A method for proteomic analysis and a substrate prepared for the method

Abstract

The invention relates to a method for proteomic analysis and a substrate prepared for the method. The substrate is prepared with immobilized antibodies using the electrospray deposition method, where a planar conductive plate is used as the substrate, onto which ions of immunoglobulin-binding protein are deposited by electrospray, then at least one antibody with affinity for the target antigen is dispensed onto the substrate with immobilized immunoglobulin-binding protein, which is anchored via a non-covalent affinity interaction with the immobilized immunoglobulin-binding protein. Subsequently, a sample containing the antigen is dispensed onto the substrate with the immobilized immunoglobulin-binding protein and bound antibody, and the prepared substrate is incubated. After incubation of the substrate, sample residues are removed, an analyte with an antigen is chemically reduced or eluted from the substrate and cleaved with trypsin, an analyte with an antigen is desalted, and the sample is analysed using a proteomic method.

Description

[0001] A method for proteomic analysis and a substrate prepared for the method

[0002] Field of the Invention

[0003] The invention relates to the field of proteomics, specifically to a method for proteomic analysis and a substrate prepared for the method.

[0004] Background of the Invention

[0005] The enrichment of a target substance, sometimes referred to as preconcentration, emerged as a concept in analytical chemistry in the second half of the 20th century, when demands began to rise for the determination of substances in extremely low concentrations, for example in the materials industry, healthcare, biochemistry, and environmental analysis. Enrichment is a process in which a target analyte is captured by specific interaction on the surface of a solid carrier, while other substances present in the sample do not have this ability. After washing the surface, the proportion of the analyte present is relatively increased compared to other components that do not interact strongly enough with the surface and are washed away with a suitable solvent or buffer.

[0006] The analyte that remains on the surface is then released or eluted back into the solution, where it is in a much purer and relatively more concentrated form than in the original sample. This process facilitates subsequent analytical determination. If a low-concentration substance is enriched from the sample on a suitable surface, it can then be determined even if it was below the detection limit of the analytical method in the original sample (A. Mizuike, 'Separations and Preconcentrations', in Trace Analysis: Physical Methods (Ed.G. H. Morrison), Interscience: New York (1965), and J. M. Rottschafer, R. J. Boczkowski and H. B. Mark Jr, Taianta, 19, 163 (1972).

[0007] Current modern chemical analysis in biological and biomedical fields relies on analyte enrichment as one of the main tools for detecting substances in extremely low concentrations or substances whose presence is masked by interferences caused by the sample matrix. In proteomics, protein enrichment is an important technique for isolating proteins from cells, detecting post-translational modifications, determining protein structural elements, or searching for binding sites. A special case of enrichment using protein interactions is affinity interaction with antibodies, i.e. special binding proteins, sometimes also called immunoglobulins. Antibodies and antibodies-based reagents have a wide range of applications in biotechnology, from immunotherapy to affinity separation.

[0008] Affinity protein arrays are substrates to which antibodies (or other affinity proteins) are anchored in a defined geometric format, which is another application of protein and, above all, antibody affinity technologies. On defined substrates of protein arrays with anchored or immobilized antibodies, it is possible to enrich the analysed antigens that have a specific binding capacity in relation to the relevant antibody. In complex samples, such as blood or serum, it would often be impossible to detect the analysed antigens in the sample without some form of enrichment due to the excessive amount of other interfering components.

[0009] An important step in the preparation of protein array substrates is the method of immobilization, or anchoring, of the affinity protein molecule to the surface of the substrate. Common methods of immobilization are based on covalent, physisorption, or ionic binding of molecules to the surface (Rusmini et al. Protein Immobilization Strategies for Protein Biochips, Biomacromolecules 2007,8, 1775-1789). Other methods include immobilization by hydrogen bonding, as described in US 8148170, or coating the surface with a soft polymer, such as polydopamine, as described in JP7498757B2.

[0010] All these methods are based on adding a solution of the protein to be immobilized to the relevant surface and then chemically reacting the solvated proteins with the surface using a buffer. This is disadvantageous because it involves immobilizing a specific antibody for the detection of a specific antigen. Such a solution is limited to only one or a few antigens, and its use is not at all variable and universal for a larger number of antigens. Therefore, if it is necessary to carry out a proteomic analysis of various antigens from complex samples such as serum or blood, the overall proteomic analysis gets more and more expensive and timeconsuming. An alternative method of protein immobilization is electrospray deposition of desolvated protein ions from the gas phase, a process called ion soft landing, as described in document CZ 305 831. This is achieved by electrospraying protein solutions at atmospheric pressure in the presence of inert gas, followed by drying to form desolvated protein ions, which are brought to the surface of the carrier substrate by high voltage. The substrate modified in this way can be used for proteomic analysis but again encounters the problem of limited universality for a larger number of antigens where it cannot be used for a larger number of different antigens. Another limitation is the fact that the stability of different antibodies may vary and must be determined separately for each molecule in order to establish appropriate storage and handling protocols.

[0011] The object of the invention is therefore to create a method for proteomic analysis and a substrate with immobilized antibodies for proteomic analysis that would eliminate the above- mentioned shortcomings and be universally applicable to a wide range of antibodies selected by the user, thereby significantly simplifying logistics and the entire business model, as it would not be necessary to supply a specific substrate with a specific antibody for each antigen to the end user. In addition, the good stability of surface-anchored immunoglobulin-binding proteins allows for easy handling and storage of protein affinity arrays based on them, without the need to deal with the properties of each individual antibody.

[0012] Summary of the Invention

[0013] The stated object is solved by means of a proteomic analysis according to this invention. In this method, a substrate with immobilized antibodies is prepared by the electrospray deposition method.

[0014] The essence of the invention is that a. a planar conductive plate is used as the substrate, onto which ions of immunoglobulin- binding proteins are deposited by electrospray, b. at least one antibody with affinity for the target antigen is dispensed onto the substrate with immobilized immunoglobulin-binding protein, c. an antibody is anchored via a non-covalent affinity interaction with the immobilized immunoglobulin-binding protein, d. a sample containing the antigen is dispensed onto the substrate with the immobilized immunoglobulin-binding protein and bound antibody, and the prepared substrate is incubated, e. after incubation of the substrate, sample residues are removed, f. an analyte with an antigen is chemically reduced or eluted from the substrate and cleaved with trypsin, g. an analyte with an antigen is desalted, h. and the sample is then analysed using a proteomic method. The term “non-covalent affinity interaction” refers to the binding between the immunoglobulin- binding protein and the antibody. It is therefore a typical protein-protein interaction based on non-covalent forces.

[0015] The term “antibody” refers to a protein or immunoglobulin that is capable of specifically binding to an antigen. As part of the immune system, it is able to identify and neutralize foreign objects in the body.

[0016] The term “antigen” refers to a substance, usually a protein, from the external environment or produced in the body itself, which, due to its (potential) harmfulness, provokes the cells of the immune system to produce one or more antibodies. Each antibody binds to a specific antigen in a highly specific interaction analogous to a lock and key.

[0017] The method described above provides a sandwich structure of a substrate with immobilized antibodies, which consists of a primary layer of an affinity protein array on a planar conductive plate with immobilized immunoglobulin-binding protein specific for antibodies of the relevant subtype and a secondary affinity layer specific for the given antigen, consisting of bound antibodies intended for the detection of the target antigen, which is specific for the given antigen. The first or primary layer composed of immunoglobulin-binding proteins is prepared by depositing molecules of this immunoglobulin-binding protein directly onto the planar surface of the conductive plate using an ion soft-landing device or an electrospray deposition device. The device uses an electrospray, inert carrier gas, and heated chamber to convert the affinity molecule of immunoglobulin-binding protein from the solution into the form of desolvated ions. The desolvated ion created by electrospray ionization from the molecule of immunoglobulin- binding protein lands onto the surface of the substrate, its charge is discharged, and it is immobilized on the surface of the substrate in its neutral molecular form. An important advantage of this solution is the fact that the preparation of the planar surface of the substrate with the first layer of immunoglobulin-binding proteins bound to the planar surface of the substrate, or to the affinity plate, takes place at atmospheric pressure, in a short time (several minutes) and with high efficiency of transfer of the molecule of affinity protein to the surface of the substrate.

[0018] Immunoglobulin-binding proteins are arranged on the substrate into a protein array, for example, of rectangular geometry. The array thus consists of several positions; for example, for a geometry 8x12, there will be 96 positions, and for a geometry 4x12, there will be 48 positions. It is possible to use all positions of immunoglobulin-binding proteins on the substrate for a single antibody, in which case the resulting antibody array will be uniform, or to combine antibodies appropriately, i.e. to place different antibodies at different positions for the detection of multiple antigens on a single substrate.

[0019] The subject of the invention is also a substrate with immobilized antibodies for proteomic analysis in the manner described above. The essence of the invention is that the substrate comprises a planar conductive plate with immobilized immunoglobulin-binding protein, to which at least one antibody with affinity for the target antigen is bound.

[0020] In a preferred embodiment, protein G, protein A, or protein L is used as the immunoglobulin- binding protein, and therefore the substrate can also be referred to as a protein GAL array. These proteins are isolated from the cell wall of Staphylococcus aureus bacteria and are capable of binding various mammalian antibodies and are commercially available in recombinant form. Landed and immobilized immunoglobulin-binding proteins have different binding capacity, generally binding preferably IgG antibodies. Protein A is more specific for antibodies from rabbits, pigs, dogs, and cats. Protein G has higher specificity for human and mouse antibodies. Protein L binds the so-called “kappa light chain”, which is also found in antibodies other than IgG and therefore also binds to the IgM, IgA, IgE, and I g D subtypes.

[0021] A rectangular-shaped planar conductive plate is preferably used, which is made of metal or contains a layer of metal oxide or conductive plastic.

[0022] The advantages of the method for proteomic analysis and the substrate with immobilized antibodies according to this invention are mainly that it has universal application for a wide range of antibodies selected by the user, significantly simplifying logistics and the entire business model, as it is not necessary to supply a specific substrate with a specific antibody for each antigen to the end user. In addition, the good stability of surface-anchored immunoglobulin-binding proteins allows for easy handling and storage of protein affinity arrays based on them, without the need to deal with the properties of each individual antibody. Explanation of drawings

[0023] The present invention will be explained in detail by means of the following figures where:

[0024] Fig. 1 shows a view of the extracted ion chromatogram of the

[0025] IMNGEADAMSLDGGFVYIAGK peptide from the transferrin protein sequence,

[0026] Fig. 2 shows a view of the statistical evaluation of transferrin protein enrichment prior to analysis using LC-MS,

[0027] Fig. 3 shows a view of the extracted ion chromatogram (XIC) of the KPVEEYANCHLAR peptide from the transferrin protein sequence,

[0028] Fig. 4 shows a view of the extracted ion chromatogram (XIC) of the IDQTVEELR peptide from the apolipoprotein A-IV sequence.

[0029] Example of the invention embodiments

[0030] Example 1 - Enrichment of human haptoglobin protein using a substrate with protein G on ITO glass

[0031] A solution of protein G at the concentration of 1 pM in 150 mM ammonium acetate was electrosprayed, and the resulting ions of protein G were soft-landed on the glass surface of the planar conductive plate coated with indium tin oxide, sold as a thin film on glass under the name “ITO glass”. Soft landing of ions is a previously known procedure that leads to the immobilization of landed ions of immunoglobulin-binding protein, specifically protein G, onto the surface of the substrate.

[0032] The surface of the substrate prepared in this way has positions with immobilized protein G, which is arranged in a defined geometric shape, in this example in a geometry of 12x4 positions. This surface of the substrate was stored at 4°C and washed twice with deionized water and dried before use. Subsequently, 3pg of transferrin antibody in 3pL of PBS buffer (1XPBS, pH = 7.4) was dispensed onto each position. Only 3pL of pure PBS buffer was dispensed onto the control positions.

[0033] The substrate with immobilized protein G, i.e. with the protein array and the dispensed antibody or buffer solution, was then left to incubate at laboratory temperature in a humidity chamber. After incubation, the surface of the substrate was washed twice with PBS buffer and twice with deionized water and left to dry at room temperature. Next, a 2pL sample of serum was dispensed onto the positions of the protein array, and the surface with the sample of serum was incubated for one hour at room temperature in a humidity chamber. The surface was then rinsed three times with PBS buffer and three times with deionized water and left to dry at room temperature.

[0034] Each position in the protein array to be analysed was eluted twice with 2 pl of HPHEB elution buffer (0.5M ammonium hydroxide, 0.5M EDTA) into a clean Eppendorf tube. Next, 16 pl of cleavage buffer (50 mM ammonium bicarbonate, pH 7.8, 5% acetonitrile) supplemented with 10 mM tris(2-hydroxyethylphosphine (TCEP) and 40 mM chloroacetamide (CAA)) was added. The tubes were incubated for 5 to 10 minutes at 70°C. The reduction / alkylation reaction was stopped by adding 10 pl of cleavage buffer without TCEP / CAA. Finally, 1 pl of 20-times diluted trypsin in cleavage buffer was added to each tube. All tubes were incubated overnight at 37°C in a humidity chamber.

[0035] The samples were then desalted using C18-StageTips according to the manufacturer’s protocol and analysed by LC-MS. The results of this analysis showed enrichment of transferrin, which is the antigen of the antibody used, from blood serum at the substrate positions for which the anti-transferrin antibody was preprepared.

[0036] Fig. 1 shows the extracted ion chromatogram (XIC) of the IMNGEADAMSLDGGFVYIAGK peptide from the transferrin protein sequence in the sample, which was enriched using an affinity surface of the substrate with protein G with deposited anti -transferrin antibody (Fig. 1 , upper panel) and from the sample that was analysed without enrichment (Fig. 1 , lower panel). Fig. 1 demonstrates that the peptide formed by trypsin cleavage of transferrin was detected only when transferrin enrichment was used prior to LC-MS analysis. Otherwise, the peptide was not detected, as evidenced by the absence of a peak in the data record.

[0037] Fig. 2 shows a view of the statistical evaluation of transferrin enrichment prior to LC-MS analysis. The y-axis shows the logarithm of the probability of correct protein identification (- Log P), while the x-axis shows the significance of the difference between the experiment with enrichment and without enrichment. The red dot corresponding to the transferrin protein shows that the probability of detection of transferrin is highest when enrichment is used (value of difference +2). Other proteins (not highlighted grey dots) are distributed evenly around the centre of the graph (value of difference approximately 0) or have a higher probability of detection without enrichment (negative value on the x-axis).

[0038] Example 2 - Enrichment of human transferrin protein using a substrate with protein A on stainless steel and cleavage on the surface

[0039] A solution of protein A at the concentration of 1 pM in 150 mM ammonium acetate was electrosprayed, and the resulting ions of protein A were soft-landed on the surface of the planar conductive plate made of stainless steel. Soft landing of ions is a previously known procedure that leads to the immobilization of landed ions onto the surface of the substrate, which leads to the immobilization of landed ions of immunoglobulin-binding protein, specifically protein A, onto the surface of the substrate.

[0040] The surface of the substrate prepared in this way with the array of protein A in a geometry of 12x8 positions was stored at 4°C. The surface was washed twice with deionized water and dried before use. Subsequently, 3pg of transferrin antibody in 3pL of PBS buffer (1XPBS, pH = 7.4) was dispensed onto selected positions. Only 3pL of pure PBS buffer was dispensed onto the control positions.

[0041] The substrate with immobilized protein A, i.e. with the protein array and the dispensed antibody or buffer solution, was then left to incubate at laboratory temperature in a humidity chamber. After incubation, the surface was washed twice with PBS buffer and twice with deionized water and left to dry at room temperature. Next, a 2pL sample of serum was dispensed onto the positions of the protein array, and the surface with the sample of serum was incubated for one hour at room temperature in a humidity chamber. The surface was then rinsed three times with PBS buffer and three times with deionized water and left to dry at room temperature.

[0042] Each position in the protein array to be analysed was then subjected to trypsin cleavage directly on the surface. Then, 2pL of cleavage buffer with reducing agent (50 mM ammonium bicarbonate, pH 7.8, 5% acetonitrile, 10 mM tris(2-hydroxyethylphosphine (TCEP) and 40 mM chloroacetamide (CAA)) was deposited onto the surface for 20 minutes at 37°C. Trypsin was then added in the cleavage buffer, but without TCEP and CAA, and the surface was left overnight at 37°C in a humidity chamber. The cleaved peptides from each point of the protein array were then dissolved with formic acid, pipetted from the plate, and analysed by LC-MS. The results of this analysis showed enrichment of transferrin, which is the antigen of the antibody used, from blood serum at the positions on the array where the anti-transferrin antibody had been preprepared.

[0043] Fig. 3 shows a view of the extracted ion chromatogram (XIC) of the KPVEEYANCHLAR peptide from the transferrin protein sequence in the sample, which was enriched using an affinity surface with protein A with deposited anti-transferrin antibody (Fig. 3, upper panel) and from the sample that was analysed without enrichment (Fig. 3, lower panel). Fig. 3 demonstrates that the peptide formed by trypsin cleavage of transferrin was detected only when transferrin enrichment was used prior to LC-MS analysis. Otherwise, the peptide was not detected, as evidenced by the absence of a peak in the data record in Fig. 3.

[0044] Example 3 - Enrichment of human apolipoprotein A-IV protein using a substrate with protein L on conductive plastic

[0045] A solution of protein L at the concentration of 1 pM in 150 mM ammonium acetate was electrosprayed, and the resulting ions of protein L were soft-landed on the surface of the planar conductive plate made of conductive plastic. Soft landing of ions is a previously known procedure that leads to the immobilization of landed ions of immunoglobulin-binding protein, specifically protein L, onto the surface of the substrate.

[0046] The surface of the substrate prepared in this way with the array of protein L in a geometry of 10x10 positions was stored at 4°C. The surface was washed twice with deionized water and dried before use. Subsequently, 3pg of antibody against apolipoprotein A-IV in 3pL of PBS buffer (1XPBS, pH = 7.4) was dispensed onto each position. Only 3pL of pure PBS buffer was dispensed onto the control positions.

[0047] The substrate with immobilized protein L, i.e. with the protein array and the dispensed antibody or buffer solution, was then left to incubate at laboratory temperature in a humidity chamber. After incubation, the surface was washed twice with PBS buffer and twice with deionized water and left to dry at room temperature. Next, a 2 pL sample of serum was dispensed onto the positions of the protein array, and the surface with the sample of serum was incubated for one hour at room temperature in a humidity chamber. The surface was then rinsed three times with PBS buffer and three times with deionized water and left to dry at room temperature.

[0048] Each position in the protein array to be analysed was eluted twice with 2 pl of HPHEB elution buffer (0.5M ammonium hydroxide, 0.5M EDTA) into a clean Eppendorf tube. Next, 16 pl of cleavage buffer (50 mM ammonium bicarbonate, pH 7.8, 5% acetonitrile) supplemented with 10 mM tris(2-hydroxyethylphosphine) (TCEP) and 40 mM chloroacetamide (CAA) was added. The tubes were incubated for 5 to 10 minutes at 70°C. The reduction / alkylation reaction was stopped by adding 10 pl of cleavage buffer without TCEP / CAA. Finally, 1 pl of 20-times diluted trypsin in cleavage buffer was added to each tube. All tubes were incubated overnight at 37°C in a humidity chamber.

[0049] The samples were then desalted using C18-StageTips according to the manufacturer’s protocol and analysed by LC-MS. The results of this analysis showed enrichment of apolipoprotein A-IV, which is the antigen of the antibody used, from blood serum at the positions of the array for which the antibody against apolipoprotein A-IV was preprepared.

[0050] Fig. 4 shows a view of the extracted ion chromatogram (XIC) of the IDQTVEELR peptide from the apolipoprotein A-IV sequence in the sample, which was enriched using an affinity surface with protein L with deposited anti-apolipoprotein A-IV antibody (Fig. 4, upper panel) and from the sample that was analysed without enrichment (Fig. 4, lower panel). Fig. 4 demonstrates that the peptide formed by trypsin cleavage of apolipoprotein A-IV was detected only when apolipoprotein A-IV enrichment was used prior to LC-MS analysis. Otherwise, the peptide was not detected, as evidenced by the absence of a peak in the data record in Fig. 4.

[0051] Industrial applicability

[0052] The method for proteomic analysis and the substrate prepared for the method according to this invention can be used in particular in the isolation of antibodies from blood, plasma, or serum samples, as so-called serological tests, in searching for new antibodies in biological samples, preparing antibody arrays for targeted detection of specific antigens, searching for biomarkers, testing and classifying allergens, but also in various other applications depending on the problem being solved.

Claims

CLAIMS1. A method for proteomic analysis in which a substrate with immobilized antibodies is prepared by electrospray deposition method, characterized in that a. a planar conductive plate is used as the substrate, onto which immunoglobulin- binding protein ions land and are immobilized by electrospray deposition, b. at least one antibody with affinity for the target antigen is dispensed onto the substrate with immobilized immunoglobulin-binding protein, c. an antibody is anchored via a non-covalent affinity interaction with the immobilized immunoglobulin-binding protein, d. a sample containing the antigen is dispensed onto the substrate with the immobilized immunoglobulin-binding protein and bound antibody, and the prepared substrate is incubated, e. after incubation of the substrate, sample residues are removed, f. an analyte with an antigen is chemically reduced or eluted from the substrate and cleaved with trypsin, g. an analyte with an antigen is desalted, h. and the sample is then analysed using a proteomic method.

2. The method according to claim 1 , characterized in that protein G or protein A or protein L is used as the immunoglobulin-binding protein.

3. The method according to claim 1 or 2, characterized in that a rectangular planar conductive plate is used, which is made of metal or contains a layer of metal oxide or conductive plastic.

4. A substrate with immobilized antibodies for proteomic analysis according to any of claims 1 to 3, characterized in that it comprises a planar conductive plate with immobilized immunoglobulin-binding protein to which at least one antibody with affinity for the target antigen is bound.

5. The substrate according to claim 4, characterized in that the immunoglobulin-binding protein is selected from the group: protein G, protein A, protein L.

6. The substrate according to claim 4 or 5, characterized in that the planar conductive plate is rectangular in shape and is made of metal or contains a layer of metal oxide or conductive plastic.

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