Proximity Labeling Mass Spectrometry
Proximity labeling mass spectrometry is a powerful technique for investigating protein interactions and their spatial distribution within cells. This method involves labeling adjacent biomolecules to facilitate their identification and quantification via mass spectrometry. By merging chemical labeling with high-resolution mass spectrometry, this approach enables the detection of protein-protein interactions, protein localization, and dynamic changes within cellular environments. The central step in proximity labeling mass spectrometry is "proximity labeling," where labeling molecules are conjugated to specific proteins or nucleic acids. These molecules then react biochemically to label neighboring target molecules within a defined spatial range. The labeled molecules are subsequently detected and analyzed using mass spectrometry, with an emphasis on the efficiency and specificity of the labeling reaction to ensure accurate labeling and detection of target molecules. This method provides highly sensitive detection of low-abundance proteins in complex biological samples, offering extensive quantitative and qualitative insights.
Proximity labeling mass spectrometry has broad applications. In cancer research, it can elucidate cancer cell-specific protein interaction networks, offering potential drug targets for targeted therapies. In neuroscience, it aids in uncovering nerve cell communication mechanisms and synaptic dynamics. In immunology, it helps study immune cell signaling pathways, enhancing the understanding of immune responses. This technology is also promising in discovering novel diagnostic markers, vaccine development, and environmental science.
Common Proximity Labeling Methods
1. Biotin Ligase-Mediated Proximity Labeling (BioID)
In BioID, the target protein is fused with the biotin ligase BirA*, which labels nearby proteins (within ~10 nm) with biotin. After labeling, biotinylated proteins are enriched using streptavidin magnetic beads and identified via mass spectrometry.
2. Peroxidase-Mediated Proximity Labeling (APEX)
In APEX, the target protein is fused with peroxidase APEX. In the presence of hydrogen peroxide, APEX converts substrates like biotin-phenol into reactive intermediates that covalently bind to adjacent protein residues, achieving biotin labeling. Enrichment and identification are conducted using streptavidin beads and mass spectrometry.
Proximity Labeling Mass Spectrometry Experimental Process
1. Construct Fusion Protein Expression Vector
Generate a fusion protein expression vector by linking the target protein gene with the labeling enzyme gene (e.g., BirA* or APEX).
2. Cell Transfection or Gene Expression
Introduce the fusion protein vector into cells to enable intracellular expression of the fusion protein.
3. Labeling Reaction
Introduce appropriate substrates and reagents, such as biotin for BioID or hydrogen peroxide and biotin-phenol for APEX, to initiate a labeling reaction catalyzed by the labeling enzyme, marking proteins adjacent to the target protein.
4. Protein Enrichment and Purification
Use techniques like streptavidin magnetic beads to specifically enrich and purify biotin-labeled proteins, removing non-labeled proteins and other contaminants.
5. Mass Spectrometry Analysis
Analyze the purified protein samples with mass spectrometry to detect protein mass and sequence information. Compare with protein databases to identify labeled proteins.
Compared to traditional methods like yeast two-hybrid and co-immunoprecipitation, proximity labeling mass spectrometry allows in situ labeling within cells, reflecting natural protein interactions and spatial distributions more accurately. MtoZ Biolabs offers professional proximity labeling mass spectrometry services, helping researchers efficiently analyze complex biological samples. Our experienced team provides tailored solutions, ensuring high result accuracy and reliability. We welcome researchers to collaborate with us in exploring the intricacies of life sciences.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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