High-performance liquid chromatography-electrospray ionization mass spectrometry of single- and double-stranded nucleic acids using monolithic capillary columns.
Monolithic capillary columns were prepared by copolymerization of styrene and divinylbenzene inside a 200-microm i.d. fused silica capillary using a mixture of tetrahydrofuran and decanol as porogen. With gradients of acetonitrile in 100 mM triethylammonium acetate, the synthesized columns allowed the rapid and highly efficient separation of single-stranded oligodeoxynucleotides and double-stranded DNA fragments by ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC). Compared with capillary columns packed with micropellicular, octadecylated poly-(styrene/divinylbenzene) particles, an improvement in column performance of approximately 40% was obtained, enabling the analysis of an 18-mer oligodeoxynucleotide with a column efficiency of more than 190000 plates per meter. The chromatographic separation system was on-line-coupled to electrospray ionization mass spectrometry (ESI-MS). To improve the mass spectrometric detectabilities, 25 mM triethylammonium bicarbonate was utilized as an ion-pair reagent at the cost of only little reduction in separation performance and acetonitrile was added postcolumn as the sheath liquid through the triaxial electrospray probe. High-quality mass spectra of femtomole amounts of 3-mer to 80-mer oligodeoxynucleotides were recorded showing very little cation adduction. Double-stranded DNA fragments ranging in size from 51 to 587 base pairs were separated and detected by IP-RP-HPLC-ESI-MS. Accurate mass determination by deconvolution of the mass spectra was feasible for DNA fragments up to the 267-mer with a molecular mass of 165 019, whereas the spectra of longer fragments were too complex for deconvolution because of incomplete separation due to overloading of the column. Finally, on-line IP-RP-HPLC tandem MS was applied to the sequencing of short oligodeoxynucleotides.
Premstaller
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A
Institute of Analytical Chemistry and Radiochemistry, Leopold-Franzens-University, Innsbruck, Austria.
Oberacher
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H
Huber
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CG
D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000032:analysis; D004277:DNA, Single-Stranded / Q000032:analysis; D019075:Electrophoresis, Capillary; D008855:Microscopy, Electron, Scanning; D009696:Nucleic Acids / Q000032:analysis*; D009841:Oligonucleotides / Q000032:analysis; D021241:Spectrometry, Mass, Electrospray Ionization; D013056:Spectrophotometry, Ultraviolet
D004277:DNA, Single-Stranded; D009696:Nucleic Acids; D009841:Oligonucleotides; D004247:DNA