maenas (intertidal zone) and Segonzacia mesatlantica - Station ...

The Structural Analysis of Large Noncovalent Oxygen Binding Proteins Current Protein and Peptide Science, 2008, Vol. 9, No. 2 165
below). Crossed immunoelectrophoresis suggested that a
particular type of subunit, subunit beta, is necessary for dodecamer
assembly in most species, but that a few groups
have independently evolved the ability to produce dodecamers
from the other subunit types [106].
The quality of the MALLS analysis depends on the efficiency
of the separation process: two badly-resolved molecular
species will yield an average molecular mass with high
polydispersity. In addition, a limitation of the MALLS
method is that complexes with the same number of subunits
but of slightly different types will differ only very slightly in
mass. They cannot be resolved by SEC and thus MALLS
will only give an average mass for all complexes of the same
order. ESI-MS can provide further characterization in this
case.
ESI-MS has also been used recently for investigation of
crustacean Hc. Noncovalent ESI-MS can resolve different
molecular species in hemolymph without any prior separation.
However, the sample preparation is a critical step in the
ESI-MS procedure and Hc samples are desalted thoroughly
in 10 mM ammonium acetate through at least 7 cycles of
concentration/dilution on centrifugal filters before analysis;
an incomplete desalting causes low signal-to-noise ratio.
Under the adjusted conditions of noncovalent ESI-MS
[62, 73] (see before), the different species are clearly visible
as several Gaussian peak distributions along the m/z axis as
presented in Fig. (3a). These distributions are well separated
between different aggregation states (hexamers versus dodecamers);
for a given aggregation state several close distributions
are sometimes detected, corresponding to a slight mass
difference. In Fig. (3a), two distributions are observed for
hexamer (a,b) and dodecamer (c,d). As previously noted,
such discrimination is not possible using the average mass
determined by MALLS because of the very close mass and
coelution of these complexes. Thus ESI-MS is a complementary
method enabling thorough investigation of the complex
native masses.
In addition to being a technique allowing high mass accuracy
measurements, ESI-MS can also detect species present
in low amount or hardly or not resolved by MALLS due to
coelution of different complexes. In Fig. (3a) a higher aggregate
is observed with a mass of 1 374 136 Da (18-meric
form). Higher masses corresponding to 24-mers and even
30-mers can be also be found [62]. It is known that aggregation
can occur in the ESI when sample concentration is too
high but it is unlikely that the assemblies observed here
could be artifacts at least for the 18-mer as it can be observed
by MALLS under low flow rate (0.25 ml/min, personal observation).
However, the very low detected amount of these
“unusual” complexes raises the question of their biological
significance ; since no study by electron microscopy was
performed on them, it is still possible that they arise from
random post-translational cross-linking.
For such high masses, charge state assignation may be
difficult due to peak enlargement and the consecutive proximity
of standard deviation values calculated for subsequent
charge states [62, 74]. Even the use of deconvolution programs
like MaxEnt cannot resolve the problem. The precision
of the determined masses decreases with increasing
masses but is nonetheless sufficient to unambiguously determine
the number of subunit for each distribution as the
uncertainty is inferior to the subunit average mass.
Fig. (2). SEC-MALLS analysis obtained for a Rimicaris exoculata hemolymph sample.
Analysis was performed with a Superose 6-C column. Optical density at 280nm (full curve) and estimated molecular weight (dots) are represented
as a function of the eluted volume. Insert: cumulative molecular weight curve (reprinted from [105], with permission).
86