Elucidation of nucleic acid-drug interactions by tandem mass spectrometry.

In continuation of the long tradition of mass spectrometric research at the University of Bern, our group focuses on the characterization of nucleic acids as therapeutic agents and as drug targets. This article provides a short overview of our recent work on platinated single-stranded and higher-order nucleic acids. Nearly three decades ago the development of soft ionization techniques opened a whole new chapter in the mass spectrometric analysis of not only nucleic acids themselves, but also their interactions with potential drug candidates. In contrast to modern next generation sequencing approaches, though, the goal of the tandem mass spectrometric investigation of nucleic acids is by no means the complete sequencing of genetic DNA, but rather the characterization of short therapeutic and regulatory oligonucleotides and the elucidation of nucleic acid-drug interactions. The influence of cisplatin binding on the gas-phase dissociation of nucleic acids was studied by the means of electrospray ionization tandem mass spectrometry. Experiments on native and modified DNA and RNA oligomers confirmed guanine base pairs as the preferred platination site and laid the basis for the formulation of a gas-phase fragmentation mechanism of platinated oligonucleotides. The study was extended to double-stranded DNA and DNA quadruplexes. While duplexes are believed to be the main target of cisplatin in vivo, the recently discovered DNA quadruplexes constitute another promising target for anti-tumor drugs owing to their regulatory functions in the cell cycle.

Comprehensive analysis of oil sands processed water by direct-infusion Fourier-transform ion cyclotron resonance mass spectrometry with and without offline UHPLC sample prefractionation.

Oil sands processed water (OSPW) is the main byproduct of the large-scale bitumen extraction activity in the Athabasca oil sands region (Alberta, Canada). We have investigated the acid-extractable fraction (AEF) of OSPW by extraction-only (EO) direct infusion (DI) negative-ion mode electrospray ionization (ESI) on a 12T-Fourier transform ion cyclotron resonance mass spectrometer (FTICR-MS), as well as by offline ultrahigh performance liquid chromatography (UHPLC) followed by DI-FTICR-MS. A preliminary offline UHPLC separation into 8 fractions using a reversed-phase C4 column led to approximately twice as many detected peaks and identified compounds (973 peaks versus 2231 peaks, of which 856 and 1734 peaks, respectively, could be assigned to chemical formulas based on accurate mass measurements). Conversion of these masses to the Kendrick mass scale allowed the straightforward recognition of homologues. Naphthenic (CnH2n+zO2) and oxy-naphthenic (CnH2n+zOx) acids represented the largest group of molecules with assigned formulas (64%), followed by sulfur-containing compounds (23%) and nitrogen-containing compounds (8%). Pooling of corresponding fractions from two consecutive offline UHPLC runs prior to MS analysis resulted in ~50% more assignments than a single injection, resulting in 3-fold increase of identifications compared to EO-DI-FTICR-MS using the same volume of starting material. Liquid-liquid extraction followed by offline UHPLC fractionation thus holds enormous potential for a more comprehensive profiling of OSPW, which may provide a deeper understanding of its chemical nature and environmental impact.

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 MS(3) 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.

Gas-phase dissociation of homo-DNA oligonucleotides.

Synthetic modified oligonucleotides are of interest for diagnostic and therapeutic applications, as their biological stability, pairing selectivity, and binding strength can be considerably increased by the incorporation of unnatural structural elements. Homo-DNA is an oligonucleotide homologue based on dideoxy-hexopyranosyl sugar moieties, which follows the Watson-Crick A-T and G-C base pairing system, but does not hybridize with complementary natural DNA and RNA. Homo-DNA has found application as a bioorthogonal element in templated chemistry applications. The gas-phase dissociation of homo-DNA has been investigated by ESI-MS/MS and MALDI-MS/MS, and mechanistic aspects of its gas-phase dissociation are discussed. Experiments revealed a charge state dependent preference for the loss of nucleobases, which are released either as neutrals or as anions. In contrast to DNA, nucleobase loss from homo-DNA was found to be decoupled from backbone cleavage, thus resulting in stable products. This renders an additional stage of ion activation necessary in order to generate sequence-defining fragment ions. Upon MS(3) of the primary base-loss ion, homo-DNA was found to exhibit unspecific backbone dissociation resulting in a balanced distribution of all fragment ion series.

OMA and OPA--software-supported mass spectra analysis of native and modified nucleic acids.

The platform-independent software package consisting of the oligonucleotide mass assembler (OMA) and the oligonucleotide peak analyzer (OPA) was created to support the analysis of oligonucleotide mass spectra. It calculates all theoretically possible fragments of a given input sequence and annotates it to an experimental spectrum, thus, saving a large amount of manual processing time. The software performs analysis of precursor and product ion spectra of oligonucleotides and their analogues comprising user-defined modifications of the backbone, the nucleobases, or the sugar moiety, as well as adducts with metal ions or drugs. The ability to expand the library of building blocks and to implement individual structural variations makes it extremely useful for supporting the analysis of therapeutically active compounds. The functionality of the software tool is demonstrated on the examples of a platinated double-stranded oligonucleotide and a modified RNA sequence. Experiments also reveal the unique dissociation behavior of platinated higher-order DNA structures.

Tandem mass spectrometry of modified and platinated oligoribonucleotides.

Therapeutic approaches for treatment of various diseases aim at the interruption of transcription or translation. Modified oligonucleotides, such as 2'-O-methyl- and methylphosphonate-derivatives, exhibit high resistance against cellular nucleases, thus rendering application for, e.g., antigene or antisense purposes possible. Other approaches are based on administration of cross-linking agents, such as cis-diamminedichloroplatinum(II) (cisplatin, DDP), which is still the most widely used anticancer drug worldwide. Due to the formation of 1,2-intrastrand cross links at adjacent guanines, replication of the double-strand is disturbed, thus resulting in significant cytotoxicity. Evidence for the gas-phase dissociation mechanism of platinated RNA is given, based on nano-electrospray ionization high-resolution multistage tandem mass spectrometry (MS(n)). Confirmation was found by investigating the fragmentation pattern of platinated and unplatinated 2'-methoxy oligoribonucleotide hexamers and their corresponding methylphosphonate derivatives. Platinated 2'-methoxy oligoribonucleotides exhibit a similar gas-phase dissociation behavior as the corresponding DNA and RNA sequences, with the 3'-C-O bond adjacent to the vicinal guanines being cleaved preferentially, leading to w(x)-ion formation. By examination of the corresponding platinated methylphosphonate derivatives of the 2'-methoxy oligoribonucleotides, the key role of the negatively charged phosphate oxygen atoms in direct proximity to the guanines was proven. The significant alteration of fragmentation due to platination is demonstrated by comparison of the fragment ion patterns of unplatinated and platinated 2'-O-methyl- and 2'-O-methyl methylphosphonate oligoribonucleotides, and the results obtained by H/D exchange experiments.

Tandem mass spectrometry of platinated quadruplex DNA.

Quadruplexes are higher-order structures formed by G-rich DNA strands that are involved in various processes of cell cycle regulation, such as control of telomere length and participation in gene regulation. Because of these central biological functions, quadruplex DNA represents a promising target for cancer therapy, e.g. by applying organometallic drugs, such as cisplatin. High-resolution electrospray tandem mass spectrometry is evaluated as a technique for exploring structural features of unplatinated and platinated quadruplexes. Results of experiments on tetramolecular, bimolecular and monomolecular quadruplexes provide information about the extent of platination and the binding sites of the drug. The dissociation behavior of the different types of quadruplexes is compared. Tetramolecular quadruplexes were found to weave out a strand end in order to provide a platination site, and their fragmentation is characterized by the release of an unplatinated strand and the formation of a platinated triplex. Partial opening of the structure in combination with the loss of small fragments leads to truncated quadruplex ions. For the bimolecular quadruplexes studied, strand separation is the predominant dissociation pathway. Depending on the loop sequence, cross-linking of the loops by cisplatin is demonstrated. Distinct differences in the product ion spectra of unannealed and annealed monomolecular sequences provide proof of quadruplex formation and show that platination preferentially occurs at the terminal regions.

The influence of Cisplatin on the gas-phase dissociation of oligonucleotides studied by electrospray ionization tandem mass spectrometry.

cis-Diamminedichloroplatinum(II) (cisplatin, DDP) is a cornerstone of anticancer therapy and has become one of the most widely used drugs for the treatment of various epithelial malignancies. The cytotoxicity of cisplatin is mainly based upon its affinity to adjacent guanines in nucleic acids, resulting in the formation of 1,2-intrastrand adducts. In this study the gas-phase dissociation of DNA- and RNA-cisplatin adducts is investigated by electrospray ionization (ESI) tandem mass spectrometry (MS/MS). The fundamental mechanistic aspects of fragmentation are elucidated to provide the basis for the tandem mass spectrometric determination of binding motifs and binding sites of this important anticancer drug. It is shown that the binding of cisplatin to vicinal guanines drastically alters the gas-phase fragmentation behavior of oligonucleotides. The 3'-C-O bond adjacent to the GG base pair is preferentially cleaved, leading to extensive formation of the corresponding w-ion. This observation was even made for oligoribonucleotides, which usually tend to form c- and y-ions under CID conditions. The absence of complementary ions of equal abundance indicates that oligonucleotide-cisplatin adducts are following more than one dissociation pathway in the gas-phase. Several mechanisms that explain the increased cleavage of the 3'-C-O bond and the lack of the complementary a-ion are proposed. Results of additional MS/MS experiments on methylphosphonate-oligodeoxynucleotides confirm the proposed mechanisms.

Proteomic analysis of human follicular fluid using an alternative bottom-up approach.

Human follicular fluid (hFF) is the in vivo environment of oocytes during follicular maturation in the ovaries. It contains a huge variety of compounds such as, e.g., proteins that might play an important role in follicular development and oocyte growth. Previous proteomic studies on follicular fluid have isolated and already identified a certain number of proteins. Nevertheless, only a small part of proteins present in follicular fluid have been covered so far and a large number have still not been identified. Therefore, the need for new, more resolving, and sensitive approaches in proteome research is evident. We utilized a proteomic setup based on in solution isoelectric focusing (IEF) and reversed-phase nanoliquid chromatography coupled to matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (nano-LC MALDI TOF/TOF MS) for in depth protein analysis of human follicular fluid samples of patients undergoing controlled ovarian hyper stimulation (COH) for in vitro fertilization therapy (IVF). This approach led to the significant identification of 69 proteins, where 32 have not been reported before to be found in human follicular fluid with proteomic methods. Among these findings, at least two relevant compounds essentially involved in hormone secretion regulation during the folliculogenetic process were identified: sex hormone binding globulin (SHBG) and inhibin A (INHA). To confirm these results, both proteins were further validated by immunoassays.