electrospray ionization(ESI) mass spectrometry (MS). Some of the current clinical applications of MS
are (a) DNA sequencing,^42 (b) genotyping^43 and detection of genetic variations,^44 microsatellites,^45 short
tandem repeats,^46 small insertions/deletions and (c) gene expression.
11.6.1 Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry
Matrix-assisted laser desorption MS was introduced for proteins by Karas and Hillenkamp in 1988.^47 The
nucleic acid sample is embedded in a crystalline matrix of a small molecule (e.g.3-hydroxypicolinic acid).
This target is excited by a pulse from an ultraviolet laser beam that, in high vacuum, results in intact molecules
of the sample becoming desorbed into the gas phase and ionized by the UV radiation to give (mostly) singly
charged ions. The resulting molecular ions are analysed by TOF MS, which sorts them by mass and thus
removes any need for prior separation of mixtures. A resolution of 1 per 1000 and the detection of low femto-
mole quantities of DNA can be achieved routinely.^48 Oligonucleotides ranging from 2 to 2000 nucleotides are
detected readily.^49 A typical MS measurement including acquisition and spectral interpretation takes10s.
This is the preferred method for analysis of oligonucleotides up to mass 15,000Da, i.e.approximately 50-
mers. For example the MALDI-TOF spectrum of a crude oligonucleotide synthesised by solid-phase DNA
synthesis (Section 4.1) is shown in Figure 11.17.
11.6.2 Electrospray Ionization Mass Spectrometry
In electrospray ionization MS, samples are ionized directly from micro-drops of solution and, as a result of
their multiple charge, high mass nucleic acid molecules or protein-nucleic acid complexes can be observed
at relatively low mass to charge (m/z) ratios. Several developments, especially time-of-flight analysers,
nanoflow sampling and capillary gel electrophoresis interfacing, have greatly enhanced the performance
of ESI MS. The advent of Fourier transform ion-cyclotron resonance (FT-ICR MS) has provided mass reso-
lution in the range 10^4 –10^6. While ESI MS is slower than MALDI-TOF and is more sensitive to salt in
the sample, it is the method of choice for studying species of mass up to several million Daltons. For example,
the negative mode ESI mass spectrum of the oligodeoxyribonucleotide d(GCG TTC CCC CTT TGC G)
450 Chapter 11
Figure 11.16 Representative force-extension curves of linear double-stranded DNA fragments recorded in 10 mM
Tris buffer containing 1 mM EDTA and 150 mM NaCl (black) and the same buffer containing the
intercalating drug proflavine (ca. 3mg ml^1 ) (grey)