Kay Lichtenwalter

New procedure for the use of high-performance liquid chromatography–electrospray ionization mass spectrometry for the analysis of nucleotides and oligonucleotides

Abstract A method is described which allows the combination of high-performance liquid chromatography (HPLC) and electrospray ionization mass spectrometry for the analysis of nucleotides and oligonucleotides without compromising the performance of either technique. The essential feature of the method uses 1,1,1,3,3,3-hexafluoro-2-isopropanol as an additive to the HPLC mobile phase. This novel additive results in both good HPLC separation and efficient negative ion mode electrospray ionization. The method is demonstrated for oligonucleotides samples such as synthetic homopolymers of thymidine (PolyT), of fragments based on the pBR322 plasmid sequence and analysis of phosphorothioate ester antisense oligonucleotides. The differentiation of phosphorylation states of natural nucleotides as well as nucleotide analogues, such as ziduvodine (AZT), is also demonstrated.

Approaches to functional genomics: potential of matrix-assisted laser desorption ionization--time of flight mass spectrometry combined with separation methods for the analysis of DNA in biological samples.

MALDI-TOF MS has potential as a valuable technique in DNA mapping studies and may well be complementary to other approaches to DNA analysis such as gel electrophoresis and sequencing. This study used 2,6-dihydroxyacetophenone (DHAP) mixed with diammonium hydrogen citrate (DAHC) as the matrix. In addition, recent technical advances such as time lag focussing (TLF) and better selection of matrices (such as 3-hydroxypicolinic acid (3 HPA) and picolinic acid (PA)) extended the range of DNA fragments that can be studied by this approach. The following samples were investigated: Poly-T mixture (dT 15, 19, 20, 25, 74 and 75), plasmid pBR322 derived oligonucleotides (10, 11, 12, 13, 14, 15, 19, 20 and 50 nucleotides long) and DNA fragments of 25, 36 and 37 base pairs corresponding to a fragment in the restriction map for the gene corresponding to the hexon protein of Adenovirus 2 and 5. The results were contrasted with similar analyses performed by ion-paired reversed-phase HPLC coupled to on-line electrospray mass spectrometry.