Efficient and Site-Specific Protein Bioconjugation Quantification in Mammalian Cells

Techniques for the quantification of protein bioconjugation in live cells have been limited. This new method allows for quantification to be measured in cellulo. Inverse electron-demand Diels-Alder (IEDDA) cycloaddition reactions are utilized to label biomolecules. IEDDA compatible unnatural amino acids (UAAs) and their chloroalkane-derivatized conjugates are labeled with the HaloTag enzyme, which is used to quantify the bioconjugation reaction through a gel shift of the conjugated protein of interest. This method is efficient and able to be adjusted to the specific protein of interest. It utilizes common instrumentation and techniques that are found in most labs. This tool's versatility is highlighted by the fact that a variety of UAA/bioconjugation pairs can be used at different incorporation sites in any protein of interest.