WO2007141510A1

WO2007141510A1 - Analysis technique

Info

Publication number: WO2007141510A1
Application number: PCT/GB2007/002059
Authority: WO
Inventors: Peter Jackson
Original assignee: Perkinelmer Singapore Pte Ltd
Priority date: 2006-06-06
Filing date: 2007-06-04
Publication date: 2007-12-13
Also published as: GB0611114D0

Abstract

A method of analysing analytes containing a reactive primary amino group, wherein the method comprises separating analytes by a thin layer technique, reacting the analytes with the reagent epicocconone and analysing the separated analytes by a process of mass spectrometry.

Description

Analysis Technique

Field of the Invention

This invention relates to a method of analysing analytes containing a reactive primary amino group, the analytes including, but not being limited to, peptides and proteins.

Background to the Invention

Epicocconone is a fluorescent chemical substance of the type azaphilone that is produced naturally by the fungus Epicoccus nigrum. Its structure is shown in Figure 1. Epicocconone is a small, neutral, water soluble organic molecule, molecular weight 410Da, with a weak absorbance maximum at approximately 390nm and weak fluorescence emission maximum at 530nm. Epicocconone is chemically reactive and can bind covalently to primary amino groups, for instance those present in peptides and proteins, according to the scheme shown in Figure 1 to yield an enamine. The epicocconone-protein reaction product, the enamine, is much more fluorescent than the unreacted epicocconone; it also has a red-shifted fluorescence maximum. There are two absorbance maxima for the enamine at wavelengths 390 and 520nm and a fluorescence maximum at 610nm.

Epicocconone is available commercially from a number of different sources, including Fluorotechnics Pty Ltd, GE Healthcare and Sigma. Epicocconone is produced by isolation from the fungus Epicoccus nigrum by the Australian company Fluorotechnics Pty Ltd. This company has several patents relating to the use of epicocconone. Epicocconone is available commercially from the company GE Healthcare in a liquid formulation known as Deep Purple (Deep Purple is a Trade Mark) for the detection of proteins in solution, in polyacrylamide gels, on Western blots and in live cell imaging. Sigma-Aldrich supply epicocconone as part of a protein quantitation kit known by the Trade Mark Fluoroprofile. However, the epicocconone available from Sigma-Aldrich has a lower concentration than that available from Fluorotechnics Pty Ltd and GE Healthcare. Properties of epicocconone that are favourable for various biological analyses are described in promotional information from either GE Healthcare or Fluorotechnics Pty Ltd. The properties of the epicocconone-amine reaction product that are relevant to the current invention are summarised as follows:

1. Much more fluorescent than unreacted epicocconone;

2. Red-shifted fluorescence emission spectrum;

3. Long Stoke' s shift;

4. Reversible reaction with amines making epicocconone compatible with mass spectrometry of peptides, proteins and other biological substances that comprise reactive primary amino groups; and

5. Has low toxicity as it has no heavy metals associated with it and thus may be disposed of easily.

Summary of the Invention

According to the present invention there is provided a method of analysing analytes containing a reactive primary amino group, wherein the method comprises separating analytes by a thin layer technique, reacting the analytes with the reagent epicocconone, and analysing the separated analytes by the process of mass spectrometry.

The invention is applicable to analytes (also referred to as target analytes) that include one or more reactive primary amino groups, including proteins, protein fragments, peptides, polypeptides, other biological molecules etc. The target analytes may have chemical modifications (known or unknown), such as phosphorylation, glycosylation, sulphonylation or methylation. The target analytes are separated, in either one or two dimensions, by a thin layer technique using a thin layer separation medium. Suitable thin layer separation media are well known to those skilled in the art and include thin layer chromatography (TLC) plates, solid porous membranes (for example, polyvinylidene difluoride (PVDF)), nitrocellulose, paper or other similar support or separation matrix.

Suitable separation methods are well known to those skilled in the art, and include, by way of example, chromatography, electrophoresis and planar electrochromatography (PEC). The separated target analytes can be visualised on the thin layer separation medium, e.g. TLC plate, by use of epicocconone.

In one approach, peptides, proteins or other target analytes are applied directly to a thin layer separation medium and separated thereon. The medium is allowed to dry and then treated with epicocconone to enable fluorescent visualisation of the target analyte.

In an alternative approach, the target analytes are reacted with epicocconone prior to separation, whilst either in solution or suspension or present on a TLC plate or similar thin layer separation medium. The resulting interaction between the analytes and the epicocconone will lead to the generation of fluorescent products that can be separated on the medium. The reacted and separated analytes can be detected after their separation by virtue of their fluorescence with no further reaction with epicocconone being required after the separation.

Target analytes may be caused to be present in, on or at the surface of a thin layer separation medium by direct application in liquid medium. Where appropriate the medium can be used for the separation of target analytes present in a mixture of analytes in any dimension using one of the separation methods mentioned above to produce a distributed pattern of separated analytes.

Target analytes may be caused to be present at the surface of the thin layer separation medium by transfer from a gel, or other similar media, either by contact transfer, diffusion, pressure or vacuum differential, chromatographic transfer, electrophoretic transfer or any suitable combination of these techniques.

The presence of target analytes and their spatial position hi two dimensions, in or on the thin layer separation medium, can be detected by the treatment of the medium in or on which any analyte exists, using the procedures described above.

As noted above, epicocconone is commercially available from a number of sources, and experiments have been carried out with different epicocconone formulations.

For example, experiments have been carried out using Deep Purple available from GE Healthcare. Typically, the Deep Purple formulation (having an estimated epicocconone concentration of approximately 0.17 mg/ml) is diluted prior to use. Preferably, the Deep Purple formulation is diluted with a solution containing triethanolamine and acetone/water. Preferably, the concentration of triethanolamine in solution is between 0.5 and 5%, more preferably between 0.8 and 2%, and most preferably 1% (v/v). Preferably, the ratio of water to acetone in solution is 0.5:2, more preferably 0.8: 1.5 and most preferably 1 : 1 (v/v). Other similar formulations of the triethanolamine/acetone/water solution can be used, wherein the concentrations of triethanolamine and acetone/water can be varied.

Preferably, one volume of the Deep Purple formulation is diluted with between 100 and 10 volumes, preferably 25 volumes, of a solution containing 1% (v/v) triethanolamine in acetone/water solution (1:1, (v/v)).

Further experiments have been carried out using epicocconone produced by the company Fluorotechnics Pty Ltd, this being the currently preferred source of epicocconone. The epicocconone as supplied is hi the form of a solution at a concentration of 1 mg/ml in acetonitrile and is preferably diluted prior to use.

Preferably, the epicocconone supplied by Fluorotechnics Pty Ltd is diluted with a solution containing triethanolamine and acetone/water. Preferably, the concentration of triethanolamine in solution is between 0.5 and 5%, more preferably between 0.8 and 2%, and most preferably 1% (v/v). Preferably, the ratio of water to acetone in solution is 0.5:2, more preferably 0.8:0.5 and most preferably 1 : 1 (v/v). Other similar formulations of the triethanolamine/acetone/water solution can be used, wherein the concentrations of triethanolamine and acetone/water can be varied.

Preferably the epicocconone solution supplied by Fluorotechnics Pty Ltd (1 mg/ml in acetonitrile) is diluted with a solution containing triethanolamine and acetone/water to produce a concentration of epicocconone in solution in the range of 0.625 to lOμg/ml.

Preferably, one volume of the epicocconone solution as supplied is diluted with 400 volumes of a solution containing 1% (v/v) triethanolamine in acetone/water (1:1, (v/v)) to give a concentration of epicocconone in solution of 2.50 μg/ml.

It is currently preferred to use epicocconone from Fluorotechnics Pty Ltd in preference to Deep Purple as this is cheaper and gives results of equal sensitivity.

The epicocconone is used in the form of a liquid, generally a solution. In embodiments where the epicocconone is applied to the thin layer separation medium this is conveniently achieved by applying the liquid in the form of a spray, preferably a fine mist, by immersion of the thin layer separation medium in the epicocconone solution or by close contact with a matrix containing the epicocconone solution.

The positions of the analytes after reaction with epicocconone can be detected in or on the thin layer separation medium by illuminating the medium with light of a suitable wavelength and where necessary filtering the emitted light so as to select specific wavelengths. Images of the positions of the analytes can be recorded using a suitable imaging device, typically an electronic or photographic imaging device. Preferably, the analytes are detected using a charge-coupled device (CCD) imager, preferably a cooled CCD imager. By way of example, and by way of preference, a suitable imaging apparatus is a ProXPRESS 2D Proteomic Imager (manufactured and supplied by the company PerkinElmer Life and Analytical Sciences) (ProXPRESS is a Trade Mark), which is based on a cooled charge- coupled-device camera. Imaging using such apparatus can enable not only the detection of the analytes but also their quantitative measurement and can reveal useful information that is characteristic of each, by way of example, mobility under specific separation conditions.

The imaging of the separation pattern of the analytes in or on the thin layer separation medium enables the position of analytes of interest to be determined. The exact spatial distribution of analytes detected can be matched between different thin layer separation media using suitable software. This procedure can facilitate the identification of specific analytes in complex mixtures.

Images can also be used to locate the position of analytes of interest in or on the thin layer separation medium. This can enable specific analytes to be removed from a thin layer separation medium for subsequent mass spectrometry analysis. Alternatively, the analytes can be targeted directly by the process of mass spectrometry as discussed below.

The reaction of epicocconone with primary amines is reversible and in particular is sensitive to pH; the adducts are most stable at pH 2.4 but are less stable at greater pH values. In this way, epicocconone differs from other reagents that form stable bonds when used to detect analytes that contain primary amino groups.

In one embodiment of the invention, analytes that have been detected and imaged on the thin layer separation medium by means of epicocconone can be eluted from the medium using either aqueous or non aqueous solvents or other suitable liquid media that can effect removal of the anlaytes from the medium and render them in the extracting solution.

The extracted analytes may then be subjected to mass spectrometric analysis by any suitable method, by way of example, matrix assisted laser desorption ionisation (MALDI), electrospray ionisation or other mass spectrometric techniques. Such methods are well known to those skilled in the art. One preferred method involves the use of a MALDI-TOF (time of flight) mass spectrometer, particularly the proTOF MALDI-TOF mass spectrometer, supplied by PerkinElmer Life and Analytical Sciences (proTOF is a Trade Mark). In an alternative embodiment, analytes may be ionised directly from the thin layer separation medium after separation. In this case, a solution containing either an inorganic or an organic matrix suitable for mass spectrometry (eg. MALDI) is applied to the thin layer separation medium (eg. TLC plate) at the position at which the analyte has been detected by the epicocconone and allowed to dry. The separation medium or a section of it is then introduced into the mass spectrometer, eg. being introduced into the plate holder of a MALDI-TOF machine and inserted into the instrument in the normal way. The position of the separation medium in the mass spectrometer is adjusted so that the laser or other ionising source impinges on the position on the medium of the analyte of interest that has been treated with the matrix (with the ionising source either being directed to the position or scanning over the position), so as to desorb and ionise the analyte of choice. Mass spectra of the analytes can then be obtained. Alternatively, the mass spectrometric technique of DESI (desorption electrospray ionisation) or DART (direct analysis in real time) may be used.

The separation, detection and quantitative measurement of peptides, proteins and other analytes on a thin layer separation medium (such as a TLC plate) is a common and essential analytical method for the investigation of biological systems. The detection of such analytes using epicocconone is effective in that the sensitivity and ease of use is comparable with those for reagents currently used for the detection of peptides and proteins on TLC plates, eg. fluorescamine, while also providing a method that is compatible with mass spectrometric analysis of the analytes .

A significant advantage of the use of epicocconone is the markedly improved peptide mass spectra that are obtained when the epicocconone treated separation medium is placed in a mass spectrometer for direct desorption/ionisation from the separation medium with a laser or other ionising source impinging on the position of the analyte on the medium.

The method has a particular advantage in that it facilitates the mass spectrometric analysis of detected peptides and proteins either after removal from the separation medium or when present in situ on the medium. The invention provides a new, efficient method of investigation of biological systems that can give information on the type and quantities of protein constituents of biological systems. The invention is of particular application when the biological samples comprise complex mixtures of analytes such as serum, urine, and cellular extracts or peptide digests of either individual proteins or mixtures of proteins. It will therefore assist in biological research, including identification, targeting and tracking of biological markers of diseases and pathologies, in clinical diagnosis, drug and therapeutics discovery and development.

The use of epicocconone in accordance with the present invention facilitates the mass spectrometry of analytes either after their elution from the separation medium or by direct ionisation from the separation medium by means of a mass spectrometer. This will provide an efficient method of obtaining a significant amount of information with regard to the detected analytes and biological systems.

The invention will be further described, by way of illustration, in the following Examples and with reference to the accompanying drawings. In the drawings:

Figure 1 is a reaction scheme showing reaction of epicocconone with a protein;

Figure 2A shows the mass spectra obtained for a peptide known as peptide 1, wherein after separation by TLC, the target analytes are sprayed with Deep Purple and analysed by mass spectrometry;

Figure 2B shows the mass spectra obtained for peptide 1, wherein the target analytes are sprayed with Deep Purple, and then analysed by mass spectrometry;

Figure 3 A shows mass spectra of peptides from a protein digest after detection on a TLC plate using the reagent fluorescamine;

Figure 3B shows a magnified view of the mass spectra obtained for a peptide known as peptide 4; Figure 3 C shows a magnified view of the mass spectra obtained for a peptide known as peptide 3 ;

Figure 3D shows mass spectra of peptides separated in an experiment similar to that shown in Figure 3 A;

Figure 4 shows the mass spectra of peptide 1 after detection on a TLC plate using epicocconone supplied by Fluorotechnics Pty Ltd; and

Figure 5 shows the mass spectra of a peptide known as peptide 5 after detection on a TLC plate using epicocconone supplied by Fluorotechnics Pty Ltd.

Example 1

Experiments were carried out using Deep Purple liquid formulation from GE Healthcare (product number RPN6305). This has an estimated concentration of epicocconone of 0.17 mg/ml.

A diluted formulation of the Deep Purple reagent was prepared as follows:

One volume of the Deep Purple solution was diluted with 25 volumes of a solution containing 1% (v/v) triethanolamine in acetone/water solution (1 : 1, (v/v)). The Deep Purple solution was sprayed in the form of a fine mist as evenly as possible on to a dry TLC plate. The treated TLC plate was allowed to dry and the peptides or proteins that had reacted with Deep Purple were allowed to dry in dim light for 90min at room temperature, and visualised and quantitated by use of a ProXPRESS 2D Proteomic Imager.

Two alternative approaches were used in the separation and detection of analytes.

In one approach, the target analytes were applied directly to a TLC plate and separated thereon. The TLC plate was allowed to dry and then treated with the Deep Purple epicocconone formulation to enable fluorescent visualisation of the target analyte. In an alternative approach, the target analytes were reacted with epicocconone prior to separation, whilst either in solution or present on a TLC plate. The resulting interaction between the analtyes and epicocconone led to the generation of fluorescent products that were separated on the TLC plate. The reacted and separated analytes were detected after their separation by virtue of their fluorescence.

Figure 2A shows the mass spectra obtained for a peptide known as peptide 1 which was applied directly to a TLC plate. The TLC plates used were supplied by Macherey-Nagel GmbH & Co. under the product number/name 805013/Polygram SiI G. The TLC plates were plastic-backed and had a matrix comprising silica gel. Peptide 1 had the amino acid sequence Thr-Arg-Asp-Ile-Tyr-Glu-Thr-Asp-Tyr-Tyr-Arg-Lys and had a mass of 1623.8, based on theoretical data from the supplier (Anaspec Inc.). After separation, the TLC plate was sprayed with Deep Purple. The TLC plate was then treated four times with a matrix (alpha-cyano-4-hydroxycinnamic acid, alpha-CHCA), three times with CHCA in 80% acetonitrile (MeCN), and once with CHCA in 25% MeCN. The TLC plate was then placed in a proTOF mass spectrometer, supplied by PerkinElmer Life and Analytical Sciences.

Figure 2B shows the mass spectra obtained for peptide 1 wherein the peptide was treated with Deep Purple (without separation). The peptide was applied to a TLC plate, sprayed with Deep Purple and treated as described for Figure 2A. The TLC plate was then placed in the proTOF mass spectrometer.

Comparison of the results of Figure 2 A and 2B shows that both approaches yielded similar spectra.

Example 2

By way of comparison, Figure 3A shows mass spectra of peptides from a protein digest detected on a TLC plate using the reagent fluorescamine in known manner. The peptides were obtained following trypic digestion of the protein bovine alpha-casein. The peptides were not purified individually but were separated on a TLC plate. After separation of the peptides and subsequent treatment with fluorescamine, the TLC plate was treated with the matrix alpha-CHCA, allowed to dry and then was placed in an ABI QStar Pulsar 1 mass spectrometer (QStar Pulsar 1 is a trade mark).

Figure 3B shows a magnified view of the mass spectra obtained for a peptide known as peptide 4 following treatment with fluorescamine.

Figure 3 C shows a magnified view of the mass spectra obtained for a peptide known as peptide 3 following treatment with fluorescamine.

Figure 3D shows mass spectra of peptides separated in an experiment similar to that shown in Figure 3 A. The results show more clearly the mass spectra of fluorescamine detected peptides and show a TLC plate with clear separation of the peptides and an explanation of the peaks in the spectra.

Mass spectra obtained after epicocconone detection are superior to those obtained by the use of detection agents that generate a stable covalent bond. This is shown in Figure 2A and B where the mass spectra obtained using an epicocconone reacted peptide are significantly less complex than those obtained from a fluorescamine treated TLC plate as shown in Figure 3 A, B and C.

Example 3

Experiments were also carried out using epicocconone supplied by Fluorotechnics Pty Ltd (Product number EP 001 A).

A diluted formulation of the epicocconone as supplied was prepared as follows:

One volume of the epicocconone solution (lmg/mL in acetonitrile) was diluted with 400 volumes of a solution preferably containing 1% (v/v) triethanolamine in acetone/water solution (1:1, (v/v)) to give a concentration of epicocconone of 2.50 μg/mL. The epicocconone solution was sprayed in the form of a fine mist as evenly as possible onto the dry TLC plate. The treated TLC plate was allowed to dry in dim light for 90min at room temperature. The peptides or proteins that had reacted with epicocconone were visualised and quantitated by use of a ProXPRESS 2D Proteomic Imager.

Figures 4 and 5 show mass spectra of peptide 1 as referred to above and a further peptide known as peptide 5, after detection on a TLC plate using epicocconone supplied by Fluorotechnics Pty Ltd. Peptide 5 had the amino acid sequence Asp-Leu-Asp-Val-Pro-Ile- Pro-Gly-Arg-Phe-Asp-Arg-Arg-Val-Ser-Val-Ala-Ala-Glu and had a mass of 2113.3, based on theoretical data from the supplier (Anaspec Inc.).

The peptides shown in Figures 2, 4 and 5 are stated to be 95% pure by the supplier.

The TLC plates used in the experiments which generated the results shown in Figures 3, 4 and 5 were supplied by Merck KGaA under the product number 1.05748.0001. The TLC plates were plastic-backed and had a matrix comprising silica gel 60.

A concentration of 10 ng of each peptide was applied to a TLC plate (Merck Silica 60). The peptides were detected by dipping the TLC plate into a solution comprising 5 μg/ml epicocconone and 0.25% (v/v) pyridine in water saturated ether for 32 minutes, drying the TLC plate in air, treating the TLC plate with the MALDI matrix alpha-CHCA and then using the technique of MALDI-TOF mass spectrometry using an ABI QStar Pulsar I mass spectrometer.

The procedure of Example 3 was subsequently optimised by spraying the epicocconone in solution in 1% (v/v) triethylamine in acetone/water (1 :1, (v/v)).

The difference between the masses indicated in the mass spectral analyses shown is probably due to the variation in the calibration of the mass spectrometers.

Claims

1. A method of analysing analytes containing a reactive primary amino group, wherein the method comprises separating analytes by a thin layer technique, reacting the analytes with the reagent epicocconone, and analysing the separated analytes by the process of mass spectrometry.

2. A method of analysing analytes hi accordance with claim 1, wherein the method comprises treating the analytes with epicocconone prior to separation by a thin layer technique.

3. A method of analysing analytes hi accordance with claim 1 or 2, wherein the method comprises separating the analytes by the process of thin layer chromatography.

4. A method of analysing analytes in accordance with claim 1 or 2, when the method comprises separating the analytes by the process of planar electrochromatography.

5. A method of analysing analytes in accordance with claim 1 or 2, wherein the method comprises separating analytes by the process of electrophoresis.

6. A method of analysing analytes In accordance with any one of the preceding claims, wherein the positions of the analytes after reaction with epicocconone are detected In or on a separation medium using an electronic or photographic imaging device.

7. A method of analysing analytes hi accordance with any one of the preceding claims, wherein a thin layer separation medium is placed directly Into a mass spectrometer for analysis.

8. A method of analysing analytes in accordance with any one of the preceding claims, wherein the mass spectrometry is performed using a MALDI-TOF spectrometer.

9. A method of analysing analytes in accordance with any one of claims 1 to 7, wherein the mass spectrometry is performed using the technique of DESI.

10. A method of analysing analytes in accordance with any one of claims 1 to 7, wherein the mass spectrometry is performed using the technique of DART.