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. 2003 May 1;31(9):e50.
doi: 10.1093/nar/gng050.

Analysis and Accurate Quantification of CpG Methylation by MALDI Mass Spectrometry

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Analysis and Accurate Quantification of CpG Methylation by MALDI Mass Spectrometry

Jörg Tost et al. Nucleic Acids Res. .
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Abstract

As the DNA sequence of the human genome is now nearly finished, the main task of genome research is to elucidate gene function and regulation. DNA methylation is of particular importance for gene regulation and is strongly implicated in the development of cancer. Even minor changes in the degree of methylation can have severe consequences. An accurate quantification of the methylation status at any given position of the genome is a powerful diagnostic indicator. Here we present the first assay for the analysis and precise quantification of methylation on CpG positions in simplex and multiplex reactions based on matrix-assisted laser desorption/ ionisation mass spectrometry detection. Calibration curves for CpGs in two genes were established and an algorithm was developed to account for systematic fluctuations. Regression analysis gave R(2) >or= 0.99 and standard deviation around 2% for the different positions. The limit of detection was approximately 5% for the minor isomer. Calibrations showed no significant differences when carried out as simplex or multiplex analyses. All variable parameters were thoroughly investigated, several paraffin-embedded tissue biopsies were analysed and results were verified by established methods like analysis of cloned material. Mass spectrometric results were also compared to chip hybridisation.

Figures

Figure 1
Figure 1
Evaluation of incorporation rates of terminating α-S-ddNTPs in the primer extension reaction using a primer in the forward direction. Results are similar for the primers in the reverse direction using α-S-ddATP/α-S-ddGTP for termination.
Figure 2
Figure 2
Comparison of mixtures of PCR products derived from cloned bisulphite-treated methylated and non-methylated DNA in a simplex (left) and duplex (right) experiment. The position analysed is CpG 6 in the FVIII gene. The duplex experiment was performed by simultaneous analysis of CPG 6 and 1.
Figure 3
Figure 3
Calibration curve for CpG position 4 in the FVIII gene. Mixtures of clones from bisulphite-treated completely methylated and non-methylated DNA were amplified by PCR. The correlation coefficient is given on the curve. Standard deviation is indicated by vertical bars and was calculated from 24 measurements of each mixture. The MALDI spectra represent a selection of the calibration measurements. Product masses of the alleles are: d(AptACTptAptdC), 1422 Da; d(AptACTptAptdT), 1436 Da. The extension primer was charge tagged with 6-trimethylammoniumbutyryl-N-hydroxysuccinimidyl ester. The peak marked with an asterix corresponds to an incomplete primer digestion.
Figure 4
Figure 4
Sequence of the GSTP1 amplicon. CpG positions are highlighted in yellow and the extension primers with which they are analysed are shown (blue for forward primers, green for reverse primers). Extension primers were checked for self-extension in a simplex format and cross-talk in multiplex assays. For multiplex analysis only non-overlapping extension primers were combined, as otherwise competition would occur between the primers querying different MVPs.
Figure 5
Figure 5
Calibration curves for four CpG positions in the GSTP1 fragment (Fig. 4). The correlation coefficient is given in each curve. Standard deviation is indicated by vertical bars and was calculated from 24 measurements of each calibration mixture. Primers for positions –4 and 10 contain no wild-cards; those for positions 5 and 1 contain one and two degenerate bases, respectively.
Figure 6
Figure 6
Simultaneous analysis of three MVPs (M1, M5 and M10) in three different prostate samples. Relative peak heights of the sum spectra were measured and results were obtained after calibration. Results deviate at most by 3% from the values given for simplex analysis (Table 2), which is in the range of standard deviation. (A) Sample 1, (B) sample 4 and (C) sample 5. Product masses of the alleles in the MALDI spectra are: d(TptTCTptTptdC), 1497 Da and d(TptTCTptTptdT), 1512 Da for M1; d(GptTCTptAptdC), 1559 Da and d(GptTCTptAptdT), 1574 Da for M5; d(CptCptCCTptCptdA), 1693 Da and d(CptCptCCTptCptdG), 1709 Da for M10. To ensure unambiguous calculation the masses of the alleles were shifted by the use of different charge tags (CT). The extension primer querying position M1 was charge tagged with 6-trimethylammoniumbutyryl-N-hydroxysuccinimidyl ester, while the other two were charge tagged with 6-trimethylammoniumhexyryl-N-hydroxysuccinimidyl ester.

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