Proximity-dependent biotinylation (PDB) methods combined with mass spectrometry have emerged as powerful techniques for studying proximal protein interactions and subcellular proteomes in the context of living cells and organisms. Here, CD BioSciences provides a service to efficiently identify lysosomal surface proteins and interactors using proximity biotinylation to support lysosomal proteomic studies.
Background of Lysosomal Surface Proteins and Interactors by Proximity Biotinylation Identification
The traditional method for identifying lysosomal proteins or protein-protein interactions is to obtain the protein of interest by affinity purification, and then identify its direct and indirect interacvtions by mass spectrometry. However, these methods require maintaining the integrity of protein-protein interactions or lysosomes during sample purification. PDB refers to the covalent labeling of adjacent proteins to allow their separation and identification by fusing the protein with an enzyme that produces a reactive molecule, most commonly biotin. PDB technology has been widely used to identify interaction partners of lysosomal membrane proteins in living cells. Complementing traditional analysis, this technique can be used to label live cells (or whole organisms), fixed samples, and even lysates or semi-purified constructs. Furthermore, the integrity of protein-protein interactions or lysosomes does not need to be maintained after labeling.
Fig. 1. Principles of Proximity-Dependent Biotinylation. (Samavarchi-Tehrani P, et al., 2020)
Our Lysosomal Surface Proteins and Interactors by Proximity Biotinylation Identification Services
Studying the subcellular distribution of lysosomal proteins, their assembly into complexes, and their protein interactions is essential for understanding basic lysosomal biological processes. Here, CD BioSciences combines affinity purification binding mass spectrometry (AP-MS) and PDB techniques to identify proteins located at or near the cytoplasmic surface of the lysosomal membrane by fusing biotin protein ligase (BioID) and peroxidase (APEX) to different types of decoy proteins.
Our engineers improve the properties of BioID and APEX by combining exploration of enzyme homologs, structure-guided protein engineering, and unbiased molecular evolution. We will select the best specific enzyme for you based on project requirements. Our goal is to help our clients screen and identify physiologically relevant interactors of lysosomal proteins in living cells. The strategy flow is specified as follows:
(1) Lysosomal membrane proteins are fused to BioID and APEX enzymes and expressed in the cell.
(2) Biotin phenol and H2O2 are added to APEX or biotin is added to BioID, and these intermediates target the amino acid side chains of neighboring proteins.
(3) Covalent biotinylated proteins can be enriched by streptavidin beads.
(4) Identification of the resulting peptides by mass spectrometry to provide a candidate list of proteins near the decoy.
Our Advantages
- Enables analysis of lysosomal protein interactions in their natural environment.
- Can use harsh conditions to force insoluble proteins into solution.
- No need to maintain protein-protein interactions during purification.
- Does not require an intact lysosomal structure.
- In vivo covalent biotinylation enables detection of transient interactions and low abundance proteins.
- Streptavidin-purified proteins can be identified and quantified by mass spectrometry.
We look forward to collaborating with you. You are always welcome to engage in discussions with us at any point of the project. If you are interested in our services, please feel free to contact us for more information.
References
- Samavarchi-Tehrani P, Samson R, Gingras A C. (2020) Proximity dependent biotinylation: key enzymes and adaptation to proteomics approaches[J]. Molecular & Cellular Proteomics. 19(5): 757-773.
- Nguyen-Tien D, Suzuki T, et al. (2022) Identification of the interacting partners of a lysosomal membrane protein in living cells by BioID technique. STAR Protoc. 3(2):101263.