Chemical Derivatization of Affinity Matrices Provides Protection from Tryptic Proteolysis

Abstract

The enrichment of biotinylated proteins using immobilized streptavidin has become a staple methodology for affinity purification-based proteomics. Many of these workflows rely upon tryptic digestion to elute streptavidin-captured moieties from the beads. The concurrent release of high amounts of streptavidin-derived peptides into the digested sample, however, can significantly hamper the effectiveness of downstream proteomic analyses by increasing the complexity and dynamic range of the mixture. Here, we describe a strategy for the chemical derivatization of streptavidin that renders it largely resistant to proteolysis by trypsin and thereby dramatically reduces the amount of streptavidin contamination in the sample. This rapid and robust approach improves the effectiveness of mass spectrometry-based characterization of streptavidin-purified samples making it broadly useful for a wide variety of applications. In addition, we show that this chemical protection strategy can also be applied to other affinity matrices including immobilized antibodies against HA epitopes.

Variability in Streptavidin–Sepharose Matrix Quality Can Significantly Affect Proximity-Dependent Biotinylation (BioID) Data

“…Chemical derivatization of arginine and lysine residues in streptavidin (via methylglyoxal treatment or reductive methylation, respectively) renders streptavidin resistant to trypsin digestion, decreasing streptavidin peptide contamination in enzymatic elution protocols . This approach may allow users to circumvent variability across matrix lots but would of course only mask the underlying issue of streptavidin shedding.…”

Protease‐resistant streptavidin for interaction proteomics

“…We applied this approach to different use cases and demonstrate that it enhances the sensitivity to identify protein–DNA interactions (in ChIP‐SICAP), protein–protein interactions (BioID), and protein–lipid interaction and to profile cell surface proteins (Fig A). Furthermore, we show that our strategy to generate protease‐resistant streptavidin beads compares favorably to a recent similar approach (Barshop et al , ). In conclusion, we demonstrate that our protease‐resistant beads represent a powerful tool for many applications of MS‐based interaction proteomics.…”

“…During the preparation of this work, Barshop et al published a similar strategy for streptavidin modification, although using different chemistry (Barshop et al , ). In a direct comparison of both methods (Fig EV2C), prS beads resulted in 25‐fold less streptavidin contamination (ratio of the intensities of the streptavidin peptides; Dataset EV1‐comparing with Barshop et al —Mock beads).…”

A Conserved Long Intergenic Non-coding RNA Containing snoRNA Sequences, lncCOBRA1, Affects Arabidopsis Germination and Development

“…Pierce High Capacity Streptavadin Agarose beads (Thermo Fisher Scientific; Carlsbad, CA, United States) were first chemically treated to protect the streptavidin from tryptic proteolysis in preparation for mass spectrometry to reduce streptavidin signal as previously described ( Barshop et al, 2019 ).…”