Use of SEC-MALS. (Size Exclusion Chromatography - Multi Angle. Light Scattering) for protein quality and characterization

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1 Use of SEC-MALS (Size Exclusion Chromatography - Multi Angle Light Scattering) for protein quality and characterization

2 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.

3 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

4 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

5 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.

6 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.

7 Normalized intensity Molar mass (Da) Homogeneous and heterogeneous of a sample 1.0 LS UV Heterogeneous sample Homogeneous sample Volume (ml)

8 Normalized intensity Molar mass (Da) Characterizing oligomeric states BSA (66.5 kda) sample LS UV Dimer kda Monomer kda Time (min) 10 4

9 Normalized absorbance at 280 nm Amplification of aggregate intensity 1.0 SEC alone Monomer Volume (ml)

10 Normalized intensity Molar mass (Da) Amplification of aggregate intensity SEC-MALS Aggregate (0.3%) Monomer (99.7%) 1.0 LS UV kda Volume (ml)

11 Normalized intensity Molar mass (Da) Detecting presence of aggregates protein quality 1.0 LS UV Aggregate (2.3%) Trimer (3.8%) 10 6 Monomer (93.9%) Volume (ml) 10 3

12 Absorbance Downstream application in industry measure aggregation percent 5% 0.5% 0.01%

13 Normalized absorbance at 280 nm Proteins with the same mass elute differently in SEC due to their shape SEC alone Volume (ml)

14 Normalized intensity Molar mass (Da) Proteins with the same mass elute differently in SEC due to their shape 1.0 LS UV SEC-MALS Volume (ml)

15 Normalized absorbance at 280 nm Calculating mass of disordered protein using a calibration curve SEC alone ml (~45 kda) Protein Mw = 17 kda Volume (ml)

16 Normalized intensity Molar mass (Da) Calculating mass of disordered protein using MALS SEC-MALS 1.0 LS UV kda Volume (ml) Protein Mw = 17 kda

17 Normalized scattered intensity Mass (Da) / Radius (nm) Calculation of mass and radius Mass Radius Volume (ml)

18 Studying protein-protein interactions Dieck et, al. FEBS Letters 584 (2010) MDM2 (2-125) P53 (1-93) A mixture

19 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

20 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.