Identification of myelin basic protein interactome using TurboID proximity labeling proteomics

ABSTRACT: The protein environment of myelin basic protein (MBP), its uncharged form, and cyclindependent kinase inhibitor 1 (p21Cip1) were analyzed using two different approaches to identify protein partners thereof. The first one was a state-of-the-art enzymatic approach (TurboID), which utilizes an engineered biotin ligase that uses ATP to convert biotin to reactive biotin-AMP that covalently attaches to nearby proteins, followed by enrichment of biotin-labeled proteins on streptavidin beads and MS analysis of the resulting protein pool. Classical immunoprecipitation with Flag-tag fused to the target protein in combination with MS analysis was used as a reference method. As a result, two pools of potential interactors of MBP and uncharged MBP were identified. It was shown that TurboID, although giving a high level of background biotinylation, is a more reproducible technique compared to the immunoprecipitation, possibly due to higher affinity during the fraction enrichment process and easier maintaining of uniform conditions for the process, as well as interaction conditions being closer to in vivo conditions.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell, Cell Culture

SUBMITTER: Georgij Arapidi  

LAB HEAD: Georgij Arapidi

PROVIDER: PXD039467 | Pride | 2023-01-25

REPOSITORIES: Pride