Proximity Proteomics Service

What Is Proximity Proteomics?

Proximity proteomics combines proximity labeling with mass spectrometry to identify proteins located near a target protein or within a specific cellular microenvironment. A labeling enzyme is targeted to a protein of interest to label nearby proteins. The labeled proteins are then enriched and identified by LC-MS/MS.

 

Low, T. Y. et al. Cell. Mol. Life Sci. 2021.

Figure 1. Principle of Proximity Proteomics

 

The resulting protein profile reflects the local molecular environment of the target protein. It may include direct interaction partners, indirectly associated proteins, and proteins that occupy the same cellular region without directly binding to the target. This approach is therefore useful for investigating weak or transient protein associations and characterizing protein neighborhoods that may be difficult to capture using conventional affinity purification methods.

 

When Proximity Proteomics Is a Good Fit

1. Weak or transient protein associations: Identify candidate proteins involved in associations that may be difficult to preserve during conventional affinity purification and washing.

 

2. Membrane and poorly soluble protein studies: Characterize neighboring proteins in specific cellular environments, particularly when biochemical extraction may compromise the recovery of associated protein complexes.

 

3. Subcellular protein neighborhood mapping: Profile the local protein environment surrounding a target protein or within a defined subcellular compartment.

 

4. Comparison across experimental conditions: Compare proximity-labeled protein profiles across conditions to identify proteins with changes in relative abundance.

 

5. Candidate protein screening: Generate candidate protein lists for subsequent interaction studies or functional validation.

 

Proximity Proteomics Service at MtoZ Biolabs

MtoZ Biolabs provides Proximity Proteomics Service integrating proximity labeling, biotinylated protein enrichment, LC-MS/MS identification, and downstream data analysis. Our service supports the following project types:

1. Target-Centered Protein Neighborhood Mapping: Identify candidate proteins surrounding a specific bait protein, followed by background filtering and candidate prioritization using appropriate negative controls.

 

2. Differential Proximity Proteomics Before and After Drug Treatment: Compare the enrichment of proximity-labeled proteins between drug-treated and control groups to identify candidates with altered enrichment, supporting investigations of drug-associated protein neighborhood remodeling and signaling pathway changes.

 

3. Protein Neighborhood Analysis of Specific Subcellular Compartments and Membrane Proteins: Identify candidate neighboring proteins within organelles, at cellular membranes, or around specific signaling complexes to characterize compartment-specific protein composition and investigate potential regulatory relationships.

 

For samples that have already undergone labeling, lysis, and enrichment, the corresponding controls, labeling quality control results, sample condition, and reference database must be reviewed before sample acceptance.

 

Proximity Proteomics vs Co-IP-MS: How to Choose?

 

Comparison Criteria	Proximity Proteomics	Co-IP-MS
Research focus	Local protein environments surrounding a target protein and candidate neighboring proteins	Proteins co-enriched with a target protein and their associated complexes
Weak or transient associations	Particularly useful for capturing proximity relationships that may be difficult to preserve during conventional biochemical extraction and washing	Weak or transient associations may be lost during cell lysis and washing
Main limitations	Proximity labeling does not establish direct interactions and may label proteins that are not direct interaction partners	Co-immunoprecipitation does not establish direct interactions, and some weak or transient associations may not be preserved
Typical applications	Weak or transient protein association studies, subcellular protein neighborhood mapping, and dynamic protein network analysis	Protein complex composition analysis, studies of relatively stable protein associations, and candidate interaction partner screening

 

These methods provide complementary evidence. When a bait protein and a candidate prey protein have already been identified, forward and reciprocal Co-IP experiments can assess whether the proteins co-enrich with one another. If the goal is to establish direct binding, an appropriate orthogonal validation method, such as a binding assay using purified proteins, may be required.

 

Analysis Workflow

1. Project Assessment and Experimental Design: Review the target protein, expression system, sample format, experimental conditions, control design, and reference database.

 

2. Construct Generation and Expression: When included in the agreed project scope, prepare the specified labeling construct and establish the corresponding expression system.

 

3. Proximity Labeling: Apply the selected labeling strategy to label proteins near the target protein within the experimental system.

 

4. Labeling Quality Control: Assess labeling efficiency by Western blot before proceeding to enrichment.

 

5. Streptavidin Enrichment: Capture biotinylated proteins in preparation for downstream proteomic analysis.

 

6. LC-MS/MS Analysis and Database Searching: Analyze enriched proteins using an Orbitrap-series high-resolution mass spectrometer and search the acquired data against the selected reference database.

 

7. Data Processing and Reporting: Compile protein and peptide identification results, relative abundance information, and the agreed comparative or functional analysis results.

 

Service Advantages

1. Flexible Labeling Strategies

Select an appropriate proximity labeling technology based on the target protein, experimental system, and required spatial or temporal resolution.

 

2. High Sensitivity

Detect low-abundance proteins in proximity-labeled samples, including candidates associated with transient protein relationships that may be challenging to capture using conventional techniques.

 

3. Unbiased Detection

Identify candidate neighboring proteins without requiring a specific antibody for each protein being detected, enabling broad characterization of the target-centered protein environment.

 

4. Targeted Enrichment

Use streptavidin-based enrichment to capture biotinylated proteins before LC-MS/MS analysis, helping reduce the contribution of non-labeled proteins to the analyzed sample.

 

5. One-Time Charge

Transparent pricing with no hidden fees or additional charges.

 

Applications

• Protein Interaction Studies: Identify candidate proteins within the local environment of a target protein.
• Subcellular Protein Neighborhood Mapping: Characterize protein environments associated with specific cellular compartments or localization sites.
• Membrane Protein Studies: Investigate neighboring proteins within membrane-associated environments.
• Cell Signaling Studies: Compare proximity-labeled protein profiles across different biological conditions.
• Dynamic Protein Association Studies: Investigate changes in local protein composition during biological processes.

 

Instrumentation and Analysis Platforms

• Mass Spectrometry Platform: Orbitrap-series high-resolution mass spectrometers.
• Mass Accuracy: Below 5 ppm, according to the service specifications.
• Data Acquisition: DDA or DIA, depending on project requirements.
• Data Analysis Software: MaxQuant or DIA-NN, selected according to the data acquisition strategy.
• Reference Database: UniProt or an appropriate customer-provided protein database.

 

Sample Submission Requirements

 

Item	Requirements
Accepted Sample Formats	Labeled cell pellets, or protein solutions that have undergone proximity labeling, lysis, and enrichment
Recommended Cell Input	At least 2 × 10⁷ cells per sample for transient or stable expression systems
Minimum Cell Input	1 × 10⁷ cells per sample; suitability depends on sample quality and project requirements
Required Controls	Appropriate negative controls based on the experimental design, including an empty-vector or labeling-enzyme-only control and a no-biotin control, as applicable
Labeling Validation	Western blot results confirming labeling are required for applicable workflows
Reference Information	Target protein information and an appropriate protein reference database
Buffer Compatibility	SDS concentration below 0.1%
Storage and Shipping	Cell pellets must be stored frozen and shipped on dry ice
Unsuitable Samples	Samples with negative Western blot labeling results, severe degradation, or excessive salt or detergent contamination

 

Before Submission: Confirm whether the project includes construct generation and proximity labeling or whether the submitted samples have already undergone labeling, lysis, and enrichment. For samples with pre-completed labeling, the corresponding negative controls and existing labeling quality control results must be provided.

 

Deliverables

 

Deliverable	Description
Protein and Peptide Identification Results	Excel-format protein and peptide identification tables, including sequence coverage and identification scores
Relative Abundance Data	Protein and peptide intensity data to support relative abundance comparisons, where applicable
Raw Mass Spectrometry Data	Raw mass spectrometry data files
Comparative Analysis	Protein abundance comparisons across experimental conditions when included in the project scope
Functional Analysis	Optional GO/KEGG enrichment analysis and protein-protein interaction network construction
Final Report	PDF report summarizing the analytical workflow and key findings

 

FAQs

1. Can proximity proteomics prove that two proteins directly bind to each other?

No. Proximity proteomics identifies proteins labeled near a target protein. These may include direct interaction partners, indirectly associated proteins, and other proteins occupying the same local environment. The results provide evidence of spatial proximity rather than proof of direct binding. Appropriate orthogonal validation is required to establish direct interactions.

 

2. What controls are required for proximity proteomics?

The experimental design should include appropriate negative controls, such as an empty-vector or labeling-enzyme-only control and a no-biotin control, depending on the labeling system. These controls help distinguish target-associated labeling from background labeling. The final control design should be confirmed during project assessment.

 

3. How should a proximity labeling system be selected?

Selection depends on the target protein, cellular localization, required labeling radius, and timescale of the biological process being investigated. Some systems are suited to labeling over longer time windows, whereas peroxidase-based systems can support rapid labeling but require reaction triggers such as hydrogen peroxide and biotin-phenol.

 

Discuss Your Project with MtoZ Biolabs

Please share your target protein, sample type, labeling status, available controls, and research objectives. MtoZ Biolabs will assess your project and recommend an appropriate workflow.

 

Contact MtoZ Biolabs to discuss your project and request a quote.

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