Molecular Mass Determination of Oligonucleotides

Practical Considerations CareM control of instrument parameters and sample purity is essential for the success of ESI-MS analysis of oligonucleotides. The following approaches have been used successfiiUy to purify oligonucleotide samples and suppress the alkali metal adduction  

Oligonucleotides are precipitated from an ammonium acetate-containing ethanol solution [8]. Upon precipitation, the alkali cations are replaced with ammonium ions to provide a significant improvement in signal intensity. A 48-mer oligonucleotide was analyzed by ESI-MS after this precipitation step, and the mass spectrum did not show any Na+ ion clustering [8]. 

The addition of strong organic bases such as trimethylamine (TMA), TEA, diisopropylamine, piperidine, or imidazole in place of ammonium acetate is another effective means to suppress the signal from alkali-adducted ions [10-12]. This strategy is particularly beneficial for phosphorothioate oligomers [10]. 

RP-HPLC either alone or in a mixed-mode format can effectively remove salts from oligonucleotide samples to provide an improvement in spectral quality [13,14]. When RP-HPLC is combined with FT-ICRMS, the mass of oligomers can be measured within 0.001% accuracy. 

Microdialysis is also a feasible option for purifying DNA fragments [15]. In this procedure the sample is injected into a regenerated hollow cellulose fiber and a countercurrent flow of the dialysis solvent is maintained through the annular space between a Teflon tube and the cellulose fiber. 

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S. C. Pomerantz, J. A. Kowalack, and J. A. McCloskey, Determination of oligonucleotide composition from mass spectrometricaUy measured molecular weight, J. Am. Soc. Mass Spectrom. 4, 203-209 (1993). 

Mass spectrometric measurements of oligonucleotides and nucleic acids involve the determination of molecular mass, or of the masses of dissociation products of gas-phase ions, which can then be... 

MALDI-MS has found applications in the mass determination of molecules ranging from small peptides, oligosaccharides, and oligonucleotides, to large proteins and synthetic polymers. Polymeric constituents in coal ranging from several hundred molecular weight to 16,000 have been mass analyzed by MALDI, and synthetic polymers of MW up to 70,000 have been reported. 

Covey, T.R. Bonner, R.F. Shushan, B.I. Henion, J.D. The Determination of Protein, Oligonucleotide, and Peptide Molecular Weights by lon-Spray-MS. Rapid Commun. Mass Spectrom. 1988, 2, 249-256. 

Mass spectrometry not only allows precise determination of the molecular weight of oligonucleotides, but also in a direct or indirect way the determination of their sequences. 

The error on the measured molecular mass obtained by MALDI varies between 0.01 to 0.05 % for small oligonucleotides. This error increases to 0.5 % or even more for large nucleotides. Indeed, at higher masses the signals from adducts or resulting from the loss of a base are more abundant and cannot be resolved, any more, causing a broadening of the peak. The error on the centroid determination increases. One could consider that the maximum size that can be analysed reasonably by MALDI is around 50 bases for DNA. For RNA more than 100 bases have been analysed with success. [164]... 

The simplest and also most common application of mass spectrometry is the accurate determination of molecular weight to verify the presence of an oligonucleotide of a... 

T.R. Covey, R.F. Bonner, B.I. Shushan, J.D. Henion, The determination of protein, oligonucleotide and peptide molecular weights by ESI-MS, Rapid Commun. Mass Spectrom., 2 (1988) 249. 

MS does not only allow precise determination of the molecular mass of oligonucleotides with their high molecular weight, but does permit sequencing of nucleic acids when using extremely small amounts of sample material. 

The monoisotopic mass is meaningful for low-mass compounds because the elemental composition can be determined from a well-defined isotopic pattern of the molecular ion. The nominal and monoisotopic masses can both be correlated with the most abundant peak in the isotopic cluster. As the mass of a compound, however, increases, the isotopic pattern becomes more synunetrical and extends over many masses [21]. Also, the monoisotopic peak becomes difficult to identify. For high-mass compounds (e.g., proteins and oligonucleotides), the molecular ion profile measured coalesces and becomes a single asymmetric peak. For such compounds, the average mass value is accepted as the molecular mass. 

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