WO2007139471A2

WO2007139471A2 - Method for chromatography

Info

Publication number: WO2007139471A2
Application number: PCT/SE2007/000514
Authority: WO
Inventors: Bengt Bjellqvist
Original assignee: Ge Healthcare Bio-Sciences Ab
Priority date: 2006-05-31
Filing date: 2007-05-28
Publication date: 2007-12-06
Also published as: WO2007139471A3

Abstract

The present invention relates to a method for chromatography. More closely, the method relates to a novel way of separating proteins by chromatography with results that previously only have been achieved by electrophoresis. The invention relates to a method for chromatography of proteins, comprising the following steps: a) solubilising a protein sample in a solubilising agent; b) adsorbing solubilised proteins on a chromatographic column; c) eluting proteins from the column with a gradient of surfactant solvent; and d) collecting fractions comprising eluted proteins. The invention also relates to a kit and fraction collector for use in the method.

Description

Title: Method for chromatography

Field of the invention

The present invention relates to a method for chromatography. More closely, the method relates to a novel way of separating proteins by chromatography with results that previously only have been achieved by electrophoresis.

Background of the invention

Separation and identification of proteins or polypeptides has been and still is of major interest and importance within research and pharmaceutical industry. For example, the discovery of novel protein biomarkers can form the basis for entirely new drugs for treatment of various medical conditions.

Several methods for separation of proteins or polypeptides exist, for example chromatographic and electrophoretic methods.

Sodium dodecyl sulphate (SDS)-electrophoresis is a variant of zone electrophoresis, which separates polypeptides according to their molecular weight. The SDS masks the charge of the proteins themselves and the formed anionic complexes have in free solution approximately identical electrophoretic mobilities independent of the size of the polypeptide. The molecular weight dependence is generated with the use of a sieving media, polyacrylamide gel is the media most commonly used for the purpose. A common and advantageous approach in connection with SDS electrophoresis is to utilise gradient gels containing varying concentrations of polyacrylamide where the a polyacrylamide concentration increase in the transport direction of the SDS-protein complexes from the sample application point towards the cathode. The mobilities of the protein will steadily decrease during the transport through the gel as a result of the variation of the sieving effect. SDS-protein complexes will remain stacked and move in narrow sharp zone localised at the boundary between the zone containing the gel buffer anion and the separation zone as long as the SDS-protein complex has a mobility higher than the weak acid present in the separation zone. As a consequence complexes corresponding to high molecular weight will destack already at low polyacrylamide concentration in an early state of the experiment. Low molecular weight complexes will remain stacked to close to the end of the experiment.

Prior to SDS-electrophoresis the protein sample is normally solubilised in an SDS- solution with an SDS concentration exceeding the critical micelle concentration (CMC) and with a SDS/protein ratio of > 1,4 gram SDS/protein. Normally a reducing agent is also added to the solution used to solubilise the proteins. Examples of reducing agents used in this context are mercaptoethanol, dithiothreitol and tris(2-carboxyethyl) phosphine. The sample is often heated to 90 to 95 ⁰C in order to ascertain reduction of all -S-S- bridges. The result is the formation of micelle like aggregates distributed along the unfolded polypeptide chains (necklace model). An important advantage of SDS electrophoresis is that the sample preparation method seems to result in complete solubilisation of all the proteins present in a sample. Furthermore, a quick heating of the sample in an SDS containing solution is a simple and fast way to denature present proteases in order to minimize proteolytic breakdown of the peptide chains. Another advantage is that the presence of SDS at concentrations comparable or higher than CMC, prevents the formation of polypeptide aggregates as well as loss of peptides due to adhesion to hydrophobic surfaces with which the sample is brought into contact.

In spite of the benefits of SDS-electrophoresis, it would in some cases be desirable to avoid the gel based system and instead be able to perform the same kind of protein separation but in a liquid based system.

Today there is no chromatographic method for protein separation which may replace SDS- electrophoresis with comparable results. Summary of the invention

The present invention provides a method called surfactant chromatography which is an alternative to presently used chromatographic protein pre-fractionation methods prior to digestion and analysis by for example mass spectrometry.

Surfactant chromatography according to the present invention is a chromatographic analytical alternative to SDS-electrophoresis. The advantages obtained according to the present invention are that all types of proteins will be solubilised, that protein losses are negligible and that the background increase caused by unspecific protease activity during sample preparation will be kept to a minimum.

In a first aspect the invention relates to a method for chromatography of proteins, comprising the following steps: a) solubilising a protein sample in a solubilising agent; b) adsorbing solubilised proteins on a chromatographic column; c) eluting proteins from the column with a gradient of surfactant solvent; and d) collecting fractions comprising eluted proteins.

In a preferred embodiment of the method, the protein sample is solubilised by a solution containing a surfactant. A wide variety of surfactants, such as anionic, cationic and non- ionic, can be used in the solubilisation step, but preferably an anionic surfactant. The surfactant should be present in a concentration equal or higher than CMC as well in the solubilisation and in all other steps preceeding the adsorption of proteins and surfactant on the chromatographic column. Preferred surfactants are alkyl sulphates, alkyl sulphonates and alkanoic acids with alkyl chains containing 8 to 16 carbon and more optimal 10 to 16 carbon atoms. The hydrophobic part of the surfactant could also contain aromatic groups as in alkylbensene sulphonates The chain length should not be too short in order to avoid the need to use large surfactant amounts to maintain concentrations equal or higher than the CMC in the solubilisation step and additional steps preceeding the adsorption of proteins and surfactant on the chromatographic column.

Without the presence of sufficient amounts of charged surfactant, protein loss will occur in contact with almost any type of surface. For example, the walls of test tubes and pipette tips will lead to severe losses of peptides when highly diluted protein samples are handled in the absence of surfactants.

As with SDS-electrophoresis, a reducing agent is normally included in the solubilisation solution. A wide variety of reducing agents can be used. The reducing agent should be strong enough to allow the reduction of all -S-S- bridges present in the sample and the amounts high enough to maintain reducing conditions in all handling steps. Examples of suitable reducing agents are compounds containing -SH groups such as dithiothreitol, dithioerythritol, mercaptoethanol and glutathione, phosphines like tris(2-carboxyethyl) phosphine and tris hydroxyl propyl phosphine, but also other reducing compounds such as sulphites. While a majority of -S-S- bridges are reduced already at room temperature, it is preferred, like in SDS-electrophoresis, to heat the sample to 90-95⁰C for 3-5 minutes for a rapid and complete reduction of all -S-S- bridges present in the sample. The application of a sample to the column and the separation is normally done under reducing conditions in order to avoid regeneration of -S-S- bridges. The reducing agents used could be the same as those used in the solubilisatioon step. As with SDS-electrophoresis, an alternative way to avoid formation of -S-S- bridges is to react the -SH groups with an excess of chemicals like iodoacetamid, acrylamid,or vinyl pyridine or to run the experiments in the presence of an excess of disulfides like di-(2-hydroxyethyl)-disulfide.

The chromatographic column is filled with a hydrophobic chromatography media. Examples of hydrofobic ligands are isopropyl, butyl, octyl and phenyl ligands bound to different types of chromatographic media such as porous silica, agarose or other suitable polymeric natural or synthetic beads. A wide variety of surfactants can be used for elution of the proteins from the column. The essential point is that the CMC of the surfactant is high enough and the CMC for the experimental conditions used should preferably be higher than 5 gram/liter and more preferably higher than 10 gram/liter. Example of surfactants suitable to use when elution is made with buffered water solutions at room temperature are hexyl sulphate, heptyl, sulphate, octyl sulphate nonyl sulphate and decyl sulphate, but also sulphonates and alkanoic acids with corresponding length of the alkyl chain as well as benzene sulphonates with none or short alkyl chains such as butyl benzene sulphonate. Use of higher temperatures or elution with water-ethanol, water -propanol or water acetonitril solutions result in higher CMC values and for this type of conditions it is also possible to use detergents with longer alkyl chains such as dodecyl sulphate or tetra decyl sulphate.

Preferably the gradient of surfactant solvent goes from c=0 to CMC. i.e. from 0% up to the critical micelle concentration for surfactant used.

The results from surfactant chromatography can be analysed based on conventional UV detection. For differential studies, for example of a normal versus diseased sample, tagging of the samples may be performed with for example CyDyes™ . The resulting fractions from the surfactant chromatography may also be applied to PVDF or nitrocellulose membranes to allow immunodetection.

In a preferred embodiment of the method, the fractions in d) are collected in wells or tubes provided with dried gel in which the fractions are allowed to swell. Preferably, the gel adheres to the wells or tubes. A wide variety of different gels such as Sephadex, poly vinylalcohol gels and poly acrylamid gels may be used as long as the pores of the re-swollen gel are large enough allow the peptide chains to enter the gel. Thus, any hydrogel having the desired properties may be used. In a preferred embodiment polycrylamide gels are used. In this embodiment, proteins in the chromatography fractions are precipitated in the gel and the gel is washed. For later identification the proteins may be digested after step d). Preferably, the proteins are loaded onto an RPC (reversed phase) column after the digestion. For identification, the digested proteins are preferably subjected to MS/MS. Alternatively, identification is done my MALDI-MS.

For differential analysis of proteins in different or the same sample, the protein sample(s) are labelled with one or more labelling reagent(s) before step a).

In a second aspect, the invention relates to a kit comprising a hydrophobic media column, a surfactant and means for generating a surfactant gradient. The media and surfactant are preferably as described above.

In a third aspect, the invention relates to a fraction collector, comprising receptacles, such as tubes or wells, provided with dried swellable gel. Preferably a hydrogel has been dried onto the tubes or wells in such a way to adhere the gel to the bottom and/or the walls or the tubes or wells. The preferred gel is a polyacrylamide gel.

Detailed description of the invention The present invention will now be described in detail in relation to a specific example which is not to be regarded as limiting for the invention.

Example

The protein separation method according to the invention is based on exchange between a surfactant containing polar solvent and a hydrophobic solid phase. The analytical procedure contains the following steps:

1. Solubilise sample proteins in SDS-DTT solution at 95°C.

2. Adsorb SDS and the proteins on a hydrophobic chromatographic column. 3. Wash away SDS.

4. Elute with an alkyl sulphate (or alkyl sulfonate) gradient going from c=0 to CMC, select an alkyl sulphate with suitable CMC at the temperature used.

5. Collect fractions in wells, wherein the bottom and optionally the walls have been covered with a thin layer of dried polyacrylamide gel. Allow the sample to swell into the gel.

6. "Precipitate" the proteins in the gel and wash away the alkyl sulphate and buffer ions.

7. Proceed with tryptic digestion and peptide extraction followed by RPC and MS/MS

One very important feature of the chromatography method of the present invention is that it is compatible with protein solubilisation in SDS-DTT. This is the only technique which allows solubilisation of all types of proteins, from integral membrane proteins to hydrophilic cytosolic proteins. Furthermore, all proteins present in a given sample are solubilised. SDS-DTT solubilisation may be used after microwave heating or as an alternative direct transfer of the sample to a hot SDS-DTT solution can be used. Thus, SDS- DTT solubilsation is the method, which gives the best possibilities to minimise background resulting from protease activity during the sample preparation steps.

Proteins in SDS solutions are incorporated in micelles. As the SDS-micelles are small each protein chain will pass through a number of micelles (the "necklace model"), where the micelles are connected with short stretches of hydrophilic amino acid residues. Due to the presence of the surfactant no loss of protein to for example test tube walls is expected and, thus, all of the solubilised protein will be bound to the column.

Provided that the reducing conditions are maintained during chromatography, it should be possible to desorb all proteins bound to the column. Thus, surfactant chromatography represents a method which allows all proteins to be solubilised and analysed by chromatography without any protein losses and with a minimum background caused by unspecific proteolytic digestion during sample preparation. Furthermore, the present invention can be applied to any type of protein even very hydrophobic proteins as well as very complex samples. Besides the surfactant chromatography according to the present invention, the only existing analytical method which has this type of performance is SDS- electrophoresis.

Claims

1. A method for chromatography of proteins, comprising the following steps: a) solubilising a protein sample in a solubilising agent; b) adsorbing solubilised proteins on chromatographic media; c) eluting proteins from the column with a gradient of surfactant-comprising solvent; and d) collecting fractions comprising eluted proteins.

2. Method according to claim 1, wherein the solubilisation surfactant is an alkyl sulphate, an alkyl sulphonate or alkanoic acids.

3. Method according to claim 1 or 2, wherein the sample is solubilised in the presence of a reducing agent comprising -SH groups.

4. Method according to claim 3, wherein the reducing agent is selected from dithiothreitol, dithioerythritol, mercaptoethanol and glutathione, phosphines like tris(2-carboxyethyl) phosphine and tris hydroxyl propyl phosphine, and sulphites.

5. Method according to claim 1 or 2, wherein -SH groups in the sample are reacted with an excess of iodoacetamid, acrylamid,or vinyl pyridine or in the presence of an excess of disulfides like di-(2-hydroxyethyl)-disulfide.

6. Method according to one or more of the above claims, wherein the sample is heated to at high temperature, 9O-95C⁰.

7. Method according to one or more of the above claims, wherein the chromatographic media is hydrophobic.

8. Method according to one or more of the above claims, wherein the elution surfactant is selected from hexyl sulphate, heptyl, sulphate, octyl sulphate nonyl sulphate and decyl sulphate, sulphonates and alkanoic acids, benzene sulphonates, such as butyl benzene sulphonate, or surfactants longer alkyl chains such as dodecyl sulphate or tetra decyl sulphate.

9. Method according to one or more of the above claims, wherein the gradient of surfactant solvent goes from c=0 to CMC.

10. Method according to one or more of the above claims, wherein the fractions in d) are collected in wells or tubes provided with dried gel in which the fractions are allowed to swell.

11. Method according to claim 10, wherein the gel is selected from a dextran based gel such as Sephadex, or polyvinylalcohol or polyacrylamide.

12. Method according to claim 11 , wherein the proteins are precipitated in polyacrylamide gel and the gel is washed.

13. Method according to one or more of the above claims, wherein the proteins are digested after step d).

14. Method according to claim 13, wherein the proteins are loaded onto an RPC column after the digestion.

15. Method according to claim 13 or 14, wherein the digested protein are subjected to

MS/MS.

16. Method according to one or more of the above claims, wherein the protein sample(s) are labelled with one or more labelling reagent(s) before step a).

17. A kit comprising a hydrophobic media column, a surfactant and means for generating a surfactant gradient.

18. A fraction collector, comprising receptacles, such as tubes or wells, provided with dried swellable hydrogel.

19. Fraction collection according to claim 18, wherein a hydrogel has been dried onto the tubes or wells in such a way to adhere the gel to the bottom and/or the walls or the tubes or wells.