Biotin and streptavidin form one of the strongest non-covalent biological bonds. Due to the extraordinary stability of the complex, biotin labeling of molecules has emerged as a widely used technique for numerous applications. However, the stability of the biotin-streptavidin complex poses a major problem when biotinylated molecules need to be recovered. Harsh elution conditions are required to dissociate the complex using heat or chemical reagents. Here we describe a simple and mild method for quantitative dissociation of biotin-streptavidin complexes which is compatible with downstream applications such as mass spectrometry.
Materials and Methods
Biotinylated BSA and transferrin were used alone or in spike-in experiments with protein lysates from HeLa cells. Different elution conditions were applied to release biotinylated proteins and peptides from streptavidin-coated dynabeads (Life Technologies) and from MSIA D.A.R.T.’s streptavidin (Thermo Fisher). Protein eluates were separated by 1D PAGE and band intensities were compared using ImageJ. For peptide elution proteins were digested with trypsin and peptides were incubated with the streptavidin surfaces. Mass spectrometry was used as read-out.
The high affinity of streptavidin to biotin makes complex formation essentially irreversible unless the biotin-streptavidin complex is exposed to harsh conditions such as heat or detergents. We developed a one-step elution strategy using an organic solvent and compared our workflow with different elution protocols. Biotinylated BSA and transferrin or biotinylated tryptic peptides were bound to either streptavidin coated magnetic beads or streptavidin coated tips. Elution of biotinylated molecules with the organic solvent was almost quantitative as shown by 1D PAGE and mass spectrometry. In contrast to other elution methods which require further purification steps, removal of the organic solvent from the eluates is simply achieved by lyophilisation in a speed-vac concentrator.
Our quantitative elution protocol is directly applicable to mass spectrometry without the need for any further workup procedures.