USE OF MATRIX-ASSISTED LASER DESORPTION/
IONIZATION TIME-OF-FLIGHT MASS SPECTROMETRY
FOR MULTIPLEX GENOTYPING
Klaus Meyer1and Per Magne Ueland
Bevital AS, c/o Section for Pharmacology, University of Bergen,
3. Genotyping Using Mass Spectrometry...........................................
4.2. Analysis of Nucleic Acids by MALDI-TOF-MS ...........................
5. Multiplex Genotyping by MALDI-TOF-MS ....................................
5.1. SNP-Genotyping Using Primer Extension Assays ..........................
5.2. SNP-Genotyping Using Nonprimer Extension Assays......................
5.3. Genotyping of Other Polymorphic Variants................................
5.4. Molecular SNP-Haplotyping...............................................
5.5. Quantification of Mixed DNA.............................................
6. Conclusions .....................................................................
After completion of the human genome project, the focus of geneticists has
shifted to elucidation of gene function and genetic diversity to understand the
mechanisms of complex diseases or variation of patient response in drug treat-
ment. In the past decade, many different genotyping techniques have been
described for the detection of single-nucleotide polymorphisms (SNPs) and
other common polymorphic variants. Matrix-assisted laser desorption/ioniza-
tion time-of-flight mass spectrometry (MALDI-TOF-MS) is among the most
1Corresponding author: Klaus Meyer, e-mail: email@example.com
Copyright 2011, Elsevier Inc.
All rights reserved.
ADVANCES IN CLINICAL CHEMISTRY, VOL. 53
powerful and widely used genotyping technologies. The method offers great
flexibilityinassaydesign andenables highlyaccurate genotyping at high sample
been combined with MALDI (hybridization, ligation, cleavage, and primer
lar applications. This combination enables rapid and reliable multiplexing of
large-scale studies in fine-mapping and verification of genome-wide scans. In
contrast to standard genotyping, more demanding approaches have enabled
genotyping of DNA pools, molecular haplotyping or the detection of free circu-
used in novel applications as DNA methylation analysis, expression profiling,
and resequencing. This review gives an introduction to multiplex genotyping by
MALDI-MS and will focus on the latest developments of this technology.
Understanding the basis of human genetic variations is a major task in
medical research. Various diseases have been related to changes in gene
sequences caused by different types of polymorphisms and mutations, which
can alter the structure and function of a protein. Among these variants,
attention has been focused in the recent years on single-nucleotide polymorph-
in a given population and are found in the human genome at a frequency of
about one in 1000 base . Public databases as the general catalog of genetic
polymorphism maintained by NCBI [2,3] actually consist of over 143 million
reported SNPs,of which 19 million reference SNP clusters have been validated
a small portion of SNPs changes the coding sequence of a protein or affects
ofSNPsdoesnot changethe aminoacidsequenceandcanserve asmarkers for
disease causing genes in genome-wide association studies. The international
HapMap  has to date registered more than 3.1 million so-called tag-SNPs,
which tag common haplotype blocks and help to reduce the number of geno-
types screened in genome-wide association studies. The number of registered
tag-SNPs will further increase with completion of the 1000 Genomes Projects
(www.1000genomes.org), which is aimed to achieve a nearly complete catalog
to determine and their interpretation can be highly automated.
Various methods for genotyping of SNPs have been developed in the past
few years and validated with respect to accuracy and sensitivity, flexibility of
the assay design, throughput, and costs per genotype [5–8]. Methods based on
MEYER AND UELAND
mass spectrometry provide very reliable platforms for genotyping . Among
these, matrix-assisted laser desorption/ionization time-of-flight mass spec-
trometry (MALDI-TOF-MS) belongs to the most powerful techniques for
high-throughputmultiplexing ofSNPs[7,10–16] andhasalsobeenusedforthe
detection of other common variations, such as restriction fragment length
polymorphisms (RFLPs) , short tandem repeats (STRs) [18,19], insertions
and deletions , and copy number variations (CNVs) . The ability of
sensitive and (semi-) quantitative allele determination has enabled pooled
DNA analysis [22,23] and noninvasive prenatal fetal diagnosis .
3. Genotyping Using Mass Spectrometry
A characteristic feature of mass spectrometry is that the detection is based
on an intrinsic physical property (mass to charge ratio), which contributes to
highly accurate data. Traditional methods use indirect methods like fluores-
cence or radioactive reporter tags, which have to be labeled to the target
molecule. While the number of different tags is limited and thus only
supports low-level multiplex applications per reaction, MS provides multiple
data points per experiment and enables high-level multiplex genotyping.
In addition, latest mass spectrometers provide sensitive analysis with limits
down to the femtomole and attomole range [25,26].
The breakthrough of MS for the analysis of large biomolecules was intro-
duced by the development of new gentle ionization methods, which prevent
the decay of the molecules during the ionization process. Nowadays, two
ionization methods are commonly used for biomolecules: electrospray ioni-
zation (ESI)  and MALDI . ESI is a much softer ionization method
than MALDI and allows the characterization of very large biomolecules of
up to 108Da . In combination with ion trap, QTOF or FT-ICR MS, the
technique has been successfully used for the analysis of nucleotides, especial-
ly intact PCR products, plasmid, or phage DNA . However, MALDI has
become the technique of choice for of MS-based multiplex genotyping .
The method is less dependent on highly purified samples than ESI, and the
direct combination of MALDI with TOF analyzers enables robust and rapid
genotyping with throughputs of several thousand samples per day .
The MALDI process can be separated into three fundamental steps of
analyte/matrix preparation, desorption of the upper matrix monolayers, and
GENOTYPING BY MALDI-MS
of analyte molecules between the matrix molecules, either in the bulk or within
the crystal surface, and separation of the analyte molecules from each other.
There is no single MALDI matrix or protocol that is suited to all analytical
questions, and a proper choice of both is crucial for the analytical outcome
[32–34]. In addition, various factors as the analyte-to-matrix ratio, type of
solvent and additives, amount of salts, and characteristic of the target surface
influence the crystalline morphology and, thus, the quality of mass spectra.
During the MALDI analyses, a short UV- or IR- laser pulse of a few
nanoseconds irradiates the crystals and leads to desorption/ionization of the
matrix and analyte molecules into the gas phase (Fig. 1). The wavelength of
the laser should be close to the absorption maximum of the matrix molecules.
The processes of material desorption and ionization of the matrix and
analyte molecules are intertwined and take place on a micrometer geometri-
cal and nanosecond time scale. While the mechanisms of desorption have
been partly understood by comparison of experimental data with simulations
[35,36], no unifying theory exists that explains the formation of the gas-phase
ions [37–39]. This might be caused by the large variety of factors including
type of analyte molecules, matrices, preparation, and experimental condi-
tions, which influence the process of ionization.
Variousanalyzersas orthogonalTOF,FT-ICR,QIT,QTOF, QIT-TOF,
or Orbitrap  have been coupled to MALDI, but axial TOF (including
reflectron TOF, TOF/TOF) has become the most common type for MALDI
spectrometers. Axial TOF analyzers, as illustrated in Fig. 1, are ideally suited to
typically accelerated by 20 kV into the flight tube, and as all ions have the same
kinetic energy E ¼ zeU ¼ 1/2mv2, they travel with different velocities v and
arrivethedetectorattimet ¼ L(m/2E)1/2whereListhelengthoftheflighttube.
For MALDI-TOF-MS instruments, there are two primary sources of error
related to the flight time. The first is caused by the initial velocity distribution
of MALDI ions and is compensated in current TOF instruments mainly by
pulsed-ion extraction (also called delayed extraction) instead of using a static
acceleration field . The second source of errors is introduced by energy
dispersion due to nonflat sample morphology and is compensated by a one-
or two-stage reflectron analyzer. Here, ions with higher energy travel a longer
way through the reflector than ions with lower energy and the ion packets can
be refocused on a second detector. Modern research-grade instruments are
equipped with both delayed extraction and reflectron TOF-MS .
4.2. ANALYSIS OF NUCLEIC ACIDS BY MALDI-TOF-MS
Even though modern MALDI-TOF instruments are well suited for high-
level multiplexing applications in genomics, determination of nucleic acids
by MALDI is generally difficult when compared to peptides. Assay
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development requires accurate evaluation of the instrumental aspects,
matrix, purification, sample preparation and analysis, and primer design in
order to achieve highest degree of multiplexing at sufficient signal quality.
4.2.1. Instrumental Aspects
The mass range, the mass resolution, and the mass accuracy are central
parameters of a mass analyzer that define the limits of an MS-based assay.
Common MALDI-TOF-MS instruments use UV lasers, which allow the
identification of oligonucleotides, for example, sequencing ladder products,
in a mass range of up to 30,000 Da (ca. 100 nucleotides) . This upper mass
ReflectronFlight tube Source
FIG. 1. MALDI-TOF-MS. MALDI-TOF instruments basically consist of three major com-
ponents: a vacuum chamber housing the source and the flight tube, a pulsed UV laser, and a fast
oscilloscope. The sample placed on a stage inside the source is irradiated by a short UV-laser
pulse of a few nanoseconds. The matrix/analyte molecules are desorbed/ionized, and accelerated
by an electric field of about U ¼ 20 kV into a field-free drift path inside the flight tube. The
oscilloscope is started by the laser pulse for measurement of the flight time. As all molecules are
accelerated to the same kinetic energy E ¼ zeU ¼ 1/2mv2, ions of different masses (m1< m2<
m3) are separated from each other during the flight and hit the detector DLinafter t ¼ L(m/2E)1/2
(L ¼ length of the flight path). Typically 20–100 laser shots are accumulated for a MALDI
spectrum, and the detector signals are registered by the oscilloscope. Linear time-of-flight MS
(registration at DLin) has become the standard mode for most genotyping applications by
MALDI-TOF-MS. Mass resolution can be optimized by application of delayed ion extraction
(DE). Reflectron TOF MS instruments can further increase mass resolution (at the expense of
signal intensity) by reflecting the ions at the end of the flight tube back on a second detector DRef.
GENOTYPING BY MALDI-MS
limit is caused by size-dependent fragmentation of DNA during desorption,
which results in loss of signal intensity toward higher masses. RNAs are more
stable than DNA in MALDI because the additional 20-hydroxyl group
stabilizes the glycosidic bond and reduces depurination and fragmentation
of the entire oligomer . Large DNA fragments such as PCR products of
up to 1400 bp can be detected using infrared MALDI [46,47], which is known
to be considerably softer than UV-MALDI. Further increase of the mass
range requires new types of detectors, as the sensitivity of microchannel plate
detectors, commonly used in TOF instruments, decreases with molecule
velocity. Cryogenic detectors have shown to decrease the detection limit by
several orders of magnitudes .
Although MALDI-TOF-MS allows the principal detection of small PCR
products <100 bp, mass resolving power of TOF analyzers is insufficient to
identify point mutations and SNPs by direct comparisonof the PCR products.
Hence, MALDI-based genotyping has been optimized with respect to a win-
dow from 1000 to 9000 Da . Reflectron TOF-MS instruments offer higher
majority of instruments used for multiplex genotyping are of linear type,
because these instruments have generally higher transmission, are cheaper
than reflectron analyzers and enable very compact bench top designs.
About one dozen of different matrices have been tested for the optimal
analysis of oligonucleotides by MALDI . Generally, a MALDI matrix
must be able to embed the analyte molecules, must be stable in the vacuum
and must promote desorption/ionization of the analyte. Matrices are classi-
fied by the terms ‘‘hot’’ and ‘‘cool.’’ ‘‘Hot’’ matrices results in excessive
fragmentation and thus are suitable for the detection of small nucleic acids
or RNA. The latter type is suitable for the analysis of larger oligonucleotides
and produces little fragmentation during MALDI. The use of matrix addi-
tives as sugar has been shown to decrease fragmentation and increases signal
resolution by an additional ‘‘cooling’’ effect.
Two matrices have been proven to be optimal for the analysis of nucleic
acids. A mixture of 2,3,4- and 2,4,6-trihydroxyacethophenone (THAP) is
well suited for RNA analysis , while 3-hydroxypicolinic acid (3-HPA) is
the preferred matrix for the detection of DNA  and has become the
‘‘golden standard’’ for oligonucleotide analysis so far.
4.2.3. Sample Purification
A challenging problem in the detection of oligonucleotides by MALDI is
the negative charge of the sugar-phosphate backbone in solution. The high
affinity to sodium and potassium from different buffers used results in
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adducts formation during the ionization process and lowers the quality and
intensity of the signals. Several homogenous and heterogeneous purification
protocols have been applied to remove the salt from reaction products and
matrices. These include dialysis , ethanol precipitation , and reversed-
phase  or size-exclusion chromatography . The use of biotin-linked
terminators in combination with streptavidin-coated magnetic particles is an
elegant and effective solid phase purification, as this method results in highly
cleaned reaction products by removing salts, PCR as well as unextended
primer products . However, for high-throughput applications with a
capacity of many thousands of samples per day multiple washing steps by
liquid handlers represent a bottleneck, and consumption of coated magnetic
particles substantially increases assay costs. Alternatively, samples can be
incubated with NH4þ-conditioned ion-exchange resins for 1 h, which replace
alkali-metal ions by ammonium ions [56,57]. Optimization of resin types and
dilution of the sample can decrease incubation time to 10 min . Although
the method is not as efficient as the usage of magnetic particles, this homog-
enous purification scheme enables easy, inexpensive, and rapid removal of
salt adducts for various high-throughput genotyping assays. Other
approaches involve on-target purification using commercial polymers, self-
assembled monolayers, ultrathin polymer films, and surface coatings with
nucleic acid-binding properties [59–62]. These techniques may introduce
several problems as spreading of sample droplets and low homogeneity,
but are promising procedures for highly purified samples at medium
4.2.4. Sample Preparation and Analysis
The universal preparation method for MALDI matrices is the droplet
procedure, where matrix and sample are mixed together and spotted onto a
MALDI target plate. Alternatively, the matrix can be dispensed first and the
sample is spotted onto the dried matrix crystals. This method, which is
the standard preparation for 3-HPA, results in matrix/sample spots of ca.
1–2 mm diameter and requires searching for a ‘‘sweet spot,’’ as crystal
morphology and variation in the local matrix-to-sample ratio dramatically
influences signal intensity and quality. Several preparation techniques have
been tested to enable homogenous crystallization for fast and automated
MALDI analyses. The so-called thin-layer method uses other matrices than
3-HPA, which are soluble in organic solvents [63,64]. After the matrix is
spotted or sprayed onto the plate, the sample is dispensed onto the matrix. As
the sample is diluted in a water-based solvent, the matrix is not completely
dissolved and the analyte is homogenously embedded into the crystal surface.
However, the majority of techniques have focused on optimized dried droplet
preparation for 3-HPA. These methods have the ability to produce a single,
GENOTYPING BY MALDI-MS
homogenous matrix crystal by the application of different miniaturization
techniques. Two commercial methods are widely used, available as part of
different assay formats developed by Bruker Daltonics  and Sequenom
. Bruker uses Teflon coated sample plate with arrays up to 1536 hydro-
philic spots. The spot size varies from 200 to 800 mm and arrays can be
spotted by matrix of choice using classical automated liquid handlers. Sam-
ples are upconcentrated on the anchor spots during solvent evaporation. The
plate can be washed and reused many times before the coating is degraded.
Sequenom’s SpectroCHIPs are based on silicon dioxide and are prestruc-
tured with 384 spots with 4 nl 3-HPA. The chips can be used once only and
require a special nanoliter dispensing device, which dispenses 384 samples in
less than 15 min. On-target plate can hold up to 10 SpectroCHIPs and
represents the actually highest number of sample loading for MALDI-
MALDI analyses are routinely controlled by fuzzy controlled data acqui-
sition software, allowing fast sample analysis under optimal conditions. The
laser beam is directed over the matrix/sample spot on a defined track in a
spiral or S-curve while laser intensity is held on an adequate level to obtain
high-quality spectra. Acquisition time is 3–5 s per sample and 384 samples
are analyzed within 30 min. As only a small amount of the matrix spots is
consumed after irradiation by typically 20–50 laser shots, samples can be
genotyped twice if higher assay accuracy is required. Commercial software
environments as Bruker’s Compass/GenoTools or Sequenom’s MassAR-
RAY Typer packages enable highly automated sample tracking, data acqui-
sition and analysis, and quality control and facilitate rapid genotyping of
several hundreds to 100,000 genotypes per day.
5. Multiplex Genotyping by MALDI-TOF-MS
5.1. SNP-GENOTYPING USING PRIMER EXTENSION ASSAYS
Due to its robustness, flexibility, and easy design, primer extension has
become the most widely used format for SNP-genotyping—not only in mass
spectrometry [67,68]. Several MALDI-based primer extension formats have
been published using single- or multibase extension; they differ by speed,
cost, complexity of sample preparation, quality of allele separation, and level
of multiplexing. These methods usually begin with the amplification of a
target region by PCR. A primer for detection of the SNP is then annealed
next to the polymorphic side and extended by a polymerase. The extension is
terminated either on the polymorphic side or a few bases behind. PCR
reagents and products, which can disturb the extension reaction in
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heterogeneous and homogenous assays, are often removed by either washing
or enzymatic cleavage, respectively.
Although poorly discussed in the literature, optimization of multiplex
genotyping assays based on primer extension is always a crucial step and
strongly determines the efficiency, robustness, and accuracy of the method.
Even, if primer characteristics can be roughly predicted by various primer
calculation and simulation programs, assay development remains an empiri-
cal process and efforts are generally proportional to the number of SNPs
investigated per reaction . Common problems include unbalanced tem-
plate PCR amplification, self-priming, or annealing to an incorrect location,
that is, on a pseudogene. In the case of incompatible PCR primers, reactions
can be run in parallels for different sets of PCR-templates and the products
are mixed together before the allele-specific reaction is performed. The design
of extension primers can become even more difficult, as the lengths of the
extension primers influence the primer annealing temperatures and have to
be different and evenly distributed in a mass window of ca. 5000 Da. This
step of optimization can be done either manually or by sophisticated soft-
ware solutions as Sequenom’s MassARRAY designer, which support quick
assay development with success rates of >95% (15-plex) .
The first and most simple approach was a single-base extension assay
called PinPoint developed by Haff and Smirnov in 1997 , which termi-
nates the extension by conventional ddNTPs on the polymorphic site
(Fig. 2). Allele discrimination is based on the mass difference between the
four terminators (9 Da for ddA/ddT; 15 Da for ddT/ddC; 16 Da for ddA/
ddG; 24 Da for ddC/ddA; 25 Da for ddT/ddG; and 40 Da for ddC/ddG). The
level of multiplexing could be increased to 20 by careful assay design with
‘‘mass-tuning’’ of the extension primers . Typical mass spectra of a 20-
plex PinPoint assay are illustrated in Fig. 3. Sample purification is based
either on reversed-phase columns or on ion-exchange resins. However, the
PinPoint suffers from ambiguous identification of A/T heterozygotes in
routine analyses due to the low mass difference of 9 Da between the extension
products for masses >5000 Da. Thus, this approach has mainly been used in
assays, which did not analyze A/T transversions (lowest frequency of ca. 7%
of all single nucleotide changes ), or if a short primer length below
5000 Da was compatible with assay design. A simple way to increase the
mass difference for A/T, but also other heterozygotes, is the usage of
ddNTPs/dNTPs mixtures or mass-modified terminators.
The PROBE assay was introduced by Little and coworkers in 1997 [74,75]
and uses one ddNTP and three dNTPs for extension reaction (Fig. 2). This
multibase extension approach enables clear distinction between all reaction
products, in the way that mixtures are optimized to always result in at least
one nucleotide (ca. 300 Da) mass difference between two allele-specific
GENOTYPING BY MALDI-MS
extension products. However, this method can also lead to misinterpretation
of genotyping results due to polymerase-pausing artifacts, which can have
the same mass as ‘‘real’’ termination products. This weakness can be mini-
mized by the use of three ddNTPs and one dNTP, which results in mainly
short extensions as demonstrated by the VSET assay . Another drawback
of the PROBE assay is the use of magnetic beads for primer immobilization,
which restricts throughput and increases cost and was the reason that the
PROBE, but also other heterogeneous assays as the Solid Phase Capture
Single-Base Extension  and the GenoSNIP , were replaced by homog-
enous techniques. However, heterogeneous assays produce highly purified
Singlebase extensionMultibase extension
FIG. 2. SNP-genotyping by primer extension assays. Primer extension can be performed as
single- or multibase extension reactions. All types of reactions require purified PCR products.
Single-base extension by the PinPoint and iPLEX assays represent the simplest approaches.
Here, a primer is annealed next to the mutation site (X) and extended by a ddNTP. While the
PinPoint uses natural ddNTPs and suffers from insufficient allele separation of A/T heterozy-
gotes and mass interferences by salt adducts, the iPLEX reaction is performed using mass-
modified terminators, which enable unambiguous allele identification. The GOOD assay repre-
sents the most advanced single-base extension assay and includes charge-tagging and backbone
neutralization. Primers are modified by photocleavable o-nitrobenzyl moieties for primer
shortening and charge-tagging. Extension reaction uses synthetic alpha-thio ddNTPs. After
extensionprimersare cleavedby UV-lightandare alkylated in the laststeptoavoidcationization
by salt adducts.Multibase extension assays use mixtures of ddNTPs and dNTPs (gray tones) and
have the advantage of larger mass differences between signals of different alleles or primers and
salt adducts. The PROBE and the MassEXTEND use three dNTPs and one ddNTP for allele
determination. Dependent on the nucleotide sequence, primers can be extended by several bases
before reactions are terminated by a ddNTP. The length of extension products is reduced in
average by usage of three ddNTPs and one dNTP as demonstrated by the VSET assay.
MEYER AND UELAND
extension products and, thus, have demonstrated the highest level of multi-
plexing so far with 50 SNPs per reaction .
A very popular homogenous multiplexing assay for SNP-genotyping by
MALDI is the MassEXTEND assay by Sequenom [58,78]. It is an improve-
ment of the PROBE and uses ion-exchange resins for desalting. In addition,
the assay uses sample dilution with deionized water for optimization of the
crystallization and desorption/ionization process, as the homogenous assay
design cannot completely remove contaminations from buffers and reaction
components. The MassEXTEND is routinely performed with up to 12
simultaneous SNPs, but higher multiplexing levels can be obtained by careful
design of the extension primers. The assay is performed with microarrayed
MALDI targets and nanoliter dispensing devices. The reduction of the
processed sample volume lowers assay costs and makes the MassEXTEND
a very cost-effective choice for 2–10 plexes when processed in 384 plates.
60007000 80009000 10,0005000
123 4 56 78 9 1011 12131415
1919 9 9
FIG. 3. MALDI-TOF spectra of a primer extension assay. Typical MALDI-TOF-MS spectra
of a primer extension reaction are illustrated. The upper panel shows the spectrum of a 20-plex
primer mixture before extension reaction, which spans a mass range from 4500 to nearly
10,000 Da. Primers are numbered from 1 to 20 and standards for mass calibration are labeled
(S). The lower panel shows the spectrum after reaction (PinPoint), and wild type, heterozygous,
and homozygous mutant genotypes are assigned. Peaks of residual reaction primers are
connected to the upper spectrum.
GENOTYPING BY MALDI-MS
For higher multiplexing of SNPs, Sequenom’s iPLEX represents a more
cost-effective approach and routinely genotypes between 25 and 29 loci per
reaction [70,79]. In contrast to the MassEXTEND, this assay is a single-base
extension format and uses exclusively mass-modified ddNTPs. The assay
creates mass differences large enough to differentiate between all bases
compared to natural ddNTPs (ddA/ddT: 56 vs. 9 Da; ddC/ddT: 80 vs.
16 Da; and ddG/ddT: 40 vs. 25 Da). In contrast to the MassEXTEND, the
iPLEX produces smaller mass gaps between primers and extension products
and, therefore, enables a higher multiplexing level of up to 40 SNPs per
While the iPLEX assay is the result of advanced desalting techniques and
new types of terminators, other approaches aim to handle the difficulties of
oligonucleotides detection at the level of primer synthesis. These approaches
use neutralization of the sugar-phosphate backbone to avoid purification
from salts , charge-tagging to increase signal–noise ratio , and primer
cleavage to utilize the lower mass range . These concepts have been
merged and realized in different versions of a homogenous approach called
‘‘GOOD assay’’ by Sauer et al. [82,83]. Common to other assay formats, the
target region is amplified by PCR and the mutation site identified by primer
extension. The extension primer carries a charge-tag close to the 30end and a
phosphorthioate bridge on the 50side of the charge tag. Primer extension is
performed using alpha-thio ddNTPs. Digestion by 50-specific phosphodies-
terase cleaves the primer down to the first phosphorthiodate, which results in
a short oligonucleotide including a charge tag and the SNP-specific nucleo-
tide. Finally, alkylation by methyl iodide of the phosphorthiodate bridge
neutralizes the backbone andprevents cationization by sodium or potassium.
Recently, the assay protocol has been improved by less-toxic reagents for
alkylation and photocleavable o-nitrobenzyl moieties for primer shortening
and charge-tagging . So far, the GOOD assay has not become as popular
as the MassEXTEND or iPLEX due to relatively low multiplexing level of
<10 SNPs per reaction.
5.2. SNP-GENOTYPING USING NONPRIMER EXTENSION ASSAYS
Several MALDI-based methods for genotyping were developed already in
the 1990s, using other techniques than primer extension such as hybridiza-
tion, ligation, or cleavage reaction (Fig. 4). Although many of these methods
include novel approaches to overcome various limitations related to amplifi-
cation by PCR or nucleic acid analysis by MALDI, none of these methods
have so far been widely used for high-throughput genotyping. This might be
related to either heterogeneous assay design or usage of expensive or toxic
MEYER AND UELAND
Peptide nucleic acid (PNA) hybridization probes have several advantages
in MALDI-based genotyping [85,86]. PNAs are more resistant to fragmen-
tation and, due to the uncharged backbone, less prone to alkali-metal adduct
formation than DNA. They have been successfully applied in DNA sequence
determination, including SNP-genotyping. However, complicated reaction
conditions, expensive PNA probes, and the need for streptavidin-coated
magnetic beads have so far hindered the use of PNAs in high-throughput
approaches. In a recent work, Boontha et al. have described an improved and
simplified procedure using pyrrolidinyl-PNA probes . In combination
with ion-exchange capture, this PNA-based method has demonstrated po-
tential of rapid and inexpensive high-throughput multiplex genotyping.
FIG. 4. SNP-genotyping by nonprimer extension assays. Common nonprimer extension
assays are based on hybridization, ligation, or cleavage reactions. For the hybridization assay,
two different peptide nucleic acid (PNA) probes are designed for the template sequence and the
matching probe is hybridized to the PCR products. The DNA–PNA hybrid is immobilized on a
solid support and the unbound probe removed by washing. Ligation assays use two different
allele-specific oligonucleotides, which are enzymatically ligated with a second primer when fully
complementary to the target sequence. The invader assay uses a ‘‘probe’’ and an ‘‘invader’’
oligonucleotide, which both hybridize to the target DNA. The probe is the downstream oligo-
nucleotide, the invader the upstream oligonucleotide, which includes a noncomplementary
nucleotide at the 30end (N). Both oligonucleotides form a duplex, which includes the polymor-
phic site and a noncomplementary 50overhang called ‘‘flap.’’ The invader nucleotide creates a
sequence overlap that is recognizedby a DNA repair enzyme. The flap is released by the cleavage
reaction and detected. For each SNP, genotyping is performed using two probes with different
sequences in the flap. The Incorporation and Complete Chemical Cleavage (ICCC) assay uses a
chemically labile nucleotide (L), which replaces one of the four dNTPs during PCR. The
generated amplicon is cleaved after PCR at the incorporation sites and the genotype can be
determined from the specific cleavage products in the mass spectrum.
GENOTYPING BY MALDI-MS
Oligonucleotide ligation was also tested in the early reports on the use of
MALDI-MS for SNP-genotyping. Here, two oligonucleotides are ligated
when fully complementary to the target sequence and the genotype is identi-
fied by mass determination of the ligation product . To date, this tech-
nique has not been developed beyond this prove-of-principle study.
The invader assay uses two sequence-specific oligonucleotides, a ‘‘probe’’
and an ‘‘invader’’ oligonucleotide, which hybridize to the target DNA .
The probe is the downstream oligonucleotide, the invader the upstream oligo-
nucleotide. Both oligonucleotides form a duplex, which includes the polymor-
phic site and a noncomplementary 50overhang called ‘‘flap.’’ The invader
nucleotide creates a sequence overlap that is recognized by a DNA repair
enzyme. The flap is released by the cleavage reaction and detected. For each
SNP,genotypingisperformedusing two probeswithdifferent sequencesinthe
flap. The process is isothermally run near melting temperature using thermo-
stable enzymes and has the main advantage that it does not require PCR
thermocycling for amplification. Griffin et al. combined the invader assay for
genotyping with MALDI with the so-called ‘‘Invader Squared assay’’ .
Here, thecleavedflapservesasaninvaderoligonucleotidefora secondinvader
reaction, which is performed with a biotin-labeled flap for sample desalting
using streptavidin-coated magnetic beads. So far, the heterogeneous assay
design and the relatively large amount of required genomic DNA have hin-
dered the MALDI-based Invader assay to become as popular as the commer-
cial Invader assays using fluorescent probe detection .
The Incorporation and Complete Chemical Cleavage (ICCC) assay intro-
duced by Wolfe et al. uses a chemically labile nucleotide, which replaces one of
the incorporation sites and the genotype can be determined from the specific
one reaction, the assay does not require a cleanup step and is well suited for
automation. Inaddition, double-strand amplicons are usedfor genotyping and
cleavage products are generated from both directions. This process delivers
redundant information for genotype determination and provides highly accu-
throughput applications due tothe useoftoxic agentsfor cleavagereaction.
5.3. GENOTYPING OF OTHER POLYMORPHIC VARIANTS
In addition to SNP-genotyping, various MALDI-based assay formats have
include RFLPs , sequence variation as insertions or deletions (indels) ,
STRs , and alternative splicing variants (ASVs) , as well as CNVs .
MEYER AND UELAND
RFLPs are DNA-based variations at restriction sites, which alter the
fragment length pattern after cleavage by a restriction endonuclease enzyme.
RFLPs are of biallelic nature and can be defined as a subset of SNPs in
the recognition sequence for a restriction site. Primer extension based
assays are difficult to develop for RFLP genotyping, as the polymorphic
region is characterized by multiple closely located polymorphisms. The
restriction fragment mass polymorphism (RFMP) assay developed by Kim
et al.  enables genotyping of polymorphic regions by incorporation of IIS
restriction endonuclease recognition sites during PCR amplification and
cleavage of the amplicons using IIS restriction endonuclease. This homoge-
nous assay allows rapid genotyping also for SNPs and offers high accuracy
by simultaneous genotyping of both DNA strands. The RFMP has been
mostly applied in screening studies for genotyping of hepatitis B, C,
and papilloma viruses, but also for SNP identification from pooled human
Sequence variations by insertion or deletion of one or more nucleotides
represent another important class of polymorphisms. Similar to SNPs,
insertions and deletions can alter the structure or the expression of a
protein. Single-base indels are easy to genotype using a simple primer
extension approach. Longer sequences can be determined by semiquanti-
tative probing of the junction or a specific SNP inside the insertion .
More importantly, genotyping of indels is a powerful application for
determination of STRs and ASVs. STRs or microsatellite are stretches
of DNA, which consist of repeating units of two, three, or four nucleo-
tides, and the different repeat length represents the alleles of the poly-
morphism. STRs are valuable markers for genetic linkage studies, but
also in cancer diagnostics, as microsatellite instability is related to defi-
ciency of the mismatch repair system. Determination of indels/STRs in
repeating sequences is difficult, as partly hybridization often occurs in the
repetitive sequence. Two different approaches have been developed using
primer extension as well as cleavage reaction for allele identification. The
primer extension method by Bonk et al.  uses primers that directly
anneal at the 50end of short mononucleotide repeats, and extension
results in specific peak patterns. For cleavage reaction, two different
formats have been published. In the method by Krebs et al., RNA
fragments were produced by cutting transcripts using ribozyme  or
G-specific ribonuclease . The second approach by Sasayama et al.
uses in vitro transcription of PCR products and cleavage of the 50and 30
ends of the indel site by artificial RNA cutters .
Alternative splicing isa significant contributor to transcriptomediversity, as
ca. 74% of the human-multiexon genes are alternatively spliced. McCullough
et al.  presented a quantitative primer extension approach, which allows
GENOTYPING BY MALDI-MS
the identification of different splicing isoforms as exon insertion/deletion,
splicing acceptor and donor, and a mutually exclusive exon. Here, reverse
transcribed cDNA was PCR amplified using primers that flank the site of
alternative splicing. Extension primers were designed to extend from one to a
few bases into the sequence differences and ASVs could be determined by the
weight of extension product using MALDI.
CNVs are segments of DNA ranging in size from thousands to millions of
DNA bases, which differ among individuals by the number of copies. The
most well known CNV is the Trisomy 21, a disorder caused by gene duplica-
tion in chromosome 21. CNVs play animportant role in disease susceptibility
and have been related to risk modification in, for example, HIV infection and
lung cancer. Different primer extension approaches have been established for
relative, absolute, and allele-specific determination of CNVs. For relative
quantification, a previously identified SNP is genotyped in a given popula-
tion by a single-base extension assay as PinPoint  or iPLEX  and the
relative allele signals are investigated by a scatter plot. As the allele ratio for a
heterozygous individual is 1:1, heterozygotes with allele ratios above or
below 1:1 indicate a potential CNV. Different statistical tools have been
developed for identification of CNV from SNP data [106,107]. Absolute
CNV quantification has been established on Sequenom’s MassARRAY
platform in combination with real-competitive PCR . Here, a competitor
DNA is spiked in at known concentration and is coamplified during PCR. As
the competitor includes an artificial marker, no SNP site is required in this
assay. This approach enables multiplexing of up to 20 CNVs and can also be
combined to allele-specific PCR.
5.4. MOLECULAR SNP-HAPLOTYPING
In diploid organisms, a collection of SNPs found on a single chromosome
can be more informative than individual SNPs for the determination of a
phenotypic outcome. These haplotype structures represent powerful markers
and can provide additional statistical power in mapping disease genes, as
they reflect the sequence of a transcribed protein. The knowledge of haplo-
types helps to decrease the number of SNPs that is needed to be genotyped in
association studies. Haplotypes can be determined using SNP genotype data
and computational algorithms as the EM  and PHASE . However,
these programs can fail and have a limited degree of accuracy, especially
when many heterozygous SNPs occur in close proximity. An alternative to
these methods is the direct molecular haplotyping. Several techniques have
been developed for the direct determination of haplotypes including mass
spectrometry, which has the advantage of multiplexing several SNPs. Three
MALDI-TOF based approaches have been recently published using
MEYER AND UELAND
Sequenom’s Mass EXTEND or the GOOD assay. In the first approach, two
homologous DNAs are separated by single-molecule dilution to about one
genome copy per PCR aliquot, which is genotyped by the Mass EXTEND
assay . Amplicons are about 100 bp long and amplified with 90–95%
efficiency. The haplotyping efficiency per reaction is 40–45%, and thus four
replicates should be sufficient to achieve 90% efficiency. This method is well
suited to multiplex SNPs that are far apart from each other. A major draw-
back of the approach is the sensitivity to contaminations and dependence on
template integrity, as genomic DNA fragments of about 25 kb are commonly
seen. The two other techniques include the GOOD assay for SNP identifica-
tion in combination with either allele-specific PCR or clone-based systematic
haplotyping. Allele-specific PCR is performed using primers that hybridize
with the 30end on the polymorphic site and enable the amplification of the
desired allele . The multiple SNPs used as PCR anchors have first to be
typed or are already available from a database. Using a brute-force approach
allele-specific PCR enables simple and effective molecular haplotyping.
However, this method can suffer from coamplification of the undesired allele
due to primer mismatch. In addition, size of amplicons is limited to about
1000 bp and long-range haplotyping requires unfavorable walking from
fragment to fragment. In contrast to allele-specific amplification, clone-
based systematic haplotyping enables the study of large genomic regions of
>30 kb . Cosmid/fosmid libraries were constructed from fragmented
isolated DNA covering 10% of the genome and individual clones were
genotyped if tested positive in a pool of 96 clones.
5.5. QUANTIFICATION OF MIXED DNA
MALDI-MS has been shown to enable quantification of proteins and
peptides . For oligonucleotides, the strong heterogeneity of the 3-HPA
crystal morphology makes quantification a demanding challenge, although
the more homogenous structure of nucleic acids, consisting of four similarly
building units, provides some compensation. The key to achieve high repro-
ducibility and, thus accurate quantification of nucleic acids, is to analyze a
large fraction of the sample/matrix by rastering the laser over many sample
spots and/or shrinking the preparation to a ‘‘single’’ crystal using miniaturi-
zation. Especially, Sequenom’s silicon chip based MassARRAY technique
has been proven to minimize sample heterozygosity so that the highest
impact on accuracy and reproducibility is caused by DNA preparation.
Quantitative MALDI analyses in genomics are based on the calculation of
the ratio of allele-specific primerextension products, which enables the relative
and absolute quantification of allele frequencies. A popular genotyping appli-
cation using relative allele frequencies is screening of pooled DNA to validate
GENOTYPING BY MALDI-MS
ethnic groups [113–115]. Pooling decreases the number of necessary genotypes
in large-scale studies and represents a shortcut to identify associations between
genetic loci and phenotypes. Only SNPs that show significant difference be-
tween two pools of, that is, case and controls are selected for individual
information as genotypes and haplotypes is lost. Ross et al. were the first who
described the relative quantification of allele-specific extension products in
mixed DNA by MALDI and found that frequencies down to 5% could be
accessed routinely . Further studies demonstrated a limit of detection of
about 2%and a standard deviation of 2–3%for allelefrequenciesbetween 10%
caused by unbalanced amplification during PCR. When allele frequencies
determined from the pooled samples were compared to the real frequencies
factors like pool preparation or preferential DNA amplification. Skewed dis-
tributions of two alleles are another frequent phenomena in individual DNA
samples and is caused by unbalanced amplification during PCR and different
incorporation rates of ddNTPs during primer extension. This deviation from
the expected 1:1 ratio has been reported for natural and mass-modified termi-
nators  and can be calculated into a correction factor which improves allele
frequency estimation from DNA pools . Several comparative pooling
studies have shown that multiplex primer extension by MALDI-TOF-MS
represents an accurate and cost-effective technique for allele frequency estima-
tion in DNA pools. Results by MALDI performed as well as or better than
RFLP, Pyrosequencing, Taqman, SNuPe, or SNaPshot, and all methods were
suitedfor determinationofSNP allelefrequenciesinDNA pools [119–121]. As
first genome-wide study, Buetow et al. applied allele-frequency estimations by
MALDI-TOF-MS in DNA pools and genotyped 9000 SNPs of 95 individuals
. Other groups have confirmed thatthisapproach allows for identification
of genetic contributors to complex diseases [122–125].
Recently, the quantitative feature of MALDI-TOF-MS and its high sensi-
tivity and specificity when combined with PCR allow the detection of genetic
variants in fetal DNA from maternal plasma and make MALDI a valuable
tool for noninvasive prenatal diagnosis [126,127]. During pregnancy, fetal
DNA amounts to 3–6% of the total DNA in maternal plasma [128,129].
Several studies have described that detection of fetal point mutations by
conventional PCR-based assays is difficult due to the high amount of mater-
nal DNA sequences [127,130]. Ding and coworkers were the first showing
that fetal single gene differences could be correctly discriminated in maternal
plasma using MALDI-TOF-MS for typing of b-thalassemia mutations .
In addition to the MassEXTEND assay, they combined MALDI with the
MEYER AND UELAND
so-called single allele base extension reaction (SABER) protocol. The advan-
tage of the SABER assay over the MassEXTEND is higher sensitivity, as
only the fetal-specific allele is extended in the primer extension reaction. The
SABER assay has also been successfully proven for noninvasive fetal blood
group genotyping to determine fetal RhD status with respect to improved
management of RhD-negative pregnant women . In addition to single
point mutations, the MassEXTEND assay can also be used in noninvasive
detection of CNVs in fetal DNA for the identification of abnormalities in
chromosome numbers as Trisomy 21 [133,134]. Enrichment of cell-free fetal
DNA by size fractionation can be applied prior to MALDI analysis in order
to increase assay sensitivity . On the basis of these assays, Sequenom has
recently launched a commercial prenatal screening technology called SEQur-
eDX , which will facilitate diagnosis of high-risk familial disorders by
typing both fetal DNA as well as fetal RNA in maternal blood. Besides
prenatal diagnosis, detection of free circulating DNA may also enable new
methods for monitoring of cancer, diabetes, trauma, and stroke .
During the past years, MALDI-TOF-MS has been proven to be a versatile
tool for rapid multiplex genotyping in different field of applications from
pharmacogenetic and genomics [138–141], disease association studies [125,
142–147], clinical diagnostic testing [14,15,140,148], controlling in agricul-
ture [149,150] and breeding [151–154], and bacterial and viral typing
[155,156]. Determination of the molecular mass offers high flexibility in
assay design for investigation of SNPs and other polymorphic sequence
variants by analysis of DNA and RNA fragments, using commercial systems
from Bruker or Sequenom, or custom-built platforms. Especially, when
combined to primer extension assays, MALDI has been demonstrated to
provide highly accurate and robust genotyping.
Although MALDI-based assay have been successfully applied in whole-
genome scans for discovery of susceptibility genes, the method has lost its
position in the past years. Multiplexing of about 40 SNPs per reaction
enables throughput levels of 150,000 genotypes per day, which have difficul-
ties to compete with levels of up to one million genotypes per sample
generated by commercially available microarray genotyping technologies
from Affymetrix or Illumina, respectively . Even, if the level of multi-
plexing could be doubled by optimal utilization of the mass range accessible
by nowadays instruments, assay throughput by MALDI-MS remains on a
low level. As a complementary method to array and bead-based technolo-
gies, the advantage of MALDI-TOF-MS is the high throughput of up to
GENOTYPING BY MALDI-MS
3840 individual samples per day. This makes MALDI-TOF-MS highly suit-
ed for fine-mapping studies and replication of findings from genome-wide
scans and an interesting alternative to other medium multiplexing platforms
as SNPlex from Applied Biosystems.
Due to the decrease in price for genotyping of down to actually 1–10 cents
per SNP , platforms can only compete by implementation of new applica-
tions, improvement of assay efficiency to decrease genotyping costs, and
development of simple-to-use/low-maintenance equipment. Several more
demanding MALDI-based applications have been developed in the recent
years for genomic research using its high sensitivity and ability to quantify.
Besides the simple genotyping of SNPs, gene expression analysis , DNA
methylation analysis [158,159], and resequencing  demonstrate the po-
tential of MALDI-TOF-MS as flexible technique for DNA/RNA analysis.
Assay costs could be further reduced by development of new tools for design
of more efficient high-level multiplex PCRs as well as by reduction of sample
volumes as already demonstrated by Sequenom’s iPLEX and SpectroCHIP
techniques. Today PCR and primer extension reaction take place in 5 ml
volumes while only 10 nl are require for MALDI analysis. Integration of
the PCR and primer extension processes into microfluid devices  or
directly onto the MALDI-chip  could further lower sample volumes
and would increase the level of automation. Ideally, primer design, sample
processing, and analysis will merge together in one instrument to facilitate
fully automated genotyping. Development of one-push-button systems
on the basis of compact platforms as Bruker’s Microflex, Sequenom’s
MassCOMPACT, or Waters’s microMX will strengthen the position
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