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154 Current Protein and Peptide Science, 2008, Vol. 9, No. 2 Bruneaux et al.
tion mass spectrometry (ESI-MS) is well suited for the detection
of noncovalent protein complexes and opened a new
era in protein characterization due to the high accuracy of
molecular mass determination [50, 51].
Compared to MALDI, ESI is a gentle method of ionization-desorption
of the sample at atmospheric pressure. In the
last decades, it was successfully applied to biomolecules
when it was made possible to transfer macromolecules into a
gas phase. The inherently mild ionization has allowed the
successful examination of noncovalent interactions of proteins
with ligands, cofactors and prosthetic groups, including
peptides, with other proteins and of the quaternary structure
of proteins [5, 50-54]. Until ten years ago, observation of
large noncovalent multi-protein complexes in aqueous solutions
at neutral pH has been limited by the upper m/z range
of about 4000 available on most commercial mass spectrometers.
The development of orthogonal time-of-flight
(ToF) mass analyzers [55, 56] and their commercial exploitation
has dramatically extended the experimentally available
m/z range to at least 25000. At the present time, the most
appropriate mass spectrometers for studying proteins and
protein complexes under native conditions have a time-offlight
analyzer [56], with a theoretically unlimited m/z range
in addition to parallel detection ensuring high sensitivity
[55]. Hybrid instruments of the quadrupole-time-of-flight
geometry are also well-suited for the analysis of native proteins
and protein complexes [57]. In addition, nanospray
development [58] has allowed the introduction of small
amounts of sample and enabled the determination of the
masses of very large protein complexes such as GroEL [59],
the intact MS2 virus capsid [60] and ribosome [61].
Under standard denaturing conditions, the protein is dissolved
in a mixture of water and organic solvent such as acetonitrile
or methanol, with a trace of an added organic acid
(e.g. formic acid). The resulting solution has a pH~3.0. Under
these conditions noncovalent bonds are broken, the protein
sample is protonated and generates an m/z spectrum that
has a wide distribution of multiply charged ions from which
the mass of the protein can be calculated according to the
formula m /z = M + nH +
, where M is the molecular mass of
n
the protein and n is the number of protons associated with it.
The mass accuracy of the ESI-MS analysis under these denaturing
conditions is typically 0.01% of the protein mass using
a modern mass spectrometer. In addition, as ESI-MS
analyses proteins from a liquid phase, the solvent conditions
can be adapted so that the protein can be studied under more
physiological conditions, for example in water alone or more
typically in an aqueous, volatile buffer at pH 6-8. The use of
a buffered solution at neutral pH is a typical method permitting
preservation of the native state of the protein in solution,
which is required for successful analysis of noncovalent
macromolecular complexes. The solvent must not contain
too much inorganic salts which would result in low signal/noise
ratio, but must be of enough ionic strength to maintain
the assembly. Besides, the use of a buffer can add a variety
of metallic or other ions or small molecules to the analyte
that may complicate the spectra due to formation of adduct
with the protein. The choice of buffer and data interpretation
are thus of paramount importance [62]. The two most
commonly used buffers are ammonium acetate and ammonium
hydrogen carbonate, due to their pH range and volatility,
at concentrations of 1-100mM. In such cases, as the protein
remains in its native, folded state, the generated m/z
spectrum exhibits a narrower distribution of charge states. In
general the protein is less charged because protonation is less
efficient than in its denatured state.
Noncovalent analysis requires adjusted parameters of the
ESI source such as increased pressure in the nebulization
chamber, decreased declustering potential and extended m/z
detection range. For respiratory pigments, samples are usually
dissolved in aqueous ammonium acetate 10 mM, pH 6.8
[62]. It has been shown that the experimental masses decreased
slightly with increased declustering potential (60 to
160 V) and were generally 0.1 to 0.2 % higher than the calculated
masses, due probably to complexation with cations
and water molecules [63].
Such data not only allow the mass of the protein to be
calculated, but also enable an assessment of the protein’s
conformation to be made due to the preservation of its tertiary
structure during the analysis [64]. Co-populated protein
conformers can often be identified if each population gives
rise to a unique charge state distribution [5, 65, 66]. The
evaluation of quaternary structures such as noncovalently
bound macromolecular protein complexes and the interaction
of proteins with DNA, RNA and other ligands are also
within reach. The first examples of non-covalently bound
protein complexes monitored by ESI-MS were reported in
1991 [5, 52, 67], just two years after ESI-MS development.
Since then, there has been significant progress in this field:
many instrumental developments have been made and a
wealth of examples of quaternary structures increasing in
size and complexity has accumulated.
The high precision of the method also allows determination
of post-translational modifications such as glycosylation
[68, 69]. By using reducing conditions, occurrence of intraand
inter-chain disulfide bridges can be investigated and
monomers from covalent complexes identified. Carbamidomethylation
of free cysteine (Cys) can provide the number
of free Cys residues. Characteristic differences in successive
species masses can be linked with modifications such as
methylation, phosphorylation and others. As the intensity of
the detected signal depends on the ionization behavior of the
molecule, quantification from deconvoluted spectrum should
be approached with extreme care. No absolute quantification
can be made but abundance of identical species in different
samples can be compared. The technique is of great interest
when studying macromolecular complexes as it gives insight
into the subunit composition and their structural relation (disulfide
bridges) [70]. From the subunit masses models can be
constructed provided the macromolecular complex mass is
known. Noncovalent ESI-MS was recently successfully applied
to invertebrate hemoglobins [63, 71, 72] and to crustacean
hemocyanins [62, 73, 74].
An important issue in noncovalent ESI-MS is whether
the detected species are relevant to the species present in
biological conditions. Since different buffer conditions (e.g.
pH, ionic strength) are sufficient to induce dissociation or
aggregation in solution, the question of the effect of a trans-
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