In vivo proteomic mapping through GFP-directed proximity-dependent biotin labelling in zebrafish

Abstract

Protein interaction networks are crucial for complex cellular processes. However, the elucidation of protein interactions occurring within highly specialised cells and tissues is challenging. Here we describe the development, and application, of a new method for proximity-dependent biotin labelling in whole zebrafish. Using a conditionally stabilised GFP-binding nanobody to target a biotin ligase to GFP-labelled proteins of interest, we show tissue-specific proteomic profiling using existing GFP-tagged transgenic zebrafish lines. We demonstrate the applicability of this approach, termed BLITZ (Biotin Labelling In Tagged Zebrafish), in diverse cell types such as neurons and vascular endothelial cells. We applied this methodology to identify interactors of caveolar coat protein, cavins, in skeletal muscle. Using this system, we defined specific interaction networks within in vivo muscle cells for the closely related but functionally distinct Cavin4 and Cavin1 proteins.

ADRAM is an experience-dependent long noncoding RNA that drives fear extinction through a direct interaction with the chaperone protein 14-3-3

“…Following the sample preparation method of Xiong et al, 2021 , peptide extracts were analysed by nanoHPLC/MS MS/MS on an Eksigent ekspert nanoLC 400 system (SCIEX) coupled to a Triple TOF 6600 mass spectrometer (SCIEX) equipped with a PicoView nanoflow (New Objective) ion source. Full scan TOFMS data were acquired over the mass range 350–1800 and for product ion ms/ms 100–1500.…”

Identification of the centrosomal maturation factor SSX2IP as a Wtip-binding partner by targeted proximity biotinylation

“…Importantly, SSX2IP has been detected by targeting BirA-GBP to GFP-WtipN and by the direct fusion protein WtipN-BirA, increasing our confidence in the result. Based on our results and a similar study using zebrafish embryos [ 32 ], we propose that TPB is a versatile general technique that is applicable for protein interaction studies of GFP-tagged proteins. However, lack of biotinylation of Flag-GFP in our experiments suggests possible sterical hindrance of the epitope or lack of lysine residues needed for biotinylation, indicating that not all proteins are suitable targets for this approach.…”

A modified TurboID approach identifies tissue-specific centriolar components in C. elegans

“…The same GFP nanobody-TurboID promoter construct can then be used for multiple different target proteins by crossing it into the relevant GFP strain background and, when expressed alone, serves as an expression-matched control for mass spectrometry sample normalization. It should be noted that a similar strategy was recently developed for use in zebrafish [ 42 ], albeit employing a conditionally stabilised GFP-binding nanobody, with implications for sample normalization as discussed below.…”