Method for analysis of proteins

Field of the invention:

The present invention relates to a method for analysis of low abundant proteins by a combination of fluorescent labeling, affinity purification and electrophoresis. This method allows for accurate quantitative analysis of target proteins in complex samples and is less time-consuming compared to conventional analysis with Western blotting.

Background of the invention:

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/ non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are probed (detected) using antibodies specific to the target protein.

The proteins of the sample are separated using gel electrophoresis. Separation of proteins may be by isoelectric point (pi), molecular weight, electric charge, or a combination of these factors. It is also possible to use a 2-D electrophoresis gel which spreads the proteins from a single sample out in two dimensions. Proteins are separated according to isoelectric point (pH at which they have neutral net charge) in the first dimension, and according to their molecular weight in the second dimension.

In order to make the proteins accessible to antibody detection, they are moved from within the gel onto a membrane made of nitrocellulose or polyvinylidene difluoride (PVDF). The membrane is placed on top of the gel, and a stack of filter papers placed on top of that. The entire stack is placed in a buffer solution which moves up the paper by capillary action, bringing the proteins with it. Another method for transferring the proteins is called electroblotting and uses an electric current to pull proteins from the gel into the PVDF or nitrocellulose membrane. The proteins move from within the gel onto the membrane while maintaining the organization they had within the gel. As a result of this "blotting" process, the proteins are exposed on a thin surface layer for detection.

Since the membrane has been chosen for its ability to bind protein, and both antibodies and the target are proteins, steps must be taken to prevent interactions between the membrane and the antibody used for detection of the target protein. Blocking of non-specific binding is achieved by placing the membrane in a dilute solution of protein - typically Bovine serum albumin (BSA) or nonfat dry milk with a minute percentage of detergent such as Tween 20. During the detection process the membrane is "probed" for the protein of interest with a modified antibody which is linked to a reporter enzyme, which when exposed to an appropriate substrate drives a colourimetric reaction and produces a colour. For a variety of reasons, this traditionally takes place in a two-step process, although there are one-step detection methods available for certain applications.

After blocking, a dilute solution of primary antibody is incubated with the membrane under gentle agitation. After rinsing the membrane to remove unbound primary antibody, the membrane is exposed to another antibody, directed at a species-specific portion of the primary antibody. This is known as a secondary antibody. The secondary antibody is usually linked to biotin or to a reporter enzyme such as alkaline phosphatase or horseradish peroxidase. This means that several secondary antibodies will bind to one primary antibody and enhance the signal.

Chemiluminescent detection methods depend on incubation of the Western blot with a substrate that will luminesce when exposed to the reporter on the secondary antibody. The light is then detected by photographic film, and more recently by CCD cameras which captures a digital image of the western blot. The image is analysed by densitometry, which evaluates the relative amount of protein staining and quantifies the results in terms of optical density. Newer software allows further data analysis such as molecular weight analysis if appropriate standards are used.

The fluorescently labeled probe is excited by light and the emission of the excitation is then detected by a photosensor such as CCD camera equipped with appropriate emission filters which captures a digital image of the Western blot and allows further data analysis such as molecular weight analysis and a quantitative western blot analysis. Fluorescence is considered to be among the most sensitive detection methods for blotting analysis.

Western blotting is used extensively for isolation and detection of specific proteins. However, the method is time-consuming and improvements and/or alternative techniques are highly desirable.

Summary of the invention

The present invention provides a simple and fast alternative to Western blotting. According to a preferred embodiment of the invention affinity purification media is used to first bind the affinity ligands and subsequently bind the target proteins of interest. Labeling the proteins, either before or after the affinity enrichment, allow for accurate quantification of the isolated proteins. The invention enables multiplexing of target proteins using several fluorescent dyes. Furthermore, a complete overview of the labeled proteins after affinity purification can be obtained by comparative analysis of starting material, proteins non-bound and proteins bound to the affinity media. This enables accurate quantification and normalization. Thus, this method is very suitable for control of protein purification processes.

Thus, the invention relates to a method for analysis of target protein(s) or group of target proteins in a sample, comprising the steps a) enriching each target protein or group of proteins by binding said target protein(s) to an affinity adsorbent having affinity-binding ligands which are directed against said target protein(s) and are immobilized directly to the adsorbent or immobilized via further affinity ligand(s) to said adsorbent; b) desorbing said target protein(s) from said ligand(s); c) separation of the target protein(s), and d) detection of said target protein(s), wherein said target protein(s) are labeled before or after step a). The detection may for example be for comparative or quantitative purposes. Preferably the dyes are fluorescent or luminescent dyes which are size and charge matched and enable multiplex analysis, for example Cy-dyes™.

In one embodiment, the adsorbent has two different types of affinity ligands: one with affinity for target protein and the other with affinity for a sample endogenous internal standard or house- keeping protein later used for normalization; and wherein the target protein and internal standard/house-keeping protein are labeled with the same or different dyes.

In another embodiment, the target protein and internal standard/house-keeping are run on two separate adsorbents having different types of affinity ligands: one with affinity for target protein and the other with affinity for a sample endogenous internal standard or house-keeping protein later used for normalization, and wherein the target protein and internal standard/house-keeping protein are labeled with the same or different dyes.

Preferably, the affinity ligands are specific antibodies, preferably of IgG type, immobilized to said adsorbent via Protein A or G. The specific antibody will be the primary antibody used in conventional Western blotting.

In a preferred embodiment of the method an internal standard is labeled and separated with the target proteins in step c); and in a step d) target proteins signals are normalized to said labeled internal standard, such as protein standard (spike), endogenous house-keeping protein or starting material. The internal standard may be labeled with the same or a different dye compared with the target proteins. In a further preferred method the starting material, affinity non-bound proteins and target (= affinity bound) proteins are separated in step c), and in step d) comparative quantitative analysis of labeled proteins is performed between starting material, affinity non-bound proteins and bound proteins. The starting material, affinity non-bound proteins and target (= affinity bound) proteins may be labeled with the same or different dyes. If the same dyes are used, then the components are separated separate from each other, for example in different lanes on an electrophoretic gel.

Preferably the separation in step c) is ID or 2D electrophoresis but may also by other types of separation such as chromatography.

The affinity ligands may be antibodies or antibody fragments, Protein A, Protein G, substrate analogues, metal ions, carbohydrates, DNA or RNA, lectins, interacting protein (protein-protein interactions) or molecule with affinity for a target protein or group of target proteins. Preferably steps a) -c) are performed on a small scale, e.g. using magnetic bead based enrichment, spin-column, micro-well plates, capillary electrophoresis, or in small scale liquid chromatography columns, preferably the affinity adsorbent comprises magnetic beads.

It will be an enormous benefit to combine an immuno-affinity step and fluorescence detection and thereby avoid the need for transfer to a membrane, blocking and washing of a membrane. It is well known that Western blotting contains many steps which are sources of experimental variation affecting the reproducibility and robustness of analysis. The method according to the invention drastically reduces the experimental time and the number of steps involved. The consumption of affinity ligands is also reduced, which make the experiment more cost effective.

Brief description of the drawings:

Fig 1 Schematically shows an overview of the method according to the present invention;

Fig 2 shows two workflows (A and B) of the present invention; and

Fig 3 shows data from an experimental comparison between the method of the present invention (A) and conventional Western blotting (B). Detailed description of the invention

Affinity ligands, such as antibodies, are often expensive and available in small amounts. Therefore, it is important that the affinity purification step is optimized and performed on a small scale, e.g. using magnetic bead based enrichment, spin-column, micro-well plates, capillary electrophoresis, or in small scale liquid chromatography columns. In possible embodiments of the invention, Protein G or Protein A media is used to bind IgG types of antibody to produce a selective affinity media.

The protein may be labeled for fluorescence quantification with for example CyDyes. Labeling proteins before the separation has the advantage that multiplexing experiments, i.e. simultaneous analyses of several samples, are possible. If the labeled samples are-mixed and run on the same affinity column it will be possible to compare abundance levels of a specific protein compared to a standard sample. The use of an internal standard (e.g. spike proteins, pooled internal standard or endogenously expressed internal standard /house- keeping protein) permits linking of results from many sample separations, e.g. in a single micro-well plate up to 96 different samples can be quantified because the standard is present in each sample, and separated together with the sample in each well. Correction for variation in sample loading between-lane normalization and in-lane normalization relative quantification of protein of interest can be achieved by using the internal standards. Normalized target signals may be related to pre-labeled starting material and/or non- bound (supernatant magnetic beads or flow through) samples run in separate lanes (all labeled with the same dye). This relation will enable reliable quantitative information on variations of target protein % in starting material between different samples.

Absolute quantification is also possible if the signal of protein of interest is compared to signals from a purified known amount of target protein labeled and detected in parallel (in a standard curve). The final electrophoresis step confirms successful separation and size of the protein of interest and at the same time permit quantification relative to an internal standard using a fluorescence scanner and image analysis software.

A proposed workflow for separation and quantification of a specific protein is described in Fig. 1. This figure shows an overview of steps involved in new alternative method for specific protein analysis. Proteins in samples (1, 2 ...n) are labeled with Dyes (1, 2 ...n) either before (solid line) or after (dotted line) the enrichment step using affinity ligands (LI, L2 ...L _n ) and the proteins are analyzed by SDS PAGE and fluorescence detection. If the proteins are labelled before enrichment the samples can be mixed prior to enrichment in the same tube or column. When the proteins are labelled after enrichment the samples are enriched separately in different tubes or columns, labeled and analyzed by SDS PAGE and fluorescence detection. Multiplexing possibilities enable accurate normalization and relative quantification of specific proteins with comparable but improved results compared to traditional fluorescent Western blotting.

The target protein is enriched using immobilized antibodies and labeled with CyDyes. The samples are separated using ID SDS PAGE and the multiplex fluorescent signals are quantified

Two experimental workflow examples of the new alternative method for analysis of specific proteins are described in Fig 2. Workflow A shows labeling before enrichment. Sample 1 and 2 are pre- labeled with Dye 1 and Dye 2, respectively, followed by simultaneous enrichment on column or bead with immobilized affinity ligands for specific proteins. There may be 2 affinity ligands immobilized on the same column, one with affinity for specific protein of interest and the other with affinity for a sample endogenous internal standard or house-keeping protein later used for normalization

(compare traditional western blotting). After enrichment the samples are analyzed by SDS-PAGE and fluorescence detection. If Sample 1 and 2 are labeled with different dyes, the can be run in the same lane of the electrophoresis gel. If they are labeled with the same dye, the have to be run in different lanes.

In workflow B, the procedure is similar but the different samples are separately subjected to enrichment followed by labeling and analysis by fluorescent SDS-PAGE analysis. If Sample 1 and 2 are labeled with different dyes, the can be run in the same lane of the electrophoresis gel. If they are labeled with the same dye, the have to be run in different lanes.

For both procedure the starting (S), non-bound (NB) and eluate (E) material from the same sample can be run in separate lanes and the signals quantitatively compared.

Fig 3 shows data from an experimental comparison between the method of the present invention (A) and conventional Western blotting (B) with Cy 3 labeled enriched proteins and proteins targeted with ECL Plex Cy 5 using the same antibody. To summarize, the main advantages of this invention is that less primary antibody (usually expensive) is needed and the results are improved by better signal to noise. No time-consuming blotting is required for isolation and detection of a specific protein, and multiplexing is possible.

Materials and methods

The inventors have performed experiments to directly compare detection of low abundant proteins by traditional fluorescent Western blotting with the new approach involving protein immuno affinity enrichment by using immobilized antibodies coupled to magnetic Sepharose beads followed by CyDye pre-labeling. Enriched pTyr proteins were produced from a pre-cleared lysate of K562 cells in RIPA (Radio immunnoprecipitation assay buffer; Tris-HCI: 50 mM, 1% NP-40, 0.25% Na-deoxycholate, 150 mM NaCI, 1 mm EDTA, pH 7.4 + protease inhibitors) , diluted 1:3 with TBS (tris buffered saline) by using the anti-pTyr 4G10 antibody (Millipore, Billerica, MA, USA) coupled to magnetic Protein G Mag Sepharose™ beads (GE Healthcare, 320 g antibody to 40 μΙ_ beads). Incubation was performed overnight at + 4 °C. pTyr proteins were eluted from the beads using 100 mM phenylphosphate in PBS (phosphate buffered saline), concentrated and desalted in Amicon ultracentrifugation filters

(Millipore). Finally the buffer was exchanged to 2-DE labeling DIGE buffer using Viva Spin (MWCO 5 kDa, GE Healthcare). The enriched pTyr proteins were pre-labeled with Cy™ 3 minimal dye (GE Healthcare) and 0.25 g of labeled proteins were separated by ID SDS PAGE (8 x 7 cm 4-20% Tris- Glycine, 10 well, Novex, Invitrogen). The proteins were transferred onto a low-fluorescent membrane (Hybond LFP™, GE Healthcare). The membrane was blocked using a low-fluorescent blocking agent (2 % ECL Advance blocking agent (GE Healthcare) in TBS 0.1% Tween-20) and probed with ~ 4 g/mL anti-phosphotyrosine primary antibody (4G10, same antibody as for enrichment), and an ECL Plex™ Cy 5 secondary antibody (CyDye conjugated, GE Healthcare) diluted 1:2500, for detection of pTyr proteins. Multiplex ECL Plex antibody signals and CyDye pre-labeled protein signals were detected separately on the same membrane by using a fluorescent imager and different detection channels (Typhoon™ 9410 Imager, GE Healthcare) (Figure 3). Thus, we confirmed a very good agreement between enriched proteins and Western blotting signals, in spite of that the antibody was used at native conditions in the enrichment and at denaturating conditions in the Western blotting procedure. The detection and signal-to-noise ratio is improved (Figure 3 A) compared to traditional fluorescent Western blotting (Figure 3B). The results are comparable and improved and no information is lost by using this alternative approach for specific protein analysis. The Cy3 labeled enriched proteins gained a mass of ~ 0.5 kDa, which caused a visible shift towards a higher molecular weight for smaller proteins for the Cy 3 pre-labeled protein compared to the ECL Plex Cy 5 signals.

In one example, an enriched sample labeled with Cy3 was separated by ID SDS PAGE and transferred to a membrane. The Cy3 label is covalent and will transfer along with the protein onto the membrane. This membrane was then probed with the same anti- phospho-tyrosine primary antibody used in the enrichment procedure and targeted with ECL Plex Cy5. The results show that the same bands are detected using enrichment and Cy3 labeling as with ECL Plex Cy5 Western blotting. There is an improvement in signal to noise using this new method. For this antibody the detection and quantification is actually improved using this new method compared to Western blotting.