More Than Charged Base Loss — Revisiting the Fragmentation of Highly Charged Oligonucleotides

Tandem mass spectrometry is a well-established analytical tool for rapid and reliable characterization of oligonucleotides (ONs) and their gas-phase dissociation channels. The fragmentation mechanisms of native and modified nucleic acids upon different mass spectrometric activation techniques have been studied extensively, resulting in a comprehensive catalogue of backbone fragments. In this study, the fragmentation behavior of highly charged oligodeoxynucleotides (ODNs) comprising up to 15 nucleobases was investigated. It was found that ODNs exhibiting a charge level (ratio of the actual to the total possible charge) of 100% follow significantly altered dissociation pathways compared with low or medium charge levels if a terminal pyrimidine base (3' or 5') is present. The corresponding product ion spectra gave evidence for the extensive loss of a cyanate anion (NCO^–), which frequently coincided with the abstraction of water from the 3'- and 5'-end in the presence of a 3'- and 5'-terminal pyrimidine nucleobase, respectively. Subsequent fragmentation of the M-NCO^– ion by MS³ revealed a so far unreported consecutive excision of a metaphosphate (PO₃ ^–)-ion for the investigated sequences. Introduction of a phosphorothioate group allowed pinpointing of PO₃ ^– loss to the ultimate phosphate group. Several dissociation mechanisms for the release of NCO^– and a metaphosphate ion were proposed and the validity of each mechanism was evaluated by the analysis of backbone- or sugar-modified ONs.

Electron stimulated desorption of anions from native and brominated single stranded oligonucleotide trimers

We measured the low energy electron stimulated desorption (ESD) of anions from thin films of native (TXT) and bromine monosubstituted (TBrXT) oligonucleotide trimers deposited on a gold surface (T = thymidine, X = T, deoxycytidine (C), deoxyadenosine (A) or deoxyguanosine (G), Br = bromine). The desorption of H(-), CH(3)(-)/NH(-), O(-)/NH(2)(-), OH(-), CN(-), and Br(-) was induced by 0 to 20 eV electrons. Dissociative electron attachment, below 12 eV, and dipolar dissociation, above 12 eV, are responsible for the formation of these anions. The comparison of the results obtained for the native and brominated trimers suggests that the main pathways of TBrXT degradation correspond to the release of the hydride and bromide anions. Significantly, the presence of bromine in oligonucleotide trimers blocks the electron-induced degradation of nuclobases as evidenced by a dramatic decrease in CN(-) desorption. An increase in the yields of OH(-) is also observed. The debromination yield of particular oligonucleotides diminishes in the following order: BrdU?>?BrdA?>?BrdG?>?BrdC. Based on these results, 5-bromo-2(')-deoxyuridine appears to be the best radiosensitizer among the studied bromonucleosides.     

Investigation of the initial fragmentation of oligodeoxynucleotides in a quadrupole ion trap: Charge level-related base loss

The charge state distribution and CID fragmentation of two series of deprotonated oligodeoxynucleotide (ODN) 9-mers (5'-GGTTXTTGG-3' and 5'-CCAAYAACC-3', X/Y = G, C, A, or T) have been studied in detail in an ion trap in an effort to understand the intrinsic properties of DNA in vacuo. The distribution of charge states (-2 to -6) is similar for both the X- and Y-series, with the most abundant being the -4 charge state. The T-rich X-series prefers higher charge states (-6 and -5) than does the Y-series. Calculations show that phosphate groups located nearest a thymine are more acidic than those near an adenine, cytosine, or guanine, thus explaining why the X-series prefers higher charge states. We use the term "charge level" to define the ratio of the charge state to the total number of phosphate groups present in the ODN. We find, consistent with previous studies, that the initial step of fragmentation is loss of nucleobase either as an anion or as a neutral. We observe the former for ODNs with charge levels greater than 50% and the latter for ODNs with charge levels below 50%. The overall anionic base loss follows the trend A(-) > G(-) approximately T(-) > C(-); electrostatic potential calculations indicate that this trend follows delocalization of electron density for each anion, with A(-) being the most stabilized through delocalization. For neutral base loss, thymine (TH) is rarely cleaved, while the preferences for AH, GH, and CH loss vary. Proton affinity (PA) calculations show that a nearby negatively charged phosphate enhances the PA of proximally located nucleobases; this PA enhancement probably plays a role in promoting neutral base loss. The trends differ by charge level. At a charge level of 37.5% (-3 charge state), AH loss is preferred over CH and GH loss, regardless of sequence. However, at a charge level of 25% (-2 charge state), the terminal bases are preferentially lost over the internal bases, regardless of identity. By reconstructing the ODN sequences from structurally informative (a-BH) and w ions, we are able to identify the charge locations for the -3 and -2 charge states. For the -3 charge state, one charge resides on each "most terminal" phosphate, with the third being in the middle. For the -2 charge state, each charge resides on the penultimate phosphate groups. We compare our data to earlier experiments in an effort to generalize trends.     

Fragmentation of photomodified oligodeoxynucleotides adducted with metal ions in an electrospray-ionization ion-trap mass spectrometer

We report the effect of metal-ion adduction on the fragmentation of oligodeoxynucleotides (ODNs) bearing DNA photoproducts. When protons on backbone phosphates of ODNs are completely replaced with metal ions, cleavages occur readily within the photoproduct moiety, whereas those cleavages do not occur in photomodified ODNs in which the phosphates are associated with protons. For example, thymine/adenine (TA*) photoproducts revert to their undamaged precursors upon collisional activation, the pyrimidine(6-4)pyrimidone product and its Dewar valence isomer show a characteristic neutral loss of C4H3NO3, and dimeric adenine photoproducts show a distinctive loss of NH2CN from the adenine six-membered ring. The product-ion mass spectra of photodamaged ODNs that are adducted to metal ions are complementary in terms of structure information to those spectra of ODNs in which the phosphates are associated with protons. The results also demonstrate that the energy required for strand cleavages is higher for ODNs adducted with metal ions than that for ODNs bound with protons. Furthermore, the loss of a pyrimidine is more favorable than the loss of a purine in the fragmentation of ODNs associated with metal ions.     

Peculiarities of the Mass Spectrometric Detection of Anthocyanins in High-Performance Liquid Chromatography

The dependence of the degree of fragmentation of anthocyanin “molecular” ions on the cone voltage of a mass spectrometric electrospray ionization detector was studied. It was found that the voltage required for the fragmentation of 50% of original “molecular” ions, E _f (0.5), increased with the number of glycoside residues. The fragmentation of glycosides proceeds with the removal of the entire residue regardless of their structures. In the case of 3,5-diglycosides, two types of fragment ions formed due to the loss of glycosidic residues from different positions; the ratio of their peak intensities is reciprocal to the ratio of the masses of residues eliminated. The values of E _f (0.5) for monoglycosides (190 V), diglycosides (229 V), triglycosides (267 V), and for some acylated cyanidin-3,5-diglycosides are determined. These results were given for the gradient separation of anthocyanins by reversed-phase HPLC in aqueous acetonitrile containing 10 vol % of formic acid using quadrupole mass spectrometric detection.     

Oligo-2'-deoxyribonucleotides containing uracil modified at the 5-position with linkers ending with guanidinium groups

We report here the synthesis and binding studies of oligo-2'-deoxyribonucleotides (ODNs) containing 2'-deoxyuridines, modified at the 5-position by linkers ending with either one or two guanidinium groups. Comparison was made with ODNs containing 2'-deoxyuridines modified at the 5-position with linkers ending with either two or one amino groups. One or two modified 2'-deoxyuridines were incorporated into pyrimidine strands, and their influence on the stability of duplex (with both DNA and RNA targets) and triplex structures was studied. The strongest stabilization was obtained with modified ODNs containing guanidinium groups. This result confirms that the reduction of the global negative charge number on one strand is an important parameter in the stability of duplex and triplex structures.     

Fragmentation of electrospray-produced oligodeoxynucleotide ions adducted to metal ions

In this article, we describe the unique fragmentations of oligodeoxynucleotides (ODNs) whose phosphate groups are completely depleted of protons and replaced with metal ions. The production of the ubiquitous [a(n) - base] ions still occurs, but no longer by transfer of an acidic phosphate proton to an adjoining 3' base. Nor is the extent of the reaction determined by the proton affinity of that base. Rather, the reaction now occurs via a cleavage 3' to both pyrimidines and purines; cleavage 3' to pyrimidine is more favorable than that 3' to purine. We also demonstrate that an ODN is more stable in the gas phase when its phosphate groups are bound to metal ions than when its phosphate groups are attached to hydrogens. This study also provides further evidence for the ODN fragmentation mechanism that involves H transfer to a nucleobase. To establish the structural utility of this new fragmentation, we applied it to distinguishing small ODNs containing a photomodified cis,syn-cyclobutane pyrimidine dimer from the parent ODNs, a system that cannot be distinguished by collisional activation of precursor species that do not contain metal ions.     

To improve the quantification sensitivity of large molecular weight compounds--with ginsenosides as example

High cone voltage was used to improve the quantification sensitivity of large molecular weight compounds in high-performance liquid chromatography electrospray ionization mass spectrometry (HPLC/ESI-MS), with ginsenosides as example. Investigations on the effect of cone voltage showed that within a voltage range of 30-130 V, for all the ginsenosides tested, i.e., Rb(1), Rb(2), Rc, Rd, Re, R(f) and R(g1), an increase in the applied cone voltage can significantly increase the sensitivity of the method. The maximum sensitivity in the determination decreases with the decreasing molecular weight of the ginsenosides in the order of Rb(1) > Rb(2) > Rc > Re > Rd > R(g1) > R(f). At the high cone voltage of 130 V, both molecular weight and structural information was obtained from a single mass spectrum. It can also be used for isomer differentiation and determination of O-glycosidic linkages in ginsenosides. Linear relationships between the peak area response and concentration were observed in the range of 50-2 x 10(5) ng/mL, with the correlation coefficients >0.99. The limits of detection reached down to pg for ginsenosides. The method was successfully applied to the determination of ginsenosides in commercial ginseng samples.     

Studies on Fragmentation Regularity of 5-Methoxycarbonyl-4-substit uted-6-methyl-3,4-dihydropyrimidin-2（1H）-ones by Time-of-flight Mass Spectrometry

The electron impact time-of-flight(TOF) mass spectra of the title compounds were studied to establish their fragmentation processes. With the high resolution of the TOF instrument, the exact mass for each fragment was determined. These data were used to infer the molecular formulas and the elemental compositions for all the molecular ions and fragments through software interpretation. By further applying the fragmentation regularity, the majority of ions were fully assigned. The main fragmentation pathways of the title compounds include the formation of molecular ions by the loss of R^1 groups in the 4-position and the ester groups in the 5-position. The formed ion can be further fragmented by the elimination of MeOH.     

Electron ionisation and electrospray ionisation mass spectrometric study of a series of isomeric methyl-, dimethyl- and trimethylalloxazines

Electron ionisation (EI) mass spectra and electrospray ionisation (ESI) mass spectra at different cone voltages of a series of isomeric methyl- and dimethylalloxazines are discussed, and compared with those of lumichrome, and 1- and 3-methyllumichrome. Examination of ESI mass spectra taken at a higher cone voltage and the use of isotope-labelled methanol allow us to discuss the fragmentation pathways of [M+H]+ and [M-H](-) ions. The fragmentation pathways of all of the compounds and the characteristic fragment ions formed in EI-MS are compared with published data. The influence of methyl and dimethyl substituents in the benzene ring on the fragmentation pathways leading to the loss of 43 and 45 Da upon both electron and electrospray ionisation is described.     

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.     

Bromine-induced cyclization of 1-acyl-3-(3-thienyl)-2-thioureas to 2-acylaminothieno[3,2-d]thiazoles

Three different 1-acyl-3-(3-thienyl)-2-thioureas were cyclized to 2-acylaminothieno[3,2-d]thiazoles with bromine in acetic acid whereas the corresponding 2-thienylthiourea derivatives were brominated under the same reaction conditions. The parent thieno[3,2-d]thiazole was prepared by acid hydrolysis and deamination of 2-benzoylaminothieno[3,2-d]thiazole. This new heterocyclic compound was nitrated and brominated in the 5-position.     

Charge effects for differentiation of oligodeoxynucleotide isomers containing 8-oxo-dG residues

Dissociation reactions of a series of multiply charged oligodeoxynucleotide (ODN) 12-mer anions were studied using an ion trap mass spectrometer. These mixed nucleobase 12-mers fragment first by loss of a neutral nucleobase (A, G, C, and/or 5-methyl-cytosine) followed by cleavage at 3' C-O bond of the sugar from which the base is lost to produce the complementary sequence ions, i.e., [a - B] and w type of ions. No detectable loss of 8-oxo-guanine and/or thymine from these 12-mers is observed under gentle collision conditions in the ion trap. The primary loss of a nucleobase and the subsequent backbone cleavage to generate sequence ions strongly depend on the charge state of the parent molecular ion. For low charge states (2- and 3-), product ions due to the loss of a neutral guanine base and related sequence ions are dominant in the tandem mass spectra. However, preferential loss of a neutral adenine becomes the primary reaction channel from the 5- charge state of the molecular ion. Such charge state dependent fragmentation behavior was utilized to determine the site of 8-oxo-dG residue in a series of structural isomers. The position of 8-oxo-dG residue can be simply determined from the fragmentation pattern of 3- charge state, but not of 5- charge state. It is suggested that in addition to specific modification that affects the N-glycosidic bond strength, total charge content of an ODN is an important factor for determining the differential fragmentation behavior.     

Differentiation of fluoronitroaniline isomers by negative-ion electrospray mass spectrometry

Tetra- and trifluoronitroanilines were studied by electrospray ionization mass spectrometry. These compounds gave signals only in the negative-ion mode. It was found that the so-called 'in-source' fragmentation, induced by cone voltage increase, enables differentiation of isomers. For para-nitroanilines, in contrast to ortho derivatives, the loss of NO(2) was the most favored process and other fragment ions were characterized by low abundances. For trifluoro conjugates the substitution pattern of aromatic ring by fluorine atoms also affected their fragmentation patterns. For example, in 2,3,6-trifluoro-4-nitroaniline, in contrast to 2,3,5-trifluoro-4-nitroaniline, efficient NO loss, followed by HF loss, took place.     

Fragmentation of Electrospray-Produced Deprotonated Ions of Oligodeoxyribonucleotides Containing an Alkylated or Oxidized Thymidine

Alkylation and oxidation constitute major routes of DNA damage induced by endogenous and exogenous genotoxic agents. Understanding the biological consequences of DNA lesions often necessitates the availability of oligodeoxyribonucleotide (ODN) substrates harboring these lesions, and sensitive and robust methods for validating the identities of these ODNs. Tandem mass spectrometry is well suited for meeting these latter analytical needs. In the present study, we evaluated how the incorporation of an ethyl group to different positions (i.e., O ², N3, and O ⁴) of thymine and the oxidation of its 5-methyl carbon impact collisionally activated dissociation (CAD) pathways of electrospray-produced deprotonated ions of ODNs harboring these thymine modifications. Unlike an unmodified thymine, which often manifests poor cleavage of the C3′–O3′ bond, the incorporation of an alkyl group to the O ² position and, to a much lesser extent, the O ⁴ position, but not the N3 position of thymine, led to facile cleavage of the C3′–O3′ bond on the 3′ side of the modified thymine. Similar efficient chain cleavage was observed when thymine was oxidized to 5-formyluracil or 5-carboxyluracil, but not 5-hydroxymethyluracil. Additionally, with the support of computational modeling, we revealed that proton affinity and acidity of the modified nucleobases govern the fragmentation of ODNs containing the alkylated and oxidized thymidine derivatives, respectively. These results provided important insights into the effects of thymine modifications on ODN fragmentation.

Characterization of labeled oligonucleotides using enzymatic digestion and tandem mass spectrometry

A simple and powerful method for the determination of labeling sites on oligodeoxynucleotides (ODN) has been developed. The method is based on the finding that nuclease P1 (NP1) digestions of label-containing ODNs produce site-specific products: 5′-labeled ODNs produce label-nucleotide (L-N); 3′-labeled ODN produces phosphorylated label (pL); and a label in between the ODN termini produces pL-N. Mass spectrometry spectra of these products from the digestion mixture can be easily utilized for structural verification of labeled ODNs such as DNA probes. We also developed a method for the determination of the labeling sites of ODNs with unknown label structures. In this method, NP1 digestion products generate site-specific fragmentation patterns upon collision-induced dissociation. These patterns can be easily recognized and used for the identification of labeling sites of ODNs with unknown label structures. When an ODN is internally labeled, phosphodiesterase digestion may be used to determine the exact labeling site (sequence location). It was demonstrated that these methods can be applied for ODNs with single or multiple labels, and for ODNs with the same or different labels within an ODN.     

Study of polybrominated diphenyl ethers using both positive and negative atmospheric pressure photoionization and tandem mass spectrometry

Atmospheric pressure photoionization (APPI) was assessed for the mass spectrometric analysis of polybromodiphenyl ethers (PBDEs) on the basis of a set of 17 standard compounds. Positive and negative ionization modes were both investigated. M(+.) ions were formed under positive ion conditions whereas the negative ion mode yielded [M-Br+O](-) ions. The behavior of these APPI-produced ions towards collisional activation was studied using an ion trap mass spectrometer. In positive ion mode, the loss of Br(2) was one of the major fragmentation pathways, and was favored for ortho-substituted PBDEs. Conversely, the loss of COBr(.) occurred only for non-ortho-substituted congeners. The collisional excitation of [M-Br+O](-) ions in the ion trap also led to the loss of Br(2), to the elimination of HBr, and to the formation of product ions by cleavage of the ether bond. The formation of para-quinone radical anions was observed for PBDEs ranging from penta- to hepta-congeners, whereas brominated aromatic carbanions were formed preferentially for the most brominated PBDEs studied in this work (hepta- or deca-BDEs). M(+.) ions did not undergo this fragmentation process.     

Template-directed photoreversible ligation of DNA via 7-carboxyvinyl-7-deaza-2′-deoxyadenosine

An efficient template-directed photoligation of oligodeoxynucleotide (ODN) using 7-deaza-2′-deoxyadenosine derivative ^VZA is described. When ODN containing ^VZA at the 5′ end was photoirradiated with ODNs containing a pyrimidine base at the 3′ end in the presence of template ODN, rapid and efficient ligation (cycloaddition reaction) was observed without any byproduct formation. ODNs containing ^VZA showed an extremely high reactivity as compared with those reported in previous photoligations.     

Characterization of microcystins using in-source collision-induced dissociation

The efficiency of the in-source collision-induced dissociation (in-source CID) technique for the structural characterization of microcystins (MCYSTs) was evaluated. Microcystins that did not contain arginine underwent facile fragmentation to produce characteristic product ions at relatively low cone voltage and could be fully characterized based on their mass spectra. On the other hand, cyclic peptides possessing arginine residues, such as MCYST-RR, -LR, -YR and nodularin, were considerably more stable under in-source CID conditions and required higher cone voltage to induce fragmentation. This behaviour is explained in terms of the mobile proton model for peptide fragmentation that can be used as an indication for the presence of arginine when unknown microcystins are analyzed. In-source CID was applied to the characterization of microcystins released into water from a Microcystis aeruginosa culture (UTCC299) (UTCC: University of Toronto Culture Collection of Algae and Cyanobacteria). Six microcystins were detected in extracts from UTCC299: I, [D-Asp(3)]MCYST-LR; II, MCYST-LR; III, isomer of MCYST-LR; IV, isomer of methyl MCYST-LR; V, [D-Asp(3), Glu(OCH(3))(6)]MCYST-LR; and VI, [D-Glu(OCH(3))(6)]MCYST-LR. In-source CID provided mass spectral patterns similar to those obtained by CID in the collision cell of the mass spectrometer but was more sensitive for the analysis of microcystins.     

Structural analysis of oligosaccharides by a combination of electrospray mass spectrometry and bromine isotope tagging of reducing-end sugars with 2-amino-5-bromopyridine

Model reducing-end oligosaccharides were successfully labeled by a brominated aromatic amine reagent, 2-amino-5-bromopyridine (ABP), through reductive amination. Using either a combination of liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) with in-source fragmentation or liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS), sequence information corresponding to the model oligosaccharides was revealed with little ambiguity via the diagnostic unique twin peaks arising from the bromine isotopes, for both the molecular ions of the derivatized oligosaccharides and their fragments. No fragment ions arising from loss of the bromine atom were observed.