P156

Abstract

Background

Identification of DNA-based biomarkers of cancer cells is highly promising and rapidly developing direction that can advance early detection and therapy of malignancies. DNA adducts are felicitous markers of cancer, because their chemical structure is significantly different from that of mutated or methylated DNA, that allows to determine them with high precision using mass spectrometry. The aim of this work is to develop the methodology of sample preparation and its mass spectrometric analysis. Samples were prepared from the blood plasma and from the tumor tissue from lung cancer patients and from blood of healthy individuals.

Materials and methods

DNA was isolated from the blood plasma and tissue by using column method (BioSilica, Russia) The final yield from 1 ml of blood was 100 ng. DNA samples were subjected to acid hydrolysis (1 M HCl) at 70 °C. After 3 h, the hydrolysis was stopped by cooling on ice for 5 min and later on adding an equivalent amount of an alkali and a phosphate buffer solution (pH 7). To assess the extent of hydrolysis of the samples they were analysed by electrophoresis on a 1.2% agarose gel in Tris-acetate buffer. The samples were extracted at cartridge HF Bond Elut-C18 100 mg, 1 ml (Agilent Technologies, USA) and eluted in several fractions with a gradual increase of methanol in the eluent. Stream of nitrogen was applied to dry the extract. The samples were subjected to mass spectrometric analysis after pre-separation by UHLC Ultimate 3000 RS (Dionex, USA) in a column Dionex Acclaim RSLC 120 C18 (2.1 × 50 mm 120 A, 0.2 μm) flow rate of 0.5 ml/min using as eluents 0.1% solution of formic acid in water (A) and 0.1% solution of formic acid in atsetontrile (B). Elution was carried out in gradient mode: (%B): 0–3min (5%), 3–28min (5–95%), 28–30 min (95%), 30–31 min (95–5%), 31–35 min (5%). Mass spectrometry was carried out on ESI-qTOF ultrahigh resolution Maxis 4G (Bruker, Germany) in the positive ion detection mode range 50–1000 m/z, 2 Hz with the following settings electrospray ion source: CV 3800 V, Nebulizer gas 1 bar, Dry Gas 8 l/min, Dry Temp: 200 °C.

Results

It was found that the DNA which was cleaved with acid hydrolysis in the result contained single DNA bases. The samples were stable at 4 °C for at least 7 days. The optimal eluent for solid phase extraction of DNA is 80% solution of methanol in water. The number of DNA adducts was evaluated by the integrated value of the mass spectrometric response detector. It was shown that most amount of adducts 4-hydroxy-1-(3-pyridyl)-1- butanone and N3-(2-carbamoyl-2-hydroxyethyl)adenine was found in DNA samples derived from tumor tissue. The adduct N7-(2-carbamoyl-2-hydroxyethyl)guanine was found in tumor tissue samples and DNA derived from plasma, as well as in all samples of healthy tissue.

Conclusion

The established protocol of DNA sample preparation followed by analysis using a mass spectrometer high resolution allows to detect the content of the DNA adducts in small DNA probes (100 ng). We found, that acid hydrolysis is cheaper and more practical in comparison to enzymatic digestion in order to generated samples containing single DNA bases without damaging the structure of adducts.

This research was supported by Federal Targeted Programme for Research and Development in Priority Areas of Development of the Russian Scientific and Technological Complex for 2014–2020, “Development of molecular signatures for early detection of lung cancer” (No. 14.575.21.0064 from 05.08.2014, RFMEFI57514X0064) and supported by Tomsk State University, Competitiveness Improvement Program”. Work was conducted with the application of the Tomsk regional common use center technical equipment acquired thanks to a grant of the Russian Ministry of the Agreement No.14.594.21.0001 (RFMEFI59414X0001).