Application of Electrospray Ionization Mass Spectrometry to the Analysis of Oligodeoxynucleotides

Abstract

Electrospray mass spectrometry is a powerful technique for the analysis of oligodeoxynucleotides providing accurate molecular masses from the multiply charged negative ions formed by the electrospray volatilization/ionization process. Fragment ions are generally absent unless formed by secondary collisions near the exit of the source where pressures are relatively high. Use of the electrospray technique for the analysis of oligodeoxynucleotides is superior to other ionization techniques for verification of site-specific incorporation of modified deoxynucleotides, especially for oligomers containing more than ten deoxynucleotides where FAB/MS is not effective. ESI spectra may be generated on 10-100 picomoles of an oligomer on a single quadrupole mass spectrometer system, and mass accuracy of ±0.01% is attainable with careful calibration of the instrument. Spectra are presented for oligomers containing modified nucleobases and sugar moieties, as well as a hybrid DNA/RNA oligomer in which an O-methyl moiety is positioned at the 2'-position of adenosine. The technique is also effective for the elucidation of mechanisms by which unstable oligomers may disintegrate into unknown products, and the known sensitivity of 8-oxo-2-deoxyguanosine-containing oligomers to the postsynthetic ammonia deprotection procedure has been examined. ESI/MS was used to characterize the major products of oligomer decomposition. Deoxynucleotide sequence of an oligomer cannot be determined from the electrospray mass spectrum. However, MS/MS experiments were conducted on multiply charged oligodeoxynucleotide molecular ions generated by ESI on a triple quadrupole instrument. Product ion spectra from collision induced dissociation are complex, and product ions may differ in charge state from the precursor ion. However, families of ions were identified from which the sequence of the oligomer was verified.

Oxidative Nucleobase Modifications Leading to Strand Scission

“…A further advantage of working with short, synthetic oligonucleotides is their ease of analysis by electrospray ionization mass spectrometry (ESI-MS). , The technique provides molecular ions without fragmentation, although care must be taken to remove hard cations (Na + and K + ) since they will considerably complicate the spectrum . In the example shown in Figure , it was assumed that the high MW band (lanes 2−4) represented a 1:1 adduct of the oligodeoxynucleotide and complex 50 .…”

Characterization of the Alkaline Degradation Products of an Oligodeoxynucleotide Containing 8-Oxo-7,8-dihydro-2‘-deoxyguanosine by Electrospray Ionization Mass Spectrometry

“…In addition, preliminary studies on the identification of the degradation products obtained when the unprotected oligonucleotide, 5′-GT-TCAXTTGC-3′, was digested under the same conditions were described (22). We report here the identification of the alkaline degradation products of an 8-oxo-dGuocontaining oligomer resulting from treatment with concentrated ammonia.…”

“…It is recognized that synthetic oligodeoxynucleotides containing 8-oxo-dGuo are sensitive to aerial oxidation in 1 N NaOH or concentrated NH 4 OH (14,21), and they degrade under the conditions required for nucleobase deprotection and release from the CPG support after synthesis (28% ammonia at 55 °C, 16 h). Under these conditions, scission of the sugar-phosphate backbone occurs at 8-oxo-dGuo sites (22); however, protection is afforded by an antioxidant such as β-mercaptoethanol (14,15). The mechanism for this decomposition has not been specified.…”

“…We have used ESI/MS to identify the dimethoxytritylprotected degradation products obtained from the oligomer 5′-DMT-GTTCAXTTGC-3′, (where X ) 8-oxo-dGuo) when it was digested with 28% ammonia in the absence of β-mercaptoethanol (27). In addition, preliminary studies on the identification of the degradation products obtained when the unprotected oligonucleotide, 5′-GT-TCAXTTGC-3′, was digested under the same conditions were described (22). We report here the identification of the alkaline degradation products of an 8-oxo-dGuocontaining oligomer resulting from treatment with concentrated ammonia.…”