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Spatial/proximity interaction
Spatial/proximity interaction studies aim to capture the physical proximity between spatially adjacent molecules (protein‑protein, protein‑nucleic acid, protein‑small molecule, etc.) within living cells or tissues, thereby revealing their functional associations and regulatory networks in specific subcellular compartments or microenvironments. This approach is critical for understanding the spatial specificity of signal transduction, dynamic assembly of protein complexes, and spatiotemporal remodeling of disease‑related interaction networks. Key technologies include DNA probe‑based proximity ligation methods (Proximity Labeling and PLA/Proximity Ligation Assay), enabling in situ visualization and quantification at the single‑molecule level; and cross‑linking mass spectrometry (CL‑MS), which covalently fixes spatially proximal amino acid residue pairs and, combined with mass spectrometry, precisely resolves protein three‑dimensional structures and interaction interfaces.
1. Proximity Labeling (PL)
1.1 Introduction
Proximity Labeling (PL) is a revolutionary proteomics technology that enables in situ capture of protein composition and interaction networks within defined spatial ranges in living cells or tissues. By fusing an engineered enzyme to a protein of interest (POI), short‑lived reactive intermediates are generated upon substrate addition to covalently tag proximal biomolecules. These tagged proteins are then enriched by streptavidin beads and identified by high‑sensitivity mass spectrometry, revealing the “molecular neighborhood” of the target protein in its native cellular environment. PL overcomes fundamental limitations of traditional co‑immunoprecipitation and affinity purification mass spectrometry, which depend on stable protein associations and detergent‑resistant complexes, providing a transformative solution for studying weak, transient, and membrane protein interactions.
The core of PL lies in the fusion of a POI with an engineered enzyme. Upon addition of a specific substrate, the enzyme catalyzes the formation of reactive intermediates—such as biotinoyl‑5’‑AMP generated by biotin ligases (BioID series), or biotin‑phenoxyl radicals generated by peroxidases (APEX series). These intermediates possess extremely short half‑lives (microsecond to millisecond scale) and become inactivated after diffusing approximately 10‑20 nm, ensuring that only proteins in spatial proximity to the target are covalently labeled. Following labeling, biotinylated proteins are efficiently enriched using streptavidin‑coated magnetic beads, digested with trypsin, and analyzed by high‑resolution mass spectrometry for identification and quantification.
| Platform | Enzyme Source | Radius | Time | Size |
|---|---|---|---|---|
| BioID | E. coli BirA mutant (BirA) | ~10 nm | 18‑24 h | 35 kDa |
| BioID2 | Humanized biotin ligase (BirA homolog) | ~10 nm | ~hours | 27 kDa |
| TurboID | Yeast display‑directed evolved BirA | ~10 nm | ~10 min | 35 kDa |
| miniTurbo | Truncated optimized TurboID variant | ~10 nm | ~10 min | 28 kDa |
| APEX/APEX2 | Soybean ascorbate peroxidase | ~20 nm | ~1 min | 28 kDa |
| Split‑TurboID | Split TurboID (N‑terminal + C‑terminal) | ~10 nm | Conditional | ~15 kDa |
1.2 Products
1.3 Applications
| Platform | Optimal Applications |
|---|---|
| BioID | Steady‑state interactome analysis; long‑term cultured cells without strict timing requirements |
| BioID2 | Spatially constrained subcellular regions (synapses, cilia) |
| TurboID | Dynamic interaction processes; in vivo applications; time‑resolved experiments |
| miniTurbo | Small protein tag requirements; viral vector delivery |
| APEX/APEX2 | Sub‑minute dynamic processes; experiments requiring rapid “freezing” of cellular states |
| Split‑TurboID | Validation of specific protein pairs; conditional labeling (optogenetic, small‑molecule inducible) |
References:
- Hussain MA, Hafeez AH, Noor I, Shakoor A, Hussain H, Gholizadeh F, Sohail H. Mapping the dynamic plant interactome: from in vitro assays to in vivo quantitative approaches. Plant Methods. 2026 Jul 12. doi: 10.1186/s13007‑026‑01571‑0. Epub ahead of print. PMID: 42437933.
- Bao X, Jia H, Zhang X, Tian S, Zhao Y, Li X, Lin P, Ma C, Wang P, Song CP, Zhu X. Mapping of cytosol‑facing organelle outer membrane proximity proteome by proximity‑dependent biotinylation in living Arabidopsis cells. Plant J. 2024 Apr;118(1):7‑23. doi: 10.1111/tpj.16641. Epub 2024 Jan 23. PMID: 38261530.
- Rayaprolu S, Bitarafan S, Santiago JV, Betarbet R, Sunna S, Cheng L, Xiao H, Nelson RS, Kumar P, Bagchi P, Duong DM, Goettemoeller AM, Oláh VJ, Rowan M, Levey AI, Wood LB, Seyfried NT, Rangaraju S. Cell type‑specific biotin labeling in vivo resolves regional neuronal and astrocyte proteomic differences in mouse brain. Nat Commun. 2022 May 25;13(1):2927. doi: 10.1038/s41467‑022‑30623‑x. PMID: 35614064; PMCID: PMC9132937.
- Lin Z, Schaefer K, Lui I, Yao Z, Fossati A, Swaney DL, Palar A, Sali A, Wells JA. Multiscale photocatalytic proximity labeling reveals cell surface neighbors on and between cells. Science. 2024 Jul 19;385(6706):eadl5763. doi: 10.1126/science.adl5763. Epub 2024 Jul 19. PMID: 39024454; PMCID: PMC12517702.
2. Chemical Crosslinking Mass Spectrometry (CL‑MS)
2.1 Introduction
Chemical Crosslinking Mass Spectrometry (XL‑MS) introduces covalent crosslinks between proteins or protein complexes, followed by mass spectrometry analysis to identify protein‑protein interactions and elucidate macromolecular complex architectures.
Core Principle: Bifunctional chemical crosslinkers (e.g., BS3, DSS, glutaraldehyde) react with specific amino acid residues (primarily lysine ε‑amino groups, but also cysteine, tyrosine, etc.) on two proteins, forming a “molecular bridge”. After enzymatic digestion (typically trypsin), the crosslinked peptides are identified by high‑resolution MS, revealing spatial proximity and interaction sites.
2.2 Products
2.3 Applications
| Field | Applications |
|---|---|
| Protein complex architecture | Map subunit topology; provide distance restraints for 3D modeling |
| Ppi network mapping | Identify direct physical interactions; distinguish true binding from indirect proximity |
| Dynamic conformation | Capture conformational changes across different functional states |
| Membrane protein studies | Elucidate structures of hard‑to‑crystallize membrane protein complexes |
| PTM‑dependent interactions | Study how phosphorylation, ubiquitination, etc. modulate interactions |
| Drug target validation | Identify drug‑target binding and target interaction networks |
References:
- Herzog F, Kahraman A, Boehringer D, Mak R, Bracher A, Walzthoeni T, Leitner A, Beck M, Hartl FU, Ban N, Malmström L, Aebersold R. Structural probing of a protein phosphatase 2A network by chemical cross‑linking and mass spectrometry. Science. 2012 Sep 14;337(6100):1348‑52. doi: 10.1126/science.1221483. PMID: 22984071.
- Chen ZA, Pellarin R, Fischer L, Sali A, Nilges M, Barlow PN, Rappsilber J. Structure of Complement C3(H2O) Revealed By Quantitative Cross‑Linking/Mass Spectrometry And Modeling. Mol Cell Proteomics. 2016 Aug;15(8):2730‑43. doi: 10.1074/mcp.M115.056473. Epub 2016 Jun 1. PMID: 27250206; PMCID: PMC4974347.
- Ding YH, Gong Z, Dong X, Liu K, Liu Z, Liu C, He SM, Dong MQ, Tang C. Modeling Protein Excited‑state Structures from “Over‑length” Chemical Cross‑links. J Biol Chem. 2017 Jan 27;292(4):1187‑1196. doi: 10.1074/jbc.M116.761841. Epub 2016 Dec 19. PMID: 27994050; PMCID: PMC5270465.
- Gutierrez CB, Block SA, Yu C, Soohoo SM, Huszagh AS, Rychnovsky SD, Huang L. Development of a Novel Sulfoxide‑Containing MS‑Cleavable Homobifunctional Cysteine‑Reactive Cross‑Linker for Studying Protein‑Protein Interactions. Anal Chem. 2018 Jun 19;90(12):7600‑7607. doi: 10.1021/acs.analchem.8b01287. Epub 2018 Jun 5. PMID: 29792801; PMCID: PMC6037416.
3. PLA（Proximity Ligation Assay）
3.1 Introduction
PLA is an antibody‑based, ultra‑sensitive in situ technique for detecting protein‑protein interactions. Two target proteins are recognized by specific primary antibodies, followed by binding of secondary antibodies conjugated with DNA oligonucleotide probes. When the two targets are within <40 nm, the probes are ligated into a circular template by a DNA ligase. Rolling circle amplification (RCA) generates long DNA products, which are detected by fluorescent oligonucleotide hybridization, with each interaction event appearing as a countable, discrete fluorescent focus.
3.2 Products
3.3 Applications
| Application | Description |
|---|---|
| In situ PPI validation | Directly validates whether two known proteins are in spatial proximity within intact cells or tissue sections, without requiring overexpression or cell lysis |
| Protein phosphorylation detection | Uses a phospho‑specific antibody paired with a total protein antibody to detect site‑specific phosphorylation and its association with interacting proteins |
| Post‑translational modification site analysis | Combines modification‑specific antibodies (e.g., acetylation, ubiquitination) to map spatial relationships between modification sites and interaction interfaces |
| Tissue section interaction mapping | Detects protein interactions in FFPE or frozen tissue sections, preserving pathological structural information for clinical samples |
| Single‑cell interaction quantification | Achieves semi‑quantitative statistics of interaction events by counting fluorescent foci per cell, enabling analysis of inter‑cellular heterogeneity |
| Drug target occupancy assessment | Detects changes in target‑effector protein interactions upon drug binding to evaluate drug occupancy rates and mechanisms of action |
References:
- Söderberg O, Gullberg M, Jarvius M, Ridderstråle K, Leuchowius KJ, Jarvius J, Wester K, Hydbring P, Bahram F, Larsson LG, Landegren U. Direct observation of individual endogenous protein complexes in situ by proximity ligation. Nat Methods. 2006 Dec;3(12):995‑1000. doi: 10.1038/nmeth947. Epub 2006 Oct 29. PMID: 17072308.
- Jarvius M, Paulsson J, Weibrecht I, Leuchowius KJ, Andersson AC, Wählby C, Gullberg M, Botling J, Sjöblom T, Markova B, Ostman A, Landegren U, Söderberg O. In situ detection of phosphorylated platelet‑derived growth factor receptor beta using a generalized proximity ligation method. Mol Cell Proteomics. 2007 Sep;6(9):1500‑9. doi: 10.1074/mcp.M700166‑MCP200. Epub 2007 Jun 12. PMID: 17565975.