Optimization of Protein Painting Procedure for Removal of Paint Molecules from Small or Nonglobular Proteins Utilizing the Rab6/Km23-1 Complex

Abstract

Protein painting is a technique that maps solvent-accessible protein surfaces through molecular dyes. However, small proteins struggle with this technique due to the viscosity of size-exclusion chromatography, a key processing step prior to mass spectrometry analysis. Hence, we hypothesized that if we use Sephadex G-15 Columns which have a lower viscosity gel filtration system instead of the commonly used G-25 Columns, then it will increase small or nonglobular protein recovery. We used proteins Rab6, Km23-1, and their complex for protein painting with and without the columns. After processing and analyzing the proteins via mass spectrometry, we found a significant loss in Peptide Spectrum Matches via G-15 Columns. Regardless of the loss, the paint coverage with the G-15 Columns was at least 75%. This suggests that the columns were able to capture some paint molecules and even unfolded/aggregated proteins, thus giving cleaner results. Data also indicates that Rab6 had an overall greater abundance ratio in the Rab6/Km23-1 complex than Km23-1 in the Rab6/Km23-1 complex, which implies that the G-15 Columns did well in recovering Rab6 in complex form as compared to Km23-1. Using an even lower viscosity gel filtration system such as the Sephadex G-10 Columns and improving protein handling can help mitigate the loss of the proteins and obtain a high abundance ratio for even smaller proteins like Km23-1. Painting small proteins like Rab6 and Km23-1 can help determine protein-to-protein interactions and aid in drug development for cancer and neurological disorders like Alzheimer’s, which is why optimizing this technique is essential.