Mass-spectrometric approaches for DNA-based genetic screening.

In the current issue of Clinical Chemistry, Braun et al. [1] describe a mutational analysis method that combines primer oligo base extension (PROBE) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). This application further extends the use of MS for genetic analysis and suggests a possible role in the clinic. However, what is the long-term potential for MALDI, based on the relative strengths and weaknesses of MS analysis of DNA [2]? The development of soft-ionization techniques for MS has, without question, revolutionized protein chemistry. MS is now one of the definitive methods for characterization of synthetic and isolated peptides and proteins. In addition to accurately determining an intact mass, one can obtain primary structure information by several different MS sequencing strategies, and secondary and tertiary structure can be evaluated by hydrogen-exchange experiments. The use of MS for DNA analysis, although first demonstrated for nucleosides in 1962 [3], is far less advanced. Potential applications of MS are (a) detection of DNA modifications, (b) DNA fragment mass determination, and (c) DNA sequencing. Both fast atom bombardment (FAB) and electrospray ionization (ESI) collisioninduced dissociation/tandem MS have been applied for identification of DNA modification sites. For example, the carcinogen N-nitrosopiperidine reacts with DNA to produce ethenodeoxyguanosine adducts. These adducts have been characterized by use of liquid chromatography (LC)ESI-MS/MS of enzymatically hydrolyzed DNA [4]. In similar fashion, oxidative modification of DNA resulting in the production of 8-hydroxydeoxyguanosine has been detected by LC-ESI-MS/MS after enzymatic hydrolysis [5]. FAB-MS/MS has been used to detect polycyclic aromatic hydrocarbon-DNA adducts [6]. One of the disadvantages of ESI is that cation (Na, K) adduction to the polynucleotide backbone limits the size of DNA analysis and analysis of oligonucleotide mixtures—although online microdialysis can minimize sodium adducts [7]. MALDI offers a potential advantage over ESI and FAB, in that biomolecules of large mass can be ionized and analyzed readily. In theory, restriction-enzyme-digested fragments of DNA could be amplified by polymerase chain reaction (PCR) and detected directly by MALDIMS. A 500-nucleotide DNA has been characterized by MALDI-MS with a mixed matrix of picolinic acid and 3-hydroxypicolinic acid [8]. This size is critical, because MALDI-MS should be able to analyze DNA fragments as large as 500 bases to compete with other DNA-sequencing methods. In general, MALDI analysis of DNA is not so straightforward [2]. Potential problems with MALDI-MS analysis that need to be considered are (a) lack of resolution of high-Mr DNA fragments, (b) DNA instability, and (c) interference from Sanger sequencing reagents. Longer oligonucleotides can give broader, less-intense signals, because MALDI imparts large kinetic energies to ions of high m/z. Novel matrices, such as trihydroxyacetophenone, combined with ammonium acetate can improve DNA resolution [9]. Resolution can also be improved by using ion-“trapping” methods or a time-lag focusing TOF-MS [10]. DNA fragmentation during MS includes elimination of a nucleic base and cleavage of a phosphate ester bond, as well as phosphate rearrangements [11]. Fragmentation was found to be a problem during DNA sequencing of M13 bacteriophage DNA [12]. The use of 7-deazapurines during Sanger DNA sequencing reactions can offer better oligonucleotide stability and sensitivity [13]. MALDI-MS sequence analysis can be performed by using Sanger sequencing chemistry and overlaying the results from the four individual sequencing reactions [14]. The combination of MALDI-MS and the Sanger sequencing method has been used for M13 bacteriophage DNA, with sequence being determined for DNA as much as 35 bases long [12]. Impurities from Sanger sequencing solutions that can interfere with MALDI analysis include deoxynucleotide triphosphates (dNTP), dideoxynucleotide triphosphates (ddNTP), polymerase, and buffers. Impurities can be removed by affinity purification methods, with a probe strand attached to either agarose or magnetic beads [15]. Separating the DNA-containing supernatant from the beads can limit sample recovery. Alternatively, one can use a solid-phase sequencing approach to immobilize sequencing ladders on a synthetic template. The template is labeled with biotin and bound to streptavidin-coated magnetic beads [16]. Solid-phase sequencing, in combination with MALDI-MS, has been used for fragments as large as 63 bp [16]. Ultimately, the solid-phase approach could be microfabricated to create “DNA chips” for sequencing. Given the above caveats, MALDI-MS has been used in impressive fashion for the detection of genetic polymorphisms. A primary example is the cystic fibrosis transmembrane conductance regulator (CFTR) gene, in which a 3-bp deletion in exon 10 can occur, resulting in loss of Phe at codon 508 (DF508). To test for this mutation, Cháng et al. used the DNA template from a patient to PCR-amplify fragments of 98 and 95 bp from exon 10 [17]. After enzymatic digestion of the fragments, differences could be somewhat resolved. A more definitive result was achieved by using primers for nested amplification to span the deletion, which generated 59or 56-bp fragments that were easily assigned by MALDI-MS [17]. In similar fashion, PCR-generated 72and 75-bp synthetic oligonucleotides corresponding to CFTR DF508 were resolved by MALDI-MS [18]. CFTR mutants were also analyzed by MALDI-MS after DNA extraction from buccal cells, PCR amplification, and generation of restriction enzyme fragments [19]. PCR products from Legionella genomic DNA were characterized by MALDI-MS, such that a 168-bp PCR product unique to L. pneumophila was detected [20]. The work of Braun et al. [1] describes an improvement Editorial