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314 Heegaard et al.
Especially useful for applications involving binding interactions should be
information-rich detector systems such as mass spectrometry (MS) and nuclear
magnetic resonance (NMR) spectroscopy, but the experience with and practice
of CE-NMR (52) is still limited. CE-MS in the form of CE coupled with electrospray
ionization (ESI) mass analysers (see Note 3) (53,54) has been of utility
in affinity studies of proteins (55–59). Also, ionization on surfaces using laserdesorption
(MALDI) has been CE-interfaced (60), but ESI is suitable for on-line
work and is more commonly used. The major issue is the junction between the
separation capillary and the spray capillary/needle and the CE-buffer compatibility
with the ionization process (61–63). Three general types of CE-ESI-MS
interfaces have been developed: the sheathless interface, the liquid junction or
split-flow interface and the more commonly used coaxial sheath-flow interface.
Buffers for CE-MS applications are typically 10–30 mM aqueous high vapour
pressure (volatile) acids such as formic and acetic acid or aqueous ammonium
acetate or ammonia for positive and negative ionization modes, respectively
(53). These types of buffers display minimal ionization suppression and adduct
formation, but are not very well suited for working with separations in the pH
4–8 range. Although sheath–flow interfaces are relatively simple, the sheathless
interfaces give higher detection sensitivity (see Note 4). However, they may
be technically demanding (53). Split–flow interfaces (54), however, overcome
the problems with analyte dilution, decrease in resolution, intricate fabrication
and bubble formation inside capillaries associated with the other types of
interfaces.
Evolving CE–detector combinations of potential utility for ACE of proteins
in addition to NMR (64,65) include Fourier transform infrared spectroscopy
(66), Raman spectroscopy (67,68), flame-heated furnace atomic absorption
spectrometry (69), electrothermal atomic absorption spectroscopy (70), X-ray
(71) and surface plasmon resonance (72,73).
4. Discovery and Mapping of Ligand-Binding Sites
If a given protein has a well-defined ligand-binding function, CE may be
used as an adjunct technique to map binding site(s) in that protein. For linear
binding sites, the standard approach will be to cleave the protein into tryptic
fragments and then perform CE peak profiling in the presence and the absence
of ligand in the electrophoresis buffer. In Fig. 3, the approach is shown with
serum amyloid P component and its ligand heparin (see Note 5). Changes
in the tryptic digest peptide peak profile are indicative of ligand interactions,
and after identification of ligand-binding peptides – e.g. by CE-MS or by
purification by HPLC followed by MS and spiking analysis by CE – the
identified peptide may be purified or synthesized and quantitative binding