Proximity Labeling Mass Spectrometry Service

Affinity purification (AP), co-immunoprecipitation (co-IP), protein crosslinking, and yeast two-hybrid (Y2H) methods are often combined with mass spectrometry (MS)-based proteomics to identify protein-protein interactions (PPIs) in various biological systems. AP-MS is also integrated with nucleic acid sequencing techniques, such as chromatin immunoprecipitation sequencing and RNA immunoprecipitation sequencing, to identify protein-nucleic acid interactions. Although AP-MS is an effective method for capturing high-affinity protein interactions, nonspecific binding and the inability to detect transient interactions are major limitations. The Y2H system can effectively provide quantitative estimates of binding affinity, with a detection threshold of at least approximately 25 μM KD. A newer approach, the next-generation Y2H system (Y2H-next-generation interaction screening), uses deep sequencing to identify candidate interactors. While Y2H-next-generation interaction screening offers methodological advantages, the complex datasets generated by this technique pose unique bioinformatics and statistical challenges for analysis.

Proximity labeling provides a viable method for characterizing strong and transiently associated protein complexes, which may be linked through relatively weak interactions and extended protein clusters where not all proteins within the same multiprotein complex directly interact with each other. The latter is crucial because the overall proximity of proteins within a network, rather than just direct binding partners, determines their biological function. Furthermore, PL offers a method for identifying PPIs within specific cell types or spatially restricted intracellular compartments. For all these reasons, proximity labeling mass spectrometry has now become the preferred technique for exploring PPIs.

Technical Principles

In general, proximity labeling operates by expressing a chimeric assembly of the target protein and a modified enzyme, enabling the substrate to be enzymatically converted into a highly reactive intermediate that binds to affinity tags, such as biotin. These activated substrates diffuse in a distance-dependent manner and covalently label nearby endogenous proteins. Since the labeling efficiency depends on the local density of the intermediate and the target protein, and declines with increasing distance from the modified enzyme, proteins closer to the enzyme are more likely to be labeled than those farther away. As the yield of labeled proteins or peptides in any proximity labeling study is influenced by multiple factors (e.g., local protein concentration, number and distribution of exposed modifiable residues, digestion efficiency, peptide ionization, and fragmentation), the relationship between affinity and yield is not straightforward. Before subsequent identification and characterization, the labeled proteins or peptides derived from digested proteins are enriched using affinity-based methods (e.g., streptavidin or anti-biotin antibodies).

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Mathew, B. et al. Mol Cell Proteomics. 2022.

Figure 1. The Principle of Commonly Used Proximity Labeling Method

Services at MtoZ Biolabs

MtoZ Biolabs, an integrated Chromatography and Mass Spectrometry (MS) Services Provider, provides advanced proteomics, metabolomics, and biopharmaceutical analysis services to researchers in biochemistry, biotechnology, and biopharmaceutical fields. Our ultimate aim is to provide more rapid, high-throughput, and cost-effective analysis, with exceptional data quality and minimal sample consumption. Combining advanced proximity labeling techniques with high-resolution mass spectrometry, MtoZ Biolabs provides Proximity Labeling Mass Spectrometry Service to offer precise solutions for studying protein-protein interactions. The service includes enzyme selection, fusion protein construction and validation, experimental optimization, and high-quality data analysis. It captures both stable and transient interacting proteins while mapping the spatial distribution and dynamic networks of proteins in complex biological environments, enabling in-depth insights into protein functions and interaction mechanisms.

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Ummethum, H. et al. Front Genet. 2020.

Figure 2. The Workflow of Proximity Labeling Mass Spectrometry

Service Advantages

1. Customized Selection of Proximity Labeling Enzymes for Diverse Applications

MtoZ Biolabs' proximity labeling mass spectrometry service flexibly selects proximity labeling enzymes (e.g., BioID, TurboID, or APEX2) based on specific research requirements. For redox-sensitive proteins or particular intracellular environments, the service optimizes enzyme selection and experimental conditions to ensure physiological relevance and reliability of the labeling process.

2. Fusion Protein Function Validation for Accurate Results

The proximity labeling mass spectrometry service at MtoZ Biolabs provides validation of the function and localization of proximity labeling enzymes fused with target proteins, ensuring that the fusion protein behaves consistently with the endogenous protein. This prevents interference with protein function, localization, or interaction networks, guaranteeing high-quality data.

3. Quantitative Mass Spectrometry Combined with Precise Data Analysis

MtoZ Biolabs applies quantitative mass spectrometry techniques (e.g., ratio-based or statistical analyses) alongside rigorous control experiments to reduce false positives, enhance data specificity, and enable precise analysis of protein networks and subcellular localization.

4. Addressing Proximity Labeling Limitations to Enhance Data Depth

To overcome specific limitations of PL techniques (e.g., substrate biases or undetected proteins), our proximity labeling mass spectrometry service provides professional optimization and data analysis strategies, including the combination of multiple labeling enzymes and efficient processing of complex proteomic datasets, ensuring comprehensive and in-depth results.

Case Study

1. Proximity Extracellular Protein-Protein Interaction Analysis of EGFR Using AirID-Conjugated Fragment of Antigen Binding

This study utilizes AirID, a novel biotin ligase, fused with a fragment of antigen-binding (Fab) to analyze proximity extracellular protein-protein interactions (PPIs) of epidermal growth factor receptor (EGFR). The method enables high specificity and efficiency in labeling proteins near EGFR in the extracellular environment. AirID generates biotinylated proteins in proximity to the Fab-conjugated EGFR without disrupting its native functions. These labeled proteins are subsequently enriched and identified through mass spectrometry. The study highlights the advantage of AirID in exploring extracellular PPIs, providing insights into EGFR-associated interaction networks in complex biological systems. Proximity labeling mass spectrometry service enables precise analysis of protein-protein interactions by utilizing advanced proximity labeling techniques, offering valuable data for protein interaction studies.

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Yamada, K. et al. Nat Commun. 2023.

Figure 3. Proximity Extracellular Interactome of Over-Expressing EGFR by EGFR- FabID on Cells

2. Molecular Spatiomics by Proximity Labeling

This study introduces the concept of Molecular Spatiomics through proximity labeling technology to map spatially resolved PPI networks within complex cellular environments. Combined with mass spectrometry, proximity labeling identifies surrounding proteins based on their spatial localization and proximity to the target protein. This approach captures both stable and transient protein interactions, generating high-resolution spatial interaction maps. By integrating advanced bioinformatics tools, Molecular Spatiomics reveals the dynamic organization and functional architecture of protein networks across different cellular compartments, contributing to a deeper understanding of protein behavior and cellular processes in their native environments. Proximity labeling mass spectrometry service performs high-resolution analysis of protein-protein interactions in native cellular environments, revealing the spatial distribution and dynamic changes of protein networks, and providing essential data support for protein function and cellular process research.

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Kang, MG. et al. Acc Chem Res. 2022.

Figure 4. Direct Mass Analysis of PL-Modified Residues

Applications

1. Analysis of Mammalian Cell Signaling Pathways

Proximity labeling mass spectrometry service enables high-resolution analysis of protein-protein interactions (PPIs) in mammalian cells, uncovering dynamic proximity relationships within key signaling pathways.

2. Plant Stress Resistance Research

In plant systems, proximity labeling mass spectrometry service helps analyze protein interaction networks under stress conditions, such as drought or salinity, revealing underlying regulatory mechanisms.

3. Microbial Metabolic Pathway Studies

Proximity labeling mass spectrometry service facilitates the investigation of protein interactions within microbial metabolic pathways, providing insights into the fine regulation of metabolic networks.

4. Subcellular Protein Network Analysis

Using proximity labeling mass spectrometry, protein complexes within specific organelles or subcellular compartments can be mapped, revealing the unique characteristics of intracellular microenvironments.

5. Disease-Associated Protein Interaction Networks

In disease models, proximity labeling mass spectrometry identifies protein interaction networks related to pathological processes, supporting disease mechanism studies and drug target discovery.

6. Dynamic Protein Complex Assembly Mechanisms

Proximity labeling mass spectrometry service aids in studying the assembly and disassembly of dynamic protein complexes, shedding light on their behavior across different physiological states.