REVIEW 147 Molecular Biotechnology 2004 Humana Press Inc. All rights of any nature whatsoever reserved. 10736085/2004/26:2/147163/$25.00 *Author to whom all correspondence and reprint requests should be addressed: Christian Jurinke, Sequenom, Inc., Johns Hopkins Court, San Diego, CA 92121. E-mail: cjurinke@sequenom.com Abstract MALDI-TOF Mass Spectrometry A Versatile Tool for High-Performance DNA Analysis Christian Jurinke,* Paul Oeth, and Dirk van den Boom 1. Introduction In the past decade matrix-assisted laser desorp- tion/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) has become one of the most powerful tools for the analysis of bio- molecules. The scope of this review is to provide an overview on the developments and most recent accomplishments in this area of research, focus- ing on the analysis of nucleic acids. 1.1. MALDI-TOF Mass Spectrometry The general principle of MS is to produce, separate, and detect gas-phase ions. Traditionally, thermal vaporization methods are used to transfer molecules into the gas phase. The classical meth- ods for ionization are electron impact (EI) and chemical ionization (CI). Most biomolecules, however, undergo significant decomposition and fragmentation under the conditions of both meth- ods. Consequently, the application of MS to nucleic acid analysis had been limited to mol- ecules the size of dinucleotides (1). Analysis of oligonucleotides with a mass range of up to 3000 Dalton (about 10 nucleotides) became feasible with the development of plasma desorption (PD) methods (2). Until the invention of soft ioniza- tion techniques such as electrospray ionization mass spectrometry (ESI-MS) and MALDI-MS, mass spectrometric tools were not widely consid- ered for routine applications in biological sciences. MALDI as a principle for analysis of large bio- molecules was introduced by Karas and Hillenkamp (3). Briefly, in MALDI-MS, the sample is em- bedded in the crystalline structure of small organic compounds (matrix) and deposited on a conduc- tive sample support. The cocrystals are irradiated with a nanosecond laser beam, for example, an ul- traviolet (UV) laser with a wavelength of 266 or 337 nm. The energies introduced are in the range of 1 10 7 5 10 7 W/cm 2 . The laser energy causes structural decomposition of the irradiated crystal and generates a particle cloud (the plume) from which ions are extracted by an electric field. The mechanism behind the process of desorption is not fully understood. It may best be described as a con- version of laser energy to vibrational oscillation of the crystal molecules. This results in the disin- tegration of the crystal. Following acceleration through the electric field, the ions drift through a field-free path and finally reach the detector (e.g., a Matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) has developed during the past decade into a versatile tool for biopolymer analysis. The aim of this review is to summarize this development and outline the applications, which have been enabled for routine use in the field of nucleic acid analysis. These include the analysis of mutations, the resequencing of amplicons with a known reference sequence, and the quantitative analysis of gene expression and allelic frequencies in com- plex DNA mixtures. Index Entries: MALDI-TOF; SNP analysis; genotyping; quantitative MALDI-TOF; gene-expression analysis; resequencing; SNP discovery; DNA pooling. 07_JW621_Jurinke_147-163 1/19/04, 9:49 AM 147 MOLECULAR BIOTECHNOLOGY Volume 26, 2004 148 Jurinke, Oeth, and van den Boon secondary electron multiplier or channel plate). Ion masses (mass-to-charge ratios [m/z]) are typi- cally calculated by measuring their TOF, which is longer for larger molecules than for smaller ones (provided their initial energies are identical). Be- cause predominantly single-charged, nonfrag- mented ions are generated, parent ion masses can easily be determined from the resulting spectrum without the need for complex data processing. The masses are accessible as numerical data for direct processing and subsequent analysis. TOFs mea- sured during a typical MALDI experiment are in the range of several microseconds. 1.2. Sample Preparation for Nucleic Acid Analysis The quality of spectra in terms of resolution, mass accuracy, signal-to-noise ratio, and sensi- tivity is highly dependent on sample preparation and the choice of matrix compounds (4). Adduct formation and fragmentation are predominantly influenced by sample purification and matrix composition. Both are described in detail in the following sections. In brief, the purification pro- cess should result in a sample that is properly con- centrated, free of crystallization-disturbing agents (such as detergents, urea or dimethyl sulfoxide [DMSO]) and adduct-forming agents (such as non- volatile cations). The standard method for sample preparation is the so-called dried-droplet method (5). It relies simply on pipetting a small volume of the sample (usually about 0.5 L) to a drop (about 1 L) of matrix solution and allowing the mixture to dry. This procedure has been modified for high- throughput applications that involve automated MALDI measurement (6).The vast amount of ap- plications of MALDI-TOF-based methods for the analysis of peptides, proteins, glycosides, or noncovalent interactions is beyond the scope of this review. For an overview the reader is referred to some excellent reviews in this field (7,8). 1.3. MALDI-TOF DNA Analysis The application of MALDI-MS to the analysis of nucleic acids had for years remained a field of small impact to the bioscience community. Nucleic acids are far more difficult to analyze under MALDI conditions than peptides, so applications had been limited. Because of their negatively charged phosphate backbone, nucleic acids are es- pecially susceptible to adduct formation. They tend to form salt adducts with cations present in the surrounding medium. In biochemical reac- tions, these are predominantly sodium and potas- sium ions. Adduct formation results in a broader distribution of the signal: A main signal with the protonated analyte may be accompanied by sig- nals resulting from multiple adduct formation. For example, every sodium ion attached to the analyte molecule will cause an additional signal with a mass of plus 23 Dalton. Consequently, adduct for- mation lowers sensitivity and analytical accu- racythe total amount of ions is distributed over a multitude of ion species, and the mass differ- ences between those may become too small to be resolved. Approaches to overcome adduct forma- tion are ion-exchange procedures based either on solid-phase (9) or cation-exchange resin methods (5). Chemical modification of the phosphate back- bone has also been proposed to prevent adduct formation (10). The addition of ammonium-con- taining additives like diammonium citrate (11) or tartrate (12) to the matrix reduces cation heteroge- neity of analytes. The use of millimolar solutions of ammonium hydroxide during the conditioning process has also a beneficial effect (13). The ratio- nale for exchanging cations for ammonium is that the latter is a volatile cation that is released as ammonia in the gas phase leaving the analyte mol- ecules as free acids. Based on the chemical nature of nucleic acids, fragmentation reactions can occur during the MALDI process. The predominant effect of these reactions is depurination. Protonation of the nucleobases A or G (at posi- tion N7) induces polarization of the N-glycosidic bond between sugar and nucleobase and finally results in nucleobase elimination. Subsequent to depurination, further fragmentation occurs via backbone cleavage. In addition to depurination, the elimination of C has also been reported (14). This reaction is mediated through protonation at N3. Hillenkamp and coworkers have demon- strated that ribonucleic acid (RNA) is less suscep- 07_JW621_Jurinke_147-163 1/19/04, 9:49 AM 148 MOLECULAR BIOTECHNOLOGY Volume 26, 2004 MALDI-TOF Mass Spectrometry 149 tible to fragmentation under MALDI conditions than deoxyribonucleic acid (DNA). The proposed reason is the lack of the 2' hydroxy group in the ribose sugar moiety (15). Approaches to compen- sate for the detrimental influences of fragmenta- tion have been contributed from instrument developers as well as biochemists. It has been demonstrated by several groups that introduction of 7-deaza nucleobases, where N7 is replaced by C, is helpful in suppressing depurination reactions (16,17) because the proton acceptor site is re- moved. An important contribution to analyzing DNA with MALDI-TOF was also the introduc- tion of delayed extraction instruments by Wiley and McLaren (18). Compared to static extraction, which employs a permanent electrical field for ion acceleration, the delayed extraction accumulates ions over a time range of some nanoseconds be- fore the extraction voltage is applied. The effect is a compensation of the initial differences in kinetic energies of the analyte molecules because mol- ecules with higher initial velocities expand further during the delay time and experience a lower po- tential during acceleration. To some extent this may also compensate for positional variations of molecules that occur owing to an inhomogeneous distribution in the matrix cocrystals. Another mile- stone was the introduction of favorable matrices for DNA analysis. Especially, 3-hydroxypicolinic acid (3-HPA) and some of its derivatives (19) pre- dominate in the field of DNA analysis. Mixtures of 2,3,4'- and 2,4,6'-trihydroxy-acetophenone (THAP) are mainly used for RNA analysis (20). A promising development is the application of infrared (IR)-MALDI for the analysis of DNA molecules of up to 2.1 kb by Hillenkamp and co- workers (21). 2. Qualitative DNA Analysis The developments described in the previous section enabled investigations in various areas of mass spectrometric DNA analysis. The analysis of amplification products generated during poly- merase chain reaction (PCR) and sequencing reac- tions has been an early focus. For those applications the most critical issue was sample preparation. The broad terminus sample preparation is gen- erally used to describe all steps necessary to get the DNA products in a suitable form for mass spectrometric analysis, as described in the previ- ous section. It is beneficial to separate the prod- ucts from the educts because all available DNA molecules will be subjected to the ionization pro- cess. To prevent competition for available charges, an excess of primer, for example, out of a PCR reaction should be avoided. Finally, the products have to be presented to the matrix in a concentra- tion and volume ratio that results in a favorable matrix-to-analyte ratio. The amount of sample delivered should be adjusted to allow for homog- enous sample crystallization on the target. Meth- ods devised to purify samples are, for example, ethanol precipitation (22) and affinity-based pu- rification via streptavidin systems, such as mag- netic beads (23,24), or reversed-phase columns (25).The applications described for MALDI-TOF mass spectrometric PCR product analysis cover a broad range. The earliest reports from researchers analyzing PCR products (26) still used purifica- tion methods that were not adaptable for high throughput. Applications that have been enabled for DNA analysis via MALDI-TOF include oli- gonucleotide sequence analysis (11,27) and PCR or LCR product detection (28,29). Also the se- quencing of PCR products (30), mutation detection (31,32), and applications for clinical diagnostics (33,34) have been described. Furthermore, the qualitative analysis of in vitro transcripts has been reported (35). The use of streptavidin-coated beads supported a variety of different applica- tions. The solid-phase approach provides ease of sample conditioning and concentration. The re- covery of the DNA from the solid support is also a very efficient process. Double-stranded DNA can be fractionated in this way, because either the sense or the antisense strand or both strands to- gether can be eluted from the beads (29). 2.1. SNP Analysis An important field is the research focused on the study of single nucleotide polymorphisms (SNPs). SNPs can be defined as biallelic variants within a population occurring with an allelic fre- 07_JW621_Jurinke_147-163 1/19/04, 9:49 AM 149 MOLECULAR BIOTECHNOLOGY Volume 26, 2004 150 Jurinke, Oeth, and van den Boon quency higher than 1% (with 1% as a more or less arbitrary threshold). SNPs can be seen as a gener- alization on genetic variations, covering also re- striction fragment length polymorphisms (RFLPs). The importance of SNPs to our under- standing of genetic diversity is recognized today by many academic and commercial organizations. Consequently, this is leading to SNP discovery efforts within the human genome. Commonly used methods for SNP analysis on mass spectro- metric platforms are based on primer extension protocols (36). The format used by our group is a homogenous assay, an advance over the use of streptavidin-coated beads because no removal of supernatants is involved. The reaction protocol consists of several component additions (37). The PCR is subjected to shrimp alkaline phos- phatase (SAP) treatment to inactivate remaining nucleotides. Following inactivation by SAP, a re- action cocktail is added consisting of an extension primer annealing adjacent to the mutated site, thermosequenase, and a deoxyribonucleotide dNTP/dideoxynucleotide (ddNTP) mixture. The PCR product now serves as templates for a primer extension reaction. Depending on the chosen nucleotide mixture, two products with distinct masses are generated (see Fig. 1). These products are purified through the addition of ion-exchange resin and subsequently dispensed on a chip array that is preloaded with the components necessary for MALDI sample preparation. This format can be used for the analysis of deletion, insertion, and point mutations, short tandem repeat (STR), and SNP analysis, and it allows for the detection of compound heterozygotes. For SNP analysis the primer-binding site is placed adjacent to the polymorphic position. A specific extension product is generated for each allele. In the case of heterozygosity, both prod- ucts are generated simultaneously. In the example given in Fig. 1B, both elongation products are ex- pected to differ in mass by one nucleotide. The two SNP alleles appear as two distinct mass sig- nals. Careful assay design makes a high-level multiplexing of MassEXTEND reactions pos- sible. Figure 2 provides an example of a nineplex. Pinpoint assays that result from a single base extension (38) can also be performed with this approach. Gut and coworkers have devised a scheme for genotyping of SNPs that is similar to the afore- mentioned but in addition relies on the chemical modification of the short DNA fragments that are generated through a 5'-phosphodiesterase digest (39). The same approach has been used for haplo- typing of SNPs. For this purpose an allele-specific PCR protocol is employed (40). This so-called GOOD assay, though scientifically elegant, has in practical applications been hampered because chemicals with a certain carcinogenic potential (like methyliodide) are involved in the alkylation reaction. Recently, an approach based on the use of specially synthesized methylphosphonate prim- ers has been devised that avoids the use of alky- lating reagents (41). Another interesting approach for SNP geno- typing is the use of the so-called invader assay. This approach uses three oligonucleotide probes for each analyzed SNP. Two probes are allele spe- cific but have a noncomplementary region 5' of the SNP, and a third oligonucleotide (the invader oligo) invades at least one nucleotide into the du- plex formed by the allele-specific oligo. The now unpaired region of the allele-specific oligo is cleaved with a flap endonuclease (FEN). The cleaved short oligonucleotide can now be purified through a biotinstreptavidin system. This reac- tion has the potential to be performed directly from genomic DNA. However, because the amount of material generated in this case is very low, a sec- ondary reaction has to be done on top of the first. For this purpose the cleaved-off oligo from the first step serves as an invader oligo for a pair of other synthetic oligonucleotides that mimic the targeted allele. The noncleaved primary probe needs to be captured for this purpose by another oligo (called the arrestor). The secondary cleav- age product is finally purified via a streptavidin biotin-based solid support. This approach has been employed for genotyping and even quantita- tive analysis (42,43). However, because this method involves the synthesis of multiple oligo- nucleotides per SNP, the setup costs for this ap- proach are rather high. 07_JW621_Jurinke_147-163 1/19/04, 9:49 AM 150 MOLECULAR BIOTECHNOLOGY Volume 26, 2004 MALDI-TOF Mass Spectrometry 151 Fig. 1. Schematic representation of primer extension assay as used in multiple applications in combination with MALDI-TOF. A primer adjacent to the mutation site is extended in an allele specific manner. The extension products differ in mass and are analyzed with MALDI-TOF MS. The spectra provided in A,B, and C represent raw data generated from a heterozygous sample (B) and the two homozygotes (A,C). 2.2. Microsatellite Analysis Microsatellites are another widely used type of genetic marker. SNPs are increasingly used in tar- get gene discovery programs, owing to their high abundance (about 1 in 500 bases). However, be- cause they are only of a biallelic nature, their suit- ability for forensic examinations is still a topic of research. Microsatellite DNA typically comprises between 4 and 25 tandemwise repeated nucleotide units from 1 to more than 7 bases. More often, these markers are referred to as STRs. Various types of STRs have been described, with di- tri- and tetranucleotide repeats being the most promi- nent. The abundance of STRs is a factor of 10 to 20 lower than that of SNPs, but owing to their highly polymorphic nature, STRs have been suc- cessfully used in a variety of different applications. Besides their importance in forensic applications, which make use of the interindividual differ- ences in STR length, linkage studies, positional cloning efforts, as well as indirect analysis of monogenetic disorders have successfully employed STR analysis (4446). Moreover, neurological dis- orders have been linked to STR instabilities (47), and tumors have been characterized by micro- satellite marker-based detection of loss of het- erozygosity (LOH) (48). The analysis of STRs is conventionally performed by PCR amplification 07_JW621_Jurinke_147-163 1/19/04, 9:49 AM 151 MOLECULAR BIOTECHNOLOGY Volume 26, 2004 152 Jurinke, Oeth, and van den Boon sample is compared to a so-called allelic ladder that serves as a standard. For MALDI-TOF analysis of microsatellites, a primer extension-based approach can be applied. For this approach a ddNTP composition is cho- sen, which terminates the polymerase extension at the first nucleotide not present within the repeat (51). Length determination of a CA repeat is con- ducted with a ddG or ddT termination mix. Even imperfect repeats harboring insertion or deletion mutations can be analyzed with this approach. Figure 3 displays raw data from the analysis of a human STR marker in a heterozygous DNA sample. Both alleles differ by 4 CA repeats. The DNA polymerase slippage during amplification generates a pattern of stutter fragments (marked with an asterisk in Fig. 3). In the case of heterozy- gotes, which differ in just one repeat, the smaller allele has higher intensities than the larger allele, because allelic and stutter signals are added to- gether. Recently, the use of ribozymes to shorten the final products for analysis has been reported Fig. 2. Raw data of a nineplex obtained with primer extension protocols as outlined in the text. Fig. 3. Depicted are raw data generated from a primer extension assay for microsatellite analysis. The asterisk marks signals that are generated through tem- plate slippage of the DNA polymerase, so-called stut- ter signals. using fluorescent-tagged primers. Products are separated by gel or capillary electrophoresis (49,50). For the purpose of determining the re- peat length, the gel electrophoretic mobility of the 07_JW621_Jurinke_147-163 1/19/04, 9:49 AM 152 MOLECULAR BIOTECHNOLOGY Volume 26, 2004 MALDI-TOF Mass Spectrometry 153 (52). However, none of the devised MALDI-based methods compete with the existing technologies. Despite all promising developments, the field of STR analysis by MS still remains a formidable challenge (53). 2.3. DNA Sequencing The work focusing on analyzing DNA sequenc- ing reactions with MALDI-TOF MS has started with attempts to translate known sequencing formats to mass spectrometric readouts. Several biochemical schemes have been developed, which generate DNA sequencing ladders of sufficient yield and purity to suit the specific requirements for the analysis by MALDI-TOF MS (30,5456). Following the concept of conventional dideoxy sequencing, the nested set of truncated sequences originating from a primer can be analyzed by MALDI-TOF MS. The mass difference between the DNA fragments can be used to calculate the nucleotide sequence. Owing to the nearly expo- nential decay in sensitivity of MALDI-TOF MS, with increasing mass the read length is limited. Despite very promising results for solid-phase- based sequencing and cycle sequencing, the 100- bp barrier was never overcome on a routine basis. In addition to sensitivity issues, the mass resolu- tion of conventional axial-TOF instruments is in- sufficient for accurate sequence determination. Lack of discrimination between polymerase paus- ing signals, generated by secondary structures of the template, and real termination signals sig- nificantly limit sequence analysis in the higher mass range. Sensitivity, mass resolution, and mass accuracy issues contribute to the fact that analysis of dideoxy sequencing ladders by MALDI-TOF MS has not yet been implemented for high- throughput sequencing applications. Even though the data acquisition time for MALDI spectra is only about 2 s per 20 indi- vidual spectra, the short read length prohibits MALDI-TOF from competing with conventional approaches. Several alternative formats have been devised to overcome this read length limi- tation. Rather than using a primer extension- based method, which yields a ladder of DNA fragments with increasing sizes, the following schemes rely on the generation of short, base-spe- cific fragments. Base-specific cleavage of nucleic acids represents a paradigm shift in DNA sequenc- ing by MS. The principle resembles more closely the original approach of Maxam and Gilbert for DNA sequencing (57). These methods, however, are not suitable for de novo sequencing. They rather represent identification or resequencing methods, where an experimentally determined sequence is cross-compared to a known reference sequence. One example involves an enzymatic DNA-based fragmentation approach. In this approach, PCR products are generated containing 2'-deoxyuridine 5'-triphosphate (dUTP) instead of dTTP. After strand separation and incubation with uracil-DNA- glycosylase (UDG), alkaline and heat treatment fa- cilitates DNA cleavage at each T position (58,59). To discriminate between the signal patterns of the two amplicon strands, their separation is neces- sary. The strand separation is currently performed using streptavidin-coated magnetic beads, as de- scribed in previous sections. An approach based on chemical cleavage uses P3'-N5'-phosphoramidite- containing DNA (60). Either 2'-deoxycytidine 5'- triphosphate (dCTP) or dTTP is replaced by their analog P-N-modified nucleoside triphosphates. They are introduced into the target sequence dur- ing a post-PCR primer extension reaction. Acidic reaction conditions produce base-specific cleav- age fragments, which are analyzed by MALDI- MS. However, the required acidic conditions generate unwanted depurination byproducts. A base loss of adenine and guanine is routinely ob- served and needs to be suppressed by incorporat- ing 7-deaza analogs of dA and dG. Although both of these methods are robust and reasonably easy to handle, each approach is limited by the rela- tively low yield of single-stranded DNA products, which prevents minimizing the reaction volumes without a significant loss of sensitivity. Another scheme uses base-specific ribonu- cleases (RNases) for template digestion, followed by an analysis of the resulting cleavage products by MS (61). The basic principle is to generate in vitro RNA transcripts from a PCR product. The transcripts are derived from the PCR product by tagging the PCR primers with an RNA poly- 07_JW621_Jurinke_147-163 1/19/04, 9:49 AM 153 MOLECULAR BIOTECHNOLOGY Volume 26, 2004 154 Jurinke, Oeth, and van den Boon merase promoter. Following the in vitro transcrip- tion, using T7 RNA polymerase, the transcripts are incubated with RNases (62,63). Figure 4 de- picts a schematic example and raw data obtained with this approach. 3. Quantitative Analysis Many important applications in biological re- search (e.g., gene-expression analysis) require quantitative analysis of nucleic acids. The use of MALDI-TOF MS for quantitative analysis is a formidable scientific challenge. Owing to inher- ent process variables, absolute quantitation is currently not an option. Relative quantitation, however, is possible and gives rise to several in- teresting applications. For relative quantitation the ratio between two signals is measured and com- pared. Two approaches are possible: 1) The abso- lute concentration of the analyte molecules is unknown, and only their ratio is compared; 2) the concentration of one analyte (referred to as inter- nal standard) is known and used to calculate the concentration of the unknown analyte. The first approach is particularly useful for the analysis of allele distributions (frequencies) in nucleic acid mixtures (6466); the second ap- proach can, for example, be employed for quanti- tative gene-expression analysis (67). 3.1. Analysis of DNA Mixtures (Allele Frequency Determination) Ross et al. were the first to describe the quanti- tative ability of MALDI-TOF MS in conjunction with primer extension reactions to measure ratios of known DNA mixtures (68). Relative quantitation of allele frequencies is achieved by calculating the areas of the peaks associated with specific primer extension reactions. Peak characteristics (such as peak height) are generally stored when acquired on the MALDI-TOF MS, and area calculations are conducted by the users mathematical method of choice. Because SNPs are generally biallelic, the allele frequency is calculated as the ratio of the area of each allele to the total summed area of both alleles. The sum of the two alleles is always 1 in this model. Allele frequencies down to 5% can be accurately discerned using MALDI-TOF MS in DNA pools (64,69). Frequencies below 5% are routinely detected, but their accuracy must be ap- proached with caution owing to the small peak area associated with a minor allele at a 50:1 ratio relative to the major allele. Several studies have compared the quantitative abilities of different platforms used for analyzing SNPs with the goal of allele frequency estimation in DNA pools (69 71). MALDI-TOF MS measurement of primer extension reactions has been found to be as sensi- tive and reproducible as all available technologies based on these studies. Figure 5 shows some ex- ample data for the estimation of allele frequency in DNA population pools as compared to the ob- served frequency determined by genotyping all of the individuals in the population. Figure 5 is a scatterplot with allele frequencies determined for 48 assays in a DNA pool of 96 individuals vs. the observed frequency based on the genotype for all 96 individuals for each of the 48 assays. As can be seen from the coefficient of determination (R 2 ) there is not a perfect 1:1 correlation between the allele frequencies calculated from a pool of indi- viduals and the actual frequency determined by genotyping. Several factors can contribute to this inaccuracy no matter what the technology used (for review, see 71,72). However, a correction factor can be applied for each individual assay based on the peak areas observed for individual heterozygous samples from the population under investigation. Individual heterozygotes have two alleles at a 1:1 ratio for any given biallelic SNP. Based on this, the peak areas observed for each allele on the MALDI-TOF MS should be equal. If they are not, then a skewing of one allele over the other has occurred at some point in the pro- cess (whether it be at the level of PCR or analyte ionization during MALDI-TOF or at some other Fig. 4. (opposite page) The upper part of the figure provides an overview on the fragments generated dur- ing the analysis of a particular G/A SNP. The lower part of the figure provides data for the T-forward and C-forward reactions, respectively. These products are most difficult to discriminate because they have the same length but differ only by 16 Daltons owing to their different sequence composition. The arrows point at the discriminative signals as generated in the two separate reactions. 07_JW621_Jurinke_147-163 1/19/04, 9:49 AM 154 MOLECULAR BIOTECHNOLOGY Volume 26, 2004 MALDI-TOF Mass Spectrometry 155 Fig. 4. 07_JW621_Jurinke_147-163 1/19/04, 9:49 AM 155 MOLECULAR BIOTECHNOLOGY Volume 26, 2004 156 Jurinke, Oeth, and van den Boon point). This bias can be observed and quantified on the MALDI-TOF MS as illustrated in Fig. 6. The deviation from a 1:1 ratio of each allele in a collection of heterozygotes can be used as a cor- rection factor for the allele frequency calculated in the pooled DNA samples. Figure 7 shows a scatterplot similar to Fig. 5. The allele frequen- cies determined in the 96-individual pool have now been corrected using the formula given in Fig. 6. Note the improvement in the coefficient of determination (R 2 ) between Figs. 5 and 7 after in- cluding the correction. This type of accuracy is sufficient for many semiquantitative applications, such as estimation of SNP allele frequency in nucleic acid pools or the differential proteinDNA binding associated with allelic variants of a gene (65,66,73). The analysis of allele frequency dis- tributions is a tool to study the abundance of the particular alleles in populations and to compare these between different populations. This infor- mation can be used to identify causative genetic loci associated with complex diseases via linkage disequilibrium between two or more loci (74). SNPs have gained acceptance as a tool for con- ducting such studies because of their widespread distribution throughout genomes and general ease of measurement (75). Genotyping thousands of SNPs over hundreds to thousands of individuals, however, still remains cost ineffective. Pooling DNA populations has therefore been proposed as an alternative to individually genotyping popula- tions for association studies (71). MALDI-TOF MS has been at the forefront of this approach. Buetow et al. conducted the first genomewide analysis of gene-based SNPs using MALDI-TOF MS in conjunction with primer extension reac- tions to estimate allele frequencies in CEPH, (Utah pedigree) DNA pools of 94 individuals (64). Several independent groups further validated the accuracy and feasibility of such an approach us- ing MALDI-TOF MS measurement of peak areas in conjunction with primer extension reactions in pooled DNA populations (65,66). The ultimate goal of these association studies is the identifica- Fig. 5. Scatterplot of genotyped population allele frequencies (x-axis) vs. allele frequencies calculated using pooled population DNAs (y-axis). Forty-eight unique assays are depicted. DNA population pool consisted of 96 individual DNAs at equimolar concentrations (260 pg per individual DNA/L = 25 ng/L). Frequencies were calculated using TYPER RT software (Sequenom). The calculated allele frequency for each assay represents the average of four replicate reactions each dispensed in replicates of four onto silicon chip arrays loaded with matrix (SpectroCHIP, Sequenom). For genotyped frequencies, each of the 96 individual DNAs was genotyped for each of the 48 assays using the MassARRAY system (Sequenom). Best-fit line and coefficient of determination (R 2 ) were calculated using Excel 2000 (Microsoft). 07_JW621_Jurinke_147-163 1/19/04, 9:49 AM 156 MOLECULAR BIOTECHNOLOGY Volume 26, 2004 MALDI-TOF Mass Spectrometry 157 tion of a genetic target, which accounts for the phenotype under investigation. Association stud- ies using MALDI-TOF MS for the measurement of SNP allele frequencies have identified specific genes, again placing the platform at the forefront of this field (76,77). Because of the rapid mea- surement time for interrogation of each primer extension reaction and the semiquantitative nature of its signal, MALDI-TOF MS has been converted Fig. 6. Correction of pool frequency calculation with individual heterozygote peak ratios: MALDI-TOF MS spectra from a genotyped individual heterozygote and a pooled population sample (allelotype) are shown. Allele ratios are depicted next to the corresponding peaks. The allele ratio of the individual heterozygote reaction can be used as a correction factor for the allele frequencies determined in the pool reaction. The individual heterozy- gote should have a 0.50:0.50 (1:1) allele ratio. Any deviation from this expected ratio represents a skewing factor in that reaction. This inaccuracy can be corrected in the pool reaction. The correction formula is presented below the spectra and an example of this calculation using the values from the spectra. As can be seen from the example, the population allele frequencies calculated from the pooled DNA template reaction do not match the allele frequencies determined by genotyping all of the individuals in the population. However, after correction with the heterozygote allele ratios, the allele frequencies from the pool reaction match the genotyped population frequency exactly. into a high-throughput platform (78). This allows for genomewide scans of thousands of SNPs per day using pooled DNA samples. This approach is currently being used by Sequenom (San Diego, CA) and has identified hundreds of genes puta- tively associated with a multitude of complex ge- netic disorders (Braun, unpublished data). Several other interesting applications exist that use the semiquantitative ability of MALDI-TOF 07_JW621_Jurinke_147-163 1/19/04, 9:49 AM 157 MOLECULAR BIOTECHNOLOGY Volume 26, 2004 158 Jurinke, Oeth, and van den Boon Fig. 7. Scatterplot of genotyped population allele frequencies (x-axis) vs. allele frequencies calculated using pooled population DNAs (y-axis) as described in Fig. 5. The pooled allele frequencies have now been corrected for each of the 48 assays as described in Fig. 6. Note the improvement in the coefficient of determination (R 2 ) after correction with individual heterozygote allele ratios relative to Fig. 5. Table 1 Potential Applications for Use With Semiquantitative Analysis of Nucleic Acids on the MALDI-TOF MS Biochemistry Assay Application PCR and primer extension Allele-specific quantitation Disease association studies, Allele-specific expression Allele ratio determination Agricultural genetics in polyploidy genomes Gene copy number Genetic diagnostics, transgenic animals Loss of heterozygosity Cancer diagnostics Loss of imprinting Cancer diagnostics Viral typing Vaccine QC Competitive PCR Gene-expression analysis Quantitative gene-expression and primer extension analysis Quantitative PCR Multiple applications Gene transfer estimations Gene therapy Gene duplication/multiplication Genetic diagnostics Viral/bacterial titering Pathogen quantitation and vaccine QC Abbr: MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; PCR, polymerase chain reaction; QC, quality control. MS analysis of nucleic acids. For example, vac- cine quality control (QC) of RNA or DNA viruses can be conducted using this methodology (79). In this study, ratios of viral quasi species of the mumps virus were determined between Jeryl Lynn substrains in live, attenuated mumps/measles vac- cine. The ratio of these two substrains was deter- mined at five distinct nucleotide positions within 07_JW621_Jurinke_147-163 1/19/04, 9:49 AM 158 MOLECULAR BIOTECHNOLOGY Volume 26, 2004 MALDI-TOF Mass Spectrometry 159 the viral genome and verified with the existing QC methodology used by the Federal Drug Adminis- tration (FDA). Such determinations are of great importance for maintaining vaccine safety and ef- ficacy. Table 1 lists potential applications for semiquantitative analysis of nucleic acids using MALDI-TOF MS. 3.2. Gene-Expression Analysis Ding and Cantor recently described the use of an internal standard added to a complementary deoxyribonucleic acid (cDNA) sample for the analysis of gene expression (67). In this approach a synthetic oligonucleotide is designed that matches the sequence of the targeted cDNA re- gion in all positions but one single base. This in- ternal standard is added in a known concentration to a cDNA sample prior to PCR. The cDNA/com- petitor mixture is PCR amplified and subjected to the primer extension process described earlier. The two sequence species are coamplified with the same efficiency; therefore the ratio between inter- nal standard and cDNA is maintained. The two distinct sequence species mimic the situation of two different alleles. Because of this, primer ex- tension reactions can be applied that are the same as those for SNP allele frequency analysis as de- scribed previously. Figure 8 shows an experiment using a 90-bp sequence from the cholesteryl ester transfer protein (CETP) gene as proof of principle Fig. 8. Scatterplot of calculated allele ratios for the CETP gene. Data points represent the average of triplicate reactions each spotted onto four replicated chip elements for MALDI-TOF MS interrogation. Expected ratios are depicted as a solid line and observed values as triangles plus/minus the standard deviation. In this experiment two artificial templates (90 bp) were designed in the antisense orientation based on the sequence of the CETP gene mRNA (Accession no. AC023825). One of the templates matched this region of the CETP gene exactly. The second had a 1 bp mismatch introduced so as to mimic a mutation and serve as a second allele in a primer extension reaction. Each template and allele is coamplified at equal rates as shown in the graph. Deviation from an exact fit to expected allele frequencies represent a skew as discussed in Fig. 5 and can be corrected in the same manner using a heterozygote (in this case artificial). The concentration of each template added to the reaction is known and therefore the amount of wild-type mRNA (or cDNA) can be determined when the two alleles are at a 1:1 ratio (0.5:0.5 allele frequency). 07_JW621_Jurinke_147-163 1/19/04, 9:49 AM 159 MOLECULAR BIOTECHNOLOGY Volume 26, 2004 160 Jurinke, Oeth, and van den Boon for this approach. The 90-bp region was synthe- sized in two forms: One matched the sequence of CETP exactly for this region; the other differed by a single base pair (C to G mutation) in the middle of the oligomer. These two molecules were used as templates for competitive PCR and subse- quent primer extension reactions for measurement on the MALDI-TOF MS. The amount of each template added to each reaction was known, and therefore the expected frequency of each allele (C or G) was also known. As can be seen from the plot, the observed allele ratios track the expected allele ratios very closely. Similar results have been observed using reverse-transcribed cDNA from mRNA in conjunction with an internal standard molecule for the measurement of gene-expression levels in a variety of genes over many different human cell types (Oeth and Jurinke, unpublished data). Semiquantitative protein analysis can also be conducted with MALDI-TOF MS using the same principles described above. For a review, see Bucknall, Fung, and Duncan (80). Protein applica- tions require different matrix and sample prepara- tions than nucleic acids and are therefore beyond the scope of this current review. 4. Conclusion DNA analysis based on MALDI-TOF MS has matured during the last years into a versatile, high-performance method for qualitative and quantitative DNA analysis. Today, a broad range of applications ranging from mutation or SNP analysis to SNP discovery and quantitative gene- expression analysis is accessible. 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