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Determination of protein molecular weight: SEC-MALS and traditional calibration
The physical properties and behavior of proteins in solution depend on a variety of factors associated with the purification and composition of the protein and its inherent properties. Size Exclusion Chromatography (SEC) is a powerful tool commonly used to analyze protein recovery, molecular weight and aggregation.
The SEC works by separating the sample as it flows through the porous inert column matrix. Smaller molecules enter the pores of the packing, while larger molecules are excluded and therefore pass through the column more quickly. The result is a separation based on hydrodynamic volume, but one usually requires a molecular weight. Previously, we estimated this process by comparing the elution time of unknown proteins with the standard globular protein (of known molecular weight), which we call "traditional correction." This is usually done using a single concentration detector such as ultraviolet (UV). However, now we use a combination of a light scattering detector and a concentration detector (UV or refractive index, RI) to determine the molecular weight of the protein independent of retention time. This advancement is very useful because many proteins do not have a globular structure, making their measured molecular weight inaccurate. The addition of more detectors (such as another concentration detector), intrinsic viscosity (IV), and dynamic light scattering (DLS) detection can greatly increase the amount of information obtained by a single SEC assay.
The Malvern SEC-MALS 20 (Figure 1) system is a 20-angle light scattering device that measures protein molecular weight independent of elution volume. In this application note, some proteins are separated using SEC. Their molecular weights were determined using multi-angle light scattering (MALS) or conventional calibration methods and the differences in these results were discussed.
Figure 1: Viscotek SEC-MALS 20 System
Materials and Method
The SEC-MALS 20 system was connected to a Viscotek TDAmax system using a TDA RI detector to determine the concentration. The samples were separated along two Viscotek protein columns using phosphate buffer as the mobile phase. All protein samples were dissolved in the mobile phase. The SEMALALS 20 system was calibrated using bovine serum albumin, a protein with a well-characterized molecular weight. Column calibration was performed using a series of globular proteins (gel filtration label kit for proteins with a molecular weight of 29,000-700,000 Da, Sigma-Aldrich).
The detector and column are placed at 30 °C to ensure good separation and optimum detector baseline stability.
result
As a system validation, calibrators should be checked frequently, and BSA dimers (usually found in BSA samples) are an excellent test standard. In this case, the molecular weight of the dimer is correctly determined and the trimer present in the sample is accurately determined. The BSA chromatogram is shown in Figure 2, and the BSA assay results are shown in Table 1. Figure 2 also shows the SEC-MALS chromatogram of BSA. As an isotropic scatterer, the peak size and detector response of BSA are the same at all angles. It can be clearly seen that the molecular weight of each peak is very stable. This situation is expected to occur in proteins whose molecular weight is tightly controlled and is considered to be monodisperse. The above results and molecular weight values ​​indicate that these peaks are different oligomers of BSA.
Figure 2: Chromatogram of BSA showing RI (red) and SEC-MALS (90°) (orange) detector signals.
Figure 3: BSA SEC-MALS 20 detector signal chromatogram.
Table 1: Molecular Weights of BSA Monomers, Dimers, and Trimers
Trimer
Dimer
monomer
RV Peak - (ml)
14.31
15.37
17.05
Mn - (kDa)
203.8
135.2
66.4
Mw - (kDa)
204.4
135.3
66.5
Mw/Mn
1.003
1.001
1.001
Wt Fr (peak)
0.054
0.165
0.78
Figure 4 shows the data for the traditional calibration method. The chromatogram shows the refractive index signal of carbonic anhydrase (a standard) and the resulting calibration curve.
Figure 4: The carbonic anhydrase chromatogram overlaps with the column calibration curve.
The molecular weights of BSA dimers and trimers were determined by conventional calibration methods. The results are shown in Table 2 and can be compared with the SEMALALS data in Table 1. Since the structures of these oligomers are no longer spherical like monomers, their molecular weight cannot be accurately calculated using column calibration. When light scattering is not used, it is impossible to know whether the molecular weight measured by the column calibration method is accurate. As such, these peaks may be incorrectly identified as trimers and pentamers.
However, dimers and trimers can be correctly identified using light scattering results (Table 1) by accurately determining their molecular weight.
Table 2: Molecular weight of BSA oligomer peaks measured using column calibration.
Trimer
Dimer
RV Peak - (ml)
14.32
15.33
Mn - (kDa)
341.1
198.4
Mw - (kDa)
345.1
202.2
Mw/Mn
1.012
1.019
Wt Fr (peak)
0.021
0.103
Finally, the molecular weight of another protein (pepsin) was determined using SEC-MALS and column calibration. The chromatogram and light scattering results are shown in Figure 5. Table 3 shows the comparison of the measured molecular weights.
Figure 5: Chromatogram of pepsin showing RI (red) and SEC-MALS (90°) (orange) detector signals.
Table 3: Peptide molecular weight determined by conventional calibration and light scattering techniques
MALS
Column calibration
RV Peak - (ml)
18.33
18.63
Mn - (kDa)
34.6
38.5
Mw - (kDa)
34.7
39.6
Mw/Mn
1.004
1.028
The molecular weight of pepsin was correctly determined using SEC-MALS to be 35 kDa, while the molecular weight of the pepsin was incorrectly determined to be 40 kDa. The reason for this result is that the structure of pepsin is not spherical, and therefore it is larger than the molecular weight when it is expected to be a globular protein. Therefore, its elution time is earlier, and the molecular weight recorded by the conventional calibration method is slightly higher than the correct value. On the other hand, the molecular weight from light scattering is independent of the elution volume and can therefore be measured correctly.
discuss
This application note shows that the molecular weight of some proteins can be successfully determined using the Malvern SEC-MALS 20 system. The addition of MALS to the SEC system allows the determination of protein molecular weight independent of the elution volume or structure of the protein. The SEC-MALS system is used to correctly measure the molecular weight of pepsin and BSA oligomers for good characterization.