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MOLECULAR BIOTECHNOLOGY Volume 26, 2004

MALDI-TOF Mass Spectrometry 147


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.
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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-
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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-
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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.
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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
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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
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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-
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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.
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MALDI-TOF Mass Spectrometry 155
Fig. 4.
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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).
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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
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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
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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).
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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. The unique
combination of flexibility, accuracy, automated
analysis, and high-throughput data generation is
of benefit in many fields of biological research.
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