Dr. Ute Bahr, Goethe University Frankfurt, Institute of Pharmaceutical Chemistry

MALDI-TOF mass spectrometry is a simple and quick method of monitoring the quality of synthetic oligonucleotides, including their chemical modifications, such as biotin, fluorescein et al.

Using 3-hydroxypicolinic acid (with the addition of ammonium citrate) as a matrix this method delivers excellent spectra in both positive and negative ion mode.

1-5 pmol of nucleotide are mixed with the matrix and analysed. The preciseness of the measurement of mass through external calibration is >0.03%; through internal calibration, this can be increased to about 50 ppm.

Comparing the result to the calculated reference molecular weight is a simple way to test whether the synthesis of the oligonucleotide or the modification has been successful or false sequences and potential breaks in the chain have occurred. Up to a length of about 50 base pairs, it is possible to detect modifications and impurities due to the mass resolution of ca. 1000.

One prerequisite for high quality MALDI mass spectra is that the samples be thoroughly desalted; MALDI has prove through the established preparation protocols that it is considerably more robust, less prone to interference and faster than electrospray mass spectrometry. Since the tendency of cations to bind rises as the oligonucleotides get longer, lines tend to broaden through salt adducts for oligonucleotides longer than about 20 kDa (>65mer). Furthermore, because the bindings grow increasingly weaker, more and more bases are lost. In spite of some forfeiture in the quality of mass spectra, however, it is possible to clearly determine the molecular ion signals of intact nucleic acids and measure their mass up to about 30 kDa (100mer).

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MALDI-TOF MS
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