OTHER INSTRUMENTAL METHODS 149
sequences in proteins and nucleotide sequences in oligonucleotides can be
determined. Protein folding can be monitored by hydrogen/deuterium (H/D)
exchange. The principle applied to protein folding analyses is that backbone
amide nitrogens undergo rapid H/D exchange when they are exposed to
solvent (unfolded domains) but much slower exchange when buried in a
hydrophobic protein domain (folded). For instance, researchers have designed
methods for monitoring solvent accessibility of protein – ligand and protein –
protein interfaces using mass spectrometry.^57 The H/D exchange reaction must
be carried out with the isolated proteins as well as with the protein complex
in the protein – protein interface experiment. The exchanging regions for the
isolated versus the combined proteins are then compared. If a region is buried
by protein – protein binding, the amides in this region will be protected and
exchange slowly, revealing the interaction interface. Subsequently, the data can
be used as input for a computer docking simulation to build a computer model
of the protein – protein complex.
The instrumentation necessary for mass spectroscopic analysis of biomole-
cules does not differ substantially from that used for small organic molecules.
The mass spectrometer must have the same three fundamental parts: ioniza-
tion source, analyzer, and detector. However, the parts may be more sophisti-
cated and different from the normal LC – MS or GC – MS spectrometer. For
biomolecule analyses, the ionization source usually will be one of two types:
(1) electrospray, known as ESI; or (2) matrix - assisted laser desorption, known
as MALDI. The ESI source is well - suited to analysis of polar molecules of
molecular mass 0.1 – 10^3 kDa. Nanospray ionization is a low - fl ow - rate version
of ESI that allows use of small sample sizes and multiple experiments with
limited sample supplies. MALDI is based on bombardment of sample mole-
cules with a laser to bring about sample ionization. In this method, the sample
is mixed with an excess of a highly absorbing matrix compound. The matrix
transforms the laser energy into excitation energy that is then transferred to
the sample molecules. Once the sample molecules have been ionized, they are
transferred into the analyzer region of the spectrometer where they are sepa-
rated according to their mass ( m ) - to - charge ( z ) ratio ( m / z ). (See below for
more details about them / z ratio.) Some mass analyzers currently used in bio-
molecular applications include (1) quadrupole, (2) time - of - fl ight (TOF) mag-
netic sector, (4) Fourier transform quadrupole ion trap, and (5) Fourier
transform ion cyclotron resonance (FT - ICR). Quadrupole analyzers routinely
analyze samples withm / z ratios up to 3000. This is useful for the ESI source
as this ionization method produces ions withm / a ratios of < 3000. The TOF
analyzer depends on mass, with the smallest masses reaching the detector fi rst.
TOF is used for kinetic studies, for fast reactions, and for methods interfaced
with chromatographs for sample separation preceding mass spectrometry. The
magnetic sector analyzer depends on changes in voltage (and thus the mag-
netic fi eld surrounding the individual ions). Each of the mass analyzers men-
tioned here has a different range of coverage or accuracy for m / z ratios,
molecular mass determination, and achievable resolution. All analyzers