Proximity labeling

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Mitochondrial outer membrane proteins are identified via proximity labeling. Proximity labeling proteomics of mitochondrial outer membrane.png
Mitochondrial outer membrane proteins are identified via proximity labeling.

Enzyme-catalyzed proximity labeling (PL), also known as proximity-based labeling, is a laboratory technique that labels biomolecules, usually proteins or RNA, proximal to a protein of interest. [1] By creating a gene fusion in a living cell between the protein of interest and an engineered labeling enzyme, biomolecules spatially proximal to the protein of interest can then be selectively marked with biotin for pulldown and analysis. Proximity labeling has been used for identifying the components of novel cellular structures and for determining protein-protein interaction partners, among other applications. [2]

Contents

History

Before the development of proximity labeling, determination of protein proximity in cells relied on studying protein-protein interactions through methods such as affinity purification-mass spectrometry and proximity ligation assays. [3]

DamID is a method developed in 2000 by Steven Henikoff for identifying parts of the genome proximal to a chromatin protein of interest. DamID relies on a DNA methyltransferase fusion to the chromatin protein to nonnaturally methylate DNA, which can then be subsequently sequenced to reveal genome methylation sites near the protein. [4] Researchers were guided by the fusion protein strategy of DamID to create a method for site-specific labeling of protein targets, culminating in the creation of the biotin protein labelling-based BioID in 2012. [1] Alice Ting and the Ting lab at Stanford University have engineered several proteins that demonstrate improvements in biotin-based proximity labeling efficacy and speed. [5] [6] [7] [8]

Principles

Proximity labeling relies on a labeling enzyme that can biotinylate nearby biomolecules promiscuously. Biotin labeling can be achieved through several different methods, depending on the species of labeling enzyme.

To label proteins nearby a protein of interest, a typical proximity labeling experiment begins by cellular expression of an APEX2 fusion to the protein of interest, which localizes to the protein of interest's native environment. Cells are next incubated with biotin-phenol, then briefly with hydrogen peroxide, initiating biotin-phenol free radical generation and labeling. To minimize cellular damage, the reaction is then quenched using an antioxidant buffer. Cells are lysed and the labeled proteins are pulled down with streptavidin beads. The proteins are digested with trypsin, and finally the resulting peptidic fragments are analyzed using shotgun proteomics methods such as LC-MS/MS or SPS-MS3. [8]

If instead a protein fusion is not genetically accessible (such as in human tissue samples) but an antibody for the protein of interest is known, proximity labeling can still be enabled by fusing a labeling enzyme with the antibody, then incubating the fusion with the sample. [9] [10]

Applications

Proximity labeling methods have been used to study the proteomes of biological structures that are otherwise difficult to isolate purely and completely, such as cilia, [11] mitochondria, [6] postsynaptic clefts, [2] p-bodies, stress granules, [12] and lipid droplets. [13]

Fusion of APEX2 with G-protein coupled receptors (GPCRs) allows for both tracking GPCR signaling at a 20-second temporal resolution [14] and also identification of unknown GPCR-linked proteins. [15]

Proximity labeling has also been used for transcriptomics and interactomics. In 2019, Alice Ting and the Ting lab have used APEX to identify RNA localized to specific cellular compartments. [16] [17] In 2019, BioID has been tethered to the beta-actin mRNA transcript to study its localization dynamics. [18] Proximity labeling has also been used to find interaction partners of heterodimeric protein phosphatases, of the miRISC (microRNA-induced silencing complex) protein Ago2, and of ribonucleoproteins. [3]

Recent developments

TurboID-based proximity labeling has been used to identify regulators of a receptor involved in the innate immune response, a NOD-like receptor. [19] BioID-based proximity labeling has been used to identify the molecular composition of breast cancer cell invadopodia, which are important for metastasis. [20] Biotin-based proximity labeling studies demonstrate increased protein tagging of intrinsically disordered regions, suggesting that biotin-based proximity labeling can be used to study the roles of IDRs. [21] A photosensitizer nucleus-targeted small molecule has also been developed for photoactivatable proximity labeling. [22]

Photocatalytic-based Proximity Labeling

A new frontier in the field of proximity labeling exploits the utility of photocatalysis to achieve high spatial and temporal resolution of proximal protein microenvironments. [23] This photocatalytic technology leverages the photonic energy of iridium-based photocatalysts to activate diazirine probes that can tag proximal proteins within a tight radius of about four nanometers. [24] This technology was developed by the Merck Exploratory Science Center in collaboration with researchers at Princeton University. [24]

Related Research Articles

<span class="mw-page-title-main">Proteomics</span> Large-scale study of proteins

Proteomics is the large-scale study of proteins. Proteins are vital parts of living organisms, with many functions such as the formation of structural fibers of muscle tissue, enzymatic digestion of food, or synthesis and replication of DNA. In addition, other kinds of proteins include antibodies that protect an organism from infection, and hormones that send important signals throughout the body.

<span class="mw-page-title-main">Gene expression</span> Conversion of a genes sequence into a mature gene product or products

Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product that enables it to produce end products, proteins or non-coding RNA, and ultimately affect a phenotype. These products are often proteins, but in non-protein-coding genes such as transfer RNA (tRNA) and small nuclear RNA (snRNA), the product is a functional non-coding RNA. The process of gene expression is used by all known life—eukaryotes, prokaryotes, and utilized by viruses—to generate the macromolecular machinery for life.

<span class="mw-page-title-main">Fluorescent tag</span>

In molecular biology and biotechnology, a fluorescent tag, also known as a fluorescent label or fluorescent probe, is a molecule that is attached chemically to aid in the detection of a biomolecule such as a protein, antibody, or amino acid. Generally, fluorescent tagging, or labeling, uses a reactive derivative of a fluorescent molecule known as a fluorophore. The fluorophore selectively binds to a specific region or functional group on the target molecule and can be attached chemically or biologically. Various labeling techniques such as enzymatic labeling, protein labeling, and genetic labeling are widely utilized. Ethidium bromide, fluorescein and green fluorescent protein are common tags. The most commonly labelled molecules are antibodies, proteins, amino acids and peptides which are then used as specific probes for detection of a particular target.

<span class="mw-page-title-main">Protein isoform</span> Forms of a protein produced from different genes

A protein isoform, or "protein variant", is a member of a set of highly similar proteins that originate from a single gene or gene family and are the result of genetic differences. While many perform the same or similar biological roles, some isoforms have unique functions. A set of protein isoforms may be formed from alternative splicings, variable promoter usage, or other post-transcriptional modifications of a single gene; post-translational modifications are generally not considered. Through RNA splicing mechanisms, mRNA has the ability to select different protein-coding segments (exons) of a gene, or even different parts of exons from RNA to form different mRNA sequences. Each unique sequence produces a specific form of a protein.

In biochemistry, biotinylation is the process of covalently attaching biotin to a protein, nucleic acid or other molecule. Biotinylation is rapid, specific and is unlikely to disturb the natural function of the molecule due to the small size of biotin. Biotin binds to streptavidin and avidin with an extremely high affinity, fast on-rate, and high specificity, and these interactions are exploited in many areas of biotechnology to isolate biotinylated molecules of interest. Biotin-binding to streptavidin and avidin is resistant to extremes of heat, pH and proteolysis, making capture of biotinylated molecules possible in a wide variety of environments. Also, multiple biotin molecules can be conjugated to a protein of interest, which allows binding of multiple streptavidin, avidin or neutravidin protein molecules and increases the sensitivity of detection of the protein of interest. There is a large number of biotinylation reagents available that exploit the wide range of possible labelling methods. Due to the strong affinity between biotin and streptavidin, the purification of biotinylated proteins has been a widely used approach to identify protein-protein interactions and post-translational events such as ubiquitylation in molecular biology.

<span class="mw-page-title-main">Two-hybrid screening</span> Molecular biology technique

Two-hybrid screening is a molecular biology technique used to discover protein–protein interactions (PPIs) and protein–DNA interactions by testing for physical interactions between two proteins or a single protein and a DNA molecule, respectively.

In cellular biology, P-bodies, or processing bodies, are distinct foci formed by phase separation within the cytoplasm of a eukaryotic cell consisting of many enzymes involved in mRNA turnover. P-bodies are highly conserved structures and have been observed in somatic cells originating from vertebrates and invertebrates, plants and yeast. To date, P-bodies have been demonstrated to play fundamental roles in general mRNA decay, nonsense-mediated mRNA decay, adenylate-uridylate-rich element mediated mRNA decay, and microRNA (miRNA) induced mRNA silencing. Not all mRNAs which enter P-bodies are degraded, as it has been demonstrated that some mRNAs can exit P-bodies and re-initiate translation. Purification and sequencing of the mRNA from purified processing bodies showed that these mRNAs are largely translationally repressed upstream of translation initiation and are protected from 5' mRNA decay.

<span class="mw-page-title-main">Stress granule</span> Cytoplasmic biomolecular condensates of proteins and RNA occurring in cells under stress

In cellular biology, stress granules are biomolecular condensates in the cytosol composed of proteins and RNAs that assemble into 0.1–2 μm membraneless organelles when the cell is under stress. The mRNA molecules found in stress granules are stalled translation pre-initiation complexes associated with 40S ribosomal subunits, translation initiation factors, poly(A)+ mRNAs and RNA-binding proteins (RBPs). While they are membraneless organelles, stress granules have been proposed to be associated with the endoplasmatic reticulum. There are also nuclear stress granules. This article is about the cytosolic variety.

<span class="mw-page-title-main">ILF2</span> Protein-coding gene in the species Homo sapiens

Interleukin enhancer-binding factor 2 is a protein that in humans is encoded by the ILF2 gene.

DNA adenine methyltransferase identification, often abbreviated DamID, is a molecular biology protocol used to map the binding sites of DNA- and chromatin-binding proteins in eukaryotes. DamID identifies binding sites by expressing the proposed DNA-binding protein as a fusion protein with DNA methyltransferase. Binding of the protein of interest to DNA localizes the methyltransferase in the region of the binding site. Adenine methylation does not occur naturally in eukaryotes and therefore adenine methylation in any region can be concluded to have been caused by the fusion protein, implying the region is located near a binding site. DamID is an alternate method to ChIP-on-chip or ChIP-seq.

Alice Yen-Ping Ting is Taiwanese-born American chemist. She is a professor of genetics, of biology, and by courtesy, of chemistry at Stanford University. She is also a Chan Zuckerberg Biohub investigator and a member of the National Academy of Sciences.

The Streptavidin-Binding Peptide (SBP)-Tag is a 38-amino acid sequence that may be engineered into recombinant proteins. Recombinant proteins containing the SBP-Tag bind to streptavidin and this property may be utilized in specific purification, detection or immobilization strategies.

Edward Marcotte is a professor of biochemistry at The University of Texas at Austin, working in genetics, proteomics, and bioinformatics. Marcotte is an example of a computational biologist who also relies on experiments to validate bioinformatics-based predictions.

<span class="mw-page-title-main">Proximity ligation assay</span>

Proximity ligation assay is a technology that extends the capabilities of traditional immunoassays to include direct detection of proteins, protein interactions, extracellular vesicles and post translational modifications with high specificity and sensitivity. Protein targets can be readily detected and localized with single molecule resolution and objectively quantified in unmodified cells and tissues. Utilizing only a few cells, sub-cellular events, even transient or weak interactions, are revealed in situ and sub-populations of cells can be differentiated. Within hours, results from conventional co-immunoprecipitation and co-localization techniques can be confirmed.

<span class="mw-page-title-main">Single-cell analysis</span> Testbg biochemical processes and reactions in an individual cell

In the field of cellular biology, single-cell analysis and subcellular analysis is the study of genomics, transcriptomics, proteomics, metabolomics and cell–cell interactions at the single cell level. The concept of single-cell analysis originated in the 1970s. Before the discovery of heterogeneity, single-cell analysis mainly referred to the analysis or manipulation of an individual cell in a bulk population of cells at a particular condition using optical or electronic microscope. To date, due to the heterogeneity seen in both eukaryotic and prokaryotic cell populations, analyzing a single cell makes it possible to discover mechanisms not seen when studying a bulk population of cells. Technologies such as fluorescence-activated cell sorting (FACS) allow the precise isolation of selected single cells from complex samples, while high throughput single cell partitioning technologies, enable the simultaneous molecular analysis of hundreds or thousands of single unsorted cells; this is particularly useful for the analysis of transcriptome variation in genotypically identical cells, allowing the definition of otherwise undetectable cell subtypes. The development of new technologies is increasing our ability to analyze the genome and transcriptome of single cells, as well as to quantify their proteome and metabolome. Mass spectrometry techniques have become important analytical tools for proteomic and metabolomic analysis of single cells. Recent advances have enabled quantifying thousands of protein across hundreds of single cells, and thus make possible new types of analysis. In situ sequencing and fluorescence in situ hybridization (FISH) do not require that cells be isolated and are increasingly being used for analysis of tissues.

The term Adhesome was first used by Richard Hynes to describe the complement of cell-cell and cell-matrix adhesion receptors in an organism and later expanded by Benny Geiger and co-workers to include the entire network of structural and signaling proteins involved in regulating cell-matrix adhesion.

<span class="mw-page-title-main">Spatial transcriptomics</span> Range of methods designed for assigning cell types

Spatial transcriptomics is a method for assigning cell types to their locations in the histological sections and can also be used to determine subcellular localization of mRNA molecules. First described in 2016 by Ståhl et al., it has since undergone a variety of improvements and modifications.

CITE-Seq is a method for performing RNA sequencing along with gaining quantitative and qualitative information on surface proteins with available antibodies on a single cell level. So far, the method has been demonstrated to work with only a few proteins per cell. As such, it provides an additional layer of information for the same cell by combining both proteomics and transcriptomics data. For phenotyping, this method has been shown to be as accurate as flow cytometry by the groups that developed it. It is currently one of the main methods, along with REAP-Seq, to evaluate both gene expression and protein levels simultaneously in different species.

<span class="mw-page-title-main">Translatomics</span>

Translatomics is the study of all open reading frames (ORFs) that are being actively translated in a cell or organism. This collection of ORFs is called the translatome. Characterizing a cell's translatome can give insight into the array of biological pathways that are active in the cell. According to the central dogma of molecular biology, the DNA in a cell is transcribed to produce RNA, which is then translated to produce a protein. Thousands of proteins are encoded in an organism's genome, and the proteins present in a cell cooperatively carry out many functions to support the life of the cell. Under various conditions, such as during stress or specific timepoints in development, the cell may require different biological pathways to be active, and therefore require a different collection of proteins. Depending on intrinsic and environmental conditions, the collection of proteins being made at one time varies. Translatomic techniques can be used to take a "snapshot" of this collection of actively translating ORFs, which can give information about which biological pathways the cell is activating under the present conditions.

Deterministic Barcoding in Tissue for Spatial Omics Sequencing (DBiT-seq) was developed at Yale University by Rong Fan and colleagues in 2020 to create a multi-omics approach for studying spatial gene expression heterogenicity within a tissue sample. This method can be used for the co-mapping mRNA and protein levels at a near single-cell resolution in fresh or frozen formaldehyde-fixed tissue samples. DBiT-seq utilizes next generation sequencing (NGS) and microfluidics. This method allows for simultaneous spatial transcriptomic and proteomic analysis of a tissue sample. DBiT-seq improves upon previous spatial transcriptomics applications such as High-Definition Spatial Transcriptomics (HDST) and Slide-seq by increasing the number of detectable genes per pixel, increased cellular resolution, and ease of implementation.

References

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