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Figure 1. Analytical system suitable for nucleic acid analysis by ion-pair reversed-phase liquid chromatography to electrospray ionization mass spectrometry (ICEMS). 1, Low pressure binary gradient micropump; 2, solvent reservoirs; 3, degasser unit; 4, splitting tee-piece; 5, restriction capillary; 6,
autosampler; 7, injector; 8, needle; 9, dispensor; 10, column thermostat; 11, monolithic capillary column; 12, quadrupole/quadrupole/time-of-flight mass spectrometer; 13, electrospray ionization (ESI) source; 14, gas inlet; 15, ion optics; 16; vacuum system; 17, collision cell; 18, pusher; 19, reflectron; 20, detector.
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major parts: (i) the chromatographic
part and (ii) the mass spectrometric
part. Electrospray ionization (ESI) in
negative ion mode is used to hyphenate
the two methods and to transfer the
charged nucleic acids from solution
into the gas phase. The analytically
useful information is solely gathered
from the mass spectrometric part.
Either the molecular mass of an intact
molecule or the masses of fragment
ions obtained from a tandem mass
spectrometric experiment give a deep
insight into the composition and/or
the sequence of a nucleic acid species.
In general, sequence variations are
detected with high sensitivity by
ICEMS (34). While other indicators
like retention or migration times are
influenced by experimental conditions, the molecular mass is an intrinsic
property that is independent from the
physical environment.
The success of the mass spectrometric analysis largely depends on the
purity of the nucleic acid molecules
introduced into the mass spectrometer.
Due to partial substitution of protons
in the sugar-phosphate backbone by
variable numbers of cations, such as
sodium, potassium, or magnesium,
the multiply deprotonated molecules
are dispersed among several different
species, resulting in highly complex
spectra and poor detection limits.
Chromatography represents one of the
most efficient methods to purify and
desalt nucleic acids prior to their mass
spectrometric characterization (35).
The advantageous feature of ESI
the transfer of intact, doublestranded PCR products from solution
into the gas phaseis disadvantageous
in the context of genotyping. Base
substitutions are difficult to identify in
double-helical DNA by molecular mass
measurements, because A/T and G/C
base pairs have very similar average
masses of 615.4 atomic mass units
(amu) and 616.4 amu, respectively, and
A/T or G/C substitutions do not cause
any mass shift at all. Denaturation of
double-stranded PCR products into
the corresponding single strands prior
to their mass spectrometric characterization is advantageous because
of the division in half of the masses
of the detected species and the possibility to identify base substitutions by
Supplement to Vol. 43 No. 4 | 2007
Review
which have a retarding effect on new
developments.
Application of ICEMS for SNP
Typing
Figure 2. The steps involved in the analysis of nucleic acids by ion-pair reversed-phase liquid chromatography to electrospray ionization mass spectrometry (ICEMS).
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of eight selected individuals. Among
these samples, four were heteroplasmic
with C contents in the range of 1.9%
to 34.1%. To check the reliability of
these results, allelic proportions were
additionally determined by a cloning
assay. The results of the two assays
correlated well. In all cases, deviations
were obtained that were smaller than
5.4%. The observed assay performance
suggested that ICEMS represents one
of the most powerful assays for the
determination of allelic frequencies.
Another ICEMS-based assay was
developed for the characterization of
certain regions of the first and second
hypervariable segments (HVS-I and
HVS-II, respectively) of the mtDNA
control region (68). Both segments
contain polycytosine (C) tracts, which
display length heteroplasmy at a
substantial rate in the population. The
two sections were simultaneously
amplified and analyzed by ICEMS in 90
maternally unrelated mother-offspring
pairs. The findings were compared
with the results obtained by Sanger
sequencing of the PCR products.
Sequence electropherograms showed
a characteristic out-of-phase pattern
downstream of the heteroplasmic Cstretch regions. This is a well-known
phenomenon, which can make data
interpretation difficult in some cases.
Here, ICEMS offered the considerable advantage that length variants
were clearly separated. Deconvoluted
mass spectra resulted in easily distinguishable peak patterns directly related
to observed heteroplasmic fragment
lengths. Hence, treatment of mass
spectrometric data was a rather simple
task, which enabled the unequivocal
identification of the length variants.
Moreover, the quantitative information
inherently present in the obtained mass
spectra in the form of peak intensities
was used for the determination of
relative contents of heteroplasmic
length variants.
Recently, a rapid and informative
mtDNA profiling system has been introduced that is based on the analysis of a
23-plex PCR by ICEMS. The developed
assay can be utilized as a prescreening
technology complementing standard
control region sequencing to eliminate
multiple suspects from an inquiry or
to discriminate between stains in high
Supplement to Vol. 43 No. 4 | 2007
www.biotechniques.com BioTechniques xi
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ACKNOWLEDGMENTS
Review
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