Optimization of Protein Painting Procedure for Removal of Paint Molecules from Small or Nonglobular Proteins Utilizing the Rab6/Km23-1 Complex
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.
Copyright (c) 2022 Anum Qureshi, Ruth Zhang, Rachel Carter, Kathryn Cassels, Dr. Amanda Haymond-Still
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