Tissue- and isoform-specific protein complex analysis with natively processed bait proteins.

Tissue- and isoform-specific protein complex analysis with natively processed bait proteins. J Proteomics. 2020 Aug 24;:103947 Authors: Beyer T, Klose F, Kuret A, Hoffmann F, Lukowski R, Ueffing M, Boldt K Abstract Protein-protein interaction analysis is an important tool to elucidate the function of proteins and protein complexes as well as their dynamic behavior. To date, the analysis of tissue- or even cell- or compartment-specific protein interactions is still relying on the availability of specific antibodies suited for immunoprecipitation. Here, we aimed at establishing a method that allows identification of protein interactions and complexes from intact tissues independent of specific, high affinity antibodies used for protein pull-down and isolation. Tagged bait proteins were expressed in human HEK293T cells and residual interactors removed by SDS. The resulting tag-fusion protein was then used as bait to pull proteins from tissue samples. Tissue-specific interactions were reproducibly identified from porcine retina as well as from retinal pigment epithelium using the ciliary protein lebercilin as bait. Further, murine heart-specific interactors of two gene products of the 3',5'-cyclic guanosine monophosphate (cGMP)-dependent protein kinase type 1 (cGK1) were investigated. Here, specific interactions were associated with the cGK1α and β gene products, that differ only in their unique amino-terminal region comprising about 1...
Source: Journal of Proteomics - Category: Biochemistry Authors: Tags: J Proteomics Source Type: research