Monolithic capillary columns were prepared by transition metal-catalyzed ring-opening metathesis copolymerization of norborn-2-ene and 1,4,4a,5,8,8a-hexahydro-1,4,5,8-exo,endo-dimethanonaphthalene inside a silanized 200-microm-i.d. fused-silica capillary using a mixture of toluene and 2-propanol as porogen and Cl2(PCy3)2Ru(=CHPh) as initiator. The synthesized columns allowed the rapid and highly efficient separation of single- and double-stranded nucleic acids by ion-pair reversed-phase high-performance liquid chromatography and of proteins by reversed-phase high-performance liquid chromatography. Compared to 3-mm-i.d. analytical columns synthesized from an identical polymerization mixture, a considerable improvement in the peak widths at half-height of oligonucleotides in the order of 60-80% was obtained. Significant differences in morphology between the capillary column, where the surface of the monolith was rather soft and rugulose, and the analytical column, where the surface was very sharp and smooth, were observed, most probably due to differences in polymerization kinetics. The synthesized monoliths were successfully applied to the separation of the diastereomers of phosphorothioate oligodeoxynucleotides. To confirm the identity of the eluting compounds on the basis of their intact molecular masses, the chromatographic separation system was on-line hyphenated to electrospray ionization mass spectrometry.
While electrospray ionization mass spectrometry has shown great potential for the identification of genotypes in DNA sequences amplified by polymerase chain reaction (PCR), the quantitative determination of allele frequencies remains challenging because of the presence of cationic adducts in the mass spectra which severely impairs accuracy of quantitation. We report here on the elaboration of an optimized desalting protocol for ion-pair reversed-phase high-performance liquid chromatography-electrospray ionization mass spectrometry (ICEMS) of PCR amplicons which facilitates the genotyping of single nucleotide polymorphisms (SNPs). Chromatographic purification at temperatures between 50 and 70°C using monolithic reversed-phase columns and acetonitrile gradients in aqueous, 20 -30 mmol/l butyldimethylammonium bicarbonate enabled the mass spectrometric analysis of nucleic acid solutions containing up to 1.7 mol/l sodium chloride. A further improvement in removal of metal cations was achieved upon the addition of 5-10 mmol/l ethylenediaminetetraacetic acid (EDTA) to the sample solution prior to liquid chromatography. ICEMS was used for the semi-quantitative genotyping of SNPs amplified from the tetraploid genome of potato cultivars. Using a quadrupole ion trap mass spectrometer, allele frequencies were determined with an accuracy of 2-9% by measuring the relative intensities of the signals corresponding to the molecular mass of each of the alleles in the deconvoluted mass spectra. ICEMS results correlated well with those obtained by pyrosequencing, single nucleotide primer extension, and conventional dideoxy
Ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC) has been evaluated as a method for the fractionation and desalting of ribonucleic acids prior to their characterization by electrospray ionization mass spectrometry. Monolithic, poly(styrene-divinylbenzene)-based capillary columns allowed the rapid and highly efficient fractionation of both synthetic and biological ribonucleic acids. The common problem of gas-phase cation adduction that is particularly prevalent in the mass spectrometric analysis of ribonucleic acids was tackled through a combination of chromatographic purification and the addition of ethylenediaminetetraacetic acid to the sample at a concentration of 25 mmol/L shortly before on-line analysis. For RNA molecules ranging in size from 10 to 120 nucleotides, the mass accuracies were typically better than 0.02%, which allowed the characterization and identification of failure sequences and byproducts with high confidence. Following injection of a 500 nL sample onto a 60 x 0.2 mm column, the limit of detection for a 120-nucleotide ribosomal RNA transcript from Escherichia coli was in the 50-80 fmol range. The method was applied to the analysis of synthetic oligoribonucleotides, transfer RNAs, and ribosomal RNA. Finally, sequence information was derived for low picomole amounts of a 32-mer RNA upon chromatographic purification and tandem mass spectrometric investigation in an ion trap mass spectrometer. Complete series of fragment ions of the c- and y-types could be assigned in the tandem mass spectrum. In conclusion, IP-RP-HPLC using monolithic capillary columns represents a very useful tool for the structural investigation and quantitative determination of RNAs of synthetic and biological origin.
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