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Wykorzystanie spektrometrii mas do analizy modyfikacji nukleotydów i adduktów DNA

Hanus J., Jelonek K., Pietrowska M.

Wiadomości Chemiczne

2011

[Z] 65, 3-4

191-205

Chemically modified nucleotides, which are not normally present in genetic material, are called DN A adducts. This type of DN A modifications (damage) is directly related to processes of mutagenesis and carcinogenesis. Elevated levels of DN A adducts present in genetic material reflect exposure of humans to carcinogenic factors and are markers of increased risk of cancer [1]. For this reason different methods useful for quantitative and qualitative analyses of DN A adducts are used in the field of cancer prevention and research (Tab. 1). Enzymatically-catalyzed methylation of cytosine, observed mostly in so called CpG islands, is a frequent endogenous modification of genetic material. Such a DN A methylation is a key factor involved in regulation of gene expression, and methylation status of oncogenes and tumor supressor genes is an important biomarker of carcinogenesis. As such, analytical methods for assessment of DN A methylation are of great importance for molecular diagnostics of cancer. During the last decade significant progress has been made in methods available for quantitative, qualitative and structural analyses of biological molecules. Among intensively developed tools for bioanalyses are methods of mass spectrometry. Spectrometers that are based on two methods of ionization, namely electrospray ionization (ESI ) [30] and matrix-assisted laser desorption-ionization (MALDI ) [48], are particularly suitable for analyses of biological macromolecules: proteins and nucleic acids. Currently available mass spectrometers, together with microscale methods for sample preparation and separation, significantly increased sensitivity and accessible mass range of analyses. New generation of “user-friendly” instruments is developed to bring the techniques directly into the workplaces of biological and clinical investigators. This review demonstrates representative examples of mass spectrometry techniques used for qualitative analyses of nucleotide modifications and adducts present in genetic material of humans. In this field several methods base on spectrometers with electrospray ionization. Generated ions are separated according to their mass-to-charge ratio in an analyzer by electric fields; among different ion analyzers frequently used in this methods are single or triple quadrupole and ion traps (Fig. 1). Among other methods available for assessment of DN A adducts is so called Accelerator Mass Spectrometry (Fig. 2) [41]. The most frequently applied method for the assessment of DN A methylation is based on methylation-specific PCR reaction. Products of such PCR reactions are analyzed using MALDI mass spectrometry [54] (Fig. 3). In summary, new powerful methods of mass spectrometry that made available qualitative analyses of damage and modifications of human genetic material found their important place in modern biological and medical laboratories.