MALDI-ToF Mass Spectrometry

MOLECULAR BIOTECHNOLOGY Volume 26, 2004

MALDI-TOF Mass Spectrometry 147REVIEW

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, SanDiego, CA 92121. E-mail: [email protected]

Abstract

MALDI-TOF Mass Spectrometry

A Versatile Tool for High-Performance DNA Analysis

Christian Jurinke,* Paul Oeth, and Dirk van den Boom

1. IntroductionIn the past decade matrix-assisted laser desorp-

tion/ionization (MALDI) time-of-flight (TOF)mass spectrometry (MS) has become one of themost powerful tools for the analysis of bio-molecules. The scope of this review is to providean overview on the developments and most recentaccomplishments 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 transfermolecules into the gas phase. The classical meth-ods for ionization are electron impact (EI) andchemical ionization (CI). Most biomolecules,however, undergo significant decomposition andfragmentation under the conditions of both meth-ods. Consequently, the application of MS tonucleic acid analysis had been limited to mol-ecules the size of dinucleotides (1). Analysis ofoligonucleotides with a mass range of up to 3000Dalton (about 10 nucleotides) became feasiblewith the development of plasma desorption (PD)

methods (2). Until the invention of soft ioniza-tion techniques such as electrospray ionizationmass 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 organiccompounds (matrix) and deposited on a conduc-tive sample support. The cocrystals are irradiatedwith a nanosecond laser beam, for example, an ul-traviolet (UV) laser with a wavelength of 266 or337 nm. The energies introduced are in the rangeof 1 1075 107 W/cm2. The laser energy causesstructural decomposition of the irradiated crystaland generates a particle cloud (the plume) fromwhich ions are extracted by an electric field. Themechanism behind the process of desorption is notfully understood. It may best be described as a con-version of laser energy to vibrational oscillationof the crystal molecules. This results in the disin-tegration of the crystal. Following accelerationthrough the electric field, the ions drift through afield-free path and finally reach the detector (e.g., a

Matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) hasdeveloped during the past decade into a versatile tool for biopolymer analysis. The aim of this review is tosummarize this development and outline the applications, which have been enabled for routine use in thefield of nucleic acid analysis. These include the analysis of mutations, the resequencing of amplicons with aknown 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-expressionanalysis; resequencing; SNP discovery; DNA pooling.

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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 islonger 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 caneasily be determined from the resulting spectrumwithout the need for complex data processing. Themasses are accessible as numerical data for directprocessing and subsequent analysis. TOFs mea-sured during a typical MALDI experiment are inthe range of several microseconds.1.2. Sample Preparationfor 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 preparationand the choice of matrix compounds (4). Adductformation and fragmentation are predominantlyinfluenced by sample purification and matrixcomposition. Both are described in detail in thefollowing 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 samplepreparation is the so-called dried-droplet method(5). It relies simply on pipetting a small volume ofthe sample (usually about 0.5 L) to a drop (about1 L) of matrix solution and allowing the mixtureto dry. This procedure has been modified for high-throughput applications that involve automatedMALDI measurement (6).The vast amount of ap-plications of MALDI-TOF-based methods for theanalysis of peptides, proteins, glycosides, ornoncovalent interactions is beyond the scope ofthis review. For an overview the reader is referredto some excellent reviews in this field (7,8).1.3. MALDI-TOF DNA Analysis

The application of MALDI-MS to the analysisof nucleic acids had for years remained a field ofsmall impact to the bioscience community. Nucleicacids are far more difficult to analyze under

MALDI conditions than peptides, so applicationshad been limited. Because of their negativelycharged phosphate backbone, nucleic acids are es-pecially susceptible to adduct formation. Theytend to form salt adducts with cations present inthe surrounding medium. In biochemical reac-tions, these are predominantly sodium and potas-sium ions. Adduct formation results in a broaderdistribution of the signal: A main signal with theprotonated analyte may be accompanied by sig-nals resulting from multiple adduct formation. Forexample, every sodium ion attached to the analytemolecule will cause an additional signal with amass of plus 23 Dalton. Consequently, adduct for-mation lowers sensitivity and analytical accu-racythe total amount of ions is distributed overa multitude of ion species, and the mass differ-ences between those may become too small to beresolved. Approaches to overcome adduct forma-tion are ion-exchange procedures based either onsolid-phase (9) or cation-exchange resin methods(5). Chemical modification of the phosphate back-bone has also been proposed to prevent adductformation (10). The addition of ammonium-con-taining additives like diammonium citrate (11) ortartrate (12) to the matrix reduces cation heteroge-neity of analytes. The use of millimolar solutionsof ammonium hydroxide during the conditioningprocess has also a beneficial effect (13). The ratio-nale for exchanging cations for ammonium is thatthe latter is a volatile cation that is released asammonia 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 theMALDI process. The predominant effect of thesereactions is depurination.

Protonation of the nucleobases A or G (at posi-tion N7) induces polarization of the N-glycosidicbond between sugar and nucleobase and finallyresults in nucleobase elimination. Subsequent todepurination, further fragmentation occurs viabackbone cleavage. In addition to depurination,the elimination of C has also been reported (14).This reaction is mediated through protonation atN3. Hillenkamp and coworkers have demon-strated that ribonucleic acid (RNA) is less suscep-

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MALDI-TOF Mass Spectrometry 149

tible to fragmentation under MALDI conditionsthan deoxyribonucleic acid (DNA). The proposedreason is the lack of the 2' hydroxy group in theribose sugar moiety (15). Approaches to compen-sate for the detrimental influences of fragmenta-tion have been contributed from instrumentdevelopers as well as biochemists. It has beendemonstrated by several groups that introductionof 7-deaza nucleobases, where N7 is replaced byC, is helpful in suppressing depurination reactions(16,17) because the proton acceptor site is re-moved. An important contribution to analyzingDNA with MALDI-TOF was also the introduc-tion of delayed extraction instruments by Wileyand McLaren (18). Compared to static extraction,which employs a permanent electrical field for ionacceleration, the delayed extraction accumulatesions over a time range of some nanoseconds be-fore the extraction voltage is applied. The effect isa compensation of the initial differences in kineticenergies of the analyte molecules because mol-ecules with higher initial velocities expand furtherduring the delay time and experience a lower po-tential during acceleration. To some extent thismay also compensate for positional variations ofmolecules that occur owing to an inhomogeneousdistribution in the matrix cocrystals. Another mile-stone was the introduction of favorable matrices forDNA analysis. Especially, 3-hydroxypicolinicacid (3-HPA) and some of its derivatives (19) pre-dominate in the field of DNA analysis. Mixturesof 2,3,4'- and 2,4,6'-trihydroxy-acetophenone(THAP) are mainly used for RNA analysis (20).A promising development is the application ofinfrared (IR)-MALDI for the analysis of DNAmolecules of up to 2.1 kb by Hillenkamp and co-workers (21).

2. Qualitative DNA AnalysisThe developments described in the previous

section enabled investigations in various areas ofmass spectrometric DNA analysis. The analysisof amplification products generated during poly-merase chain reaction (PCR) and sequencing reac-tions has been an early focus. For those applicationsthe most critical issue was sample preparation.The broad terminus sample preparation is gen-

erally used to describe all steps necessary to getthe DNA products in a suitable form for massspectrometric analysis, as described in the previ-ous section. It is beneficial to separate the prod-ucts from the educts because all available DNAmolecules will be subjected to the ionization pro-cess. To prevent competition for available charges,an excess of primer, for example, out of a PCRreaction should be avoided. Finally, the productshave to be presented to the matrix in a concentra-tion and volume ratio that results in a favorablematrix-to-analyte ratio. The amount of sampledelivered 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-TOFmass spectrometric PCR product analysis cover abroad range. The earliest reports from researchersanalyzing PCR products (26) still used purifica-tion methods that were not adaptable for highthroughput. Applications that have been enabledfor DNA analysis via MALDI-TOF include oli-gonucleotide sequence analysis (11,27) and PCRor 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, thequalitative analysis of in vitro transcripts has beenreported (35). The use of streptavidin-coatedbeads supported a variety of different applica-tions.

The solid-phase approach provides ease ofsample conditioning and concentration. The re-covery of the DNA from the solid support is also avery efficient process. Double-stranded DNA canbe fractionated in this way, because either thesense 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 onthe study of single nucleotide polymorphisms(SNPs). SNPs can be defined as biallelic variantswithin a population occurring with an allelic fre-

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quency higher than 1% (with 1% as a more or lessarbitrary 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 todayby many academic and commercial organizations.Consequently, this is leading to SNP discoveryefforts within the human genome. Commonlyused methods for SNP analysis on mass spectro-metric platforms are based on primer extensionprotocols (36). The format used by our group is ahomogenous assay, an advance over the use ofstreptavidin-coated beads because no removal ofsupernatants is involved. The reaction protocolconsists of several component additions (37).The PCR is subjected to shrimp alkaline phos-phatase (SAP) treatment to inactivate remainingnucleotides. Following inactivation by SAP, a re-action cocktail is added consisting of an extensionprimer annealing adjacent to the mutated site,thermosequenase, and a deoxyribonucleotidedNTP/dideoxynucleotide (ddNTP) mixture. ThePCR product now serves as templates for a primerextension reaction. Depending on the chosennucleotide mixture, two products with distinctmasses are generated (see Fig. 1). These productsare purified through the addition of ion-exchangeresin and subsequently dispensed on a chip arraythat is preloaded with the components necessaryfor MALDI sample preparation. This format canbe used for the analysis of deletion, insertion, andpoint mutations, short tandem repeat (STR), andSNP analysis, and it allows for the detection ofcompound heterozygotes.

For SNP analysis the primer-binding site isplaced adjacent to the polymorphic position. Aspecific extension product is generated for eachallele. In the case of heterozygosity, both prod-ucts are generated simultaneously. In the examplegiven in Fig. 1B, both elongation products are ex-pected to differ in mass by one nucleotide. Thetwo SNP alleles appear as two distinct mass sig-nals. Careful assay design makes a high-levelmultiplexing 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 thisapproach.

Gut and coworkers have devised a scheme forgenotyping of SNPs that is similar to the afore-mentioned but in addition relies on the chemicalmodification of the short DNA fragments that aregenerated through a 5'-phosphodiesterase digest(39). The same approach has been used for haplo-typing of SNPs. For this purpose an allele-specificPCR protocol is employed (40). This so-calledGOOD assay, though scientifically elegant, has inpractical applications been hampered becausechemicals with a certain carcinogenic potential(like methyliodide) are involved in the alkylationreaction. Recently, an approach based on the useof 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 probesfor each analyzed SNP. Two probes are allele spe-cific but have a noncomplementary region 5' ofthe SNP, and a third oligonucleotide (the invaderoligo) invades at least one nucleotide into the du-plex formed by the allele-specific oligo. The nowunpaired region of the allele-specific oligo iscleaved with a flap endonuclease (FEN). Thecleaved short oligonucleotide can now be purifiedthrough a biotinstreptavidin system. This reac-tion has the potential to be performed directlyfrom genomic DNA. However, because the amountof 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 thefirst step serves as an invader oligo for a pair ofother synthetic oligonucleotides that mimic thetargeted allele. The noncleaved primary probeneeds to be captured for this purpose by anotheroligo (called the arrestor). The secondary cleav-age product is finally purified via a streptavidinbiotin-based solid support. This approach hasbeen employed for genotyping and even quantita-tive analysis (42,43). However, because thismethod involves the synthesis of multiple oligo-nucleotides per SNP, the setup costs for this ap-proach are rather high.

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Fig. 1. Schematic representation of primer extension assay as used in multiple applications in combination withMALDI-TOF. A primer adjacent to the mutation site is extended in an allele specific manner. The extensionproducts differ in mass and are analyzed with MALDI-TOF MS. The spectra provided in A,B, and C represent rawdata generated from a heterozygous sample (B) and the two homozygotes (A,C).

2.2. Microsatellite AnalysisMicrosatellites are another widely used type of

genetic marker. SNPs are increasingly used in tar-get gene discovery programs, owing to their highabundance (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 ofresearch. Microsatellite DNA typically comprisesbetween 4 and 25 tandemwise repeated nucleotideunits from 1 to more than 7 bases. More often,these markers are referred to as STRs. Varioustypes 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 theirhighly 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, positionalcloning efforts, as well as indirect analysis ofmonogenetic disorders have successfully employedSTR 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 isconventionally performed by PCR amplification

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sample is compared to a so-called allelic ladderthat serves as a standard.

For MALDI-TOF analysis of microsatellites, aprimer extension-based approach can be applied.For this approach a ddNTP composition is cho-sen, which terminates the polymerase extensionat 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. Evenimperfect repeats harboring insertion or deletionmutations can be analyzed with this approach.Figure 3 displays raw data from the analysis of ahuman STR marker in a heterozygous DNAsample. Both alleles differ by 4 CA repeats. TheDNA polymerase slippage during amplificationgenerates a pattern of stutter fragments (markedwith an asterisk in Fig. 3). In the case of heterozy-gotes, which differ in just one repeat, the smallerallele has higher intensities than the larger allele,because allelic and stutter signals are added to-gether. Recently, the use of ribozymes to shortenthe 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 aprimer extension assay for microsatellite analysis. Theasterisk marks signals that are generated through tem-plate slippage of the DNA polymerase, so-called stut-ter signals.

using fluorescent-tagged primers. Products areseparated by gel or capillary electrophoresis(49,50). For the purpose of determining the re-peat length, the gel electrophoretic mobility of the

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(52). However, none of the devised MALDI-basedmethods compete with the existing technologies.Despite all promising developments, the field ofSTR analysis by MS still remains a formidablechallenge (53).2.3. DNA Sequencing

The work focusing on analyzing DNA sequenc-ing reactions with MALDI-TOF MS has startedwith attempts to translate known sequencingformats to mass spectrometric readouts. Severalbiochemical schemes have been developed, whichgenerate DNA sequencing ladders of sufficientyield and purity to suit the specific requirementsfor the analysis by MALDI-TOF MS (30,5456).Following the concept of conventional dideoxysequencing, the nested set of truncated sequencesoriginating from a primer can be analyzed byMALDI-TOF MS. The mass difference betweenthe DNA fragments can be used to calculate thenucleotide 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 ofthe template, and real termination signals sig-nificantly limit sequence analysis in the highermass range. Sensitivity, mass resolution, and massaccuracy issues contribute to the fact that analysisof dideoxy sequencing ladders by MALDI-TOFMS has not yet been implemented for high-throughput sequencing applications.

Even though the data acquisition time forMALDI spectra is only about 2 s per 20 indi-vidual spectra, the short read length prohibitsMALDI-TOF from competing with conventionalapproaches. Several alternative formats havebeen devised to overcome this read length limi-tation. Rather than using a primer extension-based method, which yields a ladder of DNAfragments with increasing sizes, the following

schemes rely on the generation of short, base-spe-cific fragments. Base-specific cleavage of nucleicacids represents a paradigm shift in DNA sequenc-ing by MS. The principle resembles more closelythe original approach of Maxam and Gilbert forDNA sequencing (57). These methods, however,are not suitable for de novo sequencing. They ratherrepresent identification or resequencing methods,where an experimentally determined sequence iscross-compared to a known reference sequence.One example involves an enzymatic DNA-basedfragmentation approach. In this approach, PCRproducts are generated containing 2'-deoxyuridine5'-triphosphate (dUTP) instead of dTTP. Afterstrand 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 thetwo amplicon strands, their separation is neces-sary. The strand separation is currently performedusing streptavidin-coated magnetic beads, as de-scribed in previous sections. An approach based onchemical cleavage uses P3'-N5'-phosphoramidite-containing DNA (60). Either 2'-deoxycytidine 5'-triphosphate (dCTP) or dTTP is replaced by theiranalog P-N-modified nucleoside triphosphates.They are introduced into the target sequence dur-ing a post-PCR primer extension reaction. Acidicreaction conditions produce base-specific cleav-age fragments, which are analyzed by MALDI-MS. However, the required acidic conditionsgenerate unwanted depurination byproducts. Abase 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 bothof these methods are robust and reasonably easyto handle, each approach is limited by the rela-tively low yield of single-stranded DNA products,which prevents minimizing the reaction volumeswithout a significant loss of sensitivity.

Another scheme uses base-specific ribonu-cleases (RNases) for template digestion, followedby an analysis of the resulting cleavage productsby MS (61). The basic principle is to generate invitro RNA transcripts from a PCR product. Thetranscripts are derived from the PCR product bytagging the PCR primers with an RNA poly-

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merase promoter. Following the in vitro transcrip-tion, using T7 RNA polymerase, the transcriptsare incubated with RNases (62,63). Figure 4 de-picts a schematic example and raw data obtainedwith this approach.

3. Quantitative AnalysisMany important applications in biological re-

search (e.g., gene-expression analysis) requirequantitative analysis of nucleic acids. The use ofMALDI-TOF MS for quantitative analysis is aformidable scientific challenge. Owing to inher-ent process variables, absolute quantitation iscurrently not an option. Relative quantitation,however, is possible and gives rise to several in-teresting applications. For relative quantitation theratio between two signals is measured and com-pared. Two approaches are possible: 1) The abso-lute concentration of the analyte molecules isunknown, and only their ratio is compared; 2) theconcentration of one analyte (referred to as inter-nal standard) is known and used to calculate theconcentration of the unknown analyte.

The first approach is particularly useful for theanalysis of allele distributions (frequencies) innucleic 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 conjunctionwith primer extension reactions to measure ratiosof known DNA mixtures (68). Relative quantitationof allele frequencies is achieved by calculating theareas of the peaks associated with specific primerextension reactions. Peak characteristics (such aspeak height) are generally stored when acquiredon the MALDI-TOF MS, and area calculations areconducted by the users mathematical method ofchoice. Because SNPs are generally biallelic, theallele frequency is calculated as the ratio of thearea of each allele to the total summed area of bothalleles. The sum of the two alleles is always 1 inthis model. Allele frequencies down to 5% can beaccurately discerned using MALDI-TOF MS inDNA pools (64,69). Frequencies below 5% are

routinely detected, but their accuracy must be ap-proached with caution owing to the small peakarea associated with a minor allele at a 50:1 ratiorelative to the major allele. Several studies havecompared the quantitative abilities of differentplatforms used for analyzing SNPs with the goalof allele frequency estimation in DNA pools (6971). MALDI-TOF MS measurement of primerextension reactions has been found to be as sensi-tive and reproducible as all available technologiesbased on these studies. Figure 5 shows some ex-ample data for the estimation of allele frequencyin DNA population pools as compared to the ob-served frequency determined by genotyping all ofthe individuals in the population. Figure 5 is ascatterplot with allele frequencies determined for48 assays in a DNA pool of 96 individuals vs. theobserved frequency based on the genotype for all96 individuals for each of the 48 assays. As can beseen from the coefficient of determination (R2)there is not a perfect 1:1 correlation between theallele frequencies calculated from a pool of indi-viduals and the actual frequency determined bygenotyping. Several factors can contribute to thisinaccuracy no matter what the technology used(for review, see 71,72). However, a correctionfactor can be applied for each individual assaybased on the peak areas observed for individualheterozygous samples from the population underinvestigation. Individual heterozygotes have twoalleles at a 1:1 ratio for any given biallelic SNP.Based on this, the peak areas observed for eachallele on the MALDI-TOF MS should be equal. Ifthey are not, then a skewing of one allele overthe other has occurred at some point in the pro-cess (whether it be at the level of PCR or analyteionization during MALDI-TOF or at some other

Fig. 4. (opposite page) The upper part of the figureprovides an overview on the fragments generated dur-ing the analysis of a particular G/A SNP. The lowerpart of the figure provides data for the T-forward andC-forward reactions, respectively. These products aremost difficult to discriminate because they have thesame length but differ only by 16 Daltons owing totheir different sequence composition. The arrows pointat the discriminative signals as generated in the twoseparate reactions.

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Fig. 4.

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point). This bias can be observed and quantifiedon the MALDI-TOF MS as illustrated in Fig. 6.The deviation from a 1:1 ratio of each allele in acollection of heterozygotes can be used as a cor-rection factor for the allele frequency calculatedin the pooled DNA samples. Figure 7 shows ascatterplot similar to Fig. 5. The allele frequen-cies determined in the 96-individual pool havenow been corrected using the formula given inFig. 6. Note the improvement in the coefficient ofdetermination (R2) between Figs. 5 and 7 after in-cluding the correction. This type of accuracy issufficient for many semiquantitative applications,such as estimation of SNP allele frequency innucleic acid pools or the differential proteinDNAbinding 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 theparticular alleles in populations and to comparethese between different populations. This infor-mation can be used to identify causative geneticloci 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 widespreaddistribution throughout genomes and general easeof measurement (75). Genotyping thousands ofSNPs over hundreds to thousands of individuals,however, still remains cost ineffective. PoolingDNA populations has therefore been proposed asan alternative to individually genotyping popula-tions for association studies (71). MALDI-TOFMS has been at the forefront of this approach.Buetow et al. conducted the first genomewideanalysis of gene-based SNPs using MALDI-TOFMS 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 theaccuracy and feasibility of such an approach us-ing MALDI-TOF MS measurement of peak areasin conjunction with primer extension reactions inpooled DNA populations (65,66). The ultimategoal of these association studies is the identifica-

Fig. 5. Scatterplot of genotyped population allele frequencies (x-axis) vs. allele frequencies calculated usingpooled population DNAs (y-axis). Forty-eight unique assays are depicted. DNA population pool consisted of 96individual DNAs at equimolar concentrations (260 pg per individual DNA/L = 25 ng/L). Frequencies werecalculated using TYPER RT software (Sequenom). The calculated allele frequency for each assay represents theaverage 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 ofthe 48 assays using the MassARRAY system (Sequenom). Best-fit line and coefficient of determination (R2) werecalculated using Excel 2000 (Microsoft).

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tion of a genetic target, which accounts for thephenotype under investigation. Association stud-ies using MALDI-TOF MS for the measurementof SNP allele frequencies have identified specificgenes, again placing the platform at the forefrontof this field (76,77). Because of the rapid mea-surement time for interrogation of each primerextension reaction and the semiquantitative natureof its signal, MALDI-TOF MS has been converted

Fig. 6. Correction of pool frequency calculation with individual heterozygote peak ratios: MALDI-TOF MSspectra from a genotyped individual heterozygote and a pooled population sample (allelotype) are shown. Alleleratios are depicted next to the corresponding peaks. The allele ratio of the individual heterozygote reaction can beused 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 skewingfactor in that reaction. This inaccuracy can be corrected in the pool reaction. The correction formula is presentedbelow the spectra and an example of this calculation using the values from the spectra. As can be seen from theexample, the population allele frequencies calculated from the pooled DNA template reaction do not match theallele frequencies determined by genotyping all of the individuals in the population. However, after correctionwith the heterozygote allele ratios, the allele frequencies from the pool reaction match the genotyped populationfrequency exactly.

into a high-throughput platform (78). This allowsfor genomewide scans of thousands of SNPs perday using pooled DNA samples. This approach iscurrently 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 thatuse the semiquantitative ability of MALDI-TOF

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Fig. 7. Scatterplot of genotyped population allele frequencies (x-axis) vs. allele frequencies calculated usingpooled population DNAs (y-axis) as described in Fig. 5. The pooled allele frequencies have now been correctedfor each of the 48 assays as described in Fig. 6. Note the improvement in the coefficient of determination (R2) aftercorrection with individual heterozygote allele ratios relative to Fig. 5.

Table 1Potential 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 expressionAllele ratio determination Agricultural genetics

in polyploidy genomesGene copy number Genetic diagnostics,

transgenic animalsLoss of heterozygosity Cancer diagnosticsLoss of imprinting Cancer diagnosticsViral typing Vaccine QC

Competitive PCR Gene-expression analysis Quantitative gene-expressionand primer extension analysis

Quantitative PCR Multiple applicationsGene transfer estimations Gene therapyGene duplication/multiplication Genetic diagnosticsViral/bacterial titering Pathogen quantitation and

vaccine QCAbbr: 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 virusescan be conducted using this methodology (79). Inthis study, ratios of viral quasi species of the

mumps virus were determined between Jeryl Lynnsubstrains in live, attenuated mumps/measles vac-cine. The ratio of these two substrains was deter-mined at five distinct nucleotide positions within

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the viral genome and verified with the existing QCmethodology used by the Federal Drug Adminis-tration (FDA). Such determinations are of greatimportance for maintaining vaccine safety and ef-ficacy. Table 1 lists potential applications forsemiquantitative analysis of nucleic acids usingMALDI-TOF MS.

3.2. Gene-Expression AnalysisDing and Cantor recently described the use of

an internal standard added to a complementarydeoxyribonucleic acid (cDNA) sample for theanalysis of gene expression (67). In this approacha synthetic oligonucleotide is designed thatmatches the sequence of the targeted cDNA re-

gion in all positions but one single base. This in-ternal standard is added in a known concentrationto a cDNA sample prior to PCR. The cDNA/com-petitor mixture is PCR amplified and subjected tothe primer extension process described earlier.The two sequence species are coamplified with thesame efficiency; therefore the ratio between inter-nal standard and cDNA is maintained. The twodistinct sequence species mimic the situation oftwo different alleles. Because of this, primer ex-tension reactions can be applied that are the sameas those for SNP allele frequency analysis as de-scribed previously. Figure 8 shows an experimentusing a 90-bp sequence from the cholesteryl estertransfer protein (CETP) gene as proof of principle

Fig. 8. Scatterplot of calculated allele ratios for the CETP gene. Data points represent the average of triplicatereactions each spotted onto four replicated chip elements for MALDI-TOF MS interrogation. Expected ratios aredepicted as a solid line and observed values as triangles plus/minus the standard deviation. In this experiment twoartificial templates (90 bp) were designed in the antisense orientation based on the sequence of the CETP genemRNA (Accession no. AC023825). One of the templates matched this region of the CETP gene exactly. Thesecond had a 1 bp mismatch introduced so as to mimic a mutation and serve as a second allele in a primerextension reaction. Each template and allele is coamplified at equal rates as shown in the graph. Deviation from anexact fit to expected allele frequencies represent a skew as discussed in Fig. 5 and can be corrected in the samemanner using a heterozygote (in this case artificial). The concentration of each template added to the reaction isknown and therefore the amount of wild-type mRNA (or cDNA) can be determined when the two alleles are at a1:1 ratio (0.5:0.5 allele frequency).

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for this approach. The 90-bp region was synthe-sized in two forms: One matched the sequence ofCETP exactly for this region; the other differedby a single base pair (C to G mutation) in themiddle of the oligomer. These two molecules wereused as templates for competitive PCR and subse-quent primer extension reactions for measurementon the MALDI-TOF MS. The amount of eachtemplate added to each reaction was known, andtherefore the expected frequency of each allele (Cor G) was also known. As can be seen from theplot, the observed allele ratios track the expectedallele ratios very closely. Similar results have beenobserved using reverse-transcribed cDNA frommRNA in conjunction with an internal standardmolecule for the measurement of gene-expressionlevels in a variety of genes over many differenthuman cell types (Oeth and Jurinke, unpublisheddata).

Semiquantitative protein analysis can also beconducted with MALDI-TOF MS using the sameprinciples described above. For a review, seeBucknall, Fung, and Duncan (80). Protein applica-tions require different matrix and sample prepara-tions than nucleic acids and are therefore beyondthe scope of this current review.

4. ConclusionDNA analysis based on MALDI-TOF MS has

matured during the last years into a versatile,high-performance method for qualitative andquantitative DNA analysis. Today, a broad rangeof applications ranging from mutation or SNPanalysis to SNP discovery and quantitative gene-expression analysis is accessible. The uniquecombination of flexibility, accuracy, automatedanalysis, and high-throughput data generation isof benefit in many fields of biological research.

References1. Takeda, N., Pomerantz, S. C., and McCloskey, J. A.

(1991) Detection of ribose-methylated nucleotides inenzymatic hydrolysates of RNA by thermospray liquidchromatography-mass spectrometry. J. Chromatogr.562, 225235.

2. Viari, A., Ballini, J. P., Meleard, P., et al. (1988) Char-acterization and sequencing of normal and modifiedoligonucleotides by 252Cf plasma desorption mass

spectrometry. Biomed. Environ. Mass Spectrom. 16,225228.

3. Karas, M. and Hillenkamp, F. (1988) Laser desorp-tion ionization of proteins with molecular masses ex-ceeding 10,000 Daltons. Anal. Chem. 60, 22992301.

4. Brnsen, K. O., Gass, M. A. S., Bruin, G. J. M., et al.(1997) Influence of solvents and detergents on matrix-assisted laser desorption/ionization mass spectrometrymeasurements of proteins and oligonucleotides. RapidCommun. Mass Spectrom. 11, 603309.

5. Nordhoff, E., Ingendoh, A., Cramer, R., et al. (1992)Matrix-assisted laser desorption/ionization mass spec-trometry of nucleic acids with wavelengths in the ul-traviolet and infrared. Rapid Commun. MassSpectrom. 6, 771776.

6. Little, D. P., Braun, A., ODonnell, M. J., and Koster,H. (1997) Mass spectrometry from miniaturized arraysfor full comparative DNA analysis. Nat. Med. 3, 14131416.

7. Chaurand, P., Luetzenkirchen, F., and Spengler, B.(1999) Peptide and protein identification by matrix-assisted laser desorption ionization (MALDI) andMALDI-post-source decay time-of-flight mass spec-trometry. J. Am. Soc. Mass Spectrom. 10, 91103.

8. Griffin, T. J., Goodlett, D. R., and Aebersold, R.(2001) Advances in proteome analysis by mass spec-trometry. Curr. Opin. Biotechnol. 12, 607612.

9. Tang, K., Fu, D. J., Kotter, S., Cotter, R. J., Cantor, C.R., and Koster, H. (1995) Matrix-assisted laser des-orption/ionization mass spectrometry of immobilizedduplex DNA probes. Nucleic Acids Res. 23, 31263131.

10. Gut, I. G. and Beck, S. (1995) A procedure for selec-tive DNA alkylation and detection by mass spectrom-etry. Nucleic Acids Res. 23, 13671373.

11. Pieles, U., Zrcher, W., Schr, M., and Moser, H. E.(1993) Matrix-assisted laser desorption ionizationtime-of-flight mass spectrometry: a powerful tool forthe mass and sequence analysis of natural and modi-fied oligonucleotides. Nucleic Acids Res. 21, 31913196.

12. Lecchi, P. and Pannell, L. K. (1995) 6-Aza-2-thio-thymine: a matrix for MALDI spectra of oligonucle-otides. J. Am. Soc. Mass Spectrom. 6, 12761277.

13. Jurinke, C., van den Boom, D., Collazo, V., Lchow,A., Jacob, A., and Koster, H. (1997) Recovery ofnucleic acids from immobilized biotin-streptavidincomplexes using ammonium hydroxide and applica-tions in MALDI-TOF mass spectrometry. Anal. Chem.69, 904910.

14. Gross, J., Leisner, A., Hillenkamp, F., et al. (1998)Investigations of the metastable decay of DNA underultraviolet matrix-assisted laser desorption/ionizationconditions with post-source-decay analysis and hydro-gen/deuterium exchange. J. Am. Soc. Mass Spectrom.9, 866878.

07_JW621_Jurinke_147-163 1/19/04, 9:49 AM160



15. Nordhoff, E., Cramer, R., Karas, M., et al. (1993) Ionstability of nucleic acids in infrared matrix-assisted la-ser desorption/ionization mass spectrometry. NucleicAcids Res. 21, 33473357.

16. Kirpekar, F., Nordhoff, E., Kristiansen, K., Roepstorff,P., Hahner, S., and Hillenkamp, F. (1995) 7-Deaza pu-rine bases offer a higher ion stability in the analysis ofDNA by matrix-assisted laser desorption/ionizationmass spectrometry. Rapid Commun. Mass Spectrom.9, 525531.

17. Siegert, C., Jacob, A., and Kster, H. (1996) Matrix-assisted laser desorption/ionization time-of-flightmass spectrometry for the detection of polymerasechain reaction products containing 7-deazapurinemoieties. Anal. Biochem. 243, 5565

18. Wiley, W. C. and McLaren, I. H. (1955) Time-of-flight mass spectrometer with improved resolution.Rev. Sci. Instrum. 26, 1150.

19. Wu, K. J., Steding, A., and Becker, C. H. (1993) Ma-trix-assisted laser desorption time-of-flight massspectrometry of oligonucleotides using 3-hydroxy-picolinic acid as an ultraviolet-sensitive matrix. RapidCommun. Mass Spectrom. 7, 142146.

20. Zhu, Y. F., Chung, C. N., Taranenko, N. I., et al.(1996) The study of 2,3,4-trihydroxyacetophenoneand 2,4,6-trihydroxyacetophenone as matrices forDNA detection in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. RapidCommun. Mass Spectrom. 10, 383388.

21. Berkenkamp, S., Kirpekar, F., and Hillenkamp, F.(1998) Infrared MALDI mass spectrometry of largenucleic acids. Science 281, 260262.

22. Jannavi, R. Srinivasan, J. R., Liu, Y., et al. (1997)Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry as a rapid screening methodto detect mutations causing Tay-Sachs disease. RapidCommun. Mass Spectrom. 11, 11441150.

23. Koster, H., van den Boom, D., Braun, A., et al. (1997)DNA analysis by mass spectrometry: applications inDNA sequencing and DNA diagnostics. Nucleosides& Nucleotides 16, 563571.

24. Jurinke, C., van den Boom, D., and Koster, H. (1998)Asymmetric PCR improves streptavidin-biotin basedpurification of PCR products prior to MALDI-TOFmass spectrometric analysis. Rapid Comm. MassSpectrom. 12, 5052.

25. Gobom, J., Nordhoff, E., Mirgorodskaya, E., Ekman,R., and Roepstorff, P. (1999) Sample purification andpreparation technique based on nano-scale reversed-phase columns for the sensitive analysis of complexpeptide mixtures by matrix-assisted laser desorption/ionization mass spectrometry. J. Mass Spectrom. 34,105116.

26. Liu, Y. H., Bai, J., Zhu, Y., et al. (1995) Rapid screen-ing of genetic polymorphisms using buccal cell DNAwith detection by matrix-assisted laser desorption/

ionization mass spectrometry. Rapid Commun. MassSpectrom. 9, 735743.

27. Faulstich, K., Worner, K., Brill, H., and Engels, J. W.(1997) A sequencing method for RNA oligonucle-otides based on mass spectrometry. Anal. Chem. 69,43494353.

28. Tang, K., Taranenko, N. I., Allmann, S. L., Chang,L. Y., and Chen, C. H. (1994) Detection of 500-nucle-otide DNA by laser desorption mass spectrometry.Rapid Commun. Mass Spectrom. 8, 727730.

29. Jurinke, C., van den Boom, D., Jacob, A., Tang, K.,Wrl, R., and Kster, H. (1996) Analysis of ligasechain reaction products via matrix-assisted laser des-orption/ionization time-of-flight-mass spectrometry.Anal. Biochem. 237, 174181.

30. Koster, H., Tang, K., Fu, D. J., et al. (1996) A strat-egy for rapid and efficient DNA sequencing by massspectrometry. Nat. Biotechnol. 14, 11231128.

31. Little, D. P., Braun, A., Darnhofer-Demar, B., et al. (1997)Detection of RET proto-oncogene codon 634 mutationsusing mass spectrometry. J. Mol. Med. 75, 745750.

32. Higgins, G. S., Little, D. P., and Koster, H. (1997)Competitive oligonucleotide single-base extensioncombined with mass spectrometric detection for mu-tation screening. Biotechniques 23, 710714.

33. Jurinke, C., Zllner, B., Feucht, H. H., et al. (1998)Application of nested PCR and mass spectrometry forDNA-based virus detection: HBV-DNA detected inthe majority of isolated anti-HBc positive sera. Genet.Anal. 14, 97102.

34. Braun, A., Little, D. P., and Kster, H. (1997) Detect-ing CFTR gene mutations by using primer oligo baseextension and mass spectrometry. Clin. Chem. 43,11511158.

35. van den Boom, D., Jurinke, C., Higgins, G. S., Becker,T., and Koster, H. (1998) Mass spectrometric DNA di-agnostics. Nucleosides & Nucleotides 17, 21572164.

36. Little, D. P., Braun, A., Darnhofer-Demar, B., andKoster, H. (1997) Identification of apolipoprotein Epolymorphisms using temperature cycled primer oligobase extension and mass spectrometry. Eur. J. Clin.Chem. Clin. Biochem. 35, 545548.

37. Storm, N., Darnhofer-Patel, B., van den Boom, D.,and Rodi, C. P. (2003) MALDI-TOF mass spectrom-etry-based SNP genotyping. Methods Mol. Biol. 212,241262.

38. Ross, P., Hall, L., Smirnov, I., and Haff, L. (1998)High-level multiplex genotyping by MALDI-TOFmass spectrometry. Nat. Biotechnol. 16, 13471351.

39. Sauer, S., Lechner, D., Berlin, K., Lehrach, H., et al.(2000) A novel procedure for efficient genotyping ofsingle nucleotide polymorphisms. Nucleic Acids Res.28, E13

40. Tost, J., Brandt, O., Boussicault, F., et al. (2002) Mo-lecular haplotyping at high throughput. Nucleic AcidsRes. 30, E96

07_JW621_Jurinke_147-163 1/19/04, 9:49 AM161



41. Sauer, S., Gelfand, D. H., Boussicault, F., Bauer, K.,Reichert, F., and Gut, I. G. (2002) Facile method for au-tomated genotyping of single nucleotide polymorphismsby mass spectrometry. Nucleic Acids Res. 30, E22

42. Griffin, T. J. and Smith, L. M. (2000) Genetic identifi-cation by mass spectrometric analysis of single-nucle-otide polymorphisms: ternary encoding of genotypes.Anal. Chem. 72, 32983302.

43. Berggren, W. T., Takova, T., Olson, M. C., Eis, P. S.,Kwiatkowski, R. W., and Smith, L. M. (2002) Multi-plexed gene expression analysis using the invaderRNA assay with MALDI-TOF mass spectrometry de-tection. Anal. Chem. 74, 17451750.

44. Dib, C., Faure, S., Fizames, C., et al. (1996) A com-prehensive genetic map of the human genome basedon 5,264 microsatellites. Nature 380, 152154.

45. Sunden, S. L., Businga, T., Beck, J., et al. (1996)Chromosomal assignment of 2900 tri- and tetra-nucleotide repeat markers using NIGMS somatic cellhybrid panel 2. Genomics 32, 1520.

46. Epplen, J. T., Buitkamp, J., Epplen, C., Maueler, W.,and Riess, O. (1995) Indirect DNA/gene diagnoses viaelectrophoresisan obsolete principle? Electrophore-sis 16, 683690.

47. Wells, R. D. and Warren, S. T. (1998) Genetic Insta-bilities and Neurological Diseases. Academic Press,San Diego, CA.

48. Canzian, F., Salovaara, R., Hemminki, A., et al.(1996) Semiautomated assessment of loss of het-erozygosity and replication error in tumors. CancerRes. 56, 33313337.

49. Ziegle, J. S., Su, Y., Corcoran, K. P., et al. (1992) Appli-cation of automated DNA sizing technology forgenotyping microsatellite loci. Genomics 14, 10261031.

50. Reed, P. W., Davies, J. L., Copeman, J. B., et al.(1994) Chromosome-specific microsatellite sets forfluorescence-based, semi-automated genome map-ping. Nat. Genet. 7, 390395.

51. Braun, A., Little, D. P., Reuter, D., Muller-Mysok, B.,and Koster, H. (1997) Improved analysis of micro-satellites using mass spectrometry. Genomics 46, 1823.

52. Krebs, S., Seichter, D., and Forster, M. (2001)Genotyping of dinucleotide tandem repeats by MALDImass spectrometry of ribozyme-cleaved RNA tran-scripts. Nat. Biotechnol. 19, 877880.

53. van den Boom, D., Jurinke, C., McGinness, M. J., andBerkenkamp, S. (2001) Microsatellites: perspectivesand potentials of mass spectrometric analysis. ExpertRev. Mol. Diagn. 1, 383393.

54. Kirpekar, F., Nordhoff, E., Larsen, L. K., Krisitansen,K., Roepstorff, P., and Hillenkamp, F. (1998) Rapiddetermination of short DNA sequences by the use ofMALDI-MS. Nucleic Acids Res. 26, 25542559.

55. Nordhoff, E., Luebbert, C., Thiele, G., Heiser, V., andLehrach, H. (2000) Rapid determination of short DNAsequences by the use of MALDI-MS. Nucleic AcidsRes. 28, E86

56. Taranenko, N. I., Allman, S. L., Golovlev, V. V.,Taranenko, N. V., Isola, N. R., and Chen, C. H. (1998)Sequencing DNA using mass spectrometry for ladderdetection. Nucleic Acids Res. 26, 24882490.

57. Maxam, A. M. and Gilbert, W. (1977) A new method forsequencing DNA. Proc. Natl. Acad. Sci. USA 74, 560564.

58. von Wintzingerode, F., Bocker, S., Schlotelburg, C.,et al. (2002) Base-specific fragmentation of amplified16S rRNA genes analyzed by mass spectrometry: atool for rapid bacterial identification. Proc. Natl.Acad. Sci. USA 99, 70397044.

59. Elso, C., Toohey, B., Reid, G. E., Poetter, K., Simpson,R. J., and Foote, S. J. (2002) Mutation detection usingmass spectrometric separation of tiny oligonucleotidefragments. Genome Res. 12, 14281433.

60. Shchepinov, M. S., Denissenko, M. F., Smylie, K. J.,et al. (2001) Matrix-induced fragmentation of P3'-N5'phosphoramidate-containing DNA: high-throughputMALDI-TOF analysis of genomic sequence polymor-phisms. Nucl. Acids Res. 25, 38643872.

61. Hahner, S., Ludemann, H. C., Kirpekar, F., et al.(1997) Matrix-assisted laser desorption/ionizationmass spectrometry (MALDI) of endonuclease digestsof RNA. Nucleic Acids Res. 25, 19571964.

62. Krebs, S., Medugorac, I., Seichter, D., and Forster, M.(2003) RNaseCut: a MALDI mass spectrometry-basedmethod for SNP discovery. Nucl. Acids Res. 31, E37

63. Hartmer, R., Storm, N., Boecker, S., et al. (2003)RNase T1 mediated base-specific cleavage andMALDI-TOF MS for high-throughput comparativesequence analysis. Nucl. Acids Res., 31, E47.

64. Buetow, K. H., Edmonson, M., MacDonald, R., et al.(2001) High-throughput development and character-ization of a genomewide collection of gene-basedsingle nucleotide polymorphism markers by chip-based matrix-assisted laser desorption/ionizationtime-of-flight mass spectrometry. Proc. Natl. Acad.Sci. USA 98, 581584.

65. Werner, M., Sych, M., Herbon, N., Illig, T., Konig, I.R., and Wjst, M. (2002) Large- scale determination ofSNP allele frequencies in DNA pools using MALDI-TOF mass spectrometry. Human Mutation 20, 5764.

66. Mohlke, K. L., Erdos, M. R., Scott, L. J., et al. (2002)High-throughput screening for evidence of associa-tion by using mass spectrometry genotyping on DNApools. Proc. Natl. Acad. Sci. USA 99, 16,92816,933.

67. Ding, C. and Cantor, C. R. (2003) A high-throughputgene expression analysis technique using competitivePCR and matrix-assisted laser desorption ionizationtime-of-flight MS. Proc. Natl. Acad. Sci. USA 100,30593064.

68. Ross, P., Hall, L., and Haff, L. A. (2000) Quantitativeapproach to single-nucleotide polymorphism analysisusing MALDI-TOF mass spectrometry. Biotechniques29, 620626.

69. Le Hellard, S., Ballereau, S. J., Visscher, P. M., et al.(2002) SNP genotyping on pooled DNAs: compari-

07_JW621_Jurinke_147-163 1/19/04, 9:49 AM162



son of genotyping technologies and a semi-automatedmethod for data storage and analysis. Nucleic AcidsRes. 30, E74.

70. Shifman, S., Pisante-Shalom, A., Yakir, B., andDarvasi, A. (2002) Quantitative technologies for al-lele frequency estimation of SNPs in DNA pools. Mol.Cell. Probes 16, 429434.

71. Sham, P., Bader, J. S., Craig, I., ODonovan, M., andOwen, M. (2002) DNA pooling: a tool for large-scaleassociation studies. Nat. Rev. Genet. 3, 862871.

72. Barratt, B. J., Payne, F., Rance, H. E., Nutland, S.,Todd, J. A., and Clayton, D. G. (2002) Identificationof the sources of error in allele frequency estimationsfrom pooled DNA indicates an optimal experimentaldesign. Ann. Hum. Genet. 66, 393405.

73. Knight, J. C., Keating, B. J., Rockett, K. A., andKwiatkowski,, P. D. (2003) In vivo characterization ofregulatory polymorphisms by allele-specific quantitationof RNA polymerase loading. Nat. Genet. 33, 469475.

74. Cardon, L. R. and Bell, J. I. (2001) Association studydesigns for complex disease. Nat. Rev. Genet. 2, 9199.

75. Tabor, H. K., Risch, N. J., and Myer, R. M. (2002)Candidate-gene approaches for studying complex

traits: practical considerations. Nat. Rev. Genet. 3,391397.

76. Bansal, A., van den Boom, D., Kammerer, S., et al.(2002) Association testing by DNA poolingan effec-tive initial screen. Proc. Natl. Acad. Sci. USA 99,16,87116,874.

77. Kammerer, S., Burns-Hamuro, L., Ma, Y., et al.(2003) Amino acid variant in the kinase binding do-main of dual-specific A kinase-anchoring protein 2, adisease susceptibility polymorphism.Proc. Natl.Acad. Sci. USA 100, 40664071.

78. Jurinke, C., van den Boom, D., Cantor, C. R., and Kster,H. (2002) Automated genotyping using DNA mass-ARRAY technology. Methods Mol. Biol. 187, 179192.

79. Amexis, G., Oeth, P., Abel, K., et al. (2001) Quantita-tive mutant analysis of viral quasispecies by chip-based matrix-assisted laser desorption/ionizationtime-of-flight mass spectrometry. Proc. Natl. Acad.Sci. USA 98, 12,09712,102.

80. Bucknall, M., Fung, K. Y. C., and Duncan, M. W.(2002) Practical quantitative biomedical applicationsof MALDI-TOF mass spectrometry. J. Am. Soc. MassSpectrom. 13, 10151027.

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