WO2006070180A1

WO2006070180A1 - Detection of phosphoproteins

Info

Publication number: WO2006070180A1
Application number: PCT/GB2005/005039
Authority: WO
Inventors: Peter Jackson
Original assignee: Perkinelmer Singapore Pte Ltd
Priority date: 2004-12-30
Filing date: 2005-12-22
Publication date: 2006-07-06
Also published as: GB0428491D0

Abstract

Disclosed is a method of detecting the presence in a sample of a phosphorylated amino acid, wherein the method comprises locating a sample in or on an attachment substrate; contacting the attachment substrate with a specific detection reagent that binds specifically and selectively to a phosphorylated amino acid residue; and detecting the bound specific detection reagent.

Description

Detection of Phosphoproteins

Field of the Invention

This invention relates to detection of phosphoproteins, i.e. proteins including one or more phosphorylated amino acids.

Summary of the invention

According to the present invention there is provided a method of detecting the presence in a sample of a phosphorylated amino acid, wherein the method comprises locating a sample in or on an attachment substrate; contacting the attachment substrate with a specific detection reagent that binds specifically and selectively to a phosphorylated amino acid residue; and detecting the bound specific detection reagent.

The phosphorylated amino acid (referred to herein as "target analyte") may, for example, be present within a protein, protein fragment, polypeptide or peptide (referred to herein as "analyte" or possibly just protein for brevity). The amino acid may be phosphorylated in vitro or in vivo. If required, phosphorylation may be carried out as a preliminary step prior to location of the sample on the attachment substrate. Conveniently, in vitro phosphorylation may involve the covalent attachment of a phosphate group on an amino acid residue by methods that are known in the art. Examples of amino acid residues that may be phosphorylated include serine, threonine, tyrosine, histidine, aspartic acid, glutamic acid and tryptophan. The amino acid residue may have other modifications, including post-translational modifications (known or unknown), e.g. glycosylation or methylation.

The attachment substrate may be an electrophoretic gel or a chromatographic substrate. The gel or substrate can be used as a support medium for the separation of analytes present as- a- mixture in the sample- In-this case _r the sample is - located- in- or on the-substrate by applying the sample to the gel or substrate and then performing electrophoretic or chromatographic separation (or a combination of both), e.g. in a conventional manner, in one or two dimensions. This results in a distributed pattern of separated analytes. The location of those analytes including a phosphorylated amino acid (i.e. the target analytes) can be detected by the method of the invention. Separated analytes may be detected directly on the gel or substrate. Preferably, however, the separated analytes are transferred from an electrophoretic gel or chromatographic substrate to a membrane in known manner for detection. In this case the attachment substrate is constituted by the membrane.

Thus, one or more phosphorylated amino acids are detected whilst present in or at the surface of either a gel matrix (for example polyacrylamide) or present bound to a solid porous membrane which preferentially is a flat planar membrane or a curved planar membrane or present in or at the surface of a solid matrix in the form of a thin layer, for example particulate silica, attached to a solid substrate.

Target analytes may be caused to be present in or on or at the surface of a gel, solid matrix or solid porous membrane, either by direct application in liquid medium or by electrophoresis or chromatography through or at the surface of the gel, or solid matrix or solid porous membrane (1, 2). The gel, matrix or membrane can be used as a support medium for the separation of analytes present as a mixture in the sample by means of electrophoresis or chromatography or any known variant of these methods in one or two dimensions, so as to produce a distributed pattern of the separated analytes (2). Where necessary, the analytes can be caused to be immobilised in or on a gel, solid matrix or solid porous membrane so as to enable subsequent analyte detection procedures (2). In the case of some analytes it may not be necessary for any specific immobilisation procedure to be used.

Target analytes can be caused to be present at the surface of the solid porous membrane or solid matrix, by transfer from a gel either by contact transfer, diffusion, pressure, vacuum differential, chromatographic transfer or by- electrophoretic transfer (or any -combination of . these). The specific detection reagent is typically an antibody (which term encompasses antigen- binding variants or fragments of antibodies, such as Fv, scFv, F(ab¹)2, Fab, "diabodies" and the like as well as monoclonal and polyclonal antibodies). Preferably, the antibody has specificity for a particular phosphorylated amino acid residue. A preferred antibody may also recognise specific phosphorylated amino acids that occur within a specific sequence of amino acids that comprise a protein or peptide. Antibodies with specificity for different phosphorylated amino acid residues are commercially available. Suitable specific antibodies may be derived from any biological source, and include phage derived antibodies that recognise and bind to amino acid residues which have been modified either in vivo or in vitro by the covalent attachment of phosphate groups. Each individual antibody type can have a specific reaction with a specific phosphorylated amino acid, e.g. phosphoserine, phosphothreonine, phosphotyrosine, phosphohistidine, etc residues comprising the analyte (3). The method of treatment of the attachment substrate to enable binding of the antibodies is well known to those skilled in the art (2).

The bound detection reagent may conveniently be detected by use of a fluorescent label. Suitable fluorescent labels are well known in the art and include, e.g., fluorescein, rhodamine and Alexa dyes (Alexa is a Trade Mark of Invitrogen). Preferably, however, the fluorescent label comprises a semiconductor nanocrystal in the form of an encapsulated sphere, e.g. in the form of so-called quantum dots (as commercially available from Quantum Dot Corporation). Preferably, the labels (e.g. quantum dots) emit fluorescent light at a wavelength in the range 500 nm to 800 nm when excited with light having a suitable wavelength, typically 600 nm or less. Quantum dots have a narrow, symmetric emission spectrum and have the property of emitting fluorescent light in the wavelength range typically from approximately 500 nm to 800 nm when excited with light of a suitable wavelength preferably with wavelength of 600 nm or shorter.

Conveniently, different fluorescent labels, e.g. quantum dots, having different peak fluorescent emission wavelengths may be used. This has the advantage that different specific phosphorylated amino acid residues can be detected simultaneously by the different fluorescence emitted by different labels associated with different residues.

The fluorescent label (e.g. quantum dot) may be provided on the specific detection reagent (for example, a primary antibody that binds to a particular phosphorylated amino acid residue). Alternatively, the fluorescent label may be provided on a further reagent (such as a secondary antibody, biotin or streptavidin) which binds to the specific detection reagent. As a further possibility, the fluorescent label may be bound directly or indirectly to a streptavidin molecule which may form a complex with a biotin molecule which is bound to the specific binding reagent. For example, the label may be bound to a biotin molecule which may interact with a streptavidin molecule which is bound to the specific binding reagent. Preferably, the fluorescent label is covalently attached to the detection reagent.

Flourescent labels can be detected in a known manner, e.g. using known electronic and photographic detection devices. It is particularly preferred to use a charge coupled detection (CCD) device, e.g. a ProXPRESS 2D Proteomic Imager (ProXPRESS is a Trade Mark) from PerkinElmer.

The method of the invention may be used qualitatively or quantitatively.

The presence of target analytes, and their spatial position in two dimensions, in or on the attachment substrate can thus be detected by the treatment of the substrate in or to which any analyte is bound with a suitable specific detection reagent or reagents, preferably specific antibodies. The antibodies that are bound to phosphorylated amino acids are desirably detected (directly or indirectly) by the presence of semiconductor nanocrystals (for instance as supplied by the Quantum Dot Corporation) in the form of encapsulated spheres (known commonly as quantum dots) bound covalently to specific antibodies (1, 3- 8). It is preferable that each specific antibody type is bound to a quantum dot, the type of which has a specific peak fluorescent emission wavelength. Thus each analyte that is comprised by any of the phosphorylated amino acid residues can be detected (and quantitated if required) by the fluorescence emitted by the quantum dots bound to the antibody probe reagents. Any suitable number of quantum dots can be bound to each antibody molecule.

The positions of the fluorescent labels (e.g. quantum dot bound antibodies) that are bound either directly or indirectly to the target analytes can be detected in or on the attachment substrate by illuminating the substrate with light of a suitable wavelength, typically 600 nm or shorter, that produces fluorescence that is characteristic of each type of quantum dot. Images of the positions of the quantum dot bound antibodies can be recorded using a suitable electronic or photographic device. Preferably, the fluorescent label is detected using an imager, such as a CCD imager. More preferably, a ProXPRESS 2D Proteomic Imager (PerkinElmer Life and Analytical Sciences Inc.) may be used. The use of such apparatus, which is based on a cooled charge-coupled-device camera, can enable not only the detection of phosphorylated analytes, but also their quantitative measurement and can reveal useful information, such as the molecular weight and isoelectric point of the analytes. Thus, the present invention may provide both qualitative and quantitative information relating to an analyte of interest.

In a first embodiment of the invention, mixtures of different antibody-quantum dot types having specificity for the different phosphorylated amino acid residues are mixed together before the detection procedure. Each antibody type can be recognised individually by the wavelength of the emitted fluorescence, which is determined by each different quantum dot species that is attached to each individual antibody type. Thus, the spatial position in the distribution pattern on the attachment substrate or support medium of any one target analyte that is bound by a specific antibody can be determined and correlated exactly with the position of other target analytes reacting with any antibody of different specificity that is recognisable by- its specific fluorescence- wavelength.- In a second embodiment of the invention, the primary antibodies are developed in a different animal species for each specific phosphorylated amino acid type. For example, anti-phosphoserine antibodies may be developed in mouse and anti-phosphothreonine antibodies may be developed in rabbit and so on. The substrate to which the analytes are bound, typically following separation, is treated with antibodies, either individually or together in a mixture by procedures known to those skilled in the art. In this embodiment of the invention the primary antibodies are not bound to quantum dots. The bound primary antibodies are instead detected by incubation with secondary antibodies, wherein the secondary antibodies are specific for the primary antibody type, depending on the particular animal in which the primary antibodies were developed. The secondary antibodies that are bound to the primary antibodies can be detected by the presence of quantum dots bound covalently to the specific secondary antibodies. It is preferable that each specific secondary antibody type is bound to a different quantum dot type having a different specific peak fluorescence emission wavelength. Therefore, different phosphorylated amino acid residues can be detected and distinguished by detection of the different fluorescence emitted by the different bound quantum dot types. Any suitable number of quantum dots can be bound to each antibody molecule. The advantage of this embodiment is that it enables more sensitive detection of the target analytes.

In a third embodiment of the invention, mixtures of different secondary antibody types having specificity for different primary antibody types, are mixed together before the detection procedure. Each type of antibody can be recognised individually by the wavelength of the emitted fluorescence, which is determined by the specific quantum dot species attached to each individual antibody type. The advantage of this embodiment is that it enables more sensitive detection of the target analytes and simultaneously enables multiplexed detection of the target analytes, thereby reducing the time taken for the analysis.

In a fourth embodiment of the invention, one of the primary antibodies is biotinylated and is -detected by- the binding thereto of a- streptavidin-molecule with- covalently attached quantum dot(s) of a particular type. In an arrangement for detecting different phosphorylated amino acid residues, one biotinylated primary antibody is used, together with other primary antibodies that are detected using quantum dot bound secondary antibodies as described above in the second or third embodiments. Potentially, the reverse situation may also be employed, wherein streptavidin is bound to the primary antibody and binds to a biotin molecule, to which a quantum dot is attached.

For comparative purposes, in all of the above embodiments, all analytes (proteins) in the sample can also be detected using non-specific detection methods, so as to reveal their positions in or on a support matrix. Advantageously, the method of the present invention thus involves detection of the position in or on the substrate of all proteins in the sample, as well as detection of specific phosphorylated amino acids (target analytes) present in the sample. This total protein detection can be achieved by using a fluorescent total protein stain that binds non-covalently to the analytes, such as Sypro Ruby or Sypro Rose. Analytes can also be detected by fluorescent total protein stains that bind covalently to the analytes, by way of example, but not limited to, reactive derivatives of any one of the BODIPY dyes (Invitrogen Inc.) or 2-methoxy-2,4-diphenyl-3(2H) furanone, EVO blue 30 or NIR 664-N-succinimidyl ester (Sigma- Aldrich).

Alternatively, the analytes can be detected non-specifically by binding a coloured dye which binds either non-covalently, for example using the dye Ponceau S, or covalently, by way of example using the dye Dabsyl-N-succinimidyl ester.

Another example of a non-specific detection method involves the treatment of an analyte with a chemical linking reagent comprising a functional moiety that either binds covalently to the analyte or enables a linking function moiety of the chemical linking reagent to bind to the analyte. One example of such a functional moiety is an N-hydroxy succinimide ester. Typically, the chemical linking reagent also comprises another chemical moiety that may act as a ligand, for example biotin, a carbohydrate substance, an oligonucleotide or a specific peptide. An example of a chemical linking reagent that may be used in accordance with the present invention is EZ-Link™ NHS-biotin (Perbio Science UK Limited). The covalent attachment of a ligand to an analyte, by way of a chemical linking reagent, allows the ligand to be bound by a specific protein or specific oligonucleotide. By way of example, biotin may be bound by streptavidin; specific peptides may be bound by specific proteins; oligonucleotide aptamers may be bound by specific proteins; and specific carbohydrates may be bound by specific lectins. In accordance with the present invention, each specific protein or oligonucleotide that binds to a specific ligand attached covalently to the analyte is bound to a quantum dot that has a specific wavelength of emitted light. The analyte may be detected by illumination with light of a suitable wavelength that causes the quantum dot to emit light of a different wavelength.

In another example, the chemical linking group comprises a cleavable chemical moiety that may be cleaved by the use of a suitable chemical reagent. An example of such a cleavable moiety is a disulphide bond that may be cleaved with the reagent dithiothreitol. One example of such a reagent is EZ-Link™ Sulfo-NHS-SS-Biotin (Perbio Science UK Limited). The use of such a reagent allows the protein that binds the ligand on the chemical linking reagent and to which the quantum dots are attached to be separated from the analyte. This procedure can be carried out after the detection of all of the analytes and facilitates any subsequent detection of specific analytes in accordance with the present invention.

Preferably, the procedure for the detection and imaging of all of the analytes non- specifically is carried out prior to the procedure for detection of target analytes with antibodies or another specific detection reagent. The reagents that bind non-covalently to the analytes, whether fluorescent or not, are preferably, but not necessarily, unbound during subsequent processing for specific target analyte detection. In this case it is essential that the detection pattern is imaged before proceeding with the subsequent specific detection. Reagents that bind covalently can be imaged either before or after the subsequent specific detection. Preferably, any detection reagent that remains bound to the analytes during the specific target analyte detection does not specifically interfere with the binding of the detection reagent or the detection of light emitted by the quantum dots. Such non-specific techniques reveal the positions of all analytes in the distributed pattern.. The simultaneous probing for different analytes on a single support membrane or matrix can be described as a multiplexed procedure or multiplexing. The advantage of this approach is that it reduces the time taken to carry out an analysis when it is desired to probe a gel, solid matrix or solid porous membrane with all types of antibody.

The present invention may be used to investigate the role of protein phosphorylation in biological systems and is of particular application when the biological samples comprise a complex mixture of analytes such as serum, urine and cellular extracts.

The processes of protein phosphorylation and dephosphorylation are key metabolic events in the control of numerous cellular processes (9-11). Protein phosphorylation is regulated by enzymes known as protein kinases, which catalyse the transfer of the terminal phosphate residue of ATP to an amino acid residue of a protein. The phosphorylation of proteins is known to be reversible and the reverse reaction is catalysed by enzymes known as protein phosphatases. Research has shown that some proteins in prokaryotic organisms (such as members of the Archae and Bacteria domains) undergo covalent phosphorylation on the hydroxyl side chains of serine, threonine and/or tyrosine residues (11).

The addition of a phosphate group introduces a negative charge to the protein and may induce a conformational change. Such a change may result in activation of the protein. Dephosphorylation may return the protein to its original conformation. Consequently, protein phosphorylation may act as a 'molecular switch' which may regulate protein activity. As well as regulating the activity of a protein, the addition of a phosphate group to an amino acid residue may provide a binding site on the protein to which another protein may bind. Thus, proteins which are not associated with one another under normal conditions in the cell may be brought into close proximity. Therefore, protein phosphorylation and dephosphorylation are of great importance in many aspects of cell regulation, including cell growth, differentiation and metabolism. A greater understanding of such processes would provide important insights into the mechanisms by which diseases,- such as cancer, operate. Several-human diseases-have- now been shown to be ~ associated with the abnormal phosphorylation of cellular proteins. As a result, investigation into the role of protein phosphorylation in biological systems is currently a major area of research.

The analysis of total protein expression and the post-translational modification of proteins may be carried out using a variety of biochemical techniques that are known in the art. One such approach that allows detection of proteins following their separation by electrophoretic and/or chromatographic methods and their subsequent transfer to a support medium (such as a membrane) is Western Blotting. The detection of proteins has been greatly assisted by the use of fluorescent dyes. However, typical fluorescent dyes have excitation and emission spectra with a relatively small Stokes shift, such that the optimal excitation wavelength is close to the emission peak.

This problem may be overcome using "quantum dot" nanocrystals (Quantum Dot Corporation) in the form of encapsulated spheres, consisting of nanometer-scale crystals of semiconductor material which have been coated with an additional semiconductor shell to improve the physical properties of the material. The quantum dots are further coated with a polymer shell that allows the materials to be conjugated to biological molecules (such as antibodies) and to retain their physical properties (12).

Different quantum dots with different emission wavelengths have closely similar (or possibly identical) absorbance spectra. This unique spectral property is due to the semiconductor material that forms the core of a conjugate produced with a quantum dot nanocrystal. In spite of the broad absorbance range, the emission of fluorescent light is narrow, symmetric, and independent of the excitation wavelength. This has the advantage that the shape of the emission is independent of the excitation wavelength.

Simple multiplexed Western blotting methods using quantum dots have been used to analyse the post-translational modification and total expression of proteins. The use of such techniques provides exceptional sensitivity (1). The present invention provides a method of combining the sensitive specific probing and recognition of phosphoproteins, bound either in or on gels, solid matrices or membranes, through the simultaneous use of primary antibodies specific for different phosphorylated amino acid residues, with detection by imaging of the fluorescent light emitted from quantum dots. Thus, the present invention provides a method for discriminating between a combination of multiple antibody types using quantum dots and a suitable imaging apparatus, thereby allowing identification of different phosphorylated amino acid residues.

Typically, use of the present invention will facilitate the identification, targeting and tracking of biological markers of diseases and pathologies, in clinical diagnosis, in drug and therapeutics discovery and development.

The invention also provides a kit for the detection of a phosphorylated amino acid in a sample, the kit comprising at least one specific detection reagent that binds specifically and selectively to a phosphorylated amino acid residue; and instructions for carrying out the method of the invention.

The kit may include samples of a plurality of different specific detection reagents, each reagent binding to a different phosphorylated amino acid residue.

The or each specific detection reagent may include a fluorescent label. Alternatively, the kit may include a supply of one or more fluorescently labelled further reagents that bind to the specific detection reagent(s). In either case the fluorescent label preferably comprises a semiconductor nanocrystal in the form of an encapsulated sphere (e.g. a quantum dot).

The kit may include a reagent to detect the position in or on the substrate of all proteins in the sample. By way of example, the kit may comprise a fluorescent total protein stain that binds covalently or non-covalently to an analyte.

The invention will be -further -described-, by way of illustration, in the-following-Example. and with reference to the accompanying drawings. In the drawings: Figure 1 illustrates schematically a first embodiment of the invention; and

Figure 2 illustrates schematically a second embodiment of the invention.

Referring to the drawings, Figure 1 provides a representation of the first embodiment, wherein an analyte 2 is bound to a membrane or matrix 1. The analyte is phosphorylated such that a primary antibody 3 recognises and binds specifically to the phosphorylated amino acid residue constituting a target analyte. A quantum dot 4 is covalently attached to the primary antibody, providing a fluorescent label to enable detection of the antibody when using an imager.

Figure 2 provides an illustration of the second embodiment wherein a multiplex of three different analytes 2 are bound to the membrane or matrix 1. Three different primary antibodies 3 recognise and bind specifically to different phosphorylated amino acid residues on the analytes. In a further step, three different secondary antibodies 5 are respectively bound to the primary antibodies. In this embodiment, three different quantum dots 4 of three different colours are covalently attached to the three different secondary antibodies. The different quantum dots that are bound to the antibodies each have a specific peak fluorescence emission wavelength, allowing the user to distinguish between the different phosphorylated analytes.

Example

A sample including phosphorylated amino acids was separated and the phosphorylated amino acids detected by the following procedure.

1. A crude extract of proteins from the selected biological material is prepared in a suitable sample buffer. Typically the sample buffer will contain a chaotropic agent(s) such as urea, a reducing agent such dithiothreitol, a neutral detergent such as 3-[(3- cholamidopropyl)dimethylammmonio]-l-propanesulfonate (CHAPS)- and ampholytes- suited to the pH range selected for the separation procedure. 2. The proteins are separated by two dimensional polyacrylamide gel electrophoresis (20- PAGE). In a typical procedure, isoelectric focusing is used for the first dimension separation by the protein isoelectric points in the range pH 3 to 10 or in narrower ranges. The focussed proteins are obtained, typically, in a low concentration polyacrylamide gel which can be applied to a second gel to generate the second separation. Most commonly, sodium dodecyl sulphate-poly aery lamide gel electrophoresis (SDS-PAGE) is used in the second dimension to separate the proteins by their molecular weight. The 2D-PAGE procedure produces a two dimensional array of the proteins in the polyacrylamide gel.

3. The proteins are transferred by electrophoresis from the polyacrylamide gel to a blotting membrane, for example nitrocellulose or polyvinyl-difluoride (PVDF). The transfer electrophoresis proceeds in a direction perpendicular to the plane of the gel. This procedure is known either as either blotting, electroblotting or western blotting.

4. The membrane (the blot) to which the proteins have been transferred is blocked, to inhibit non-specific binding of proteins in subsequent steps of the protocol, by incubation in a buffer solution containing a suitable blocking agent that is compatible with detection of phosphoproteins.

5. The blot is probed by incubation in a buffer solution containing any one or any combination of the primary antibodies that are to be used. The primary antibodies can be a mixture of, for example, three antibody types; that is, antibodies with three different specificities; one being anti-phosphoserine, another anti-phosphothreonine and another anti-phosphotyrosine. Each antibody is developed in a different animal, For instance anti-phosphoserine in mouse, anti-phosphothreonine in rabbit and anti- phosphotyrosine in chicken. Alternatively, one (only) of the anti-phosphoamino acid antibodies can be developed in rabbit, mouse or chicken and can then be labelled covalently with biotin or a hapten. The concentration of the antibodies are as recommended by the suppliers. ^* 6. The blot is washed to remove excess primary antibodies or streptavidin.

7. The blot is incubated with a mixture of three secondary antibodies each one of which can bind to one only of the primary antibodies. Each type of secondary antibody is bound to one type of quantum dot. Each type of quantum dot has a different fluorescence emission spectrum by which each type of secondary antibody can be recognised. Alternatively, in the case where one of the primary antibodies is biotinylated, one of the secondary antibodies can be replaced with streptavidin bound to a specific type of quantum dot that is different from any other type of quantum dot being used in the experiment. All three primary antibodies, or the streptavidin if used, can therefore be distinguished by the specific fluorescence associated with the quantum dots bound to the secondary antibodies.

8. The blot is washed to remove excess secondary antibodies.

9. The blot is imaged using a suitable imaging apparatus either when wet for fluorescence or chemiluminescence detection, or after drying for fluorescecnce or chemifluorescence detection.

References:

1. Anon. Multiplexed Protein Expression Analysis in Western Blots with Qdot^® Conjugates

Vision [Quantum Dot Corporation publication ] 2004; 2 (3): 2 http : //www . qdots . com/li ve/upload_documents/Nov Visionpg2. pdf

2. Nagata, K., Izawa, I. and Inagaki, M. A decade of site- and phosphorylation state- specific antibodies: recent advances in studies of spatiotemporal protein phophorylation Genes to cells 2001; 6: 653-664

3. Harper, T. F. Western Blot Detection with Qdot^® Conjugates Vision [Quantum Dot Corporation publication ] 2004; 2 (3): 3-6 http://www.qdots.com/live/upload_documents/Nov Visionpgs3-6.pdf

4. Chan, W. C. W, et al. Luminescent quantum dots for multiplexed bilogical detection and imaging

Curr, Opin. Biotech. 2002; 13:40-46

5. Alivisatos, A. P. The Use of Nanocrystals in Biological Detection Nature Biotech. 2004;22:47-52.

6. Zhu et al. Quantum Dots as a Novel Immunofluorescent Detection System for Cryptosporidium parvum and Giardia lamblia

Appl. Environ. Microbiol. 2004;70:597-598.

7. Ness et al. Combined Tyramide Signal Amplification and Quantum Dots for Sensitive and Photostable Immunofluorescence Detection

J. Histochem. Cytochem. 2003 ;51 :981-987. 8. Goldman et al. Multiplexed Toxin analaysis using Four Colors of Quantum Dot Fluororeagents

Anal. Chem. 2004;76:684-688

9. Hunter, T. Protein kinases and phosphatases: the yin and phosphorylation and signaling: the yin and yang of protein

Cell. 1995;80(2):225-36.

10. Graves J. D. and Krebbs, E.G. Protein phosphorylation and signal trans duction Pharmacol Ther. 1999; 82(2-3): 111-121

11. Cozzone, A.J. Post transalational modification of proteins by revesible phsphorylation in prokaryotes

Biochimie. 1998; 80(1): 43-8

12. Qdot® Western Blotting Kit User Manual. http : //www . qdots . com/live/upload_documents/90-0093. pdf

Claims

1. A method of detecting the presence in a sample of a phosphorylated amino acid, the method comprising the steps of: locating a sample in or on an attachment substrate; contacting the attachment substrate with a specific detection reagent that binds specifically and selectively to a phosphorylated amino acid residue; and detecting the bound specific detection reagent.

2. The method of claim 1, wherein phosphorylation is carried out as a preliminary step prior to location of the sample on the attachment substrate.

3. The method of claim 1 or 2, wherein the phosphorylated amino acid is selected from serine, threonine, tyrosine, histidine, aspartic acid, glutamic acid and tryptophan.

4. The method of claim 1, 2 or 3, wherein the attachment substrate is an electrophoretic gel or chromatographic substrate.

5. The method of any one of the preceding claims, wherein the attachment substrate is constituted by a membrane.

6. The method of any one of the preceding claims, wherein the phosphorylated amino acid is detected whilst present in or at the surface of a gel matrix.

7. The method of any one of the preceding claims, wherein the phosphorylated amino acid is detected whilst bound to a solid porous membrane.

8. The method of claim 7, wherein the solid porous membrane is a flat planar membrane or a curved planar membrane or present in or at the surface of a solid matrix in the form of a thin layer attached to a solid substrate.

9. The method of any one of the preceding claims, where the specific detection reagent is an antibody.

10. The method of any one of the preceding claims, wherein the bound detection reagent is detected by use of a fluorescent label.

11. The method of claim 10, wherein the fluorescent label comprises a semiconductor nanocrystal in the form of an encapsulated sphere.

12. The method of claim 10 or 11, wherein the label emits fluorescent light at a wavelength in the range 500 run to 800 nm when excited with light having a wavelength of 600 nm or less.

13. The method of claim 10, 11 or 12, wherein the fluorescent label is provided on the specific detection reagent.

14. The method of claim 10, 11 or 12, wherein the fluorescent label is provided on a further reagent which binds to the specific detection reagent.

15. The method of claim 10, 11 or 12, wherein the fluorescent label is bound directly or indirectly to a streptavidin molecule which forms a complex with a biotin molecule which is bound to the specific binding reagent.

16. The method of claim 10, 11 or 12, wherein different specific phosphorylated amino acids can be detected simultaneously by different fluorescence emitted by different fluorescent labels associated with different amino acids.

17. The method of any one of claims 10 to 16, wherein the fluorescent label is detected using an electronic or photographic detection device.

18. The method of claim 16, wherein the fluorescent label is detected using a charge coupled detection (CCD) device.

19. The method of any one of the preceding claims, further comprising detecting the position in or on the substrate of all proteins in the sample.

20. The method of claim 19, wherein the total protein content is detected using a fluorescent protein stain such as Sypro Ruby, Sypro Rose, reactive derivatives of any one of the BODIPY dyes, 2-methoxy-2,4-diphenyl-3(2H)furanone, EVO blue 30 or NIR 664- N-succinimidyl ester.

21. The method of claim 19, wherein the total protein content of the sample is detected prior to detection of phosphorylated amino acid or acids.

22. A kit for the detection of a phosphorylated amino acid in a sample, the kit comprising at least one specific detection reagent that binds specifically and selectively to a phosphorylated amino acid residue, and instructions for carrying out the method of the invention in accordance with any one of the preceding claims.