Analysis of DNA by ‘Charge Tagging’ and Matrix-assisted Laser Desorption/Ionization Mass Spectrometry

✍ Scribed by Ivo G. Gut; William A. Jeffery; Darryl J. C. Pappin; Stephan Beck

• Publisher: John Wiley and Sons
• Year: 1997
• Tongue: English
• Weight: 200 KB
• Volume: 11
• Category: Article
• ISSN: 0951-4198
• DOI: 10.1002/(sici)1097-0231(19970115)11:1<43::aid-rcm817>3.0.co;2-h

✦ Synopsis

We have developed a method to quantitatively attach quarternary ammonium fixed charge tags to the 5' or 3'NH 2 ends of DNA using N-hydroxysuccinimidyl ester chemistry. The chemical conditions for tagging were chosen so that tagging takes place exclusively on aliphatic NH 2 groups while base amino groups remain unmodified. The charge tagging chemistry was combined with a previously developed backbone alkylation procedure for phosphorothioate DNA. The efficiency of the detection in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry of unmodified and modified DNA (phosphorothioate backbone, charge tagged, backbone alkylated, and charge tagged and backbone alkylated) was investigated using a series of different matrices. For α-cyano-4-hydroxycinnamic acid (a matrix, commonly used for the analysis of proteins, but which gives unsatisfactory results with unmodified DNA). For instance, the charge tagged and backbone alkylated DNA is detectable with a sensitivity and resolution comparable with that for peptides. The combination of charge tagging and backbone alkylation with the use of a suitable matrix improves the detectability of small oligonucleotides by MALDI by a factor greater than 100 compared to unmodified oligonucleotides.