Patent Application
20140186870
One of the greatest challenges in biology is to determine the complete molecular composition of cells and their sub-compartments. Currently, the state-of-the-art for tackling this problem is to purify compartments or macromolecular complexes of interest, and determine their components (e.g., proteins) by mass spectrometry. Such conventional proteomics methods are not capable to provide a comprehensive assessment of the proteome of living cells or of substructures within living cells, such as organelles, or macromolecular complexes. While some conventional approaches to proteomic analysis, e.g., based on mass spectrometry (MS) allow for the unbiased identification of nearly all proteins in a complex sample, they are limited by the fact that they cannot be performed on living cells. Sample preparation for current proteomics assays requires lysis of biological material, and, therefore, spatial and dynamic information cannot be analyzed.
Currently used approaches to solve this problem rely on the purification of spatially-defined components of interest from cells after lysis. For example, mitochondria, ER, and other organelles are purified by repeated centrifugation and fractionation steps, while macromolecular complexes are purified using antibodies or affinity tags. However, these purifications have drawbacks. For example, current purification methods typically lead to numerous false positives and false negatives, and many subcellular structures of live cells are not amenable to purification. For example, in neurons, it would be transformative to know the complete molecular composition of the synaptic cleft, but this structure cannot be purified. Likewise, although the mitochondrial proteome has been characterized, it would be illuminating to separately map the proteomes of the mitochondrial inter-membrane space, outer mitochondrial membrane, and contact sites between mitochondria and the endoplasmic reticulum. However, these proteomes are currently unknown because they cannot be purified.
Some aspects of this disclosure provide strategies, methods, reagents, systems, and kits for in vivo proteomics, allowing for the assessment of the proteome of cells and subcellular structures. The strategies for live cell proteomics described herein obviate the need for sample purification, and instead label the relevant proteome of interest within living cells. This is accomplished, for instance, through the use of promiscuous tagging enzymes that can be genetically targeted to any subcellular region of interest. The enzymes used catalyze reactions that lead to covalent labeling of nearby endogenous proteins. Because the methods for live cell proteomics provided herein do not rely on the purification of subcellular structures, problems and artifacts associated with organelle or macromolecular complex purification are avoided. Any subcellular region that can be accessed via genetic targeting is amenable to proteomic mapping using the strategies, methods, reagents, systems, and kits provided herein. The labeled proteome can be analyzed with any suitable downstream proteomic analysis technology, including, but not limited to mass spectrometry (MS) proteomics.
Some aspects of the invention relate to methods, materials, and compositions for tagging and analyzing molecules, e.g., proteins, in vivo. For example, some embodiments of this invention provide methods, materials, and compositions for tagging proteins in cells or in subcellular compartments, e.g., in living cells or subcellular compartments of living cells. In some embodiments, the tagged proteins are subsequently isolated and analyzed (e.g., identified). Aspects of the invention can be used to determine the proteomic composition of specific cells, cellular compartments, or macromolecular structures (e.g., protein complexes) under in vivo (e.g., physiological or perturbed) conditions. Aspects of this invention can be used to identify protein interaction partners, to map the proteome of subcellular compartments, and to map the proteome of specific cells or cell types in complex tissues or animals. Using aspects of this invention in personalized proteomics and for drug screening applications is also contemplated.
In some aspects the invention provides a method for proteomic mapping by contacting a living cell with a tagging enzyme under conditions suitable for the tagging enzyme to catalyze a reaction with a tagging substrate resulting in the tagging of proteins within the vicinity of the tagging enzyme, and isolating and analyzing the tagged proteins to create a first proteome map. In some embodiments the method further comprises contacting the living cell with a tagging substrate. In other embodiments the tagging enzyme that is contacted with the cell is a genetic construct encoding a protein tagging enzyme. The living cell may be exposed to a condition, such as exposure to a therapeutic agent, prior to or during the step of contacting with a tagging enzyme in some embodiments.
In other embodiments the method further comprises contacting a second living cell exposed to a second condition with a tagging enzyme under conditions suitable for the tagging enzyme to catalyze a reaction with a tagging substrate resulting in the tagging of proteins in the second living cell within the vicinity of the tagging enzyme, and isolating and analyzing the tagged proteins from the second living cell to create a second proteome map. In some embodiments the first and second proteome maps are compared.
Some aspects of this invention provide methods for protein tagging in live cells. In some embodiments, the method comprises contacting a living cell with a tagging enzyme and a tagging substrate under conditions suitable for the enzyme to catalyze a reaction resulting in the tagging of molecules within the vicinity of the enzyme. In some embodiments, the tagged molecules comprise protein molecules. In some embodiments, the enzyme is a peroxidase. In some embodiments, the conditions comprise the presence of a substrate of the enzyme within the subcellular compartment. In some embodiments, the substrate is a tyramide. In some embodiments, the substrate is a labeled tyramide. In some embodiments, the substrate is a biotinylated tyramide. In some embodiments, the method further comprises isolating the tagged molecules. In some embodiments, the method further comprises analyzing the isolated molecules. In some embodiments, the analyzing comprises identifying the molecules. In some embodiments, the analyzing comprises determining the amino acid sequence of the tagged proteins. In some embodiments, the analyzing is performed by mass spectrometry analysis. In some embodiments, the tagging enzyme is targeted to a subcellular compartment. In some embodiments, the subcellular compartment is endoplasmatic reticulum, golgi apparatus, synaptic plaques, nucleus, mitochondria. In some embodiments, the contacting comprises expressing the enzyme in the cell. In some embodiments, the enzyme comprises a localization signal or targeting sequences that targets the enzyme to a specific subcellular domain. In some embodiments, the localization signal is a peptide sequence, for example, a nuclear localization signal (NLS) peptide, fused to the enzyme. In some embodiments, the enzyme is expressed in the cell under the control of a cell-type specific promoter limiting expression of the enzyme to specific cell types. In some embodiments, the enzyme is expressed as a fusion to a signal peptide targeting the enzyme to a particular subcellular compartment and wherein expression of the enzyme is controlled by a cell type-specific promoter.
Some aspects of this invention provide methods for live cell protein tagging that, comprise contacting a living cell with a peroxidase under conditions suitable for the peroxidase to convert a substrate into a reactive, short-lived intermediate that covalently binds to a molecule in the vicinity of the peroxidase. In some embodiments, the substrate comprises a tag. In some embodiments, the tag is a binding agent, for example, biotin. In some embodiments, the substrate comprises a tyramide moiety. In some embodiments, the peroxidase is horse radish peroxidase. In some embodiments, the peroxidase is soybean peroxidase. In some embodiments, the peroxidase is ascorbate peroxidase. In some embodiments, the peroxidase is targeted to be localized in a specific subcellular compartment. In some embodiments, the subcellular compartment is endoplasmatic reticulum, golgi apparatus, synaptic plaques, nucleus, mitochondria, or synaptic cleft. In some embodiments, the method further comprises isolating the tagged molecules. In some embodiments, the method further comprises analyzing the isolated molecules. In some embodiments, the analyzing comprises identifying the molecules. In some embodiments, the analyzing is performed by Mass Spectrometry. In some embodiments, the contacting comprises expressing the enzyme in the cell.
Some aspects of this invention provide cells expressing a tagging enzyme fused to a localization signal targeting the tagging enzyme to a subcellular compartment. In some embodiments, the cell is contacted with a substrate of the tagging enzyme. In some embodiments, the tagging enzyme is a peroxidase. In some embodiments, the tagging enzyme is not horse radish peroxidase. In some embodiments, the tagging enzyme is soy bean peroxidase. In some embodiments, the substrate comprises a tyramide moiety. In some embodiments, the substrate is biotin tyramide. In some embodiments, the tagging enzyme is expressed from an expression construct comprising a tissue-specific promoter.
Some aspects of this invention provide methods for protein tagging that comprise (i) expressing a tagging enzyme comprising a subcellular localization signal in a living cell; (ii) contacting the subcellular compartment with a substrate of the tagging enzyme; (iii) isolating a population of tagged proteins from the cell; and (iv) characterizing the isolated protein population. In some embodiments, the characterizing comprises determining the identity of a protein in the isolated protein population. In some embodiments, the protein identity is determined by performing mass spectrometry. In some embodiments, the tagging enzyme is a peroxidase. In some embodiments, the tagging enzyme is contacted with the substrate in the presence of H2O2. In some embodiments, the substrate is a tagged tyramide. In some embodiments, the substrate is alkyne-tyramide. In some embodiments, the tagging enzyme is expressed as a fusion with a signaling peptide targeting the enzyme to a particular subcellular compartment. In some embodiments, the tagging enzyme is expressed from a tissue-specific promoter. In some embodiments, the tagging enzyme is selected from the group consisting of horseradish peroxidase, soybean peroxidase, and ascorbate peroxidase.
Some aspects of this invention provide isolated, monomeric peroxidases that mutants of a wild type peroxidases. In some embodiments, the mutant peroxidase is an ascorbate peroxidase mutant and exhibits peroxidase activity towards a substrate of a wild type ascorbate peroxidase (APX). In some embodiments, the substrate is 3,3′-diaminobenzidine. In some embodiments, the peroxidase exhibits activity towards the substrate at a level similar to or higher than the activity level of wild type APX. In some embodiments, the peroxidase is a mutant pea APX. In some embodiments, the peroxidase comprises a K17N, a K20A, and/or a R21L mutation.
Some aspects of this invention provide kits for protein tagging. In some embodiments, the kit comprises an expression construct encoding a tagging enzyme, for example, a peroxidase. In some embodiments, the kit comprises an expression construct comprising a subcellular localization signal. In some embodiments, the subcellular localization signal is a signal targeting a protein comprising the signal to the endoplasmatic reticulum, golgi apparatus, synaptic plaques, nucleus, mitochondria, or the synaptic cleft. In some embodiments, the kit includes an expression construct into which a desired tagging enzyme or localization signal can be cloned to generate a nucleic acid encoding a tagging enzyme fused to a localization signal. In some embodiments, the kit comprises an enzyme substrate. In some embodiments, the enzyme substrate comprises or is conjugated to an agent, for example, a detectable label or a reactive handle. In some embodiments, the tagging enzyme is a peroxidase. In some embodiments, the tagging enzyme is selected from the group consisting of horseradish peroxidase, ascorbate peroxidase, and soybean peroxidase. In some embodiments, the peroxidase is an engineered, mutant peroxidase. In some embodiments, the peroxidase is a mutant pea ascorbate peroxidase. In some embodiments, the peroxidase comprises a K17N, a K20A, and/or a R21L mutation. In some embodiments, the substrate comprises a tyramide moiety. In some embodiments, the substrate comprises a binding agent. In some embodiments, the substrate is biotin tyramide. In some embodiments, the kit further comprises H2O2. In some embodiments, the expression construct encodes a protein targeting signal as a fusion to the tagging enzyme.
Other advantages, features, and uses of the invention will be apparent from the Detailed Description of certain Embodiments; the Drawings, the Examples section, and the Claims.
Some aspects of the present invention are based on the development of a protein tagging strategy using a promiscuous tagging enzyme that tags a proteome of interest, with spatial and temporal control, and that can be used to label molecules, e.g., proteins, within living cells or subcellular components, or multi protein complexes. This process may be referred to as proteome mapping. The approach described herein has not previously been explored for mapping cellular and sub-cellular proteomes, and allows for the tagging and subsequent analysis of live cell proteomes, for example, of proteomes associated with subcellular structures. An important advantage of the methods of the invention is that they permit both spatial and temporal control of labeling in live cells.
Some aspects of this invention relate to the surprising discovery that promiscuous labeling of proteins can be used in the context of living cells to tag proteins of cells or subcellular structures. This is surprising in that the interior of living cells comprises highly complex environments and compartments that were not previously thought to be accessible to promiscuous labeling methodologies. A proteome may be a set of proteins expressed by a genome, cell, subcellular compartment, a multi-protein complex, tissue or organism. In some embodiments, a proteome is the entire set of proteins expressed by a genome, subcellular compartment, multi-protein complex, tissue or organism, in some embodiments, a proteome is the set of expressed proteins expressed in a given type of cells, subcellular compartment or an organism at a given time under defined conditions.
The methods of the invention allow, for the first time, the ability to analyze proteomes in an accurate and high throughput manner. As such, proteome maps can be developed not only for different cells, subcellular compartments or organisms but also for cells or organisms exposed to different conditions or environments. For example, proteomes of cells or organisms exposed to different therapeutic agents, different concentrations of therapeutic agents, and/or combinations of therapeutic agents may be mapped and analyzed independently or compared against one another to examine changes occurring within a cell or organism. Additionally, changes in cells or organisms over time in diseased states or normal states may be examined at the proteome level. The wealth of information that can be extracted from the methods of the invention is enormous.
In general, the methods and strategies for protein tagging in living cells employ a tagging enzyme (a promiscuous bond-forming enzyme), alternatively, in some instances, simply referred to as an enzyme or promiscuous enzyme, e.g., a ligase, such as a biotin ligase, to catalyze a reaction resulting in tagging proteins in the vicinity of the enzyme. A tagging enzyme may be used, for instance, that utilizes a substrate to create and release a reactive moiety, e.g., an activated adenylate ester-biotin-AMP in the case of biotin ligase, that attaches to and thus labels nearby proteins9. When it is desirable to achieve sufficient spatial resolution of labeling, or spatial restriction of the cloud of reactive moieties generated by the tagging enzyme, reactive moieties with a sufficiently short half-life may be chosen. For example, because adenylate esters generated by biotin ligase persist for many minutes, using such tagging enzymes results in insufficient spatial resolution in cells for certain applications, since biotin-AMP would diffuse across the entire cell before becoming quenched. Some methods and strategies provided herein utilize reactive moieties that have a shorter half-life, and thus a restricted labeling radius, allowing for proteome tagging at a resolution of subcellular structures, such as organelles or protein complexes. The strategies provided herein further do not rely on toxic labels, such as ruthenium and nickel complexes which catalyze photo-oxidation of tyrosine and tryptophan side chains, leading to protein-protein crosslinking10, 11. Such toxic chemistry is incompatible with the cell interior due to its requirement for cytotoxic ammonium persulfate as a co-oxidant.
In some aspects, this disclosure provides methods for labeling the endogenous proteome of defined subcellular compartments or macromolecular complexes, with nanometer spatial resolution in living cells. Proteins thus labeled, for example with a binding agent, such as biotin, can be isolated using affinity-based methods (e.g., streptavidin beads) and analyzed by any suitable downstream assay (e.g., mass spectrometry). Some of the methods for labeling the endogenous proteome of subcellular compartments rely on tagging enzymes that can be genetically targeted to a cellular region of interest. The methods provided herein, accordingly, allow for the analysis of the molecular composition of cells and their sub-compartments. The strategies and methods provided herein are useful in the context of proteomics research as well as in the context of the diagnosis of disease, e.g., as they allow for the generation of patient-specific proteomes of subcellular structures implicated in disease.
Tagging methods and techniques described herein can be used for different applications. In some embodiments, proteins within a specific subcellular compartment or region (e.g., the nucleus, endoplasmic reticulum, Golgi, mitochondria, mitochondria outer membrane, mitochondria inner membrane, mitochondria matrix space, chloroplasts, synaptic cleft, presynaptic membrane, postsynaptic membrane, dendritic spines, transport vesicles, regions of contact between mitochondria and endoplasmic reticulum, nuclear membrane, etc.) can be specifically tagged. In some embodiments, proteins within particular cell types (e.g., astrocytes, dendrocytes, stem cells, etc.) can be specifically tagged, for example, proteins within a specific cell type within a complex tissue, animal, or cell population. In some embodiments, proteins within particular macromolecular complexes (e.g., protein complexes such as ribosomes, replisome, transcription complex, spliceosome, DNA repair complex, fatty acid synthase, polyketide synthase, non-ribosomal peptide synthase, glutamate receptor signaling complex, neurexin-neuroligin signaling complex, etc.) can be tagged. In each context, the tagged proteins can be analyzed (e.g., isolated and identified) to determine the proteomic content of the specific cells, cellular compartments or regions, or macromolecular complexes of interest. This information can be used for research, diagnostic, therapeutic, and other applications. In some embodiments, the proteomic content of patient cells may be determined. For example, cells may be isolated from a patient, amplified or differentiated using IPS cell technology (induced pluripotent stem cell), contacted with a vector (e.g., a viral vector) that expresses a tagging enzyme, for example, a tagging enzyme fused to a localization signal effecting localization of the tagging enzyme in a specific subcellular compartment, labeling can be performed in the living cells, and the resulting tagged proteins can be analyzed, for example, to identify patient specific proteomic information that can be useful to assist in diagnostic, prognostic, and/or therapeutic decisions, and in drug screening assays.
Thus, in some aspects the invention is a method for proteomic mapping, which involves contacting a living cell with a tagging enzyme under conditions suitable for the tagging enzyme to catalyze a reaction with a tagging substrate resulting in the tagging of proteins within the vicinity of the tagging enzyme, and isolating and analyzing the tagged proteins to create a first proteome map.
A living cell, as used herein, refers to an intact cell naturally occurring or modified isolated from other cells, mixed with other cells in a culture, within a tissue (partial or intact) or within an organism. In some embodiments, the living cell is a cell engineered to express a tagging enzyme, for example, a peroxidase. In some embodiments, the living cell expresses a tagging enzyme that is targeted to a subcellular compartment or structure, for example, via a localization signal comprised in or fused to the tagging enzyme.
In some embodiments, the living cell is contacted with a tagging enzyme. The term contacting as used herein refers to exposure of the cell to the enzyme in such a manner that the tagging enzyme is delivered to the cell interior or exterior, depending on which region of the cell will be analyzed. In some embodiments the tagging enzyme is delivered to the interior or the cell and in some instances to specific subcellular compartments. The term contacting may refer to expression of the tagging enzyme in the cell, e.g., from the cellular genome or from an exogenous nucleic acid (e.g., a viral genome, a plasmid, an artificial chromosome). The term may also refer to delivering the tagging enzyme to the cell, e.g., by administering a tagging enzyme to a tissue.
The term tagging enzyme refers to an enzyme that catalyzes a reaction which leads to the conjugation of a tag to a set of molecules, for example, proteins, carbohydrates, or lipids. In some embodiments, a tagging enzyme catalyzes a reaction that results in promiscuous labeling of molecules, e.g., proteins, in the vicinity of the enzyme. A tagging enzyme may catalyze a reaction in which a substrate, also referred to herein as a tagging substrate, is converted into a reactive form, e.g., a radical, which reacts with and attaches to a molecule, e.g., a protein, in the vicinity of the enzyme. In some embodiments, the reactive form of the tagging substrate attaches to the molecule via the formation of a covalent bond between the tagging substrate and the molecule. The half-life of the reactive form of the tagging substrate determines how far the reactive form can travel from its point of generation before reacting with a molecule. Accordingly, the half-life of the reactive form of the tagging substrate determines the labeling radius. For example, in some embodiments, the tagging enzyme is a biotin ligase, catalyzing the addition of a biotin tag to a protein. In some embodiments, the tagging enzyme catalyzes a reaction converting a tagging substrate into a reactive form by creating a reactive moiety on the tagging substrate that is able to react with a protein, for example, able to form a covalent bond with a protein, e.g., a moiety on the peptide backbone or a moiety on an amino acid side chain of the protein. In some embodiments, the tagging enzyme, for example, a peroxidase, is engineered to be expressed and targeted to particular cells, subcellular compartments, and/or macromolecular complexes in vivo. In some embodiments, a tagging enzyme is engineered to have a novel activity towards a substrate of interest. In some embodiments, a cell contacted with a tagging enzyme is contacted with a tagging substrate under conditions suitable for the tagging enzyme to convert the tagging substrate into a reactive form that can react with and attach to molecules in the vicinity of the tagging enzyme.
In some embodiments, the tagging enzyme is a peroxidase (e.g., horseradish peroxidase, soybean peroxidase, or ascorbate peroxidase). The discovery that peroxidases can be used as efficient tagging enzymes in living cells, as disclosed herein, was surprising, in that, while horseradish peroxidase (HRP) targeted to cell surface proteins via antibodies has been reported to convert an aryl azide into a nitrene, which labeled nearby proteins12, the use of peroxidases for protein labeling in the interior of cells has not been deemed possible before, for example, since it has been reported that HRP is inactive in the cytosol because its four essential disulfide bonds have become reduced13.
Some exemplary peroxidases suitable as tagging enzyme in embodiments of this invention are described herein. In some embodiments, the tagging enzyme is a wild type peroxidase. In some embodiments, the tagging enzyme is an engineered peroxidase, for example, a peroxidase engineered to be expressed and/or active only within a subcellular compartment or structure of interest. In some embodiments, the tagging enzyme is a mutant peroxidase, e.g., a peroxidase comprising one or more mutations that enhances the catalytic activity of the peroxidase towards a tagging substrate in a subcellular compartment or structure of interest.
Representative sequences of some exemplary suitable tagging enzymes are provided below:
Additional suitable tagging enzymes will be apparent to those of skill in the art based on this disclosure. The application is not limited in this respect.
The methods of labeling, or tagging, molecules, e.g., proteins, in living cells provided herein typically include the use of a tagging substrate. A tagging substrate is a substrate of a tagging enzyme. A tagging substrate is typically provided in an inert, stable, or non-reactive form, e.g., a form that does not readily react with other molecules in living cells. Once in contact with an active tagging enzyme, the tagging substrate is converted from its stable form into a short-lived reactive form, for e.g., via generation of a reactive moiety, such as a radical, on the tagging substrate by the tagging enzyme. Some tagging substrates are, accordingly, also referred to as radical precursors. The reactive form of the tagging substrate then reacts with and attaches to a molecule, e.g., a protein, in the vicinity of the tagging enzyme. Accordingly, in some embodiments, a tagging substrate comprises an inert or stable moiety that can be converted by the tagging enzyme into a reactive moiety. The reaction of the tagging substrate with a molecule, e.g., a protein in the vicinity of the tagging enzyme, results in the tagging, or labeling, of the molecule. Typically, a tagging substrate comprises a tag, which is a functional moiety or structure that can be used to detect, identify, or isolate a molecule comprising the tag, e.g., a protein that has been tagged by reacting with a tagging substrate. Suitable tags include, but are not limited to, for example, a detectable label, a binding agent, such as biotin, or a fluorescent probe, a click chemistry handle, an azide, alkyne, phosphine, trans-cyclooctene, or a tetrazine moiety. In some embodiments, the reaction of the reactive form of the tagging substrate with a molecule, e.g., a protein, may lead to changes in the molecule, e.g., oxygenation, that can be exploited for detecting and/or isolating the changed molecules. Non-limiting examples of such tagging substrates are chromophores, e.g., resorufin, malachite green, KillerRed, Ru(bpy)32+, and miniSOG31 , which can generate reactive oxygen species that oxidize molecules in the vicinity of the respective tagging enzyme. The oxidation can be used to isolate and/or identify the oxidized molecules. In some embodiments, the reactive form of the tagging substrate crosses cell membranes, while in other embodiments membranes are impermeable to the reactive form of the tagging substrate.
A tag may be, in some embodiments, a detectable label. In some embodiments, a tag may be a functional moiety or structure that can be used to detect, isolate, or identify molecules comprising the tag. A tag may also be created as a result of a reactive form of a tagging substrate reacting with a molecule, e.g., the creation of oxidative damage on a protein by a reactive oxygen species may be a tag. In some embodiments, the tag is a biotin-based tag and the tagging enzyme, e.g., a peroxidase, generates a reactive biotin moiety that binds to proteins within the vicinity of the tagging enzyme. In some embodiments, the biotin-based tags are biotin tyramide molecules. In some embodiments, the tagging substrate is a peroxidase substrate. Structures of some exemplary tagging substrates (radical precursors) of peroxidase enzymes that are useful in some of the methods provided herein are provided below:
Additional exemplary peroxidase substrates (radical precursors) are provided below:
Additional suitable tagging substrates will be apparent to those of skill in the art, and the invention is not limited in this respect. In some embodiments, the tag is an alkyne tyramide and the peroxidase generates a reactive moiety that binds to proteins within the vicinity of the peroxidase. The alkyne subsequently can be modified, for example, by a click chemistry reaction to attach a tag (e.g., a biotin tag). The tag can then be used for further analysis (e.g., isolation and identification). It should be noted that the invention is not limited to alkyne tyramide, but that any functional group that can be chemoselectively derivatized can be used. Some examples are: azide or alkyne or phosphine, or trans-cyclooctene, or tetrazine, or cyclooctyne, or ketone, or hydrazide, or aldehyde, or hydrazine.
In some embodiments, a tagging substrate for peroxidase, for example, a biotinylated tyramide, is administered, applied, or contacted to the cells or tissue in vivo, and proteins that are located within the vicinity of the expressed peroxidase are tagged, i.e., the biotin tyramide is converted into a reactive form by the tagging enzyme, here the peroxidase, and the reactive form reacts with and attaches to proteins in the vicinity of the peroxidase, resulting in biotin-tagging of the respective proteins. In the presence of peroxide (e.g., H2O2), the peroxidase converts the substrate into a short-lived, reactive intermediate, for example, a reactive tyramide radical, that can form a covalent bond with a protein. In some embodiments, the reactive intermediate, once created, reacts with (labels) proteins that are within the vicinity of the peroxidase enzyme molecule. The term “within the vicinity” refers to the special location around the enzyme and/or substrate. In some instances it may refer to a region of the cell such as a sub-cellular region, a membrane or protein complex. Alternatively it can be defined in terms of distance from the enzyme or substrate or a region i.e., as a diameter, circumference or linear distance. For example, in some embodiments, a molecule within the vicinity of a tagging enzyme is a molecule that is positioned less than about less than about 900 nm, less than about 800 nm, less than about 700 nm, less than about 600 nm, less than about 500 nm, less than about 400 nm, less than about 300 nm, less than about 200 nm, less than about 100 nm, less than about 90 nm, less than about 80 nm, less than about 70 nm, less than about 60 nm, less than about 50 nm, less than about 40 nm, less than about 30 nm, less than about 20 nm, or less than about 10 nm away from the active site of the tagging enzyme. In some embodiments, proteins that are not within the vicinity of the enzyme are not exposed to the reactive intermediate and hence not labeled. In some embodiments, expression or targeting of the tagging enzyme to a subcellular compartment results in quantitative tagging of virtually all proteins within that compartment.
In addition to improving promiscuous labeling via peroxidase reaction engineering, the invention also includes but it is not limited to other non-peroxidase strategies for labeling, including the use of other enzymes, light-triggered labeling, and cascade reactions. For example, KatG (a mycobacterial catalase-peroxidase enzyme), CueO (a multi-copper oxidase), and bilirubin oxidase are three suitable tagging enzymes. Like peroxidases, all of these enzymes convert stable small molecule substrates into short-lived reactive species. Their advantage, however, is that they utilize O2, and not H2O2, to catalyze their respective reactions, which may be advantageous in embodiments involving cells, subcellular compartments, or structures that are sensitive to H2O2 toxicity, KatG from M. tuberculosis is believed to oxidize the anti-tuberculosis drug isoniazid (an aryl hydrazide) into an acyl radical, which then diffuses out of the KatG active site to label the NADH moiety of InhA reductase30. CueO and bilirubin oxidase convert phenols into phenoxyl radicals at physiological pH. They also lack disulfides, and have solved crystal structures, which facilitates engineering.
Photo-oxidation reactions may also be used in the methods of the invention. Chromophores such as resorufin, malachite green, KillerRed, Ru(bpy)32+, and miniSOG31 can be used as tagging substrates, as they generate reactive oxygen species, which diffuse very short distances (40 Å for singlet oxygen and 15 Å for hydroxyl radical32) before oxidizing cellular molecules and thereby damaging them. These chromophores are the basis of Chromophore Assisted Light Inactivation, or CALI, which has been applied to cellular proteins. Common products of oxidative damage to proteins are aldehydes and ketones33, which provide a handle for selective protein pull-down by hydrazine- or hydroxylamine-biotin conjugates. If photo-oxidation is performed in the presence of reducing substrates, such as phenols or anilines (e.g., diaminobenzidine, used for electron microscopy), organic radicals will be generated, which can be exploited for covalent protein labeling. An advantage of this photo-oxidation approach compared to peroxidase-mediated labeling is the use of O2 instead of H2O2. In addition, hydroxyl radicals generated in type I photo-oxidation (by chromophores such as malachite green) are much more reactive than peroxidase-generated aryloxyl radicals (BDE 119 versus 88 kcal/mol18), which should lead to greater depth of coverage.
An additional type of tagging enzyme is based on a cascade reaction for covalent labeling in cells. Enediyne antibiotic prodrugs such as calicheamicin are activated inside cells to generate highly reactive 1,4-benzenoid diradicals. The structure of these prodrugs may be modified to make them activatable instead by orthogonal enzymes such as esterases or proteases, and, thus, useful as tagging substrates. N-nitrosoamides, which are converted by proteases via a cascade mechanism into reactive carbocations (with departure of N2)34 may also be used as tagging substrates. Originally designed as protease suicide inhibitors, the carbocations were found to diffuse too rapidly from the site of generation and label neighboring molecules, making them particularly well suited for use as tagging substrates.
Thus, exemplary tagging enzymes include but are not limited to peroxidases, KatG, CueO, and bilirubin oxidases. Exemplary tagging substrates include but are not limited to peroxidase substrates, such as tyramides, chromophores such as resorufin, malachite green, KillerRed, Ru(bpy)32+, and miniSOG (EKSFVITDPRLPDNPIIFASDGFLELTEYSREEILGRNGRFLQGPETDQATVQKI RDAIRDQREITVQLINYTKSGKKFWNLLHLQPMRDQKGELQYFIGVQLDG, SEQ ID NO: 5), and enediyne antibiotic prodrugs such as calicheamicin.
Some embodiments of this invention allow in vivo protein tagging mediated by a tagging enzyme which can be genetically targeted to any part of a living cell. In some embodiments, the tagging enzyme is present and/or active in all regions of the cell. In some embodiments, the tagging enzyme is present and/or active only in a subcellular compartment of the cell. In some embodiments, the tagging substrate is an exogenous small-molecule substrate that can be added or uncaged for the desired window of time, to permit precise temporal control of labeling. In some embodiments, the tagging substrate is conjugated to a binding agent, e.g., biotin (or other purification handle), for subsequent capture, e.g., by streptavidin-coated beads. In some embodiments, the tagging enzyme converts the substrate into a highly reactive species that has the potential to label any endogenous protein, in order to achieve high depth-of-coverage, e.g., in an MS experiment. In some embodiments, the reactive species has a short half-life on that its diffusion radius before quenching is less than ˜100 nm, to ensure high specificity. In some embodiments, it is preferable for the reactive species not to cross cell membranes, to allow mapping of the proteomes of membrane-bounded structures.
In some embodiments, a tagging enzyme is engineered to be expressed and/or targeted in vivo or in situ to specific cells, cellular compartments (e.g., endoplasmic reticulum, Golgi apparatus, mitochondria, nucleus, the synaptic cleft, transport vesicles, etc.), and/or macromolecular complexes (e.g., protein complexes such as ribosomes, nuclear pore complex, fatty acid synthases) of interest. In some embodiments, a tagging enzyme is engineered to tag proteins that are located within a limited distance of the tagging enzyme. As a result, in some embodiments, proteins that are located within the targeted cell, cellular compartment, and/or macromolecular complex (e.g., protein complex) are specifically tagged relative to other proteins that are not located near the tagging enzyme. It should be appreciated that the tagging process itself does not need to be protein specific. For example, in some embodiments, it is the specific localization of the tagging enzyme that results in the specific tagging of a subset of proteins of interest. In some embodiments, proteins that are present within the vicinity of the tagging enzyme may be tagged for further analysis. In some embodiments, all proteins present within the vicinity of the tagging enzyme may be tagged. Various versions of the methodology offer a range of labeling radii, from ˜500 nm to less than 10 nm, e.g., tagging radii of about 500 nm, about 400 nm, about 300 nm, about 250 nm, about 200 nm, about 100 nm, about 90 nm, about 80 nm, about 70 nm, about 60 nm, about 50 nm, about 40 nm, about 30 nm, about 20 nm, about 10 nm, about 5 nm, about 2.5 nm, or about 1 nm.
In some embodiments, the reactive moiety produced by the tagging enzyme, e.g., the peroxidase, can be inactivated by contacting it with a quenching agent, e.g., with water. As a result, the reactive moiety can have a short half-life and only modify proteins that are located within a short distance of the site of production (the peroxidase) before being inactivated. Accordingly, the zone of tagging can be limited by the diffusion rate of the reactive form of the tagging substrate, or the activated tagging moiety, and the half-life of the reactive form of the tagging substrate, or the activated tagging moiety.
In some embodiments, only proteins that are located within about 10 nm of the tagging enzyme are tagged. For example, in some embodiments using a peroxidase and a biotinylated peroxidase tagging substrate, e.g., a biotinylated tyramide, only proteins that are located within about 10 nm of the peroxidase are biotinylated. However, it should be appreciated that the zone of biotinylation may be altered depending on the enzyme and/or substrate structure used for tagging. Thus the labeling range can be adjusted from about 500 nm to <10 nm.
While the methods for protein tagging and the related reagents, materials and compositions described herein are well suited for use in living cells and tissues, it should be appreciated that their use is not so limited, but that they can also be applied to fixed cells and tissues, for example, to fixed cells and tissues obtained from a subject, e.g., in a clinical setting.
In some embodiments, only proteins that are located within about 10 nm of the peroxidase are biotinylated. However, it should be appreciated that the zone of biotinylation may be altered depending on the enzyme and/or substrate structure used for tagging. Thus the labeling range can be adjusted from about 500 nm to <10 nm.
While the methods for protein tagging and the related reagents, materials and compositions described herein are well suited for use in living cells and tissues, it should be appreciated that their use is not so limited, but that they can also be applied to fixed cells and tissues, for example, to fixed cells and tissues obtained from a subject, e.g., in a clinical setting.
The methods of the invention are particularly useful for analyzing proteins. The terms “protein,” “peptide” and “polypeptide” are used interchangeably herein, and refer to a polymer of amino acid residues linked together by peptide (amide) bonds. The terms refer to a protein, peptide, or polypeptide of any size, structure, or function. Typically, a protein, peptide, or polypeptide will be at least three amino acids long. A protein, peptide, or polypeptide may refer to an individual protein or a collection of proteins. One or more of the amino acids in a protein, peptide, or polypeptide may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a lipid group, a linker for conjugation, functionalization, or other modification, etc. A protein, peptide, or polypeptide may also be a single molecule or may be a multi-molecular complex. A protein, peptide, or polypeptide may be just a fragment of a naturally occurring protein or peptide. A protein, peptide, or polypeptide may be naturally occurring, recombinant, or synthetic, or any combination thereof.
The term “amino acid,” as used herein, includes any naturally occurring and non-naturally occurring amino acid. There are many known non-natural amino acids any of which may be included in the polypeptides or proteins described herein. See, for example, S. Hunt, The Non-Protein Amino Acids: In Chemistry and Biochemistry of the Amino Acids, edited by G. C. Barrett, Chapman and Hall, 1985. Some non-limiting examples of non-natural amino acids are 4-hydroxyproline, desmosine, gamma-aminobutyric acid, beta-cyanoalanine, norvaline, 4-(E)-butenyl-4(R)-methyl—N-methyl-L-threonine, N-methyl-L-leucine, 1-amino-cyclopropanecarboxylic acid, 1-amino-2-phenyl-cyclopropanecarboxylic acid, 1-amino-cyclobutanecarboxylic acid, 4-amino-cyclopentenecarboxylic acid, 3-amino-cyclohexanecarboxylic acid, 4-piperidylacetic acid, 4-amino-1-methylpyrrole-2-carboxylic acid, 2,4-diaminobutyric acid, 2,3-diaminopropionic acid, 2,4-diaminobutyric acid, 2-aminoheptanedioic acid, 4-(aminomethyl)benzoic acid, 4-aminobenzoic acid, ortho-, meta- and para-substituted phenylalanines (e.g., substituted with —C(═O)C6H5; —CF3; —CN; -halo; —NO2; —CH3), disubstituted phenylalanines, substituted tyrosines (e.g., further substituted with —C(═O)C6H5; —CF3; —CN; -halo; —NO2; —CH3), and statine. In the context of amino acid sequences, “X” or “Xaa” represents any amino acid residue, e.g., any naturally occurring and/or any non-naturally occurring amino acid residue.
Some methods described herein are useful to identify and characterize the proteomes of defined subcellular zones (organelles, synapses, macromolecular complexes) of living cells. In some embodiments, in vivo analysis of localized proteomes combines the advantages of imaging (spatial and temporal resolution, compatibility with live cells) with those of mass spectrometry-based proteomics (multiplexed detection of endogenous proteins) without their respective limitations (limited multiplexing and not endogenous proteins, for imaging—and loss of spatial information and information recorded after cell lysis for mass spectrometry) as illustrated in
Existing methods for analyzing the subcellular localization of proteins involve subcellular fractionation. However, this approach is messy and prone to contamination (for example, proteins get lost during purification, or contaminating proteins get included in the analysis). In addition, the biochemistry changes during long purification processes making it difficult to analyze dynamic states. Furthermore, fractionation methods require a lot of material making it difficult to analyze patient-derived samples. It also should be appreciated that certain cellular regions or zones just cannot be purified (e.g., synapses).
In contrast, methods described herein allow for rapid in vivo tagging (e.g., in living cells or tissues), starting from small amounts of cells. Because in some embodiments, labeling is performed on living, intact cells, subcellular compartments are intact and undisrupted, reducing the possibility of contamination or loss of protein components. In some embodiments, promiscuous enzymes are used in live cells to biotinylate endogenous proteins in a spatially-defined manner. See
The methods provided herein can also be used to discover endogenous components of macromolecular complexes, for example, of replisomes, ribosomes, transcription complexes, spliceosomes, and DNA repair complexes, complexes for fatty acid synthesis or for non-ribosomal peptide synthesis, neurexin-neuroligin signaling complexes, and glutamate receptor signaling complexes.
The methods provided herein can also be used to determine the proteome of specific cell types within complex tissues or heterogeneous cell populations, or of specific subcellular structures or organelles within specific cells in complex tissues or populations. For example, the methods provided herein can be used to assess the proteome of specific cell types in the brain, such as astrocytes, or of adult stem cells. The methods are particularly useful for analyzing the proteome of rare cells within complex cell populations.
Some aspects of the invention relate to an in vivo method of localizing protein tagging in order to analyze the proteins within a particular cell, cellular region or compartment, and/or macromolecular complex. Accordingly, some aspects of the invention are useful to identify specific proteomes associated with particular cells, cellular regions or compartments, and/or macromolecular complexes
Some embodiments of this invention use a peroxidase targeted to a specific cell type or subcellular structure and utilizes a tyramide substrate to label proteins within its immediate vicinity. For example, in some embodiments, horseradish peroxidase (HRP) is directed to a cell or subcellular compartment, and an HRP substrate, biotin-tyramide, is added along with H2O2 HRP oxidizes biotin-tyramide, which reacts locally and covalently with proteins in the vicinity of the enzyme. Deposited biotin can then be visualized by staining with fluorophore-conjugated streptavidin or biotin-labeled proteins can be isolated and further analyzed.
Those of skill in the art will appreciate that any peroxidase exhibiting peroxidase activity within the cell, cell type, or subcellular compartment or structure of interest can be used in embodiments of this invention. As described in more detail in the Examples section, specific peroxidases, e.g., HRP, soybean peroxidase, and ascorbate peroxidase, as well as mutant peroxidases, can be used in living cells for spatially-restricted biotinylation of endogenous proteins. These peroxidases were used in connection with some methods provided herein to obtain preliminary proteomes of the endoplasmic reticulum (ER) and mitochondrial matrix in living mammalian cells.
Additional exemplary protein tagging methods and results obtained by using such methods are described in more detail in the Examples section and in
The term “agent,” as used herein, refers to any molecule, entity, or moiety that can be conjugated to a protein, peptide, carbohydrate, lipid, or other biomolecule. For example, an agent may be a protein, an amino acid, a peptide, a polynucleotide, a carbohydrate, a detectable label, a binding agent, a tag, a metal atom, a contrast agent, a catalyst, a non-polypeptide polymer, a synthetic polymer, a recognition element, a lipid, a linker, or chemical compound, such as a small molecule. In some embodiments, the agent is a binding agent, for example, a ligand or a ligand-binding molecule, streptavidin, biotin, an antibody or an antibody fragment. In some such embodiments, the agent is a lipid, a carbohydrate, or a small molecule. Additional agents suitable for use in embodiments of the present invention will be apparent to the skilled artisan. The invention is not limited in this respect.
Peroxidases are enzymes that catalyze the one-electron oxidation of phenol-type substrates into highly-reactive phenoxyl radicals (
Peroxidases cannot readily be exploited for intracellular protein labeling because of their expression in mammalian cells and because of their lack of activity sufficient to achieve effective labeling. For example, Horseradish peroxidase (HRP), the most widely used peroxidase in biotechnology, has been successfully expressed and shown to be active in the secretory pathway of mammalian cells13. However, HRP has four structurally essential disulfide bonds that are reduced in the cytosol, destroying its activity. Some aspects of this invention address the problem of peroxidase expression and activity in mammalian cells, for example, by using different peroxidases, e.g., ascorbate peroxidase (APX), that lacks disulfide bonds. APX exhibits high activity in the mammalian cytosol with a range of substrates, including biotin-phenol (
The term “antibody”, as used herein, refers to a protein belonging to the immunoglobulin superfamily. The terms antibody and immunoglobulin are used interchangeably. With some exceptions, mammalian antibodies are typically made of basic structural units each with two large heavy chains and two small light chains. There are several different types of antibody heavy chains, and several different kinds of antibodies, which are grouped into different isotypes based on which heavy chain they possess. Five different antibody isotypes are known in mammals, IgG, IgA, IgE, IgD, and IgM, which perform different roles, and help direct the appropriate immune response for each different type of foreign object they encounter. In some embodiments, an antibody is an IgG antibody, e.g., an antibody of the IgG1, 2, 3, or 4 human subclass. Antibodies from mammalian species (e.g., human, mouse, rat, goat, pig, horse, cattle, camel) are within the scope of the term, as are antibodies from non-mammalian species (e.g., from birds, reptiles, amphibia) are also within the scope of the term, e.g., IgY antibodies.
Only part of an antibody is involved in the binding of the antigen, and antigen-binding antibody fragments, their preparation and use, are well known to those of skill in the art. As is well-known in the art, only a small portion of an antibody molecule, the paratope, is involved in the binding of the antibody to its epitope (see, in general, Clark, W. R. (1986) The Experimental Foundations of Modern Immunology Wiley & Sons, Inc., New York; Roitt, I. (1991) Essential Immunology, 7th Ed., Blackwell Scientific Publications, Oxford). Suitable antibodies and antibody fragments for use in the context of some embodiments of the present invention include, for example, human antibodies, humanized antibodies, domain antibodies, F(ab′), F(ab′)2, Fab, Fv, Fc, and Fd fragments, antibodies in which the Fc and/or FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; antibodies in which the FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; antibodies in which the FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; and antibodies in which the FR and/or CDR1 and/or CDR2 regions have been replaced by homologous human or non-human sequences. In some embodiments, so-called single chain antibodies (e.g., ScFv), (single) domain antibodies, and other intracellular antibodies may be used in the context of the present invention. Domain antibodies, camelid and camelized antibodies and fragments thereof, for example, VHH domains, or nanobodies are also encompassed in the term antibody. Further, chimeric antibodies, e.g., antibodies comprising two antigen-binding domains that bind to different antigens, are also suitable for use in the context of some embodiments of the present invention.
The term “antigen-binding antibody fragment,” as used herein, refers to a fragment of an antibody that comprises the paratope, or a fragment of the antibody that binds to the antigen the antibody binds to, with similar specificity and affinity as the intact antibody. Antibodies, e.g., fully human monoclonal antibodies, may be identified using phage display (or other display methods such as yeast display, ribosome display, bacterial display). Display libraries, e.g., phage display libraries, are available (and/or can be generated by one of ordinary skill in the art) that can be screened to identify an antibody that binds to an antigen of interest, e.g., using panning. See, e.g., Sidhu, S. (ed.) Phage Display in Biotechnology and Drug Discovery (Drug Discovery Series; CRC Press; 1st ed., 2005; Aitken, R. (ed.) Antibody Phage Display: Methods and Protocols (Methods in Molecular Biology) Humana Press; 2nd ed., 2009.
The term “binding agent,” as used herein refers to any molecule that binds another molecule with high affinity. In some embodiments, a binding agent binds its binding partner with high specificity. Examples for binding agents include, without limitation, antibodies, antibody fragments, receptors, ligands, aptamers, and adnectins.
The term “click chemistry” refers to a chemical philosophy introduced by K. Barry Sharpless of The Scripps Research Institute, describing chemistry tailored to generate covalent bonds quickly and reliably by joining small units comprising reactive groups together (see H. C. Kolb, M. G. Finn and K. B. Sharpless (2001). Click Chemistry: Diverse Chemical Function from a Few Good Reactions. Angewandte Chemie International Edition 40 (11): 2004-2021). Click chemistry does not refer to a specific reaction, but to a concept including, but not limited to, reactions that mimic reactions found in nature. In some embodiments, click chemistry reactions are modular, wide in scope, give high chemical yields, generate inoffensive byproducts, are stereospecific, exhibit a large thermodynamic driving force >84 kJ/mol to favor a reaction with a single reaction product, and/or can be carried out under physiological conditions. In some embodiments, a click chemistry reaction exhibits high atom economy, can be carried out under simple reaction conditions, use readily available starting materials and reagents, uses no toxic solvents or use a solvent that is benign or easily removed (preferably water), and/or provides simple product isolation by non-chromatographic methods (crystallisation or distillation).
The term “click chemistry handle,” as used herein, refers to a reactant, or a reactive group, that can partake in a click chemistry reaction. In some embodiments, a reactive handle is a click chemistry handle. For example, a strained alkyne, e.g., a cyclooctyne, is a click chemistry handle, since it can partake in a strain-promoted cycloaddition. In general, click chemistry reactions require at least two molecules comprising click chemistry handles that can react with each other. Such click chemistry handle pairs that are reactive with each other are sometimes referred to herein as partner click chemistry handles. For example, an azide is a partner click chemistry handle to a cyclooctyne or any other alkyne. Exemplary click chemistry handles suitable for use according to some aspects of this invention are described herein. Other suitable click chemistry handles are known to those of skill in the art. For two molecules to be conjugated via click chemistry, the click chemistry handles of the molecules have to be reactive with each other, for example, in that the reactive moiety of one of the click chemistry handles can react with the reactive moiety of the second click chemistry handle to form a covalent bond. Such reactive pairs of click chemistry handles are well known to those of skill in the art and include, but are not limited to, those described in Becer, Hoogenboom, and Schubert, Click Chemistry beyond Metal-Catalyzed Cycloaddition, Angewandte Chemie International Edition (2009) 48: 4900-4908; and H. C. Kolb, M. G. Finn and K. B. Sharpless (2001). Click Chemistry: Diverse Chemical Function from a Few Good Reactions. Angewandte Chemie International Edition 40 (11): 2004-2021, the entire contents of each of which are incorporated herein by reference.
The term “conjugated” or “conjugation” refers to an association of two molecules, for example, two proteins or a protein and an agent, e.g., a small molecule, with one another in a way that they are linked by a direct or indirect covalent or non-covalent interaction. In certain embodiments, the association is covalent, and the entities are said to be “conjugated” to one another. In some embodiments, a protein is post-translationally conjugated to another molecule, for example, a second protein, a small molecule, a detectable label, a click chemistry handle, or a binding agent, by forming a covalent bond between the protein and the other molecule after the protein has been formed, and, in some embodiments, after the protein has been isolated. In some embodiments, two molecules are conjugated via a linker connecting both molecules. For example, in some embodiments where two proteins are conjugated to each other to form a protein fusion, the two proteins may be conjugated via a polypeptide linker, e.g., an amino acid sequence connecting the C-terminus of one protein to the N-terminus of the other protein. In some embodiments, two proteins are conjugated at their respective C-termini, generating a C—C conjugated chimeric protein. In some embodiments, two proteins are conjugated at their respective N-termini, generating an N—N conjugated chimeric protein.
The term “detectable label” refers to a moiety that has at least one element, isotope, or functional group incorporated into the moiety which enables detection of the molecule, e.g., a protein or peptide, or other entity, to which the label is attached. Labels can be directly attached (i.e., via a bond) or can be attached by a linker (such as, for example, an optionally substituted alkylene; an optionally substituted alkenylene; an optionally substituted alkynylene; an optionally substituted heteroalkylene; an optionally substituted heteroalkenylene; an optionally substituted heteroalkynylene; an optionally substituted arylene; an optionally substituted heteroarylene; or an optionally substituted acylene, or any combination thereof, which can make up a linker). It will be appreciated that the label may be attached to or incorporated into a molecule, for example, a protein, polypeptide, or other entity, at any position. In general, a detectable label can fall into any one (or more) of five classes: a) a label which contains isotopic moieties, which may be radioactive or heavy isotopes, including, but not limited to, 2H, 3H, 13C, 14C, 15N, 18F, 31P, 32P, 35S, 67Ga, 76Br, 99mTc (Tc-99m) 111In, 123I, 125I, 131I, 153Gd, 169Yb, and 186Re; b) a label which contains an immune moiety, which may be antibodies or antigens, which may be bound to enzymes (e.g., such as horseradish peroxidase); c) a label which is a colored, luminescent, phosphorescent, or fluorescent moieties (e.g., such as the fluorescent label fluorescein-isothiocyanate (FITC); d) a label which has one or more photo affinity moieties; and e) a label which is a ligand for one or more known binding partners (e.g., biotin-streptavidin, FK506-FKBP). In certain embodiments, a label comprises a radioactive isotope, preferably an isotope which emits detectable particles, such as β particles. In certain embodiments, the label comprises a fluorescent moiety. In certain embodiments, the label is the fluorescent label fluorescein-isothiocyanate (FITC). In certain embodiments, the label comprises a ligand moiety with one or more known binding partners. In certain embodiments, the label comprises biotin. In some embodiments, a label is a fluorescent polypeptide (e.g., GFP or a derivative thereof such as enhanced GFP (EGFP)) or a luciferase (e.g., a firefly, Renilla, or Gaussia luciferase). It will be appreciated that, in certain embodiments, a label may react with a suitable substrate (e.g., a luciferin) to generate a detectable signal. Non-limiting examples of fluorescent proteins include GFP and derivatives thereof, proteins comprising fluorophores that emit light of different colors such as red, yellow, and cyan fluorescent proteins. Exemplary fluorescent proteins include, e.g., Sirius, Azurite, EBFP2, TagBFP, mTurquoise, ECFP, Cerulean, TagCFP, mTFP1, mUkG1, mAG1, AcGFP1, TagGFP2, EGFP, mWasabi, EmGFP, TagYPF, EYFP, Topaz, SYFP2, Venus, Citrine, mKO, mKO2, mOrange, mOrange2, TagRFP, TagRFP-T, mStrawberry, mRuby, mCherry, mRaspberry, mKate2, mPlum, mNeptune, T-Sapphire, mAmetrine, mKeima. See, e.g., Chalfie, M. and Kain, S R (eds.) Green fluorescent protein: properties, applications, and protocols Methods of biochemical analysis, v. 47 Wiley-Interscience, Hoboken, N.J., 2006; and Chudakov, D M, et al., Physiol Rev. 90(3):1103-63, 2010, for discussion of GFP and numerous other fluorescent or luminescent proteins. In some embodiments, a label comprises a dark quencher, e.g., a substance that absorbs excitation energy from a fluorophore and dissipates the energy as heat.
The term “linker,” as used herein, refers to a chemical group or molecule covalently linked to a molecule, for example, a protein, and a chemical group or moiety, for example, a click chemistry handle. In some embodiments, the linker is positioned between, or flanked by, two groups, molecules, or moieties and connected to each one via a covalent bond, thus connecting the two.
Definitions of specific functional groups and chemical terms are described in more detail below. For purposes of this invention, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed., inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties, groups, and reactivities, are as described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and March March's Advanced Organic Chemistry, 5th Edition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; and Carruthers, Some Modern Methods of Organic Synthesis, 3rd Edition, Cambridge University Press, Cambridge, 1987; the entire contents of each of which are incorporated herein by reference.
The term “alkyne,” used herein interchangeably with the term “alynyl,”, refers to a monovalent group derived from a straight- or branched-chain hydrocarbon having at least one carbon-carbon triple bond by the removal of a single hydrogen atom. In certain embodiments, the alkynyl group employed in the invention contains 2-20 carbon atoms (C2-20alkynyl). In some embodiments, the alkynyl group employed in the invention contains 2-15 carbon atoms (C2-15alkynyl). In another embodiment, the alkynyl group employed contains 2-10 carbon atoms (C2-10alkynyl). In still other embodiments, the alkynyl group contains 2-8 carbon atoms (C2-8alkynyl). In still other embodiments, the alkynyl group contains 2-6 carbon atoms (C2-6alkynyl). In still other embodiments, the alkynyl group contains 2-5 carbon atoms (C2-5alkynyl). In still other embodiments, the alkynyl group contains 2-4 carbon atoms (C2-4alkynyl). In still other embodiments, the alkynyl group contains 2-3 carbon atoms (C2-3alkynyl). In still other embodiments, the alkynyl group contains 2 carbon atoms (C2alkynyl). Representative alkynyl groups include, but are not limited to, ethynyl, 2-propynyl (propargyl), 1-propynyl, and the like, which may bear one or more substituents. Alkynyl group substituents include, but are not limited to, any of the substituents described herein, that result in the formation of a stable moiety. The term “alkynylene,” as used herein, refers to a biradical derived from an alkynylene group, as defined herein, by removal of two hydrogen atoms. Alkynylene groups may be cyclic or acyclic, branched or unbranched, substituted or unsubstituted. Alkynylene group substituents include, but are not limited to, any of the substituents described herein, that result in the formation of a stable moiety.
The term “amino,” as used herein, refers to a group of the formula (—NH2). A “substituted amino” refers either to a mono-substituted amine (—NHRh) of a disubstituted amine (—NRh2), wherein the Rh substituent is any substituent as described herein that results in the formation of a stable moiety (e.g., an amino protecting group; aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclic, aryl, heteroaryl, acyl, amino, nitro, hydroxyl, thiol, halo, aliphaticamino, heteroaliphaticamino, alkylamino, heteroalkylamino, arylamino, heteroarylamino, alkylaryl, arylalkyl, aliphaticoxy, heteroaliphaticoxy, alkyloxy, heteroalkyloxy, aryloxy, heteroaryloxy, aliphaticthioxy, heteroaliphaticthioxy, alkylthioxy, heteroalkylthioxy, arylthioxy, heteroarylthioxy, acyloxy, and the like, each of which may or may not be further substituted). In certain embodiments, the Rh substituents of the di-substituted amino group (—NRh2) form a 5- to 6-membered heterocyclic ring.
The term “azide” or “azido,” as used herein, refers to a group of the formula (—N3).
The function and advantage of these and other embodiments of the present invention will be more fully understood from the Examples below. The following Examples are intended to illustrate the benefits of the present invention and to describe particular embodiments, but are not intended to exemplify the full scope of the invention. Accordingly, it will be understood that the Examples are not meant to limit the scope of the invention.
A non-limiting study of the ER proteome was performed as illustrated in
Various peroxidase/substrate combinations were evaluated for use in the provided methods of in vivo proteomics. The following assays were used to measure the performance of the different combinations: In vitro generation of dityramide fluorescence; Cellular peroxidase activity assay with Amplex Red; Cellular peroxidase activity assay with streptavidin-fluorophore staining.
Peroxidases evaluated included Horse Radish Peroxidase (HRP), HRP mutants, soy bean ascorbate peroxidase (sbAPX), sbAPX mutants, peaAPX, peaAPX point mutants, Arabidopsis APX, maize APX, cytochrome C peroxidase, laccase, tyrosinase. All of the above listed peroxidases are useful in some aspects of this invention.
Substrates evaluated included: biotin-tyramide, biotin-linker-tyramide, biotin-dopamide, biotin-methoxytyramide, biotin-nitrotyramide, alkyne-tyramide, alkyne-linker-tyramide. All of these substrates are useful in some aspects of this invention.
In some embodiments, the preferred peroxidase/substrate combination is sbAPX, for example, sbAPX W41A mutant in combination with a tyramide substrate, for example, alkyne-tyramide.
Further, wild-type HRP in combination with biotin tyramide, and sbAPX(W41A) in combination with hexy-tyramide are preferred tagging enzyme/substrate combinations in some embodiments.
In some embodiments, mass spectrometry is used to analyze tagged molecules, for example, tagged proteins. In some embodiments, heavy/light-biotin-tyramide substrate is used in combination with mass spec.
In some embodiments, the tagging methods and reagents provided herein are also useful for applications involving fixed cells. For example, in some embodiments, STORM imaging has been successfully performed, e.g., imaging of vimentin on fixed cells. In some embodiments, AP-myc-vimentin was used in combination with SA-HRP or vimentin-APX.
It should be appreciated that the labeling radius may be affected by the half-life of the reactive moiety created by the tagging enzyme. For example, different substrates create different reactive moieties with different half-life, and thus different labeling radii. Further, endogenous factors may influence half-life and labeling radius, for example, endogenous GSH, ascorbate, beta-carotene, etc., may shorten tyramide radical half-life and, thus, the labeling radius of tyramide-based reactive moieties, thus creating a more focused labeling hot spot.
In some embodiments, a protein of interest is fused to a tagging enzyme. For example, in some embodiments, a peroxidase useful in aspects of this invention is fused to a protein of interest to identify a molecule (e.g., a protein) that the protein of interest interacts with (e.g., binds to). In some embodiments, the protein of interest is fused to the protein of interest, e.g., expressed in a cell from a recombinant expression construct encoding the enzyme/protein of interest fusion. The cell is then contacted with a suitable substrate, for example, a tyramide substrate described herein, and labeled proteins, which are proteins in the vicinity of the enzyme, and, thus, of the protein of interest, are identified according to methods described herein.
In some embodiments, HRP is functional in the ER and/or Golgi, but not in other subcellular compartments. Accordingly, in some embodiments HRP is not functional when targeted with a nuclear localization signal (NLS), nuclear export signal (NES), or mitochondrial localization sequence. Some aspects of this invention provide that soybean peroxidase, for example, a soybean peroxidase described herein, is active and can be used according to aspects of this invention in subcellular compartments in which HRP is inactive.
The ER proteome was analyzed using a peroxidase targeted to the ER. A SILAC experiment was performed using HRP-ER+biotin-tyramide, and 279 proteins were isolated and identified that are enriched in +HRP-ER cells compared to untransfected negative control cells. For these 279 proteins, 67% are classified as “ER” by the Gene Ontology Cell Component (GOCC) database. This indicates that the technique described herein is characterized by a high specificity in identifying bona fide ER proteins. For comparison, of the entire human proteome, 7% of proteins are classified as “ER” according to GOCC. For comparison, the best previously available ER proteomic database (Gilchrist Cell 2006), 16% are classified as “ER” according to GOCC.
It was also investigated how many known ER proteins were captured in the above experiment. Compared to a list of 25 bona fide ER proteins analyzed by Rapoport et al. (Cell 2010), 21 were detected with the above methodology. Further, peroxidase was targeted to the mitochondria, and activity according to cellular peroxidase activity assays was detected in mitochondria.
In some embodiments, the methods provided herein can be used to analyze mitochondrial proteomes. In some embodiments, the localization of mito-APX is determined, for example, as compared to mitotracker +/− H2O2 and tyramide. In some embodiments, endogenous peroxidase activity is controlled for under labeling conditions described herein. In some embodiments, a SILAC experiment is performed, comparing cells with and without mito-APX expression
In some embodiments, the mitochondrial matrix proteome for cells from healthy versus diseased subjects is compared. For example, the proteome from healthy subjects is compared to subjects diagnosed with MDS patient (mito DNA depletion syndrome). In some embodiments, such comparisons are used in the evaluation of drug treatments, in the screening of drugs, or in the monitoring of the efficacy of a specific treatment schedule in a patient undergoing such treatment.
In some embodiments, sub-proteomes of subcellular compartments are analyzed, for example, mitochondrial sub-proteomes, such as the mitochondrial outer membrane (OM) proteome, both facing cytosol and facing inside. In conventional proteomic analyses of mitochondrial proteomes (e.g. mitocarta), outer membrane proteins were depleted and likely missed.
Wild-type (wt) aspartate peroxidase (APX), for example, soybean APX, is dimeric. This property makes it disadvantageous as a reporter enzyme for some applications since dimeric tags can perturb the trafficking of the endogenous proteins to which they are appended. To address this disadvantage, mutants of APX were engineered that are monomeric, as confirmed by gel filtration chromatography (GFC,
Some aspects of this invention provide for engineered monomeric APX enzymes that are useful in the proteomics methods described herein. Methods using such engineered monomeric enzymes are also provided.
Several experiments were performed to determine the suitability of HRP and APX and mutants of these enzymes for in vivo protein tagging, for example, in exemplary protein tagging strategies as provided herein, e.g., as illustrated in
It was observed that (1) APX expresses well in all cell compartments tested (cytosol, nucleus, ER, mitochondria, cell surface), and HRP expresses well in the secretory pathway (ER, Golgi, cell surface), as has previously been shown13. (2) Both peroxidases catalyze covalent biotinylation when biotin-phenol and 1 mM H2O2 are added to live cells for 1 minute (
Based on the promising characteristics of peroxidase-mediated biotinylation described above, proteomics experiments were conducted. We targeted the ER proteome because although many attempts have been made to map its proteome via subcellular fractionation3, and published datasets are inconsistent, likely due to high rates of false positives and false negatives in ER-derived microsomes. We also targeted the proteome of the mitochondrial matrix, which has only been mapped crudely by mitochondrial fractionation followed by hypotonic lysis to remove the outer mitochondrial membrane22.
APX was fused to a mitochondrial matrix targeting sequence, and HRP (because it has higher activity in the ER than APX) was fused to a KDEL tag to induce retention in the ER. Both constructs were introduced into HEK cells with lipofectamine, and labeling was performed by pre-loading the cells for 30 min with biotin-phenol, then adding 1 mM H2O2 for 1 min to allow biotinylation. Thereafter, cells were lysed, with simultaneous quenching of peroxidase activity. Negative controls with H2O2, biotin-phenol, or peroxidase omitted did not show labeling, demonstrating that endogenous mammalian peroxidases do not contribute to background under our labeling conditions23. We used SILAC labeling of untransfected control samples to subtract back round due to non-specific bead binding.
Living HEK cells expressing ER-targeted HRP or mitochondrial matrix-targeted APX were labeled with biotin-phenol and H2O2, before lysis and analysis of biotinylated proteins by MS (
These experiments demonstrated that peroxidases can be used in living cells to biotinylate endogenous proteomes for MS identification. Preliminary characterization suggests that specificity is high for these membrane-bounded compartments, although depth-of-coverage could be improved.
The data presented herein demonstrate that the peroxidase-mediated protein tagging strategies provided herein are applicable to proteomic mapping experiments in some subcellular compartments. The successful labeling of the endoplasmic reticulum (ER) and mitochondrial matrix proteomes as described herein represent a critical step towards the proteomic mapping of subcellular regions that are impossible to purify and hence inaccessible to any current technology. Similar to these two subcellular compartments, the contact sites between biological membranes are impossible to purify with conventional methods. In some embodiments, the technology provided herein is applied to three such contact sites (
Mapping of the synaptic cleftome with synapse-targeted ascorbate peroxidase (APX) is illustrated in
Mapping the synaptic cleftome. The synaptic cleft is responsible for electrical coupling between neurons in the brain, and undergoes differentiation and remodeling in response to developmental state, environment, and activity/usage. Only a couple dozen synaptic cleft proteins are known, and these have mostly been discovered in a slow, labor-intensive, one-by-one manner. The methods and strategies provided herein are applied to tag and identify the complete inventory of endogenous proteins in the synaptic cleft by fusing either HRP or APX peroxidase to the extracellular domains of well-characterized synaptically-localized transmembrane proteins such as neurexin and neuroligin. A recent study targeted fragments of GFP to the synaptic cleft via fusions to these same proteins25. Whereas a peroxidase-neuroligin-1 fusion will traffic to excitatory synapses, a neuroligin-2 fusion will traffic to inhibitory synapses, allowing us to compare their proteomes. In some embodiments, each type of synaptic cleft is analyzed before and after neurotransmitter stimulation, and at earlier versus later stages of development.
To enhance labeling specificity, a membrane impermeant biotin-phenol conjugate is used, such as a carboxylated variant bearing a permanent negative charge, that will react with surface pools of the peroxidase fusion construct, but not intracellular pools in the ER or Golgi. Initially labeling is performed on cultures of dissociated rat cortical neurons, transfected with HSV virus. Subsequently, tissue from transgenic mice expressing the peroxidase fusion construct will be analyzed.
The delivery of biotin-phenol and H2O2 into some tissues presents a challenge. Some aspects of this invention provide strategies that address this problem by partially homogenizing the transgenic tissue, as if performing a synaptosome preparation7, and then adding the labeling reagents. To eliminate background from ER and Golgi pools of peroxidase that would be exposed by this protocol, a “split peroxidase” reporter is used in some embodiments, that reconstitutes across synaptic membranes and therefore gives activity only in the cleft, similar to the mGRASP split-GFP reporter25. A split HRP was engineered and it has been confirmed to reconstitute its fragments in the ER of live cells. In addition to identifying synaptic proteins, studies using the strategies provided herein provide information about the topology of membrane proteins in the synapse. The results obtained so far show that the biotin phenoxyl radical does not cross membranes, so surface-exposed/extracellular amino acids should be labeled, while intracellular amino acids should not (
Membrane-anchored APX peroxidase, facing the extracellular space, will label only surface-exposed amino acids of endogenous membrane proteins, because the phenoxyl radical does not cross membranes (
In some embodiments, biotinylated peptides and proteins are isolate and sequence directly, for example, by affinity purification and subsequent MS analysis. The information thus obtained is used, in some embodiments, to infer which regions of synaptic proteins face into the cleft.
Mapping the proteome of mitochondria-ER contact sites. The methods and strategies for protein tagging provided herein are, in some embodiments, used to map the proteome of contact sites between mitochondria and ER. A handful of proteins—Mff, Drp1, and mitofusin-2—have been discovered at these sites in mammalian cells, and shed light on the diverse possible roles of mitochondria-ER junctions in the regulation of mitochondrial division26, calcium and phospholipid exchange, and protein import into mitochondria8.
Three different approaches are contemplated: (1) fuse APX to proteins already known to be present at these junctions; (2) separately map the proteomes of the mitochondrial outer membrane and the ER membrane, and then intersect these two lists (3) develop and use a split-APX reporter, in which half of APX will be targeted to the outer mitochondrial membrane, and the other half of APX will be targeted to the ER membrane (facing cytosol) (
Mapping the proteome of the mitochondrial intermembrane space. In some embodiments, the in vivo proteomics methodology provided herein is employed to analyze the proteome of the mitochondrial inter-membrane space (IMS). During apoptosis, the mitochondrial outer membrane undergoes a permeability transition, and contents of the IMS, including caspases, Smac/Diablo, and cytochrome c, release into the cytosol27. We have targeted our APX peroxidase to the IMS using a localization sequence from Smac/Diablo. By mapping the IMS proteome, it is possible to gain insight into how mitochondria control programmed cell death. Furthermore, proteins embedded in the mitochondrial inner and outer membranes can be detected that function in protein translocation, calcium exchange, and oxidative phosphorylation.
In addition to proteomic mapping of spatially-defined zones of cells, the strategies and methods for in vivo protein tagging can be used to uncover the molecular composition of macromolecular complexes, in their intact state within living cells. In this context, it is preferable to use enzyme/substrate: pairs with a labeling radius that is relatively small, e.g., less than 5-10 nm.
In some embodiments, a peroxidase, e.g., APX, is fused to a known protein component of a macromolecular complex (e.g., similar to GFP-tagging), and then promiscuous, spatially-restricted biotinylation is exploited to tag and identify the unknown protein members of the macromolecular complex. The strategies provided herein can be used to map protein and other components of, for example, ribosomes, the neurexin-neuroligin trans-synaptic adhesion complex, the calcium channel complex, and other macromolecular complexes.
Another class of applications for which this technology is suitable is proteomic mapping of specific cell types within complex tissue. For example, the astrocyte proteome has been elusive because pure astrocyte cultures are not physiological, and astrocyte dissociation and purification from brain tissue greatly disrupts its physiology. According to some aspects of this invention, it is preferable to determine the proteome of healthy astrocytes embedded in their native context—the brain—and clearly distinguish this proteome from that of the surrounding neurons. To this end, transgenic mice that express cytosolic APX under control of an astrocyte-specific promoter, such as the GLT-1 promoter are used.
Labeling specificity is determined by the labeling radius, which in turn is determined by radical half-life. In some embodiments, the labeling radius is modulated by the presence or sequestration of radical quenchers, such as ascorbate and trolox, which are non-toxic membrane-permeable radical scavengers. Such radical quenchers can be added at different concentrations to cells being subjected to an in vivo protein tagging procedure as described herein. Changes in labeling radius can be measured by super-resolution STORM imaging of biotinylated proteins after cell fixation, as in
Another approach to reducing radical lifetime is to explore other peroxidase substrate structures. Aside from phenols, peroxidases are known to oxidize and generate radical species from anilines, hydrazines, hydroxamic acids, indoles, amines, beta-diketones, and other compounds, each with different half-life, reaction efficiency, and, thus, labeling radius. Furthermore, since phenoxyl radicals are electron-deficient, electron withdrawing groups such as nitro, perchloro, or cyano further destabilize them, thus further decreasing the labeling radius. In some embodiments, the use of alternate peroxidase substrate structures may necessitate the use of peroxidases with higher oxidizing power than APX, such as myeloperoxidase or engineered peroxidases with enhanced activity. Such enhanced peroxidases are provided herein and additional enhanced peroxidases will be apparent to the skilled artisan or can be generated by the skilled artisan without more than routine experimentation. For example, enhanced peroxidases can be created using yeast display evolution, previously employed to engineer the enantioselectivity of HRP29.
To improve specificity, it may be preferable in some embodiments to reduce the toxicity of labeling conditions. For example, in some embodiments, cells are treated with 1 mM H2O for 1 minute to initiate peroxidase labeling. In some cell types, this treatment may initiate early apoptotic events that might alter proteomic compositions within the cells. In some embodiments, this problem is addressed by using glucose, oxygen, and transfected glucose oxidase to generate H2O2 only locally in the compartments of interest.
To improve the sensitivity, or depth of coverage, of the technology provided herein, it is preferable in some embodiments to maximize the phenoxyl radical conjugation yield, and expand the scope of the reaction to amino acid side chains besides tyrosine, tryptophan, and cysteine. The more reactive radical products described herein can be employed to achieve this goal. In addition, tuning enzyme kinetics, and hence the rate of radical generation from the enzyme active site, can be exploited to modulate labeling yields.
The methods were performed as shown in
Electron microscopy was performed on the cells by APEX-catalyzed diaminobenzidine polymerization, followed by OsO4 staining 23(B). It was determined based on the electron micrographs that APEX-mito was localized in the mitochondrial matrix of HEK cells.
The biotinylated proteins were also examined by fluorescence imaging 23(C). Mitochondrial matrix-targeted APEX in HEK was visualized by anti-V5 staining. Proteins biotinylated by APEX were stained with streptavidin. Super-resolution STORM images showed streptavidin and APEX localization patterns in cells. A gel showing the results of the described methods is shown in
Eight different biotin substrate structures were tested in the methods of the invention using APX (25(B) top row) and HRP (25(B) bottom row) as enzymes. The structures for the tested biotin substrates are shown in
W41 mutants of monomeric APX having increased intracellular activity were examined and the imaging results are shown in
An assay scheme of the method performed is shown in
A SILAC labeling scheme, as shown in
Members of mitochondrial proteomes were identified using the methods provided herein.
All publications, patents and sequence database entries mentioned herein, including those items listed above, are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above description, but rather is as set forth in the appended claims.
In the claims articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention also includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.
Furthermore, it is to be understood that the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the claims or from relevant portions of the description is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Furthermore, where the claims recite a composition, it is to be understood that methods of using the composition for any of the purposes disclosed herein are included, and methods of making the composition according to any of the methods of making disclosed herein or other methods known in the art are included, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise.
Where elements are presented as lists, e.g., in Markush group format, it is to be understood that each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It is also noted that the term “comprising” is intended to be open and permits the inclusion of additional elements or steps. It should be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements, features, steps, etc., certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements, features, steps, etc. For purposes of simplicity those embodiments have not been specifically set forth in haec verba herein. Thus for each embodiment of the invention that comprises one or more elements, features, steps, etc., the invention also provides embodiments that consist or consist essentially of those elements, features, steps, etc.
Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. It is also to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values expressed as ranges can assume any subrange within the given range, wherein the endpoints of the subrange are expressed to the same degree of accuracy as the tenth of the unit of the lower limit of the range.
In addition, it is to be understood that any particular embodiment of the present invention may be explicitly excluded from any one or more of the claims. Where ranges are given, any value within the range may explicitly be excluded from any one or more of the claims. Any embodiment, element, feature, application, or aspect of the compositions and/or methods of the invention, can be excluded from any one or more claims. For purposes of brevity, all of the embodiments in which one or more elements, features, purposes, or aspects is excluded are not set forth explicitly herein.
This application claims priority under 35 U.S.C. §119(e) to U.S. provisional patent application, U.S. Ser. No. 61/497,155, filed Jun. 15, 2011, and entitled In Vivo Proteomics/Live Cell Proteomics, the entire contents of which are incorporated herein by reference.
This invention was made with U.S. Government support under grant NIH 1 DP1 OD003961-01 awarded by the National Institutes of Health. The U.S. Government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US2012/042817 | 6/15/2012 | WO | 00 | 2/18/2014 |
| Number | Date | Country | |
|---|---|---|---|
| 61497155 | Jun 2011 | US |
