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  Methods and compositions for tagging via azido substratesUSPTO Application #: 20060166286Title: Methods and compositions for tagging via azido substrates Abstract: The invention provides methods and compositions for azide tagging of biomolecules. In one embodiment of the invention, proteins are tagged by metabolic incorporation of prenylated azido-analog substrates. Examples of such analogs are azido farnesyl diphosphate and azido farnesyl alcohol. The azido moiety in the resulting modified proteins provides an affinity tag, which can be chemoselectively captured by an azide-specific conjugation reaction, such as the Staudinger reaction, using a phosphine capture reagent. When the capture agent is biotinylated, the resulting conjugates can be detected and affinity-purified by streptavidin-linked-HRP and streptavidin-conjugated agarose beads, respectively. The invention allows detection and isolation of proteins with high yield, high specificity, and low contamination without harsh treatment of proteins. (end of abstract)
Agent: Fulbright & Jaworski L.L.P. - Austin, TX, US Inventors: Yingming Zhao, John R. Falck USPTO Applicaton #: 20060166286 - Class: 435007500 (USPTO) Related Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Antigen-antibody Binding, Specific Binding Protein Assay Or Specific Ligand-receptor Binding Assay, Involving Avidin-biotin Binding The Patent Description & Claims data below is from USPTO Patent Application 20060166286. Brief Patent Description - Full Patent Description - Patent Application Claims
BACKGROUND OF THE INVENTION [0002] 1. Field of the Invention [0003] The invention relates generally to the field of biochemistry. More specifically, the invention relates to tagging of biomolecules using synthetic azido substrates. [0004] 2. Description of Related Art [0005] Protein isoprenylation is a general term of which protein farnesylation and protein geranylgeranylation are examples. It is a type of post-translational modifications involving the covalent attachment of polyisoprenoids, for example, a 15-carbon farnesyl or 20-carbon geranylgeranyl isoprenoid, typically through a thioether bond to a C-terminal cysteine residue of proteins (Fu, 1999). To date, three enzymes are known to isoprenylate proteins, viz., protein farnesyltransferase (FTase) (Reiss, 1990), protein geranylgeranyltransferase type I (GGTase-I) (Seabra, 1991) and protein geranylgeranyltransferase type II (GGTase-II) (Moores, 1991). FTase utilizes farnesyl diphosphate (FPP) and selectively alkylates the cysteine residue fourth from the C-terminus in a conserved isoprenylation motif designated the "CAAX box", where "C" is a cysteine residue, "A" as an aliphatic residue, and "X" is either S, M, Q, A, or T (single-letter amino acid codes). GGTase-I and GGTase-II are responsible for linking a geranylgeranyl group, from geranylgeranyl diphosphate (GGPP), to a cysteine residue in the C terminal CAAX (where X is L or F), CC, or CXC motifs of the proteins (Fu, 1999). Isoprenylation promotes membrane association of the target proteins and protein-protein interactions, and is essential for the function of the modified proteins (Fu, 1999; Tamanoi, 2001). [0006] A variety of proteins are farnesylated, including the Ras superfamily G-proteins. The post-translational modification is required for the activation of Ras proteins and their transforming potential (Fu, 1999). For this reason, farnesyltransferase has been hypothesized as an anti-tumor drug target. Farnesyltransferase inhibitors (FTIs) that inhibit FTase have been developed as potential cancer therapeutic agents and a few FTI compounds are currently under clinical evaluation (End, 2001; Tamanoi, 2001). [0007] Efficient methods for the detection and quantification of protein prenylation are needed for the analysis of the dynamics of protein prenylation. Metabolic incorporation of radio-isotope labeled farnesyl pyrophosphate (FPP) has been used to detect farnesylated proteins, but is expensive and inconvenient (Melkonian, 1999; Gibbs, 1999). Anti-farnesylation antibodies have also been developed, but they have not been widely used, largely due to limited binding affinity and specificity (Lin, 1999; Baron, 2000). Neither approach is able to adequately enrich the farnesylated proteins from a complex protein mixture. [0008] Global profiling of farnesylated proteins under diverse cellular environments with FTI treatments would reveal those farnesylated proteins with a change in farnesylation modification, and would reveal likely protein targets for several FTIs currently under clinical trials. This would allow characterization of the dynamics of farnesylated proteins in response to changes of cellular environment. Proteomics analysis is usually performed by 2D-gel/mass spectrometry- (Hanash, 2003) or ICAT/mass spectrometry-based proteomic methods (Gygi, 1999; Aebersold, 2003), which is typically limited to a few thousand of the most abundant proteins (Aebersold, 2003). Due to their low-to-medium abundant expression, farnesylated proteins are usually not detected, and, therefore, not quantified by these methods when whole-cell protein lysates are used as starting materials. Thus, efficient proteomic analyses of these proteins require an enrichment technology that is able to remove non-farnesylated proteins and reduces the complexity of the protein mixture. Unfortunately, such a method has not previously existed. There is, therefore, a great need in the art for techniques that may be used for purification or enrichment of farnesylated and similarly modified proteins. SUMMARY OF THE INVENTION [0009] In one aspect, the invention provides a method for detecting at least a first isoprenylated protein in a cell comprising: a) obtaining a synthetic isoprenyl azide substrate of at least a first protein in said cell; b) contacting the cell under conditions wherein the cell takes up and incorporates into the protein at least a first azide from the substrate; and c) detecting at least said first protein from proteins produced by said cell with a phosphine capture reagent by the Staudinger reaction. In the method, the protein may be farnesylated. Detecting may comprise isolating the first protein. In one embodiment of the method, FPP is inhibited in the cell. FPP may be inhibited, for example, by contacting the cell with an HMG Co-A reductase inhibitor, including lovastatin. In certain embodiments of the invention, the prenyl azide is an azido farnesyl diphosphate, and/or azido farnesyl alcohol. The protein may be native to said cell. [0010] In certain embodiments of the invention, the step of detecting comprises Western blot analysis. The phosphine capture reagent may be bound to a solid support, including by a photocleavable linker. The phosphine capture reagent may comprise a label, including a fluorescent, colorimetric, chemiluminescent, or radioactive label and further including antigens. An antigen may be biotin and the method may comprise affinity-purification with streptavidin- and/or avidin-conjugated beads. The beads, for example, may comprise silica gel, polystyrene, starch, sugars, or organic or inorganic matrixes. In further embodiments of the invention, a nucleophile in the Staudinger reaction is immobilized on a polymer, for example, mono-methyl polyethylene oxide, sepharose, tentagel, agrogel-Wang, polysaccharide, polystyrene, polyethane, and co-polymers thereof. The method may comprise detecting a plurality of proteins. Ras is one example of a protein that may be detected. [0011] In further embodiments of the invention, a substrate includes one of the following formulas presented below. The invention also provides compositions comprising at least one of these molecules. [0012] In yet another aspect, the invention provides a method for labeling a protein in a cell, comprising: a) preparing a synthetic substrate of said protein comprising at least a first azide; and b) contacting the cell under conditions wherein the synthetic substrate is taken up and incorporated into the protein and wherein the protein is labeled with said first azide. In the method, the synthetic substrate may be prenylated. [0013] The following abbreviations have been used herein: BPPCR, biotinylated phosphine capture reagent; DMSO. Dimethyl sulfoxide; FTase, farnesyltransferase; FOH, farnesyl alcohol; F-azide, azido-farnesyl; F-azide-OH (FN.sub.3OH), azido-farnesyl alcohol; FPP, farnesyl diphosphate; FPP azide (FPPN.sub.3), azido farnesyl diphosphate; FTIs, farnesyltransferase inhibitors; GG-OH, geranylgeranyl alcohol; GGPP, geranylgeranyl diphosphate; HPLC, high-performance liquid chromatography; MS/MS, tandem mass spectrometry; and TAS, Tagging-via-Azido-Substrate. BRIEF DESCRIPTION OF THE DRAWINGS [0014] These and other features, aspects, and advantages of the present invention will become better understood with regard to the following description, appended claims, and accompanying figures. [0015] FIG. 1. Schematic representation of one embodiment of the TAS technology for solid-phase-isolation of azide-labeled farnesylated proteins. (A) Farnesyltransferase-catalyzed enzymatic reaction using FPP or FPP azide as a substrate. (B) Chemical structures of natural FPP, and a FPP-azide and the corresponding alcohols, F-OH (farnesol) and F-azide-OH. (C) A Staudinger conjugation reaction between a phosphine and an azide-containing molecule. (D) The structure of a biotinylated phosphine capture reagent and its reaction product with an azido-farnesylated protein. (E) Experimental procedure for the isolation of F-azide modified proteins. Protein i and ii, unmodified proteins; protein iii, a protein modified by natural farnesyl group; and protein iv, F-azide modified proteins. Only F-azide modified protein iv is purified and released by UV light-induced photocleavage. [0016] FIG. 2. FPP azide is a substrate for in vitro farnesylation reaction. MALDI-TOF mass spectra of RC peptide before (A) and after (B) in vitro farnesylation reaction. One hundred pmol RC-peptide (KKFFCAIS) were mixed with 3000 pmol FPP azide and 35 pmol FTase in 7 .mu.l reaction buffer and incubated at 30.degree. C. for 10 h. The F-azide modified RC-peptide was confirmed by mass spectrometry (m/z, 1191). [0017] FIG. 3. Detection of F-azide modified Ras and HDJ-2 by mobility-shift assay. Mobility-shift assay of Ras (A) and HDJ-2 (B). COS-1 cells were labeled with the indicated compounds for 24 h, the cell lysate were resolved in SDS-PAGE and probed using anti-Ras and anti-HDJ-2 antibodies, respectively. The letters, "u" and "p", indicate the unmodified and isoprenylated forms of the protein, respectively. [0018] FIG. 4. Global detection of F-azide modified proteins by Western blotting analysis. COS cells were labeled with DMSO, or FPP N3 with or without lovastatin, as indicated, for 24 h. The protein lysates from the cells were precipitated by acetone/TCA method, redissolved in a buffer containing 2% SDS and PBS. The resulting solution was conjugated to biotinylated phosphine capture reagents or not, precipitated by acetone/TCA method again. The protein pellet was redissolved in 1.times. SDS sample buffer, resolved in SDS-PAGE, and detected by Western blotting analysis using streptavidin-conjugated HRP with or without biotin. DETAILED DESCRIPTION OF THE INVENTION [0019] The invention provides Tagging-via-Azido-Substrate (TAS) technology for the tagging and isolation of selected proteins. The invention, in particular embodiments, allows detection and isolation of farnesylated proteins by metabolic incorporation of a synthetic azido-farnesyl analog, such as azido farnesyl diphosphate (FPP-azide) or azido farnesyl alcohol (F-azide-OH), as a replacement of the natural substrate, FPP, in a cellular pathway for protein farnesylation. The azido moiety in the resulting farnesyl-azide (F-azide)-modified proteins provides a tag, which can be chemoselectively captured by an azide-specific conjugation reaction, for example, the Staudinger reaction and subsequent intramolecular interception of the iminophosphorane intermediate by an ester or other suitable functionality. The resulting conjugates can be detected and/or purified, for example, using streptavidin-linked- HRP or agarose beads. Since the purification relies on covalent bond formation during a specific, efficient conjugation reaction between an azide and a phosphine capture reagent, other proteins without F-azide modification can be effectively removed by thorough washing, centrifugation or other means. [0020] TAS technology allows farnesylated proteins to be detected with high yield and high specificity. For example, it was demonstrated that (1) FPP-azide or F-azide-OH was used by cells for protein farnesylation; and (2) The F-azide modified proteins could be specifically detected by Western blotting analysis. The techniques of the invention will find wide applications for detection, quantification, and proteomics analysis of farnesylated proteins. The methods and compositions described can be extended to other post-translational modifications, including geranylgeranylation and glycosylation, expanding the scope for detection, quantification, and proteomics analysis of post-translationally modified proteins. [0021] The inventors describe the design and synthesis of azido-farnesyl substrates for protein farnesylation and ability of the compounds to enter into the cells and then incorporate into the proteins that contain farnesylation consensus sequence. The farnesyl transferase was shown to be tolerant of an azido tag on the farnesyl. Metabolic incorporation of azido-farnesyl groups into farnesylated proteins could be increased by inhibition of endogenous synthesis of FPP by blocking HMG Co-A reductase using lovastatin. The metabolic labeled, azido-prenyl-modified proteins could be conjugated to phosphine capture reagents linked to detection or affinity-purification reagents, such as biotin, or other molecules allowing ready detection and isolation of the conjugate. While FPP analogues are charged and hydrophilic, and appear to be difficult to penetrate cell plasma membrane, they were shown to be used by the cell and reverse lovastatin inhibition (Gibbs, 1999). In addition, F-OH analogs have been used to dose the cells, and the cellular machine was able to convert the compounds into FPP analogs for protein farnesylation (McGuire, 1997; Gibbs, 1999). It is believed that both FPP azide and F-azide-OH described in this work were used by the cells likewise. Continue reading... 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