Nucleic acids mass spectrum

New techniques for data analysis and improvements in instrumentation have now made it possible to carry out stmctural and conformational studies of biopolymers including proteins, polysaccharides, and nucleic acids. NMR, which may be done on noncrystalline materials in solution, provides a technique complementary to X-ray diffraction, which requires crystals for analysis. One-dimensional NMR, as described to this point, can offer structural data for smaller molecules. But proteins and other biopolymers with large numbers of protons will yield a very crowded spectrum with many overlapping lines. In multidimensional NMR (2-D, 3-D, 4-D), peaks are spread out through two or more axes to improve resolution. The techniques of correlation spectroscopy (COSY), nuclear Overhausser effect spectroscopy (NOESY), and transverse relaxation-optimized spectroscopy (TROSY) depend on the observation that nonequivalent protons interact with each other. By using multiple-pulse techniques, it is possible to perturb one nucleus and observe the effect on the spin states of other nuclei. The availability of powerful computers and Fourier transform (FT) calculations makes it possible to elucidate structures of proteins up to 40,000 daltons in molecular mass and there is future promise for studies on proteins over 100,000... 

A) The MALDI linear TOF mass spectrum of a DNA 40-mer in negative ion mode. (B) The MALDI reflection TOF mass spectrum of an RNA 195-mer in the positive ion mode. In both cases, the spectra were obtained using 3-HPA as matrix and a laser at 337 nm. Reproduced (modified) from Wu K.J., Shaler T. A. and Becker C.H., Anal. Chem., 66, 1637, 1994 and Kirpekar F., Nordhoff E., Kristiansen K., Roepstorff R, Lezius Z., Hahner S., Karas M. and Hillkamp F., Nucleic Acids Res., 22, 3688, 1994, with permission. 

At one time the idea of recording a mass spectrum of a nucleic acid would have been considered utopic and futuristic. Nucleic acids are practically nonvolatile and usually possess a molecular weight of several million atomic mass units (amu) (fi) often expressed in daltons up to 10 daltons where 1 dalton = 1.67 X 10 g. They possess their own mass spectra. In general they are esters of phosphoric acid and polyols, such as the sugars ribose and 2 -deoxyribose, which are themselves substituted with heteroaromatic purine or pyrimidine bases. Consequently, fragment ions characteristic of all these structural elements can be found in the mass spectra of nucleic acids. 

Phosphate esters show important thermal susceptibility (Fig. 1). Dialkyl phosphates, such as those found in nucleic acids (Fig. 2), decompose with the initial loss of one alkyl group, the concomitant transfer of protons, followed by the elimination of the second alkyl group and the subsequent loss of water. This thermal instability of phosphoesters has been used in the analysis of nucleic acids. Thus the pyrolysis that usually precedes the recording of a mass spectrum permits cleavage of the polymeric phosphoesters (nucleic acids), followed by phosphate extrusions, producing nucleotides or simple nucleosides as fragment ions. 

A particular advantage of ESTMS for biomolecule analysis is realized by generating the analyte ions from solution conditions that retain the secondary, tertiary and even quaternary structure of the biomoT ecules. Noncovalent binding of biomolecules has been observed in the ESI mass spectrum and, when operated under the appropriate conditions, the mass spectral data are a direct probe of the solution-phase biomolecule assembly. Protein assemblies, protein-nucleic acid complexes, duplex DNA and other non-covalently bound biomolecule assemblies have been studied using mass spectrometry. 

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