Use of SEC-MALS
(Size Exclusion Chromatography - Multi Angle
Light Scattering)
for protein quality and characterization
Methods for protein characterization
• Analytical SEC is a common method to characterize protein properties:
size, oligomers, aggregations, and purity.
• Other methods: - light scattering (LS and DLS for mass and radius)
- CD for secondary structure
- SDS-PAGE: coomassie, western blot, native gels for
protein identification and purity.
• Size exclusion chromatograph in line with multi angle light scattering is
a useful methodology to characterize proteins size and shape in native
solution conditions.
Multi Angle Light Scattering (MALS)
• When a laser light hits a macromolecule, the electric field of the light
induces an oscillating dipole that re-radiates light.
• The intensity of the radiated light depends on the magnitude of the dipole
and the macromolecule concentration.
• By measuring the intensity of light scattering, the mass of the
molecule can be calculated.
LS- intensity of scattered light
c – concentration
K – a constant for a specific solute in a solution
Quasi Elastic Light Scattering (QELS) / Dynamic Light Scattering (DLS)
• Measures the time-dependent fluctuations in the intensity of the scattered
light caused by random motion of the macromolecules in the solution.
• The fluctuations are related to the rate of diffusion which is related to the
radius of the molecule.
• Stokes-Einstein equation:
R- radius
k- Boltzmann constant
T-temperature
D- diffusion coefficient
η- viscosity
𝑘𝑇
𝑅=
6𝜋𝐷𝜂
The instrument
• Mini DAWN TREOS Wyatt technology.
• Triple-angle MALS, 60 mW Laser, mass range: 1Da – 1000 KDa
• For continuous flow detection or for stand-alone unit in batch mode.
SEC vs. SEC-MALS
SEC
• Separation by size.
• Calculating Mw based on
calibration curves of globular
proteins.
• Different molecules with the
same size will elute together –
undetectable heterogeneous of a
sample.
SEC-MALS
• Separation by size.
• Calculating Mw and radius from
the light scattering equations –
much more accurate.
• Calculate the Mw during the
elution peaks- indicate
homogeneous of a sample.
• Detect low amount of
aggregation – large molecules
amplify the intensity of LS.
Homogeneous and heterogeneous of a sample
LS
UV
1.0
Heterogeneous
sample
6
10
0.6
Homogeneous
sample
0.4
5
10
0.2
0.0
7.0
7.5
8.0
8.5
9.0
Volume (ml)
9.5
10.0
10.5
11.0
Molar mass (Da)
Normalized intensity
0.8
Characterizing oligomeric states
BSA (66.5 kDa) sample
6
10
1.0
LS
UV
Dimer
135 2 kDa
0.6
5
Monomer
66.3 0.4 kDa
10
0.4
0.2
4
0.0
10
14.5
15.0
15.5
16.0
Time (min)
16.5
17.0
17.5
Molar mass (Da)
Normalized intensity
0.8
Amplification of aggregate intensity
Monomer
SEC alone
10
10
Normalized absorbance at 280 nm
1.0
9
10
8
0.8
10
7
10
0.6
6
10
0.4
5
10
4
10
0.2
3
10
0.0
2
10
5
6
7
8
9
Volume (ml)
10
11
12
Amplification of aggregate intensity
Aggregate (0.3%)
SEC-MALS
Monomer (99.7%)
10
10
LS
UV
1.0
9
10
8
10
7
10
0.6
6
10
0.4
5
10
31.8 0.6 kDa
4
10
0.2
3
10
0.0
2
10
5
6
7
8
9
Volume (ml)
10
11
12
Molar mass (Da)
Normalized intensity
0.8
Detecting presence of aggregates –
protein quality
LS
UV
1.0
7
10
0.8
6
10
Trimer (3.8%)
0.6
Monomer (93.9%)
5
10
0.4
4
0.2
10
0.0
10
3
7.5
8.0
8.5
9.0
Volume (ml)
9.5
10.0
Molar mass (Da)
Normalized intensity
Aggregate (2.3%)
Absorbance
Downstream application in industry –
measure aggregation percent
5%
0.5%
0.01%
Proteins with the same mass elute differently in
SEC due to their shape
SEC alone
1.0
9
Normalized absorbance at 280 nm
+
10
0.8
8
10
7
10
0.6
6
10
0.4
5
10
4
10
0.2
3
10
0.0
2
10
6
8
10
Volume (ml)
12
14
Proteins with the same mass elute differently in
SEC due to their shape
SEC-MALS
LS
UV
9
10
Normalized intensity
0.8
8
10
7
10
0.6
6
10
0.4
5
10
4
10
0.2
3
10
0.0
2
10
6
8
10
Volume (ml)
12
14
Molar mass (Da)
1.0
Calculating mass of disordered protein –
using a calibration curve
SEC alone
10.45 ml (~45 kDa)
Normalized absorbance at 280 nm
1.0
0.8
0.6
0.4
0.2
0.0
8
9
10
11
Volume (ml)
Protein Mw = 17 kDa
12
13
Calculating mass of disordered protein –
using MALS
SEC-MALS
LS
UV
1.0
6
10
5
10
0.6
17.1
0.5 kDa
4
10
0.4
3
10
0.2
0.0
2
10
8
9
10
11
Volume (ml)
Protein Mw = 17 kDa
12
13
Molar mass (Da)
Normalized intensity
0.8
Calculation of mass and radius
8
10
1.0
Mass
Radius
7
0.8
6
10
5
10
0.6
4
10
0.4
3
10
0.2
2
10
1
10
0.0
0
10
5
6
7
8
Volume (ml)
9
10
Mass (Da) / Radius (nm)
Normalized scattered intensity
10
Studying protein-protein interactions
MDM2 (2-125)
P53 (1-93)
A mixture
Dieck et, al. FEBS Letters 584 (2010) 3269–3274
Studying protein modifications
• Use to study protein modification such as glycosylation and pegylation.
• Can be used to characterize number of modifications.
Molar mass
Hydrodynamic radius
• Can be used to study structural changes in modified proteins.
Volume
Volume
Summary
• SEC-MALS is a powerful tool to study protein structure – shape
and mass.
• Used to characterize protein oligomerization/aggregation,
protein shape, changes in protein mass or shape caused by
protein/ligand interaction or by modification or by
environmental conditions.
• Limitation: can detect low amount of large macromolecules but
needs high concentration of small macromolecules.