Nucleic acids mass spectrometry analysis

Johnson, L., Mollah, S., Garcia, B. A., Muratore, T. F., Shabanowitz, J., Hunt, D. F., and Jacobsen, S. E. (2004). Mass spectrometry analysis of Arabidopsis histone H3 reveals distinct combinations of post-translational modifications. Nucleic Acids Res. 32 6511 -6518. 

These first applications of mass spectrometry to nucleic acid or polynucleotide analysis are, in effect, a qualitative verification of the presence of base units. However, these studies initiated a quantitation of the base abundances, using the intensities of BH fragments. The method gives data very... 

K. H. Schram, Preparation of trimethylsilyl derivatives of nucleic acid components for analysis by mass spectrometry. Methods Enzymol. 193, 791-796 (1990). 

R. (2003) MALDI mass spectrometry analysis of single nucleotide polymorphisms by photodeavage and charge-tagging. Nucleic Acids Res., 31 (11), e63. 

This chapter will address the applications of protein-based bioinformatics to analysis of microorganisms introduced intact into the instrumental system for rapid processing and analysis. Strategies that require offline extraction and fractionation of proteins will not be discussed. Although the amplification of nucleic acids is a powerful approach, especially coupled with mass spectrometry,15 it requires extraction and processing, and thus is not included. 

The investigations directed at the synthesis of thymine-substituted polymers demonstrate that the type of functional groups displayed by nucleic acid bases are compatible with ROMP. Moreover, the application of MALDI-TOF mass spectrometry to the analysis of these polymers adds to the battery of tools available for the characterization of ROMP and its products. The utility of this approach for the creation of molecules with the desired biological properties, however, is still undetermined. It is unknown whether these thymine-substituted polymers can hybridize with nucleic acids. Moreover, ROMP does not provide a simple solution to the controlled synthesis of materials that display specific sequences composed of all five common nucleic acid bases. Nevertheless, the demonstration that metathesis reactions can be conducted with such substrates suggests that perhaps neobiopolymers that function as nucleic acid analogs can be synthesized by such processes. 

Willems, A. V., Deforce, D. L., Van Peteghem, C. H., and Van Bocxlaer, J. E. (2005). Analysis of nucleic acid constituents by on-line capillary electrophoresis-mass spectrometry. Electrophoresis 26, 1221-1253. 

Polymeric stationary phases have many advantages when polymerized in capillaries (diameter < 0.5 mm) as rods in the presence of porogens to yield channels for mobile phase transport. They are frequently used in the analysis of peptides, proteins, and nucleic acids when directly coupled to electro spray mass spectrometry. 

FAB and PD have been replaced by electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI) in the analytical mass spectrometry laboratory, because both of these newer techniques have a wider mass range of analysis and have lower detection limits. ESI and MALDI have become invaluable ionization techniques for nonvolatile components. This is particularly true for a wide range of biological molecules including proteins, peptides, nucleic acids, etc. Samples can be analyzed by ESI using either direct injection or introduction through liquid chromatography. 

The topic of nucleic acid analysis by MALDI-TOF MS has recently been reviewed elsewhere (10,11). The reader can refer to those reviews for an exhaustive description of the evolution of mass spectrometry in nucleic acids analysis. This review will emphasize the new trends of using MALDI-TOF MS for specific applications in molecular biology and pharmacogenomics. 

Today, mass spectrometry has become one of the most widely used analytical techniques in the life sciences [11,12], The mass spectrometric analysis of different classes of biomolecules is reviewed in this chapter peptides, proteins, nucleic acids, oligosaccharides and lipids. Several applications are detailed for each class. Metabolomics, which is the omics science of metabolism, will also be examined at the end of the chapter. 

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. 

Improvements in column technology, detector sensitivity and the development of new detection systems, have made possible the routine separation of picomole quantities of nucleic acid components in complex physiological matrices. The very sensitivity of most LC systems, however, which is invaluable in the analysis of biological samples, is often the limiting factor because of inadequate or ambiguous identification methods. Although tremendous advances have been made in the on-line combination of HPLC with spectroscopic techniques [e.g., mass spectrometry, Fourier transform infrared (FT/IR), nuclear magnetic resonance], their application has not become routine in most biochemical and biomedical laboratories. 

In spite of the incomplete coverage in classical reviews and the absence of references in chemical and biochemical texts on mass spectral studies applied to nucleic acids and their derivatives, mass spectrometry is a very promising technique for studying these compounds. The analysis of the relevant building blocks (nucleosides and nucleotides) is satisfactorily achieved, whereas the analysis of the polymers (oligonucleotides and nucleic acids) still needs refinement despite the sophistication level of the techniques used. The future looks very promising for the sequence analysis of nucleic acids and this, along with structural elucidation studies of modified bases, for example, could establish mass spectrometry as a routine technique in this area. 

Recent advances in mass spectrometry have produced a number of soft ionisation techniques such as fast atom bombardment (FAB) or electrospray ionisation. The major advantage of these techniques is that they are less likely to break the sample into small fragments and are more likely to produce a molecular ion. This is particularly important in the analysis of macromolecules such as proteins and nucleic acids. 

Newer technologies that replace assays traditionally performed by electrophoresis are emerging, some of which are attractive alternatives for the clinical laboratory as they achieve analysis of nucleic acids with less hands-on time and with far greater throughput because of automation. These include pyrosequencing, mass spectrometry, and HPLC. 

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