of cisplatin on a model protein, cytochrome c, was undertaken in this paper. The monoadduct cytochrome c—Pt(NH₃)₂(H₂O) is found to be the primary adduct produced by the cytochrome c—cisplatin interactions under native conditions. To locate the primary binding site of cisplatin, both free cytochrome c and the cytochrome c adducts underwent trypsin digestion, followed by Fourier transform mass spectrometry (FT-MS) to identify unique fragments in the adduct digest. Four such fragments were found in the adduct digest. Tandem mass spectrometry (MS/MS and MS³ indicates that two fragments are Pt(NH₃)₂(H₂O) bound peptides (Gly56-Glu104 and Asn54-Glu104) with one water associated at the peptide bond Lys79∼Met80, and the other two fragments are heme containing peptides (acety1-Gly1-Lys53 and acety1-Gly1-Lys55). The product-ion spectra of the four fragments reveal that Met65 is the primary binding site of cisplatin on cytochrome c.     

Electrospray tandem mass spectrometry of mixed-sequence RNA/DNA oligonucleotides

The fragmentation of electrospray-generated multiply deprotonated RNA and mixed-sequence RNA/DNA pentanucleotides upon low-energy collision-induced dissociation (CID) in a hybrid quadrupole time-of-flight mass spectrometer was investigated. The goal of unambiguous sequence identification of mixed-sequence RNA/DNA oligonucleotides requires detailed understanding of the gas-phase dissociation of this class of compounds. The two major dissociation events, base loss and backbone fragmentation, are discussed and the unique fragmentation behavior of oligoribonucleotides is demonstrated. Backbone fragmentation of the all-RNA pentanucleotides is characterized by abundant c-ions and their complementary y-ions as the major sequence-defining fragment ion series. In contrast to the dissociation of oligodeoxyribonucleotides, where backbone fragmentation is initiated by the loss of a nucleobase which subsequently leads to the formation of the w- and [a-base]-ions, backbone dissociation of oligoribonucleotides is essentially decoupled from base loss. The different behavior of RNA and DNA oligonucleotides is related to the presence of the 2'-hydroxyl substituent, which is the only structural alteration between the DNA and RNA pentanucleotides studied. CID of mixed-sequence RNA/DNA pentanucleotides results in a combination of the nucleotide-typical backbone fragmentation products, with abundant w-fragment ions generated by cleavage of the phosphodiester backbone adjacent to the deoxy building blocks, whereas backbone cleavage adjacent to ribonucleotides induces the formation of c- and y-ions.