Post on 04-Apr-2018
7/30/2019 Maldi Mass Spectrometry Detection of Oligonucleotides
1/3
GENOMICS
PRINCIPLEOF MALDI MASSSPECTROMETRY
Matrix-assisted laser desorption/ionisa-
tion (MALDI) mass spectrometry can
be efficiently applied for high-resolution
detection of oligonucleotides in the mass
range of about 1000-10000 Mr. The prin-
ciple of this detection method is illus-
trated in Figure 1. MALDI is routinelyused for evaluation of oligonucleotide
production. Moreover, it is applied for
accurate, high-throughput genotyping of
DNA markers such as single nucleotide
polymorphisms (SNPs), as well as detec-
tion of cytosine methylation in genomic
DNA and for quantitative gene expres-
sion analysis [1, 2]. In contrast to widely
used optical detection methods based
on fluorescence, no labelling of analytes
is required. MALDI mass spectrometry
detects an inherent physical property of
analyte ions such as oligonucleotides,namely the mass-to-charge ratio [2].
Therefore, a number of oligonucleotides
with different masses can be detected
simultaneously by MALDI.
In MALDI co-crystals formed by organic
molecules called the matrix and nucleic
acids are irradiated with a pulsed laser at
a wavelength close to a resonant absorp-tion band of the matrix. Subsequently, this
desorption process generates an energy
transfer when analyte and matrix mol-
ecules are evaporated into the gas-phase.
This triggers a proton-transfer reaction
of matrix and analyte molecules, which
mainly leads to singly charged oligonucle-
otide ions. However, the energy transfer and
protonation can also affect the oligonucle-otides themselves thus leading to fragmen-
tation [3]. The chemo-physical characteris-
tics of MALDI are not well understood, but
empirical findings resulted in developments
in this detection method, and a number of
suitable matrix-analyte preparation proce-
dures were developed. 3-hydroxypicolinic
acid, which results in little fragmentation of
MALDI mass spectrometry
detection of oligonucleotides
Matrix-assisted laser desorption/
ionisation mass spectrometry
(MALDI) is an important analyti-
cal technique in functional genom-
ics and proteomics. In this short
review essential information on
sample preparation of oligonu-
cleotides is presented, and recentadvances are discussed.
by Dr Sascha Sauer
BTi February/March 2007 11
Figure 1. Illustration of the principle of MALDI mass spectrometry. Matrices for MALDI
are deposited on flat conductive plates. Stainless steel, aluminium-nickel alloys, gold or
silicon are commonly used plate materials. The target plates are introduced in the ionisa-
tion chamber of the instrument. Oligonucleotides and matrix molecules are evaporated
into the gas-phase by a pulsed ultraviolet laser at a wavelength close to a resonant absorp-
tion band of the matrix. The gas-phase is a high vacuum of about 10-7mbar. Nitrogen
lasers with an emission maximum at 337 nm or frequency-tripled Nd:YAG lasers with
an emission maximum at 355 nm are mainly used for MALDI. After desorption, pro-
ton-transfer reactions take place leading to ionisation of matrix and analyte molecules.
Resulting ions are accelerated by an electric field. Mass analysis is commonly performed
by time-of-flight (TOF) separation. Thereby ions are guided into a flight-tube with a
vacuum of about 10-9 mbar. The ions are separated according to their mass-to-charge
ratios. Finally, the ions are detected by a microchannel plate device. The sensitivity of
current MALDI mass spectrometers is in the range of about 100 amol per analyte, and at
least one spectrum a second can be recorded. The scale of this figure is not proportional to
the instrumental set-up. The illustration was re-printed with permission from [5].
BTI_FEB_MARCH_07_P11-13.indd 1 7/03/07 12:06:5
7/30/2019 Maldi Mass Spectrometry Detection of Oligonucleotides
2/3
BTi February/March 2007 12GENOMICS
DNA, is currently the gold standard matrix
for sensitive MALDI analysis of nucleic
acids [4].
Analyte ions are accelerated by an elec-
tric field and guided by ion optics into amass analyser. Mass analysis in MALDI
is mainly performed by determination
of the time-of-flight (TOF) of an ion.
Variations in matrix preparation can
result in a shift of the starting position,
which affects TOF determination and
eventually gives a few Daltons of mass
variation from one analysis to another.
DNA SAMPLEPURIFICATIONMost of the protocols in molecular biol-
ogy involve buffered reaction solutions
that potentially contain salts, denaturants(e.g. urea) and detergents, such as Tween-
20. However, sample impurity severely
affects matrix-analyte co-crystallisation
and thus MALDI detection. Furthermore,
MALDI analysis of nucleic acids is par-
ticularly complicated by their negatively
charged sugar-phosphate backbones that
interact with sodium and potassium
counterions. Resulting adducts signifi-
cantly limit mass signal intensity and res-
olution. The affinity of alkali salt ions to
nucleic acids is high, but does not result
in complete saturation. Thus, efficientsample purification of DNA samples is
crucial in order to generate high quality
MALDI mass spectra [5].
Liquid chromatography could be applied
to purify samples for MALDI, but this
technique is too time-consuming for
genomic applications. More popular
approaches are parallel off-line proce-
dures, such as sample purification by
ethanol precipitation, dialysis, gel filtra-
tion or application of reversed-phase
materials [4]. Alternative procedures usesurfaces coated with streptavidin, which
requires DNA to be biotinylated. The
usual approach is the simple addition of
cation-exchange resins to oligonucleotide
samples, thereby replacing alkali ions by
hydrogen or ammonium ions that evapo-
rate during desorption as ammonium
gas. Although this method is automatable
and cost-efficient, it cannot efficiently
purify DNA samples from detergents.
Other purification protocols allow sam-
ple preparation directly onto a surface
with nucleic acid-binding properties thatis also suitable for MALDI detection. For
example, we have recently developed gold
microscope slides, which were coated
with dendrimer molecules containing
primary amino groups that interact with
negatively charged oligonucleotides [6].
We deposited and dried oligonucleotides
onto these slides and simply washed the
slides in a vessel filled with pure water.
After drying the slides we could detect
the purified oligonucleotides efficiently
by MALDI mass spectrometry. This pro-
cedure was applied for SNP genotypingapplications.
Oligonucleotide modification chemis-
try is another strategy used to improve
MALDI detection, while avoiding strin-
gent purification of DNA samples. For
example, the DNA charge-tagging con-
cept focused on the chemical difference
between nucleic acids and peptides. Many
peptides are uncharged, while DNA car-
ries as many negative charges as phos-
phate linkages. Charges from the DNA
linkages can be neutralised by replacingphosphates by phosphorothioate groups
and methylating them. The idea was to
generate a product with a single positive
or single negative charge, thus relying
on the matrix for desorption, but not
for ionisation. -Cyano-4-hydroxy-cin-
namic acid methyl ester is a matrix that
is tailored for the charge-tagged DNA
products carrying one fix charge. The
charge tag principle has been exten-
sively applied for the purification-free
GOOD assays for high-throughput SNP
genotyping in candidate genes [7].
MATRIXPREPARATIONMETHODSThe two most common MALDI matrix
preparation methods are called thin-
layer and dried droplet preparation.
For thin-layer preparations, low-microli-
tre volumes of matrices, such as -cyano-
4-hydroxy-cinnamic acid methyl ester
dissolved in a volatile solvent like acetone,
are deposited with a pipette or a liquid
handling robot onto the MALDI target
plate. The acetone spreads and evaporates
immediately, leaving a thin layer of smallmatrix crystals. The DNA sample is dis-
pensed onto the thin-layer in an organic
solvent such as 40% acetonitrile. DNA
molecules are then built into the surface of
the dry matrix. Alternatively, oligonucle-
otides are firstly spotted onto the surface
and the matrix is then applied.
For dried droplet preparations a matrix
solution, such as 3-hydroxypicolinic acid
in ammonium citrate, is mixed with
an oligonucleotide sample and subse-
quently deposited onto the target plate.Alternatively, the matrix can be spotted
onto the surface and oligonucleotides
added to the dry matrix thereby re-dis-
solving it. This mixture is then allowed
to dry again. DNA sample and matrix
can also be deposited in reverse order.
In general, dried droplet preparations
in microlitre volumes result in sweet
spots. Certain positions on the co-crystal
show better mass signals than other posi-
tions. This may be overcome by extensive
searching for sweet spots or by min-
iaturisation of the sample preparationto nanolitre volumes allowing complete
matrix-analyte desorption by the laser.
The company Sequenom (http://www.
sequenom.com) has developed nanodis-
penser robots to deposit low nanolitre
volumes of DNA products onto silicon
dioxide chips that are covered with an
array of ~4 nL dried 3-hydroxypico-
linic acid matrix spots [8]. The com-
pany Bruker Daltonics (http://www.bdal.
com) has developed anchor-chip tar-
gets, which are coated with hydrophobic
Teflon and up to 1536 hydrophilic spots[9]. After solvent evaporation, matrix and
DNA samples are concentrated because
of the strong water repellent nature of
the Teflon surface. This yields an up-
concentration of more than two orders
of magnitude during crystallisation com-
pared to a conventional preparation on a
flat target plate.
BTI_FEB_MARCH_07_P11-13.indd 2 7/03/07 12:06:5
7/30/2019 Maldi Mass Spectrometry Detection of Oligonucleotides
3/3
BTi February/March 2007 13GENOMICS
OUTLOOKMALDI mass spectrometry is a key bioan-
alytical technique in proteomics and genomics
[10]. Furthermore, due to its accuracy MALDI
has great potential for applications combiningdiagnostic risk profiling at the level of both
DNA and proteins. More efficient robotic sys-
tems, which enable fast and flexible disposal
of nanolitre droplets of matrix and samples,
could significantly improve MALDI detection
of nucleic acids with regard to spectra quality,
assay reproducibility and sample throughput.
Future developments in mass spectrometry
might also focus on the development of instru-
ments that could allow sensitive analysis of
molecules larger than oligonucleotides, for
example PCR products.
REFERENCES1. Jurinke C, Denissenko MF, Oeth P, Ehrich M, van
den Boom D, Cantor CR. A single nucleotide poly-
morphism based approach for the identification
and characterization of gene expression modulation
using MassARRAY. Mutat Res 2005; 573: 83-95.
2. Sauer S. Typing of single nucleotide polymor-
phisms by MALDI mass spectrometry: principles
and diagnostic applications. Clin Chim Acta 2006;
363: 95-105.
3. Christian NP, Reilly JP, Mokler VR, Wincott FE,
Ellington AD. Elucidation of the initial step of oli-
gonucleotide fragmentation in matrix-assisted laserdesorption/ionization using modified nucleic acids.
J Am Soc Mass Spectrom 2001; 12: 744-753.
4. Wu KJ, Steding A, Becker CH. Matrix-assisted
laser desorption time-of-flight mass spectrometry
of oligonucleotides using 3-hydroxypicolinic acid
as an ultraviolet-sensitive matrix. Rapid Commun
Mass Spectrom 1993; 7: 142-146.
5. Sauer S. The essence of DNA sample preparation
for MALDI mass spectrometry.J Biochem Biophys
Methods 2006 Oct 21; [Epub ahead of print].
6. Kepper P, Reinhardt R, Dahl A, Lehrach H, Sauer
S. Matrix-assisted laser desorption/ionisation mass
spectrometry analysis of DNA on microarrays. ClinChem 2006; 52: 1303-1310.
7. Sauer S, Reinhardt R, Lehrach H, Gut IG.
Single-nucleotide polymorphisms: analysis by
mass spectreometry. Nature Protoc 2006; 1:
1761-1771.
8. Little DP, Cornish TJ, ODonnell MJ, Braun
A, Cotter RJ, Koster H. MALDI on a chip:
analysis of low-to subfemtomole quantities
of synthetic oligonucleotides and DNA diag-
nostic products dispensed by a piezoelectric
pipette. Anal Chem 1997; 69: 4540-4546.
9. Schuerenberg M, Luebbert C, Eickhoff
H, Kalkum M, Lehrach H, Nordhoff E.Prestructured MALDI-MS sample supports.
Anal Chem 2000; 72: 3436-3442.
10. Sauer S, Lange BM, Gobom J, Nyarsik L,
Seitz H, Lehrach H. Miniaturization in func-
tional genomics and proteomics, Nature Rev
Genet 2005; 6: 465-476.
THEAUTHORDr Sascha Sauer,
Max Planck Institute for Molecular
Genetics,
Department of Vertebrate Genomics(Prof. H. Lehrach),
Ihnestrasse 73, 14195 Berlin,
Germany
Tel +49 30 84131565
e-mail: sauer@molgen.mpg.de
"$"IO#OAT!NGIOGENESIS3YSTEMS3TANDARDIZEDs1UANTITATIVEs!UTOMATIONCOMPATIBLE
"$"$,OGOANDALLOTHERTRADEMARKSARETHEPROPERTYOF"ECTON$ICKINSONAND#OMPANY"$!
"$"IOSCIENCES4EL&AXHELPBIOSCIENCESEUROPEBDCOM
BDBIOSCIENCESCOM
3TANDARDIZEDINVITROASSAYSFORRAPIDQUANTITATIVEANALYSISOFANGIOGENESISANDCONVENIENTSCREENINGOFANGIOGENESISMODULATINGCOMPOUNDS
s2EADYTOUSEASSAYSFORENDOTHELIALCELLMIGRATIONINVASIONANDTUBEFORMATIONs/PTIMIZEDANDSTANDARDIZEDFORMAXIMUMREPRODUCIBILITY
s2EALTIMEDETECTIONOFINVASIONANDMIGRATIONWITH"$&LUORO"LOKMEMBRANE
s!UTOMATIONCOMPATIBLESCREENINGFORPROANDANTIANGIOGENICCOMPOUNDS
s0REQUALIlEDPRIMARYENDOTHELIAL"$(56%#CELLSTOENSUREASSAYPERFORMANCE
s.%7"$&LUORESCENT$YESFORLIVECELLLABELING
Go to Hotline www.biotech-online.com & tick 11507
BTI_FEB_MARCH_07_P11-13.indd 3 7/03/07 12:06:5