Maldi Mass Spectrometry Detection of Oligonucleotides

7/30/2019 Maldi Mass Spectrometry Detection of Oligonucleotides

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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


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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.



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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: [email protected]

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Maldi Mass Spectrometry Detection of Oligonucleotides

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