This protocol describes the use of high-specificity ion-exchange chromatography with multi-angle light scattering for an accurate molar mass determination of proteins, protein complexes, and peptides in a heterogeneous sample. This method is valuable for quality assessment, as well as for the characterization of native oligomers, charge variants, and mixed-protein samples.
Total time
~3–4 hours per sample (including column equilibration, separation run, and data analysis)
Steps
1
Prepare and equilibrate ion-exchange chromatography buffer
Prepare the appropriate ion-exchange buffer solution and equilibrate the column to establish baseline conditions for protein separation by charge.
▶ 00:50
2
Execute IEX-MALS separation and detection experiment
Load protein sample onto the equilibrated IEX column, elute using salt gradient, and simultaneously collect multi-angle light scattering data for molar mass determination.
▶ 02:10
3
Analyze IEX-MALS chromatography and mass data
Process raw chromatographic and light-scattering data to determine protein molar mass, oligomeric state, and identify charge variants and contaminants in the sample.
▶ 05:38
4
Interpret results and assess protein quality metrics
Evaluate IEX-MALS results for BSA and other protein standards to confirm method optimization and assess sample homogeneity and native oligomeric composition.
▶ 08:27