WO2013087963A1

WO2013087963A1 - Method for identifying a particular protein in a cell, using a marker peptide and spectroscopy techniques and uses thereof

Info

Publication number: WO2013087963A1
Application number: PCT/ES2012/070864
Authority: WO
Inventors: Cristina Risco Ortiz; Raoul Junior De Groot
Original assignee: Consejo Superior De Investigaciones Científicas (Csic)
Priority date: 2011-12-15
Filing date: 2012-12-13
Publication date: 2013-06-20

Abstract

The invention relates to a method that can be used to view and detect a particular protein in a cell, a cell organ or a virus, using a marker peptide bound to metal particles and using correlative, optical and electronic microscopy images, in which the microscopically obtained image is analysed together with at least one elementary image obtained using a spectroscopy technique. This method can be used to study the biological action of proteins of biomedical interest as well as to locate said proteins in different intracellular organelles with considerable sensitivity.

Description

PROCEDURE TO IDENTIFY A PROTEIN OF INTEREST IN A CELL BY MEANS OF A MARKER PEPTIDE AND SPECTROSCOPY TECHNIQUES AND ITS TECHNICAL SECTOR APPLICATIONS

The present invention falls within the field of molecular biology and microscopy, and more specifically, correlative microscopy techniques in combination with the use of clonable markers, applied to the detection of metals bound to proteins of interest.

STATE OF THE PREVIOUS TECHNIQUE

Electron microscopy techniques in combination with the use of groups of gold atoms covalently linked to each other and to a small molecule, in some cases, or with the use of primary antibodies, in others, allow the mapping and visualization of cell proteins in a way not always satisfactory. Among the disadvantages of these techniques, the following stand out: the lack of application in vivo; the limited sensitivity; and the limited resolution to locate proteins in situ.

Recently, clonable markers have begun to be used, due to their considerable ability to locate almost all of the molecules of the proteins of interest within the cells. Thus, for example, metallothionein (MT), a small protein that lacks secondary structure and that folds when joining metals, when treated with gold salts, forms dense particles to electrons in vitro (Mercogliano and DeRosier, 2006, J Mol. Biol., 355: 21 1-223; Mercogliano and DeRosier, 2007, J. Struct. Biol., 160 (1): 70-82), positioning itself as an excellent clone marker for electron microscopy (WO20061 18615). Some studies show the visualization in two dimensions (2D) and three dimensions (3D) with a very high sensitivity, of chimeric proteins fused to the MT in bacteria, by means of treatments of the living cells with gold salts and their processing for their subsequent visualization in electron microscopy, although only in prokaryotic cells (Diestra E., et al., 2009, J Struct Biol, 165: 157-168; Diestra E., et al., 2009, PLoS ONE 4: e8301).

There has been a need for a long time for electron microscopy techniques, and their derivatives in more appropriate optical microscopy, associated with the use of clonable markers, which not only allow visualization of proteins in vivo in their native intracellular environment in prokaryotic cells without relying on of the antigen-antibody recognition, but could also be applied to visualization studies of individual protein molecules in living eukaryotic cells with high sensitivity and resolution. These techniques would allow the elaboration of 2D and 3D macromolecular maps of eukaryotic cells to obtain relevant information on the structures that maintain cellular functions, thus expanding their applications in biology.

Recently, the Spanish patent application P201031880 entitled "Cloning marker for use in microscopy" has provided technical solutions in the field of in vivo protein visualization in prokaryotic and eukaryotic cells, through microscopy techniques, using a clone marker as a peptide or smaller fragments thereof, which have an amino acid sequence consisting of a cysteine skeleton of the metallothionein (MT) sequence and small amino acids without charge, fused or not to a marker protein. The present PCT application claims inventive matter incorporated in the PCT application of priority ES201031880 filed on December 17, 2010 (PCT / ES1 1/070869 requested on December 15, 201 1) and which has been considered as a different invention from the priority ES201031880 and must claim its own priority dated December 15, 201 1. At the same time that this PCT application is submitted, it will proceed to request the recognition of your priority application in the Spanish Patent and Trademark Office.

DESCRIPTION OF THE INVENTION

Brief description

An object of the present invention is a useful method for the visualization and detection of a protein of interest in a cell, in a cellular organelle or in a virus by means of a marker peptide bound to metal nano-particles and by correlative microscopy images optics and electronics, hereinafter method of the invention, where the image obtained by microscopy is analyzed together with at least one elementary image obtained by means of a spectroscopy technique and by the joint analysis of different levels of observation, which are simultaneously visualized after superposition at computational level (pixel to pixel) and because said procedure comprises:

a) expressing at least one peptide in a cell, in a cell organelle or in a virus,

b) treating the cell, the cellular organelle or the virus of step (a) with a metal, and c) analyzing the cell, the cellular organelle or the virus of step (b) by microscopy and, also by at least one image elemental obtained by spectroscopy, and identify the protein of interest. A preferred object of the invention is the process of the invention where the marker peptide of a) comprises an amino acid sequence, by way of illustration and without limiting the scope of the invention, belonging to the following group: any one of the SEQ sequences ID NO: 1 to 6, any one of its fragments comprising 7 or more cisterns or any amino acid sequence consisting of any combination thereof. Another preferred object of the invention is the process of the invention where the metal of step (b) is selected from the list, by way of illustration and without limiting the scope of the invention, as follows: gold, silver, mercury, cadmium, zinc, platinum, bismuth, copper, lead or cobalt.

A particular embodiment of the invention is the process of the invention where the metal of step (b) is gold, more preferably, gold in the form of aurous chloride (AuCI), auric chloride (AuC), aurothiomalate or chlorouric acid ( HAuCU).

Another particular object of the invention is the method of the invention where the c) spectroscopy technique used belongs to the following group: Electron Energy-Loss Spectroscopy (EELS) or X-ray spectroscopy.

Another particular object of the invention is the process of the invention where elementary image of c) is an elementary image of the metal used in step (b). Another preferred object of the invention is the process of the invention where the elementary image is an elementary image of a metal belonging to the following group: elementary image of gold, an elementary image of silver, an elementary image of mercury, an elementary image of Cadmium, an elemental image of zinc, an elementary image of platinum, an elementary image of bismuth, an elementary image of copper, an elementary image of lead and an elementary image of cobalt.

Another particular object of the invention is the process of the invention where a noise extraction process is carried out, preferably, the acquisition of additional images by selecting energy losses close to the specific energy loss of the element of interest, previous and after the elementary image at the peak of the spectrum, to later subtract them from the latter.

Finally, another object of the invention is the use of the method of the invention for the identification of proteins of biomedical interest within cells, cellular organelles, viruses.

Detailed Description The present invention is based on the fact that the inventors have observed that it is possible to considerably increase the sensitivity of visualization and detection of proteins of interest by means of images of optical and electronic correlative microscopy, when analyzed together with at least one elementary image obtained by spectroscopy and the joint analysis of different levels of observation. The competitive advantage described in the present invention is that the use of elementary images allows solving the masking that occurs when using certain stains of heavy materials in preparations to identify proteins of interest by correlative microscopy techniques (see Example 1).

The identification of the protein of interest is carried out by the detection of nanoparticles or metal atoms attached to at least said protein of interest using as a point of attachment a marker peptide fused to said protein of interest.

In conventional transmission electron microscopy (MET), not all metals are well visualized as dense particles to electrons. One of the most used elements is gold since it is one of the metals that can best be visualized by this type of microscopy. Similarly, the use of the elementary image described in the present invention has the main advantage that it allows visualizing other metals other than gold, whether or not they are dense to electrons, and distinguish them when they are masked with stains. With the Elemental images Identification offers no doubt, even for gold, that it is not clearly located if it is in small quantities or if it forms nano-particles of less than 1 nm (Figure 1). The use of metals other than gold has additional advantages if elementary images can be obtained, since the affinity of MT is greater for metals other than gold: Hg2 +> Cu +> Ag +> Pb2 +> Cd2 + = Au +> Zn2 +> Co2 + (Nielson et al., (1985) Distinct metal-binding configurations in metallothionein. J. Biol. Chem. 260: 5342-5350); Schmitz et al., (1980) The Binding of Gold (I) to Metallothionein. J. Inorganic Biochemistry 12: 293-306).

The elemental image obtained by spectroscopy also provides a much greater sensitivity than the visualization of dense nano-particles to electrons in cellular electron microscopy either by direct visual verification (Figure 1) or by quantification by microscopy with energy loss spectroscopy , X-ray or STEM microscopy (scanning electron transmission microscopy; LeBeau et al., (2010) Standardless atom counting in scanning transmission electron microscopy. NanoLetters 10, 4406-4408; Krivanek et al., (1991) EELS quantification near the single -atom detection level Microsc. Microanal. Microstruct. 2, 257-267; Sousa et al., (2008) Determination of quantitative distributions of heavy-metal stain in biologival specimens by anular dark-field STEM. J. Struct. Biol. 162, 14-28). The reason is that in the elementary images obtained by spectroscopy the proteins of interest are correctly detected with very few gold atoms incorporated therein and the growth of the nano-particles is not necessary until reaching the size of 1 nm (formed by about 40-60 atoms), which is the minimum diameter for detection in cells by electron microscopy. The present invention represents important competitive or commercial advantages since the process requires less reaction time, less concentration of gold salts (cheaper process), thus reducing or eliminating a possible toxicity for those cells that could be particularly sensitive to metals (with the described protocols it has been verified that there is no toxicity in the cell lines with which it has been worked); but the main The advantage is that many more molecules are detected when viewing smaller nano-particles, all those that incorporated less than 40 gold atoms and were invisible in conventional MET (Figure 1). In the case of gold, as mentioned above, the detection of gold particles is very specific, since the energy loss spectrum allows the detection of said particles even in samples stained with salts of other heavy metals, usually used to staining the sample and normally masking them in a conventional MET image (Figure 1). In this way, a high contrast image is achieved that allows detailed study of the distribution of protein molecules together with the simultaneous visualization of cell ultrastructure. Additionally, it allows to obtain many more details about the subcellular localization of the proteins that carry the gold-marker peptide, such as the association with intracellular membranes, the orientation of the protein molecules on one side or the other of the membranes (lumen of organelles or cytosol), or the association to macromolecular complexes such as ribosomes or cytoskeleton elements, all of them invisible in the absence of conventional MET staining. Therefore, in a more preferred embodiment of the above, the elementary image is a gold image obtained by electronic energy loss spectroscopy.

The situation for each previous metallic element is different and in all cases a "noise extraction" process is carried out. A "noise extraction process" refers to a procedure that eliminates the non-specific signal that may interfere or be blurring an image obtained by a microscopic technique (according to the invention), to produce an image where only the element of interest appears . Noise extraction is always done and has been done in the example and results shown in Figure 1. It is done computationally once acquired the images of loss of energy corresponding to gold (specific), an area immediately before the spectrum and a subsequent one, which are subtracted from the specific one. For this, various methods are used, but one of the most used is to acquire images additional by selecting energy losses close to the specific energy loss of the element of interest, before and after the elementary image at the peak of the spectrum, to subsequently subtract them from the latter. As an example, and without limitation, the most commonly used method is that described by Egerton (Egerton, RF (1996) Electron Energy-Loss Spectroscopy in the Electron Microscope. Plenum Press, New York, USA).

Obtaining at least one elementary image obtained by means of a spectroscopy technique as described in the present invention, together with the use of expression of a marker peptide, treatment with cell metals and subsequent processing, together with correlative microscopy techniques - these last elements are described in the previous patent ES201031880 to which it is considered a reference for its sufficient description - it also has the following advantages already described in said patent:

- allows its application to living cells, both prokaryotic and eukaryotic, to purified cellular organelles or to viruses, allowing the visualization of proteins in their native intracellular environment without relying on antigen-antibody recognition;

- does not interfere with protein functionality due to its small size;

- can be used for the detection of one or several proteins of interest, since the simultaneous use of several copies of the marker peptide or of several fragments thereof of different length treated with metals give rise to the formation of dense particles to the electrons of different size, thus allowing multiple tides to locate more than one protein simultaneously; Y

- allows to visualize the molecules in the protein of interest with a high sensitivity and resolution (very precise location), including the position of the individual protein molecules and the structural detail in living eukaryotic cells. Thus, an object of the present invention constitutes a useful method for the visualization and detection of a protein of interest in a cell, in a cellular organelle or in a virus by means of a marker peptide bound to metal nano-particles and by images of correlative optical and electronic microscopy, hereinafter method of the invention, where the image obtained by microscopy is analyzed together with at least one elementary image obtained by means of a spectroscopy technique and by the joint analysis of different levels of observation, which are visualized simultaneously after computational superposition (pixel to pixel) and because said procedure comprises:

d) express at least one peptide in a cell, in a cell organelle or in a virus,

e) treating the cell, the cellular organelle or the virus of step (a) with a metal, and f) analyzing the cell, the cellular organelle or the virus of step (b) by microscopy and, also by at least one image elemental obtained by spectroscopy, and identify the protein of interest.

The term "microscopy" refers to a technique used to produce enlarged visible images of any structure belonging, by way of illustration and without limiting the scope of the invention, to the following group:

- optical microscopy, for example, but not limited to, bright-field, dark-field, phase contrast optical microscopy; differential interference microscopy or DIC, fluorescence or confocal; Y

- electron microscopy, for example, but not limited to scanning or transmission; or correlative microscopy.

"Electron microscopy" means both transmission electron microscopy (MET) and scanning electron microscopy (SEM) and both 2D electron microscopy and 3D electron microscopy. Finally, "correlative microscopy" means the technique based on the use of experimental approaches that allow visualizing a sample or specimen at two levels of observation, for example, but not limited to, optical microscopy and electron microscopy, that is, with microscopic techniques different that normally provide different resolutions. The correlative technique referred to in the present invention (CLEM: correlative light and electron microscopy) selects the cells that express a protein (fused to GFP and the metal-binding peptide) in the optical microscope (fluorescent) to carry them to electronic (for peptide-gold visualization); in this way the same molecules are visualized in optical and electronic, in the latter case with molecular resolution (individual molecules).

The following expressions are considered to express the same meaning: "elementary image of gold", "image of gold", "image of gold" or "map of gold".

The term "marker peptide" as used in the present invention refers to a peptide useful for the detection of proteins of interest by microscopy, preferably electronically or correlatively for their ability to bind to metal molecules that can subsequently be identified by techniques. microscopic By way of example, and without representing a limitation on the scope of the invention, a cloning marker peptide whose amino acid sequence has been designed based on the tank skeleton of the metallothionein (MT) sequence but where The rest of the amino acids have been replaced by small amino acids without charge. This definition also refers to smaller fragments of said marker peptide. In addition, any other metal affinity marker peptide can be used in the process of the invention, such as the tetra-cysteine marker (Gaietta et al., (2002) Multicolor and electron microscopy imaging of connexin trafficking. Science 296, 503 -507).

More specifically, for the execution of the process of the present invention a marker peptide based on the endogenous MT constituted by the amino acid sequence SEQ ID NO: 4 or any of its fragments comprising 7 or more tanks, preferably comprising 8 or more tanks, more preferably comprising 9 or more tanks. SEQ ID NO: 4 is equivalent to the complete MT sequence. Any other amino acid sequence comprising said SEQ ID NO4, its minor variants or combinations thereof can also be used within the framework of the process of the invention.

A possible marker peptide is constituted by SEQ ID NO1, which comprises 20 tanks (in SEQ ID NO1 the tanks were conserved by eliminating residues 1, 2, 1 1, 12, 32 and 54 to make a peptide as small as possible, the result is a 53 amino acid peptide).

Another possible marker peptide is constituted by SEQ ID NO: 2, fragment corresponding to amino acids 1 to 25 of SEQ ID NO: 1 and comprising 9 tanks (equivalent to the beta domain of MT but with 25 amino acids instead of 29 ).

Another possible marker peptide is constituted by SEQ ID NO: 3, a fragment corresponding to amino acids 26 to 53 of SEQ ID NO: 1 and comprising 1 1 tanks.

Another possible marker peptide is constituted by SEQ ID NO: 5, a fragment corresponding to amino acids 1 to 29 of SEQ ID NO: 4 and comprising 9 cysteines (equivalent to the complete beta-MT).

Another possible marker peptide is constituted by SEQ ID NO: 6, fragment corresponding to amino acids 30 to 61 of SEQ ID NO: 4 and comprising 1 1 cysteines (equivalent to the complete alpha-MT).

The marker peptide and its vanants or derivatives can be synthesized, for example, but not limited to, in vitro. For example, by synthesizing solid phase peptides or by recombinant DNA approaches. The marker peptide can be produced recombinantly, not only directly but as a fusion polypeptide together with a heterologous polypeptide, which may contain, for example, but without limiting the scope of the invention, a signal sequence or other polypeptide having a cleavage site. for a protease, for example, but not limited to, at the N-terminal end of the polypeptide.

The marker peptide used may have variants. These variants refer to limited variations in the amino acid sequence, which allow the maintenance of the functionality of the peptide. This means that the reference sequence and the variant sequence are similar as a whole, and identical in many regions. These variations are generated by substitutions, deletions or additions. These substitutions are conserved amino acids. The conserved amino acids are amino acids that have similar side chains and properties in terms of, for example, hydrophobicity or aromaticity. These substitutions are between serine (Ser) and threonine (Thr), and / or between the amino acids that make up the alanine (Ala), leucine (Leu), valine (Val) and isoleucine (lie) group. Variations can be artificially generated variations, such as by mutagenesis or direct synthesis. These variations do not cause essential modifications in the essential characteristics or properties of the peptide. Therefore, peptides or polypeptides whose amino acid sequence is identical or homologous, in the sense described in this paragraph, to the sequences described in the present invention are also included within the scope of the present invention.

In another preferred embodiment, the marker peptide is fused to a protein of interest. The term "protein of interest", as used in the present invention, refers to any peptide or polypeptide of interest whose microscopy location is to be carried out, and includes any intracellular, extracellular or viral macromolecule, formed by a linear chain of amino acids. This term includes, without limitation, both simple and conjugated proteins or heteroproteins and refers for example, but not limited to, to structural, regulatory (such as, but not limited to, hormones), transporter, defensive (such as, but not limited to, antibodies), enzymatic or contractile proteins. In another preferred embodiment, the marker peptide used in the present invention is fused to a marker protein. In a more preferred embodiment, the marker peptide is fused to a protein and a marker protein. "Marker protein" or "reporter protein" means any protein that provides, to the system where it is expressed, visually identifiable characteristics, such as, but not limited to, fluorescence or luminescence. Examples of marker proteins are, but are not limited to, β-galactosidase, luciferase, GUS (β-glucuronidase) or fluorescent proteins derived from GFP, such as, but not limited to, Green Fluorescent Protein or Green Fluorescent Protein (GFP), Red Fluorescent Protein or Fluorescent Red Protein (RFP), Cyan Fluorescent Protein or Cyan Fluorescent Protein (CFP), Yellow Fluorescent Protein or Yellow Fluorescent Protein (YFP) or Blue Fluorescent Protein or Blue Fluorescent Protein (BFP). In an even more preferred embodiment of this aspect of the invention, the marker protein is GFP, which is a 238 amino acid (26.9 kDa) protein isolated from the Aequorea victoria jellyfish that emits green fluorescence when exposed to light. blue.

Protein fusion is a technique that is frequently used in molecular biology and can be carried out by techniques known to a person skilled in the art. It is usually related to the production of proteins in living systems, such as, but not limited to, in cells (both prokaryotes, bacteria, and eukaryotes, such as yeasts, mammalian cells or insect cells), in cellular organelles or in virus. Protein fusion consists in cloning the coding sequence of a protein of interest at one end, preferably the 3 'end (carboxyl end), of another peptide. The inclusion of the coding sequence of the protein of interest must be carried out in phase, respecting the reading pattern, so as to allow production of a chimeric protein, which in the case of the present invention would consist, but not limited to, the marker peptide fused to a protein, the peptide of the invention fused to a marker protein, or the peptide of the invention fused to a protein and a marker protein.

The marker peptide used in the invention has no secondary structure and is folded by joining metal atoms. Said binding is preferably of the ionic type between the metal atoms and the cysteine residues of said peptide, although additional metal atoms may also be incorporated into the group of metal atoms already attached to the marker peptide, said additional metal atoms are attached covalently between them and the atoms that are already attached to the peptide tanks of the invention. Therefore, in another preferred embodiment, the marker peptide further comprises a group of metal atoms attached. The metals that can be attached to this marker peptide are, by way of illustration and without limiting the scope of the invention, all those useful for carrying out electron microscopy, preferably, heavy metals. Thus, in a more preferred embodiment, the metal is selected from the list comprising: gold (Au), silver (Ag), mercury (Hg), cadmium (Cd), zinc (Zn), platinum (Pt), bismuth ( B¡), copper (Cu), lead (Pb) and Cobalt (Co). In an even more preferred embodiment, the metal is gold.

The size of the "group of metal atoms" attached to the marker peptide used in the present invention is, at a minimum, that necessary for electron microscopy techniques to detect. Said size, as well as the number of metal atoms that form the group and their stability during irradiation in the electron microscope can be monitored by, for example, but not limited to, electron microscopy, mass spectrometry or spectroscopic techniques.

A preferred object of the invention is the process of the invention where the marker peptide of a) comprises an amino acid sequence, by way of illustration and without limiting the scope of the invention, belonging to the following group: any one of the SEQ sequences ID NO: 1 to 6, one any of its fragments comprising 7 or more tanks or any amino acid sequence consisting of any combination thereof. A person skilled in the art can obtain a marker peptide with these sequences SEQ ID NO: 1 to 6, its fragments or combinations, fused to other different amino acid sequences that also have affinity for metal atoms.

Another preferred object of the invention is the process of the invention where the marker peptide of a) is constituted by an amino acid sequence, by way of illustration and without limiting the scope of the invention, belonging to the following group: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, any of its fragments comprising 7 or more tanks or any amino acid sequence consisting of a any combination thereof.

Another particular object of the invention is the process of the invention where the a) marker peptide is fused to a protein, preferably a marker protein, and more preferably, is fused to the GFP protein.

Another particular object of the invention is the process of the invention where the marker peptide of a) also comprises a group of metal atoms after step b). Another preferred object of the invention is the process of the invention where the metal of step (b) is selected from the list, by way of illustration and without limiting the scope of the invention, as follows: gold, silver, mercury, cadmium, zinc, platinum, bismuth, copper, lead or cobalt. A particular embodiment of the invention is the process of the invention where the metal of step (b) is gold, more preferably, gold in the form of aurous chloride (AuCI), auric chloride (AuC), aurothiomalate or chlorouric acid (HAuCU).

Another particular object of the invention is the process of the invention where it also comprises, between steps (b) and (c), a cell fixation stage, the cellular organelle or the virus of step (b) by chemical fixation or freezing and the realization of sections of between 20 and 500 nm of the cell, the cellular organelle or the virus previously fixed.

Another particular object of the invention is the process of the invention where elementary image of c) is an elementary image of the metal used in step (b).

Another preferred object of the invention is the process of the invention where the elementary image is an elementary image of a metal belonging to the following group: elementary image of gold, an elementary image of silver, an elementary image of mercury, an elementary image of Cadmium, an elemental image of zinc, an elementary image of platinum, an elementary image of bismuth, an elementary image of copper, an elementary image of lead and an elementary image of cobalt.

Another preferred object of the invention is the process of the invention where the elementary image is a gold image, and more preferably, where the gold image is obtained by electronic energy loss spectroscopy, and even more preferably, the gold image is obtained. with spectroscopes that are coupled to electronic microscopes. Another particular object of the invention is the process of the invention where the expression of the peptide in step (a) is carried out in a cell and because the treatment of step (b) is carried out with aurous chloride (AuCI), auric chloride ( AuC), aurothiomalate or chlorouric acid (HAuCU) at a concentration of between 0.1 and 50 mM, preferably, for between 5 and 60 minutes.

Another particular object of the invention is the process of the invention where the cell of step (c) is analyzed for the visualization of at least one cell surface protein and / or at least one protein in vivo.

Another particular object of the invention is the process of the invention where it also comprises a stage of selective permeabilization of the cell membrane between steps (a) and (b), preferably by Streptolysin O.

Another particular object of the invention is the process of the invention where the cell of step (c) is analyzed for the visualization of at least one intracellular protein. Another particular object of the invention is the process of the invention where the expression of the peptide in step (a) is carried out in a cellular organelle or in a virus and because the treatment of step (b) is carried out with aurous chloride (AuCI ), auric chloride (AuC) or chlorouric acid (HAuCU) at a concentration of between 0.1 and 5 mM, preferably for between 5 and 30 minutes.

Another particular object of the invention is the process of the invention where, in step (a), two or more different sized peptides fused to different marker proteins are expressed in a cell, in a cell organelle or in a virus, and, alternatively, to marker proteins of different color. Another more particular object of the invention is the process of the invention where the cell is eukaryotic.

Another particular object of the invention is the process of the invention where a noise extraction process is performed, preferably the acquisition of additional images by selecting energy losses close to the specific energy loss of the element of interest, before and after the elementary image at the peak of the spectrum, to later subtract them from the latter.

Finally, another object of the invention is the use of the method of the invention for the identification of proteins of biomedical interest within cells, cellular organelles, viruses. DESCRIPTION OF THE FIGURES

Figure 1.- Visual comparison of the chimeric proteins of MT1 in a Rubella virus replication organelle with conventional EMT techniques and the method of the present invention. (A) Visualization of the chimeras of MT1 by means of a conventional transmission electron microscopy image showing the dense gold nano-particles to the electrons associated with MT1 (arrows). (B) Image of the organelle with MT1 stained with heavy metal salts by means of an image of conventional transmission electron microscopy, where the ultrastructure of the organelle is appreciated, which is filled with condensed membranes, but the gold nano-particles are no longer detected of MT1. (C) to (E) Method of the present invention. (C) Conventional MET display of the organelle in section of a cell stained with heavy metal salts (uranyl acetate and lead citrate). In the conventional MET image the gold nano-particles associated with the P150-MT1 molecules are masked by staining heavy materials. Colloidal gold particles of larger size (10 nm) (arrows). (D) Detection of P150-MT1 -oro molecules in the same organelle by Elemental image obtained with energy loss spectroscopy after subtraction of the non-specific background (gold map). (E) Image of elemental gold in section of the organelle stained with heavy metal salts (uranyl acetate and lead citrate) and processed by energy loss spectroscopy to obtain said gold image. The elementary image of gold has been colored in computationally green, (it is not a fluorescent signal) and has been superimposed on the ultrastructure image. This overlay shows the detailed distribution of the P150-MT1 -oro molecules in the different subdomains of the organelle. Bars: 200.

EXAMPLES OF REALIZATION

For a better understanding of the invention, the following example is attached, which is not intended to be limiting in its application.

Example 1.- Location of chimeric proteins-MT1 -oro in dense organelles of cells by spectroscopic analysis and elementary images (gold maps). A nucleotide sequence encoding a chimeric protein comprising the P150 subunit of the viral replicase fused with the mouse metallothionein peptide 1 (P150-MT1) was prepared as described in patent ES201031880, which allows the expression of said chimeric protein inside cells, organelles or viruses.

More specifically, first BHK-21 mammalian cells cultured in monolayer were transfected with a Rubella virus replicon that allows the expression of the chimeric protein P150-MT1 in vivo, incubated with 1 mM AuC for 30 minutes at 37 ° C, fixed with a mixture of 4% paraformaldehyde and 0.1% glutaraldehyde in PBS (phosphate buffer saline), they were dehydrated in increasing amounts of ethanol and infiltrated in acrylic resin, which once polymerized by heat and hardened, allowed to obtain ultra thin sections (50-70 nm) samples of the sample in an ultramicrotome; alternatively, the samples fixed with aldehydes were frozen at high speed in liquid ethane, dehydrated by cryosubstitution in methanol at -90 ° C for two days and infiltrated in acrylic resin that was polymerized and hardened with ultraviolet light. Sections were collected on electron microscopy grids for staining with saturated uranyl acetate (25 min) and lead citrate (2 min), washed with water and allowed to dry before viewing by transmission electron microscopy (MET) conventional; dense perinuclear organelles of cells (modified lysosomes where virus replicative complexes are assembled) were studied. The ultrastructural details of the organelle can be seen in Figures 1 B and 1 C but the gold nano-particles associated with the P150-MT1 molecules are masked by the staining of heavy materials. Only the largest colloidal gold particles (10 nm) added to the post-ori section are visible in a stained section (arrows, Figure 1 C). Colloidal gold particles are immunoglobulins (IgG) adsorbed to colloid spheres and are commercial preparations that are used in "immunogold" assays. They have no affinity for the marker peptide of the invention and are adsorbed by electrostatic attraction after incubating the sections with a suspension of these particles for 30 min at room temperature. Incubation of these particles with colloidal gold was carried out before staining with heavy metals and elemental analysis as an internal confirmation of specificity of the elemental analysis object of the invention: it can be seen how the colloidal gold particles are clearly detected in the Gold maps (Figures 1 C and 1 D, arrows). In a conventional MET image the gold particles associated with the P150-MT1 proteins are perfectly visualized as small electron dense structures (Figure 1A).

Subsequently, for the detection of the P150-MT1 -oro molecules that are masked in the previous staining with heavy metals, an elemental gold image was obtained by energy loss spectroscopy after non-specific background subtraction (gold map, Figure 1 D ) according to the process of the present invention. Noise extraction had to be done to obtain the map gold presented in Figure 1 D. This extraction was performed computationally once the images of energy loss corresponding to gold (specific), an area immediately before the spectrum and a subsequent one, which are subtracted from the specific one. The Egerton procedure (Egerton, RF (1996) Electron Energy-Loss Spectroscopy in the Electron Microscope, Plenum Press, New York, USA) was used for noise extraction and specific elementary mapping.

Finally, the superposition of the gold map of D) on the conventional MET image of C) showed the detailed distribution of the SO-MU -oro PI molecules in the different subdomains of the organelle (Figure 1 E). The organelle is filled with membranes with which the P150-MT1 -oro molecules are associated as can be seen in the staining thereof (Figure 1 C) and in particular in the superposition of the gold map on the ultrastructure shown by the staining (Figure 1 E). However, in the gold image obtained by the process of the invention many more P150-MT1 -oro molecules are found that are occupying the entire interior of the replication organelle (this is verified by comparing Figures 1A with 1 E). In addition, only in Figure 1 E can the precise distribution of the protein molecules bearing the MT1 marker peptide and its distribution within the organelle be appreciated. It is also observed that the molecules completely fill the organelle and are associated with the membranes exclusively, while in the conventional MET image only the molecules in which the largest nano-particles (of at least 1 nm) were formed , which are concentrated only in a lower area of the organelle (arrows in Figure 1A); In conventional MET it is also not possible to visualize the membranes with which the nano-particles are associated (arrows in Figure 1A).

Claims

one . - Useful procedure for the visualization and detection of a protein of interest in a cell, in a cellular organelle or in a virus by means of a marker peptide bound to metal particles and by correlative microscopy images, either optical or electronic, characterized in that the The image obtained by microscopy is analyzed together with at least one elementary image obtained by means of a spectroscopy technique and by the joint analysis of different levels of observation, which are simultaneously visualized after superposition at the computational level and because said procedure comprises:

a) expressing at least one marker peptide in a cell, in a cell organelle or in a virus,

b) treat the cell, cell organelle or virus of step (a) with a metal, and

c) analyze the cell, the cellular organelle or the virus of step (b) by microscopy and, also, by at least one elementary image obtained by spectroscopy.

2. - Method according to claim 1 characterized in that the marker peptide of a) is constituted by an amino acid sequence comprising an amino acid sequence belonging to the following group: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, any of its fragments comprising 7 or more tanks or any amino acid sequence consisting of any combination thereof.

3. - Method according to claim 2 characterized in that the marker peptide of a) is constituted by an amino acid sequence belonging to the following group: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, any of its fragments comprising 7 or more tanks or any amino acid sequence consisting of any combination thereof.

4. - Method according to any one of claims 1 to 3 characterized in that the marker peptide of a) is fused to a protein.

5. - Method according to claim 4 characterized in that the protein is a marker protein.

6. - Method according to claim 5 characterized in that the marker protein is the GFP protein.

7. Method according to any of claims 1 to 6, characterized in that the marker peptide further comprises a group of metal atoms after step b).

8. - Method according to claim 7 characterized in that the metal of step (b) is selected from the list comprising: gold, silver, mercury, cadmium, zinc, platinum, bismuth, copper, lead or cobalt.

9. - Method according to claims 7 and 8 characterized in that the metal of step (b) is gold.

10. - Method according to claim 9 characterized in that the gold source is aurous chloride, auric chloride, aurothiomalate or chlorouric acid.

eleven . - Method according to claims 1 to 10, characterized in that it also comprises, between steps (b) and (c), fixing the cell, the cellular organelle or the virus of step (b) by chemical fixation or freezing and making sections between 20 and 500 nm of the cell, the cell organelle or the virus previously fixed.

12. Method according to any one of claims 1 to 1, characterized in that the spectroscopy technique used belongs to the following group: electronic energy loss spectroscopy or X-ray spectroscopy.

13. - Method according to claim 12 characterized in that the elementary image of c) is an elementary image of the metal of the passage (b).

14. - Method according to claims 12 and 13 characterized in that the elementary image is an elementary image of a metal belonging to the following group: elementary image of gold, an elementary image of silver, an elementary image of mercury, an elementary image of cadmium, an elemental image of zinc, an elementary image of platinum, an elementary image of bismuth, an elementary image of copper, an elementary image of lead or an elementary image of cobalt.

15. Method according to claims 12 and 13 characterized in that the elementary image is a gold image.

16. - Method according to claim 15 characterized in that the gold image is obtained by electronic energy loss spectroscopy.

17. - Method according to any one of claims 1 to 16, characterized in that the obtaining of the elementary image is carried out with spectroscopic equipment that is coupled to electron microscopes.

18. Method according to any one of claims 1 to 17 characterized in that the expression of the peptide in step (a) is carried out in a cell and because the treatment of step (b) is carried out with aurous chloride (AuCI), chloride Auric (AuC), aurothiomalate or chlorouric acid (HAuCU) at a concentration of between 0.1 and 50 mM.

19. Method according to claim 18 characterized in that the treatment of step (b) is carried out for between 5 and 60 minutes.

20. Method according to claims 18 and 19, characterized in that the cell in step (c) is analyzed for the visualization of at least one cell surface protein and / or at least one protein in vivo.

21. Method according to claims 18 and 19, characterized in that it further comprises selectively permeabilizing the cell membrane between steps (a) and (b).

22. - Method according to claim 21 characterized in that the selective permeabilization of the cell membrane is carried out by

Streptolysin O.

23. - Method according to any one of claims 21 and 22 wherein the cell of step (c) is analyzed for the visualization of at least one intracellular protein.

24. - Method according to any one of claims 1 to 16 characterized in that the expression of the peptide in step (a) is carried out in a cellular organelle or in a virus and because the treatment of step (b) is carried out with aurous chloride , auric chloride or chlorouric acid at a concentration of between 0.1 and 5 mM.

25. - Method according to claim 24 characterized in that the treatment of step (b) is carried out for between 5 and 30 minutes.

26. - Method according to any one of claims 1 to 25 characterized in that in step (a), two or more different sized peptides fused to different marker proteins are expressed in a cell, in a cell organelle or in a virus. and, alternatively, to marker proteins of different color.

27. - Method according to any one of claims 1 to 26 characterized in that the cell is eukaryotic.

28. - Method according to any one of claims 1 to 27 characterized in that the elementary image of c) is obtained by a noise extraction process.

29. - The method according to claim 28, characterized in that the noise extraction process consists in the acquisition of additional images by selecting energy losses close to the specific energy loss of the element of interest, before and after the elementary image at the peak of the spectrum, to later subtract them from the latter.

30. - Use of the method according to any one of claims 1 to 29 for the identification of proteins of biomedical interest inside cells, cell organelles, viruses.