protein sequence. Depending on the matrix, ISD fragments are
mainly b-, c-, y-, and z- ions. The 2,5-dihydroxybenzoic acid
(DHB) and super DHB matrices contribute to the N-terminal
validation, while the 1,5-diaminonaphthalene (DAN) matrix helps
in determining the C-terminal residues. Compare with the Edman
degradation, ISD has the advantage to detect both termini and in
presence of modifications. This method can confirm 10–50 residues from both sides of the intact protein. Due to the matrix
contribution below 800 Da, the first residues cannot be determined. This weakness can be overcome by using the
T3-sequencing approach or Pseudo-MS3, where selected ISD fragments are further fragmented (MALDI-TOF/TOF) [17]. Nowadays, the all procedure intact mass and T3-sequencing can be
performed in less than 1 h.
1.3 Homogeneity
By PDZ domain homogeneity measurements, we intend to measure if the domain is monomeric or oligomeric, the state of assemblies, and if the sample contains soluble high-order assemblies and
aggregates. There is no single technique that will answer all these
issues [18], as one of the challenges is the concentration required to
make the measurement given the small molecular mass of PDZ
domains.
1.3.1 Dynamic Light
Scattering
Dynamic light scattering (DLS), because of its speed and low
sample consumption, is a very convenient method to determine
the monodispersity of the species of interest and the presence of
soluble high-order assemblies and aggregates, simultaneously
[19]. However, for PDZ domain measurement, a concentration
of at least 1 mg/ml is needed to get a reliable calculation of the
hydrodynamic radius (Rh) of the domain. It is important to emphasize that DLS does not measure molecular mass, but Brownian
motion, which is related to the Rh of the particles. With the
translational diffusion coefficient deduced from this motion, the
hydrodynamic radius can be calculated, that is, the radius of the
sphere that would diffuse with the same rate as the molecule of
interest [20]. One should note that light scattering intensity is
related to the cube of the radius, and consequently, DLS is the
method of choice to detect small quantities of large aggregates in a
PDZ sample [18]. This property makes it difficult to estimate the
relative amount of the detected specie with the added difficulty that
DLS has a weak resolving power and cannot distinguish between
protein monomer and dimer in such molecular weight range, that is
10–20 kDa (Fig. 1).
1.3.2 Analytical Size
Exclusion Chromatography
To further analyze the oligomeric states of the PDZ domains, DLS
measurement can be completed by analytical size exclusion chromatography (SEC). SEC is currently the standard separation technique to quantify protein oligomers. It separates molecules
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Ce ´ lia Caillet-Saguy et al.
mainly b-, c-, y-, and z- ions. The 2,5-dihydroxybenzoic acid
(DHB) and super DHB matrices contribute to the N-terminal
validation, while the 1,5-diaminonaphthalene (DAN) matrix helps
in determining the C-terminal residues. Compare with the Edman
degradation, ISD has the advantage to detect both termini and in
presence of modifications. This method can confirm 10–50 residues from both sides of the intact protein. Due to the matrix
contribution below 800 Da, the first residues cannot be determined. This weakness can be overcome by using the
T3-sequencing approach or Pseudo-MS3, where selected ISD fragments are further fragmented (MALDI-TOF/TOF) [17]. Nowadays, the all procedure intact mass and T3-sequencing can be
performed in less than 1 h.
1.3 Homogeneity
By PDZ domain homogeneity measurements, we intend to measure if the domain is monomeric or oligomeric, the state of assemblies, and if the sample contains soluble high-order assemblies and
aggregates. There is no single technique that will answer all these
issues [18], as one of the challenges is the concentration required to
make the measurement given the small molecular mass of PDZ
domains.
1.3.1 Dynamic Light
Scattering
Dynamic light scattering (DLS), because of its speed and low
sample consumption, is a very convenient method to determine
the monodispersity of the species of interest and the presence of
soluble high-order assemblies and aggregates, simultaneously
[19]. However, for PDZ domain measurement, a concentration
of at least 1 mg/ml is needed to get a reliable calculation of the
hydrodynamic radius (Rh) of the domain. It is important to emphasize that DLS does not measure molecular mass, but Brownian
motion, which is related to the Rh of the particles. With the
translational diffusion coefficient deduced from this motion, the
hydrodynamic radius can be calculated, that is, the radius of the
sphere that would diffuse with the same rate as the molecule of
interest [20]. One should note that light scattering intensity is
related to the cube of the radius, and consequently, DLS is the
method of choice to detect small quantities of large aggregates in a
PDZ sample [18]. This property makes it difficult to estimate the
relative amount of the detected specie with the added difficulty that
DLS has a weak resolving power and cannot distinguish between
protein monomer and dimer in such molecular weight range, that is
10–20 kDa (Fig. 1).
1.3.2 Analytical Size
Exclusion Chromatography
To further analyze the oligomeric states of the PDZ domains, DLS
measurement can be completed by analytical size exclusion chromatography (SEC). SEC is currently the standard separation technique to quantify protein oligomers. It separates molecules
94
Ce ´ lia Caillet-Saguy et al.