Proximity labeling and other novel mass spectrometric approaches for spatiotemporal protein dynamics

Abstract

Background: Proteins are highly dynamic and their biological function is controlled by not only temporal abundance changes but also via regulated protein-protein interaction networks, which respond to internal and external perturbations. A wealth of novel analytical reagents and workflows allow studying spatiotemporal protein environments with great granularity while maintaining high throughput and ease of analysis. Areas covered: We review technology advances for measuring protein-protein proximity interactions with an emphasis on proximity labeling, and briefly summarize other spatiotemporal approaches including protein localization, and their dynamic changes over time, specifically in human cells and mammalian tissues. We focus especially on novel technologies and workflows emerging within the past 5 years. This includes enrichment-based techniques (proximity labeling and crosslinking), separationbased techniques (organelle fractionation and size exclusion chromatography), and finally sortingbased techniques (laser capture microdissection and mass spectrometry imaging). Expert opinion: Spatiotemporal proteomics is a key step in assessing biological complexity, understanding refined regulatory mechanisms, and forming protein complexes and networks. Studying protein dynamics across space and time holds promise for gaining deep insights into how protein networks may be perturbed during disease and aging processes, and offer potential avenues for therapeutic interventions, drug discovery, and biomarker development.

Endothelial cilia dysfunction in pathogenesis of hereditary hemorrhagic telangiectasia

“…Several questions remain to be addressed in order to further expound on the contribution of endothelial cilia to ALK1/SMAD4 mediated vascular development: 1- Improved imaging modalities and post-processing of images to rigorously quantify and colocalize components of ALK1/ENG→SMAD4 signaling are needed to convincingly demonstrate their physical association with the endothelial primary cilium. Specifically, better imaging modalities and image analysis, as well as techniques such as rapid protein proximity labeling can be employed to accurately co-localize primary cilia with components of ALK1/SMAD4 signaling ( Pino and Schilling, 2021 ; Tameling et al, 2021 ). 2- If endothelial cilia are indeed conducive to BMP9/ALK1/SMAD4, we would predict that ECs lacking primary cilia (due, for example, to increased flow velocity or defective cilia assembly) would have significantly attenuated ALK1 signaling, as evidenced by reduced phosphorylation rates of SMAD1/5/8.…”

“…1- Improved imaging modalities and post-processing of images to rigorously quantify and colocalize components of ALK1/ENG→SMAD4 signaling are needed to convincingly demonstrate their physical association with the endothelial primary cilium. Specifically, better imaging modalities and image analysis, as well as techniques such as rapid protein proximity labeling can be employed to accurately co-localize primary cilia with components of ALK1/SMAD4 signaling ( Pino and Schilling, 2021 ; Tameling et al, 2021 ).…”

Recent Advancements in Subcellular Proteomics: Growing Impact of Organellar Protein Niches on the Understanding of Cell Biology

“…A protein−protein interaction network is used to identify the local subcellular distribution of proteins through enzymelinked proximity labeling. 85 Proximity labeling involves fusing "bait" protein with labeling enzymes to covalently label neighboring "prey" proteins. Four standard proximity labeling approaches are used in subcellular proteomics.…”

ACKR3 Proximity Labeling Identifies Novel G protein- and β-arrestin-independent GPCR Interacting Proteins

“…An inherent limitation in proximity labeling is the identification of biologically inconsequential proteins due to non-specific binding of proteins during purification, bystander effects, and endogenous biotinylation of proteins, among others (56)(57)(58). To more confidently identify biologically meaningful GIPs, we selected three other GPCR-based proximity labeling datasets performed in HEK293 cells for further analyses (Figure 2D).…”