Matrix-Assisted Laser Desorption/Ionisation, Time-of-Flight Mass Spectrometry in Genomics Research

The beginning of this millennium has seen dramatic advances in genomic research. Milestones such as the complete sequencing of the human genome and of many other species were achieved and complemented by the systematic discovery of variation at the single nucleotide (SNP) and whole segment (copy number polymorphism) level. Currently most genomics research efforts are concentrated on the production of whole genome functional annotations, as well as on mapping the epigenome by identifying the methylation status of CpGs, mainly in CpG islands, in different tissues. These recent advances have a major impact on the way genetic research is conducted and have accelerated the discovery of genetic factors contributing to disease. Technology was the critical driving force behind genomics projects: both the combination of Sanger sequencing with high-throughput capillary electrophoresis and the rapid advances in microarray technologies were keys to success. MALDI-TOF MS-based genome analysis represents a relative newcomer in this field. Can it establish itself as a long-term contributor to genetics research, or is it only suitable for niche areas and for laboratories with a passion for mass spectrometry? In this review, we will highlight the potential of MALDI-TOF MS-based tools for resequencing and for epigenetics research applications, as well as for classical complex genetic studies, allele quanti. cation, and quantitative gene expression analysis. We will also identify the current limitations of this approach and attempt to place it in the context of other genome analysis technologies

Mutational analysis of two positional candidate susceptibility genes for bipolar disorder on chromosome 12q23-q24: phenylalanine hydroxylase and human LIM-homeobox LHX5

Objectives: In the search for chromosome 12 genes potentially involved in the pathogenesis of bipolar disorder we will screen Phenylalanine hydroxylase and human LIM-homeobox LHX5 genes for sequence variants, both of which have been suggested as candidate genes. The genes lie on chromosome 12q23–24, near the Darier's disease gene, ATP2A2. We have previously reported two families in which the pattern of segregation of illness is consistent with genetic linkage between this chromosomal region and a putative highly penetrant autosomal dominant major affective disorder locus (pedigree 324, maximum LOD=2.1; pedigree 5501, maximum LOD=3.6). Methods: We screened the coding and intronic flanking regions of the phenylalanine hydroxylase and LHX5 genes for sequence variation by denaturing high-performance liquid chromatography in individuals from the pedigrees. Results: In total, nine single nucleotide polymorphisms and one 6 base pair deletion were identified. Conclusion: Our studies allowed us to conclude that none of these variants act as a highly penetrant autosomal dominant susceptibility locus for mood disorder in our families

MALDI-TOF Mass Spectrometry

<p>The analyte is deposited on a matrix crystal containing spot. A laser is fired at the matrix leading to the desorption of the analytes and their ionisation. For the majority of DNA analyses, the matrix is designed to produce positive ions. The molecules accelerate into the flight tube where they fly towards the detector on the right. Low mass molecules arrive in a shorter time than heavier ones and molecules of different mass are thus separated (1). After data processing, a spectrum is produced with relative intensity on the <i>y</i>-axis and mass in Daltons on the <i>x</i>-axis (2).</p

Example of a 29plex iPLEX Reaction

<p>For the iPLEX reactions, the primers are split into three groups according to the position of their respective mass peaks to use the whole spectrum. The concentrations of the three groups are adjusted so that peaks detected at the low mass end of the spectrum have a similar peak height to peaks at the high end of the spectrum. Three examples are highlighted in different colours in different areas of the spectrum. The unextended primer peak is marked by an asterisk and dotted arrow, while the other solid arrows indicate the two different alleles. In red, a homozygous genotype example is shown. In green and blue, two heterozygous alleles are shown. Note the similar peak heights at which extension products corresponding to the two alleles of an SNP are detected and their relative proximity. This proximity enables a higher accuracy to be achieved in the assay by applying the same level of local background subtraction and by eliminating misinterpretations resulting from small peaks, detected just above background, at the expected mass.</p

Methylation Detection Using MALDI-TOF MS

<div><p>MALDI-TOF MS spectra for the detection of different DNA methylation levels (examples shown: 100% methylated DNA; 50:50 mix of methylated and unmethylated DNA; 10% methylated DNA samples mixed with 90% unmethylated DNA) after bisulfite treatment in specific CpG positions in the promoter of <i>INK4A</i> (p16) gene (specific amplicon mapping to Chromosome 9: 21964960–21965128 on the Human Genome May 2004 assembly).</p><p>(A) Primer extension approach using iPLEX chemistry to detect DNA methylation at CpG 2 (Chromosome 9: 21965105–21965106), C call corresponding to the methylated and T call to the nonmethylated alleles.</p><p>(B) MassCLEAVE assay using T-reverse specific cleavage, detection of the methylation levels at CpG 10–11 (Chromosome 9: 21964989–21964992 on the Human Genome May 2004 assembly). Fragments detected on the reverse strand are as follows: 4484.85 Dalton 5OH-AAAAAAAC<u>CACA</u>AT-3p for the unmethylated (Unmeth) and 4516.84 Dalton 5OH-AAAAAAAC<u>CGCG</u>AT-3p with both CpG sites methylated (Meth).</p></div

Base Specific Cleavage for Mutation Detection

<div><p>(A) Illustration of the MassCLEAVE procedure.</p><p>(A1) PCR using one primer with a T7 RNA polymerase tag and one with a universal tag.</p><p>(A2) In vitro transcription using T7 RNA polymerase and a mixture of dNTPs and NTPs.</p><p>(A3) Cleavage by RNAse A. RNAse A cleaves at pyrimidines. By adding one of the pyrimidines as dCTP or dTTP only, the positions containing CTP or TTP are cleaved by RNAse A. Four cleavage reactions are set up in total.</p><p>Box to the right of (A): Bioanalyser trace showing a full-length 660 bp RNA produced by IVT (IVT) compared with negative control (CTRL). The final steps involve sample conditioning and analysis by MALDI-TOF MS. Other possibilities include RNAseT1, which cleaves at a G. For methylation analysis, only the two reverse reactions are required.</p><p>(B) Spectra and interpretation: A 396-bp fragment containing exon 6 of the <i>EXT1</i> gene was analysed by MassCLEAVE to identify a heterozygous deletion of a T at 160 bp. The informative T-forward and C-reverse reaction spectra are shown, whereby the spectrum produced by the sample under investigation is at the top and the control underneath. The numbers at the top indicate expected peak positions along the spectrum and are indicators of reaction quality. In the T-forward reaction, a new peak corresponding to the A1C6G3T1-composed primer appears at 3,425 Dalton (indicated by a red arrow). In the C-reverse reaction, a new peak at 2,699 Daltons is observed, corresponding to the T1G6C1 fragment, while the height of the 3,028 Dalton peak corresponding to the normal A1T1G6C1 fragment is reduced. The relevant normal and mutation-containing fragments are indicated and the deleted T (or A in reverse) is underlined in red. In total, 11/15 mutations in <i>EXT1</i> exon 6 and <i>EXT2</i> exon 5 (associated with hereditary multiple exostosis) were detectable with this method.</p></div

RC-PCR Experiment

<div><p>(A) A triplex assay for the genes <i>BNIP3, CA9,</i> and <i>NDRG1</i> is shown. Using a high competitor concentration (10⁻¹²M), competitor is predominantly PCR-amplified and only the primer extension products resulting from the competitor oligos (blue arrows, top graph) are detected. Using low competitor concentrations (10⁻¹⁶M), cDNA is predominantly PCR-amplified and only the primer extension products resulting from the cDNA are detected (blue arrows, bottom graph). The equivalence point, whereby an equal area of competitor and cDNA peaks is derived, would lie at a competitor concentration of between 10⁻¹² M and 10⁻¹⁶ M.</p><p>(B) Output of the TITAN software (<a href="http://www.well.ox.ac.uk/~tprice/titan" target="_blank">http://www.well.ox.ac.uk/~tprice/titan</a>) showing linear regression analysis of two samples (red and black) for BNIP3 expression. <i>x</i>-axis: competitor concentrations; <i>y</i>-axis (left) is the log 10 ratio of test (cDNA) to competitor signal. The equivalence point is given by the concentration at which <i>y</i> = 0, and is calculated by interpolation (black/red arrows). An approximated difference in expression is detected between the two samples.</p></div