Koen Vanhoutte

Liquid chromatography–mass spectrometry in nucleoside, nucleotide and modified nucleotide characterization

Abstract Macromolecules such as deoxyribonucleic acid (DNA) and ribonucleic acid as well as their constituents play an important role in all kinds of biochemical reactions in nature. Hence their isolation and identification plays a major role in biochemical analysis. Mass spectrometry (MS) has gained an important position in this field because of the development of soft ionization techniques such as fast atom bombardment (FAB), liquid secondary mass spectrometry, thermospray ionization (TSP), the atmospheric pressure ionization techniques, electrospray ionization and atmospheric pressure chemical ionization and matrix assisted laser desorption. Because of their polar nature, mixtures of nucleosides, nucleotides and oligonucleotides are wel separated by liquid chromatography (LC) and electrophoretic techniques. Therefore it is not surprising to note that a lot of effort has been put into the development of LC–MS methods for the analysis of these compounds. In this review, covering the period 1990–1996, the LC–MS analysis of nucleobases, nucleosides, nucleotides, oligonucleotides and DNA adducts by TSP, continuous flow FAB and electrospray MS is discussed.

Analysis of cyclic nucleotides and cytokinins in minute plant samples using phase-system switching capillary electrospray-liquid chromatography-tandem mass spectrometry

Using an electrospray tandem mass spectrometer as a concentration-sensitive detector, a method has been developed to quantify femtomole amounts of plant growth regulators (i.e. isoprenoid type cytokinins, zeatin, dihydrozeatin, isopentenyladenine and their respective riboside and glucoside analogues) and the second messenger adenosine 3′:5′-cyclic monophosphate (3′:5′-cAMP). Miniaturisation of the chromatographic setup using capillary high performance liquid chromatographic (HPLC) ion spray mass spectrometry increased the sensitivity to the low femtomole region. Application of automated capillary column switching allowed the introduction of large injection volumes into the HPLC system. Aliquots (25 µL) were injected into one dimension of the HPLC set-up and stacked onto a micro pre-column. By means of mobile phase switching the pre-column was back-flushed to introduce the analytes onto the analytical column. For cytokinin analysis positive electrospray ionisation was used and resulted in 2.5–25 fmol detection limits. Cyclic nucleotides were separated under ion-pair conditions using tetrabutyl ammonium bromide as ion-pair reagent and were detected under negative electrospray ionisation conditions. Here a 25 fmol detection limit was determined. Following this approach, cytokinins and 3′:5′-cAMP extracted from only mg amounts of apical shoot meristem and chloroplasts obtained from Nicotiana tabacum cv. Petit Havana SR1 were identified and quantified. Copyright

On-line nanoscale liquid chromatography nano-electrospray mass spectrometry: effect of the mobile phase composition and the electrospray tip design on the performance of a nanoflowTM electrospray probe

In earlier liquid chromatography/mass spectrometry (LC/MS) experiments using a commercially available nano-electrospray interface designed for the coupling of nano-LC (flow rate = 200 nL/min) to electrospray mass spectrometry, a sudden drop in the electrospray total ion current was observed at certain percentages of organic modifier in the mobile phase. Therefore the performance of this nano LC/MS system was evaluated for different mobile phase compositions. The uncoated, non-tapered fused silica tips (20 μm i.d.) which were delivered standard with the interface, and several other electrospray capillaries, were evaluated for different mobile phase compositions: uncoated, tapered (20→9 μm i.d.) fused silica tips; gold coated, tapered fused silica tips and stainless steel tips (70 μm i.d.). The use of tapered but uncoated fused silica tips did increase the performance of the nano-electrospray system. The best results were obtained with gold coated, tapered tips. Stainless steel tips with an i.d. of 70 μm gave no results at the applied flow rate of 200 nL/min.

A label-free potentiometric sensor principle for the detection of antibody-antigen interactions.

We report here on a new potentiometric biosensing principle for the detection of antibody-antigen interactions at the sensing membrane surface without the need to add a label or a reporter ion to the sample solution. This is accomplished by establishing a steady-state outward flux of a marker ion from the membrane into the contacting solution. The immunobinding event at the sensing surface retards the marker ion, which results in its accumulation at the membrane surface and hence in a potential response. The ion-selective membranes were surface-modified with an antibody against respiratory syncytial virus using click chemistry between biotin molecules functionalized with a triple bond and an azide group on the modified poly (vinyl chloride) group of the membrane. The bioassay sensor was then built up with streptavidin and subsequent biotinylated antibody. A quaternary ammonium ion served as the marker ion. The observed potential was found to be modulated by the presence of respiratory syncytial virus bound on the membrane surface. The sensing architecture was confirmed with quartz crystal microbalance studies, and stir effects confirmed the kinetic nature of the marker release from the membrane. The sensitivity of the model sensor was compared to that of a commercially available point-of-care test, with promising results.

A strategy for the identification of 2′-deoxynucleoside and 2′--deoxynucleotide adducts using electrospray tandem mass spectrometry

A methodology using electrospray tandem mass spectrometry (ES MS/MS) was developed to identify 2′-deoxynucleotide adducts of bisphenol A diglycidylether (BPADGE). The 2′-deoxynucleotide adducts were formed in vitro and were separated using reversed-phase high-performance liquid chromatography (RP-HPLC). The adducts were investigated by (–) electrospray liquid chromatography-mass spectrometry [(–) ES LC-MS]. (–) ES LC-MS/MS allowed the differentiation between 5′-phosphate- and base-alkylated adducts. More detailed structural information on the heterocyclic moiety was obtained by examining the (+) ES LC-MS/MS data and by comparison with the (+) ES LC-MS/MS spectra of the corresponding 2′-deoxynucleoside adducts.

Phenylglycidyl ether adducts of 2′-deoxycytidine and 2′-deoxyadenosine: Stability in solution and structure analysis by electrospray tandem mass spectrometry

The adducts of phenylglycidyl ether with 2′-deoxyadenosine (dAdo) and 2′-deoxycytidine (dCyd) exhibit structural modifications. The N-1 adduct of dAdo underwent rearrangement to the N-6 adduct; the N-3 adduct of dCyd was deaminated to the corresponding 2′-deoxyuridine adduct. These structural modifications were studied by using liquid chromatography-electrospray tandem mass spectrometry, and kinetic data for both reactions are presented. The low energy (+) collision-activated dissociation spectra of the dAdo adducts allow the two positional isomers N-1 versus N-6 to be distinguished. The structure of the latter is independently proven by an extended NMR study. For the dCyd and 2′-deoxyuridine adducts, information about the alkylation site is found in the (−) collision-activated dissociation spectra. These spectra show the presence of an unexpected N-4-alkylated dCyd in addition to the two epimeric N-3 adducts.