Proximity Labeling Mass Spectrometry Service

What Is Proximity Labeling Mass Spectrometry?

Proximity Labeling Mass Spectrometry is a proteomics technique that combines proximity labeling, affinity enrichment, and LC-MS/MS. In living cells, a labeling enzyme fused to a bait protein biotinylates nearby proteins. The labeled proteins are then enriched and identified by mass spectrometry to characterize the protein environment surrounding the target.

Ummethum, H. et al. Frontiers in Genetics. 2020.

Figure 1. Workflow of Proximity Labeling Mass Spectrometry

Proximity labeling mass spectrometry supports the identification of candidate neighboring proteins and the characterization of local protein networks and their changes across experimental conditions. It is particularly useful for investigating weak or transient protein associations. Combined with appropriate controls and quantitative analysis, this approach provides evidence for protein interaction studies and subsequent functional validation.

When Proximity Labeling Mass Spectrometry Is a Good Fit

1. Target-Centered Protein Profiling

Identify candidate proteins enriched near a target when its surrounding protein composition is not fully characterized.

2. Weak or Transient Protein Associations

Investigate nearby proteins that may be difficult to retain during conventional affinity purification.

3. Subcellular Protein Network Analysis

Characterize protein neighborhoods associated with specific intracellular compartments or subcellular locations.

4. Condition-Dependent Comparisons

Compare target-proximal protein profiles across treatments, biological states, or genetic backgrounds using appropriate controls.

Proximity Labeling Mass Spectrometry vs Co-IP-MS vs AP-MS: How to Choose?

Comparison	Proximity Labeling MS	Co-IP-MS	AP-MS
Primary Research Objective	Identify candidate neighboring proteins and characterize target-centered protein networks	Identify candidate interaction partners co-immunoprecipitated with the target protein	Identify candidate proteins co-enriched with a bait protein through affinity purification
Core Strategy	Label proteins near the target, followed by affinity enrichment and LC-MS/MS identification	Capture the target and associated proteins using a specific antibody, followed by mass spectrometry	Enrich the bait and associated proteins using affinity tags, ligands, or other suitable capture methods
Suitable Research Applications	Weak or transient associations, membrane proteins, poorly soluble proteins, and subcellular protein neighborhood profiling	Endogenous protein interaction studies, candidate interaction analysis, and protein complex characterization	Interaction screening using tagged bait proteins, candidate interactor identification, and protein complex analysis
Key Considerations	Labeling system selection, fusion construct development and validation, labeling conditions, and negative controls	Antibody specificity, immunoprecipitation conditions, and IgG or other appropriate negative controls	Bait construct and expression, affinity capture conditions, tag-only or vector controls, and nonspecific background assessment

How to Choose

1. Choose Proximity Labeling MS: When the goal is to discover proteins surrounding a target, particularly in studies of weak or transient associations and local protein environments.

2. Choose Co-IP-MS: When the bait and candidate prey are known and mass spectrometry is needed to characterize proteins co-immunoprecipitated with the bait.

3. Choose AP-MS: When affinity purification is used to screen for candidate proteins associated with a bait protein.

Service at MtoZ Biolabs

MtoZ Biolabs provides proximity labeling and LC-MS/MS analysis service tailored to different target protein characteristics and research objectives.

1. Weak or Transient Protein Interaction Analysis

Identify nearby proteins involved in weak or transient interactions that may be lost during conventional affinity purification.

 

2. Membrane and Poorly Soluble Protein Analysis

Profile neighboring proteins around membrane and poorly soluble targets using proximity labeling in living cells.

 

3. Spatiotemporal Proximity Labeling

Select labeling systems according to project requirements, including approximately 10-minute labeling with a ~10 nm radius or labeling within ~1 minute with a ~20 nm radius.

 

Analysis Workflow

1. Project Assessment

Review the target protein, organism, biological system, research objective, experimental groups, and available controls.

2. Vector Construction

Construct the target-fusion expression vector.

3. Cell Expression

Express the target-fusion protein in the selected cell system.

4. Biotin Labeling

Biotinylate proteins in the vicinity of the target protein.

5. Western Blot Detection

Evaluate biotin labeling by Western blot (WB).

6. Streptavidin-Based Enrichment

Enrich biotinylated proteins using streptavidin affinity capture.

7. LC-MS/MS Analysis

Identify enriched proteins by liquid chromatography-tandem mass spectrometry.

8. Database Searching and Data Analysis

Search acquired mass spectrometry data against the appropriate protein reference database and analyze protein identification results.

Service Advantages

1. Multiple Labeling Systems

Multiple labeling systems are available to accommodate different experimental objectives, labeling speeds, and spatial ranges.

 

2. High-Resolution Mass Spectrometry

High-resolution mass spectrometry enables broad identification of proteins surrounding the target.

 

3. One-Time Charge

Our pricing is transparent, with no hidden fees or additional charges.

 

Experimental Instruments

1. Mass Spectrometry Platform

Orbitrap-series high-resolution mass spectrometer.

2. Mass Accuracy

Below 5 ppm.

3. Data Acquisition Mode

DDA or DIA, depending on project requirements.

4. Data Analysis Software

MaxQuant or DIA-NN, as appropriate.

5. Protein Reference Database

UniProt or a customer-provided custom database.

 

Applications

1. Cell Signaling Research

Analyze changes in target-proximal proteins associated with signaling activation, inhibition, or other experimental perturbations.

2. Subcellular Protein Network Analysis

Characterize the protein composition surrounding a target within a specific intracellular compartment.

3. Dynamic Protein Association Studies

Compare local protein profiles across different biological states or experimental conditions.

4. Plant and Microbial Research

Investigate target-centered protein networks associated with plant stress responses, microbial metabolism, and other system-specific biological processes.

Sample Submission Suggestions

Submission Item	Requirements
Sample Type	Cells that have undergone proximity labeling, or protein solutions that have undergone labeling, lysis, and enrichment.
Recommended Sample Amount	At least (2 × 10⁷) cells per sample for transiently or stably transfected cells.
Minimum Sample Amount	(1 × 10⁷) cells per sample for transiently or stably transfected cells.
Experimental Controls	A negative control must be provided alongside labeled samples.
Protein Reference Database	Provide information on the appropriate protein reference database.
Storage and Shipping	Freeze cell pellets and ship on dry ice.
Buffer Requirements	SDS concentration must be below 0.1%.
Sample Quality Requirements	Samples with negative WB validation results, severe degradation, or high salt or detergent concentrations are not accepted.

Case Study

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

This study used a Fab-based proximity labeling approach to identify proteins near extracellular EGFR. Labeled proteins were enriched and analyzed by mass spectrometry, revealing candidate proteins in the EGFR-associated protein network. The study demonstrates the value of proximity labeling MS for investigating target-centered protein interactions.

Yamada, K. et al. Nature Communications. 2023.

Figure 2. Proximity Extracellular Interactome of Overexpressed EGFR Using EGFR-FabID in Cells

Deliverables

1. Raw Mass Spectrometry Data

Original mass spectrometry data files.

2. Protein and Peptide Identification Results

Excel files containing protein and peptide identification results, sequence coverage, identification scores, and intensity values.

3. PDF Analysis Report

A report summarizing the analytical results.

4. Relative Abundance Comparison

Relative abundance comparison results, where supported by the experimental design and quantitative data.

5. Optional Bioinformatics Analysis

Customized enrichment analysis and protein interaction network analysis, according to project requirements.

 

FAQs

Q1. Can proximity labeling confirm direct protein-protein binding?

No. Proximity labeling identifies proteins within the labeling range of a target. These may include direct interaction partners, indirectly associated proteins, and other proteins in the same local environment. Independent validation is required to establish direct binding when that is the research objective.

 

Q2. Why might some neighboring proteins not be identified by LC-MS/MS?

Protein identification depends on labeling efficiency, local protein abundance, accessible labeling sites, sample processing, digestion efficiency, peptide ionization, and fragmentation. The final protein list therefore does not represent every protein near the target. Targeted experimental optimization and, where justified, complementary labeling strategies may help address some sources of incomplete coverage.

 

Contact Us

Provide the target protein, biological system, research objective, fusion construct status, experimental controls, and available sample information. MtoZ Biolabs can review these details to assess the appropriate experimental scope, labeling strategy, and LC-MS/MS analysis plan.