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. 2 177
termining molecular masses, it can be used with great profit
for unambiguous determination of subunit identity by their
masses.
In the previously mentioned study of Hc from a hydrothermal
vent crab, subunit composition was also determined
[74]. In both experienced conditions (hypoxia and hyperoxia),
the same 4 polypeptide chains were detected. However,
when comparing MaxEnt deconvoluted spectra from
the two samples, a change in the abundance of one subunit
can be observed: the 75 541 Da subunit is more abundant
after hyperoxia. Since it has often been demonstrated that an
oligo-hexameric state of arthropod Hc can depend on the
presence or absence of certain subunit types, the observed
change in the 75 541 Da subunit abundance could be suggested
as a cause for the change in aggregation states within
such a short period observed in the same experiment. Once
again, even if interindividual variations could still have interfered
with condition-induced variations, this results shows
that a small change in subunit abundance can be detected by
ESI-MS and encourage to perform such analysis on individual
samples. This is made possible by the high sensitivity of
the method and would permit to distinguish between interindividual
variability and reproducible trends induced by experimental
conditions. ESI-MS is a powerful method to explore
subunit plasticity for these complexes, considering the
similarities existing between subunits and the need to identify
them precisely.
CONCLUSION: WHICH QUESTIONS DO MALLS
AND ESI-MS HELP TO SOLVE ?
All the results presented here clearly reveal the complementarities
of ESI-MS and MALLS analysis to investigate
the structure of noncovalent multimeric complexes such as
HBL-Hb and Hc.
Indeed, the molecular masses of native proteins determined
by light scattering systems agree well with known
molecular masses based on gel permeation chromatography
or analytical centrifugation, and LS technologies can be alternatives
to these conventional methods. In theory, SEC-
MALLS provides the capability of determining the “absolute”
molecular masses of proteins and their protein-protein
complexes as it is in solution. Molecular masses determined
by SEC-MALLS depend only on the readings obtained from
the downstream MALLS and refractive index (RI) detectors
and not on the SEC elution position [38-41]. As for analytical
centrifugation, the resulting average mass is independent
of any external calibration. Other advantages of
SEC-MALLS are that it is a “non-invasive” technique without
incorporation of a radioactive or fluorescent tag and it is
non-destructive. The sample may thus be recovered for use
in subsequent studies. Compared with techniques such as
analytical centrifugation, SEC-MALLS is more rapid and
samples may be analyzed easily at various pH values, ionic
strengths, and temperature and in the presence or absence of
ligands. SEC-MALLS is a particularly useful tool for studying
homoassociations and heteroassociations of proteins and
other biological macromolecules [38-41, 137] as illustrated
with AmHb dissociation/reassociation mechanism. In conclusion,
SEC-MALLS offers a powerful tool for characterization
of the biophysical properties of such proteins and their
biologically relevant complexes in solution. MALLS and
analytical centrifugation are the techniques the most likely to
keep molecules in their native state, in particular for fragile
assemblies such as annelid HBL-Hb in which the central
piece may be lost by other techniques.
Concerning protein conformation, the MALLS system
can measure the rms gyration radius (Rg) of globular proteins
but values are generally below the angular variation
detection limit of 10 nm, making the determination unreliable.
Conformational changes in the protein molecules during
aggregation/denaturation can be predicted from these
measurements. More accurate measurements of the conformation
of these molecules would require the determination
of additional parameters such as hydrodynamic radius (Rh)
and are limited by difficulties in accurately measuring Rg of
these globular proteins by MALLS.
Even if MALLS gave plausible information on the mass
of the native pigment in solution, it lacks the resolution capability
to test for the existence of similar-sized isoforms.
This can be achieved with a variety of mass spectrometric
(MS) approaches. Indeed, MS can be used to determine molecular
masses more accurately than by SEC-MALLS, but
the ESI process may break fragile noncovalent interactions
and the reliability of the method must be tested first. The
good agreement between MALLS and ESI-MS results suggests
that the interactions maintaining the complex are conserved
during ionization. Thus the species observed by ESI-
MS are likely to be representative of the species in solution
and the high mass accuracy of the method can be used to
distinguish between very close isoforms.
The utility of MS for detection and quantification of noncovalent
protein-protein interactions under native, equilibrium
conditions in solution can be limited by several factors.
Although the m/z range of the time-of-flight analyzer is
theoretically unlimited, in practice it is often difficult to detect
larger proteins and noncovalently bound macromolecular
complexes in excess of 100kDa as their flight through
technique used for improving the detection of these large
structures is collisional cooling [138-143]. However, this
seems to be protein dependent as revealed by the analysis of
hemocyanin complexes presented in this review. In this case,
ESI-MS in native mode enables to measure absolute mass of
native complexes similar to the species in solution. MALLS
has the advantage that it can be performed on-line with separative
techniques such as FPLC using a physiological buffer
as eluent, thus minimizing sample manipulation and possible
deterioration, but does not permit to distinguish isoforms.
When native masses can be determined by both techniques,
small differences (2 to 4 % for Carcinus maenas) are
often observed between MALLS and ESI-MS, with MALLS
masses often being superior to MS masses. Two hypotheses
can be proposed to explain this. First, as the two techniques
are based on different physical principles, the differences
could be inherent to the devices themselves and reflect no
real difference. On the other hand, one can consider that during
the ionization process in ESI-MS, labile solvent molecules
and ions could be separated from the protein complex
during vaporization in the nebulization chamber, while the
MALLS measurement could take into account divalent
cations or other physiological adducts and solvating layer
98