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 R = kt 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.
Normalized intensity Molar mass (Da) Homogeneous and heterogeneous of a sample 1.0 LS UV Heterogeneous sample 0.8 10 6 0.6 0.4 Homogeneous sample 10 5 0.2 0.0 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 Volume (ml)
Normalized intensity Molar mass (Da) Characterizing oligomeric states BSA (66.5 kda) sample 10 6 1.0 LS UV 0.8 0.6 Dimer 135 2 kda Monomer 66.3 0.4 kda 10 5 0.4 0.2 0.0 14.5 15.0 15.5 16.0 16.5 17.0 17.5 Time (min) 10 4
Normalized absorbance at 280 nm Amplification of aggregate intensity 1.0 SEC alone Monomer 10 10 10 9 0.8 10 8 0.6 10 7 10 6 0.4 10 5 0.2 10 4 10 3 0.0 10 2 5 6 7 8 9 10 11 12 Volume (ml)
Normalized intensity Molar mass (Da) Amplification of aggregate intensity SEC-MALS Aggregate (0.3%) Monomer (99.7%) 1.0 LS UV 10 10 10 9 0.8 10 8 0.6 10 7 10 6 0.4 0.2 31.8 0.6 kda 10 5 10 4 10 3 0.0 10 2 5 6 7 8 9 10 11 12 Volume (ml)
Normalized intensity Molar mass (Da) Detecting presence of aggregates protein quality 1.0 LS UV Aggregate (2.3%) 10 7 0.8 0.6 Trimer (3.8%) 10 6 Monomer (93.9%) 0.4 10 5 0.2 10 4 0.0 7.5 8.0 8.5 9.0 9.5 10.0 Volume (ml) 10 3
Absorbance Downstream application in industry measure aggregation percent 5% 0.5% 0.01%
Normalized absorbance at 280 nm Proteins with the same mass elute differently in SEC due to their shape 1.0 + SEC alone 10 9 0.8 10 8 0.6 10 7 10 6 0.4 10 5 0.2 10 4 10 3 0.0 10 2 6 8 10 12 14 Volume (ml)
Normalized intensity Molar mass (Da) Proteins with the same mass elute differently in SEC due to their shape 1.0 LS UV SEC-MALS 10 9 0.8 10 8 0.6 10 7 10 6 0.4 10 5 0.2 10 4 10 3 0.0 10 2 6 8 10 12 14 Volume (ml)
Normalized absorbance at 280 nm Calculating mass of disordered protein using a calibration curve SEC alone 1.0 10.45 ml (~45 kda) 0.8 0.6 0.4 0.2 0.0 Protein Mw = 17 kda 8 9 10 11 12 13 Volume (ml)
Normalized intensity Molar mass (Da) Calculating mass of disordered protein using MALS SEC-MALS 1.0 LS UV 10 6 0.8 10 5 0.6 17.1 0.5 kda 0.4 10 4 0.2 10 3 0.0 10 2 8 9 10 11 12 13 Volume (ml) Protein Mw = 17 kda
Normalized scattered intensity Mass (Da) / Radius (nm) Calculation of mass and radius 1.0 0.8 Mass Radius 10 8 10 7 10 6 0.6 10 5 0.4 0.2 10 4 10 3 10 2 0.0 10 1 10 0 5 6 7 8 9 10 Volume (ml)
Studying protein-protein interactions Dieck et, al. FEBS Letters 584 (2010) 3269 3274 MDM2 (2-125) P53 (1-93) A mixture
Molar mass Hydrodynamic radius Studying protein modifications Use to study protein modification such as glycosylation and pegylation. Can be used to characterize number of modifications. 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.