1.2.2 Assessing
Homogeneity
Depending on the context, the term “homogeneity” describes
different intrinsic properties of the sample. For example, researchers can question, if the protein is monomeric or oligomeric, if there
is only one conformational state, if there are one or many states of
assemblies, if all proteins carry the same posttranslational modifications (PTMs), or if the sample contains soluble aggregates. No technique will answer definitively all these questions in a single
experiment. In protein QC, “homogeneity” normally means that
the protein or protein complex is monodisperse, i.e., in one stable
assembly of fixed stoichiometry, and that no aggregates are present.
Due to its speed and low sample consumption, dynamic light
scattering (DLS) is a very convenient method to assess simultaneously the apparent monodispersity of the species of interest and
the presence of soluble high-order assemblies and aggregates
[11]. The phenomenon of DLS arises because of Brownian motion,
the extent of which is related to the size of the particles. When light
from a laser hits small particles, the light is scattered in all directions, and the scattering intensity at a particular location fluctuates
over time due to the Brownian motion of the particles. The autocorrelation curve of the scattering intensity provides information
on the particles’ motion—the scattering intensity will fluctuate, and
the autocorrelation will decay, more slowly for larger particles.
Using the translational diffusion deduced from this motion, a
hydrodynamic radius R h (typically a Stokes radius, i.e., the radius
of a sphere that would diffuse with the same rate as that observed)
can be calculated for each species present [3] (Fig. 3). Light scattering intensity is proportional to the mass squared. Consequently,
DLS is the method of choice to detect small quantities of large
aggregates in a sample. However, a key weakness of DLS is that it
does not have high mass resolution and will typically not distinguish
between monomeric and dimeric forms. In order to distinguish
separate species correctly, their radii must differ approximately
twofold (which corresponds to an approximately tenfold difference
in molecular weight for globular proteins). DLS only allows the
determination of the hypothetical radii and not the actual mass of
the protein. Thus, one should be aware that any indication of
molecular weight given by instrument software is based on theoretical calculations assuming a certain shape and protein density.
Because of this lack in resolution, DLS measurements can be
complemented by analytical size-exclusion chromatography (SEC),
which is currently the standard column-based chromatography
technique to quantify protein oligomers and, like DLS, a hydrodynamic technique [12]. The chromatography column contains a
matrix with many fine pores of various sizes and SEC separates
molecules according to their size because molecular species with a
greater radius can enter fewer pores and thus take a shorter path
and elute before smaller ones from the column. Aggregates, contaminants, and potentially different oligomeric states of the protein
8
Bertrand Raynal et al.
Homogeneity
Depending on the context, the term “homogeneity” describes
different intrinsic properties of the sample. For example, researchers can question, if the protein is monomeric or oligomeric, if there
is only one conformational state, if there are one or many states of
assemblies, if all proteins carry the same posttranslational modifications (PTMs), or if the sample contains soluble aggregates. No technique will answer definitively all these questions in a single
experiment. In protein QC, “homogeneity” normally means that
the protein or protein complex is monodisperse, i.e., in one stable
assembly of fixed stoichiometry, and that no aggregates are present.
Due to its speed and low sample consumption, dynamic light
scattering (DLS) is a very convenient method to assess simultaneously the apparent monodispersity of the species of interest and
the presence of soluble high-order assemblies and aggregates
[11]. The phenomenon of DLS arises because of Brownian motion,
the extent of which is related to the size of the particles. When light
from a laser hits small particles, the light is scattered in all directions, and the scattering intensity at a particular location fluctuates
over time due to the Brownian motion of the particles. The autocorrelation curve of the scattering intensity provides information
on the particles’ motion—the scattering intensity will fluctuate, and
the autocorrelation will decay, more slowly for larger particles.
Using the translational diffusion deduced from this motion, a
hydrodynamic radius R h (typically a Stokes radius, i.e., the radius
of a sphere that would diffuse with the same rate as that observed)
can be calculated for each species present [3] (Fig. 3). Light scattering intensity is proportional to the mass squared. Consequently,
DLS is the method of choice to detect small quantities of large
aggregates in a sample. However, a key weakness of DLS is that it
does not have high mass resolution and will typically not distinguish
between monomeric and dimeric forms. In order to distinguish
separate species correctly, their radii must differ approximately
twofold (which corresponds to an approximately tenfold difference
in molecular weight for globular proteins). DLS only allows the
determination of the hypothetical radii and not the actual mass of
the protein. Thus, one should be aware that any indication of
molecular weight given by instrument software is based on theoretical calculations assuming a certain shape and protein density.
Because of this lack in resolution, DLS measurements can be
complemented by analytical size-exclusion chromatography (SEC),
which is currently the standard column-based chromatography
technique to quantify protein oligomers and, like DLS, a hydrodynamic technique [12]. The chromatography column contains a
matrix with many fine pores of various sizes and SEC separates
molecules according to their size because molecular species with a
greater radius can enter fewer pores and thus take a shorter path
and elute before smaller ones from the column. Aggregates, contaminants, and potentially different oligomeric states of the protein
8
Bertrand Raynal et al.