An improved TurboID-based proximity labeling pipeline in C. elegans by biochemical depletion of endogenously biotinylated carboxylases

ABSTRACT: To review an improved proximity-labeling strategy that uses the improved E. coli biotin ligase TurboID to characterize C. elegans protein complexes, the interactome of a presynaptic active zone protein, ELKS-1, was analyzed as proof of principle. A significant constraint on the sensitivity of TurboID-based proximity labeling is the presence of abundant, endogenously biotinylated proteins that take up bandwidth in the mass spectrometer, notably carboxylases that use biotin as a co-factor. We developed ways to remove these carboxylases prior to streptavidin purification and mass spectrometry, by engineering their corresponding genes to add a C-terminal His10 tag. This allows us to deplete them from C. elegans lysates using immobilized metal affinity chromatography (IMAC).

INSTRUMENT(S):

ORGANISM(S): Caenorhabditis Elegans

SUBMITTER: WeiQiang Chen  

LAB HEAD: Mario de Bono

PROVIDER: PXD034639 | Pride | 2022-09-12

REPOSITORIES: Pride