DNA quantification approach by GE-ICP-SFMS and complementary total phosphorus determination by ICP-SFMS

Abstract

Quantification of DNA is still a great challenge for scientists in many fields. Here, we present the on-line coupling of gel electrophoresis (GE) and inductively coupled plasma-sector field mass spectrometry (ICP-SFMS) for quantitative purposes. GE conditions are chosen for optimised separations depending on the target analyte composition in terms of DNA chain length. In particular, agarose concentrations are varied in the range 0.6-2.2%, which corresponds to a separation range of DNA from 100 base pairs (bp) to genomic DNA (approximately 3 Mbp). Separated DNA compounds are directly transported at a flow rate of 100 mL min À1 to a Micromist nebuliser which is followed by ICP-SFMS with 31 P detection at a mass resolution of 4000. For their quantification different approaches are tested using external calibration by PO 4 3À standards as well as by a commercially available QuantLadder standard. Precisions lower than 3% can be achieved in any analysis: however, the indicative values for DNA concentrations given by the manufacturers cannot be found in all the samples. As to our best knowledge no certified quantitative DNA reference material exists, unfortunately we could not prove the accuracy of the developed method. Despite this, we then applied the GE-ICP-SFMS coupling to the analysis of genomic DNA in order to confirm its practicability for the separation and detection of these large biomolecules.

Analysis of mono-phosphate nucleotides as a potential method for quantification of DNA using high performance liquid chromatography–inductively coupled plasma-mass spectrometry

“…Elemental analysis is preferred over other techniques as it offers lower detection limits, a larger dynamic range, a response that is to a first approximation independent of molecular form, and potentially more accurate quantification at low concentrations by isotope dilution analysis. Both ICP-MS and ICP-OES have been utilised for the quantification of nucleotides, DNA, oligonucleotides and DNA adducts [3][4][5][6][7][8][9][10][11][12][13][14][15]. The quantification of oligonucleotides has been achieved by two different methods by Yang et al [4] and Donald et al [7], with the latter group digesting the oligonucleotides to mono-nucleosides and quantifying by isotope dilution electrospray mass spectrometry (ES-ID-MS), and comparing that value to the total 31 P signal obtained from ICP-OES and the gravimetric value.…”

A novel approach for analysis of oligonucleotide–cisplatin interactions by continuous elution gel electrophoresis coupled to isotope dilution inductively coupled plasma mass spectrometry and matrix‐assisted laser desorption/ionization mass spectrometry

“…The detailed setup of the GE unit (Mini Prep Cell with highvoltage supply PowerPac 3000, BioRad Laboratories, Munich, Germany) and the coupling are described elsewhere [20][21][22].…”

“…As the simultaneous quantification of phosphorus and platinum is required for these investigations, two different quantification procedures were applied. As ICP-MS has shown compound-independent detection sensitivity for DNA fragments [22] it was possible to quantify the oligonucleotides by external PO 4 32 calibration under the chosen experimental conditions.…”

Nano-particle labelling of nucleic acids for enhanced detection by inductively-coupled plasma mass spectrometry (ICP-MS)

“…Most elements in the periodic table can be ionised in the ICP source including biologically 15 important elements such as P and S and as a result, ICP-MS has been increasingly employed in the analysis of nucleic acids and proteins. [1][2][3] ICP-MS provides complementary information to conventional organic MS and has many additional advantages. 2,4,5 Firstly, ICP-MS offers very low limits of detection, which 20 generally range from pg/l -µg/l depending on the analyte.…”