Mass spectra of oligonucleotides

For many years, mass spectrometry has had an important role in the analysis of nucleic acids. However, older work was limited to the analysis of nucleotides because the El and Cl methods did not allow oligonucleotide analysis. With the advent of FAB and PD, small oligonucleotides comprising up to about 10 bases have been analysed. 

A second difficulty in the analysis of oligonucleotides is their easy fragmentation. As will be discussed in more detail, the oligonucleotides tend to lose their nucleic bases by a 1,2-elimination mechanism. Fragmentation of the nucleic chain at either the 5 or 3 side of the sugar having lost a base then can occur. This stability problem is more important for MALDI than for ESI analysis. 

Oligonucleotides can be analysed in both positive and negative ion modes. However, negative ion mode give better sensitivity and resolution, especially in ESI. 

The success of the analysis of oligonucleotides is strongly dependent on the choice of the matrix and the preparation of the sample. A major improvement has resulted from the introduction of new matrices specific for the oligonucleotides. An example is 3-hydroxypicolinic acid. [165] Several strategies for sample preparation have been adopted, with the principal goal of eliminating interference from the ubiquitous alkali metal ions a combination of the use of cation exchange columns to replace the alkali metal ions by ammonium and the addition of some ammonium salt to the sample. 

The response of nucleotides to MALDI depends also on their nature, their size and their composition in nucleic bases. Oligonucleotides containing only thymine give a much 

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An algorithm has been developed for the interpretation of tandem mass spectra of oligonucleotides. [201] This greatly facilitates the interpretation of mass spectra of oligonucleotides and is based on the general fragmentation scheme of these molecules. 

Figure 13.13. Negative-ion mass spectra of an eight-component oligonucleotide mixture (a) nonreacted spectrum and (b) spectrum after ion/ion reaction with O2+. The ion/ion-reacted spectrum is much simpler then the nonreacted spectrum. (Reproduced from ref. 68 by permission of the American Chemical Society, Washington, DC, copyright 2002.)...

Figure 5.1 Negative-ion mode mass spectra of a 12mer DNA oligonucleotide, demonstrating the importance of careful sample preparation and conditioning of the phosphate backbone of nucleic acids for efficient analysis by MALDI-TOF-MS. (a) At concentrations of sodium or potassium similar to those conventionally used in molecular biological assays, the 12mer shows a distribution of up to 10 potassium ions attached to the phosphate backbone.

In later work (which is described below), two-laser mass spectrometers using a pulsed CO2 ablating laser, rovibronic cooling in a jet expansion, and a pulsed multiphoton ionization laser have been used successfully for obtaining mass spectra of biological molecules including peptides, small proteins, and protected oligonucleotides. 

FIGURE 11.14 Comparison of the MALDI-TOF mass spectra of an oligonucleotide obtained on (a) a linear TOF and (b) a reflectron TOR (Reprinted with permission from reference 15). 

Another very sensitive MS technique is MALDI-TOF. Its high-resolution mass spectra allowed, for example, the identification of the 8-oxo-G lesion by its 416-Da mass shift in a oligonucleotide heptamer at picomol level (Bartolini and Johnston 2000). 

The MALDI/TOF mass spectrum of an oligonucleotide produced by PCR amplification of a part of the gene responsible for cystic fibrosis. The top spectrum is obtained from a normal individual, whereas the central and bottom spectra are obtained, respectively, from a healthy heterozygote carrier and an ill homozygote. Reproduced (modified) from Chang L., Tang K., Shell M., Ringelberg C., Matteson K.J., Allman S.L. and Chen C.H., Rapid Comm. Mass Spectrom., 9, 772, 1995, with permission. 

Ni, J.S., Pomerantz, S.C., Rozenski, J. et al. (1996) Interpretation of oligonucleotide mass spectra for determination of sequence using electrospray ionization and tandem mass spectrometry. Anal. Chem., 68 (13), 1989-99. 

Biomolecules such as proteins and oligonucleotides present mass spectra that are complicated by the presence of multiply charged families of ions. The parent species, for example, generated by a soft ionization method such as FAB of ESI, will yield several m/z peaks in which m is equal to a constant plus (protein) or minus (nucleic acid) a variable number of proton masses, while z is a variable. Egg white lysozyme, for example, yields ESI-mass spectra with fine parent peaks between m/z values of 1194 and 1791 the corresponding z values are 12-8 [see Fig. 15.9(a)]. 

Figure 14 Electrospray mass spectra at a fixed concentration of oligonucleotide and four concentrations of distamycin. The spectra show the uncom-plexed oligonucleotide at both the -7 and -8 charge states and the appearance of the distamycin oligonucleotide complex at higher concentrations of the ligand (distamycin = DstA).

FAB mass spectrometry of nucleosides, nucleotides and oligonucleotides has been reviewed the spectra of the major nucleosides of RNA and DNA have been reported in both positive-and... 

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