GOVERNMENTAL RIGHTS

[0001 ] This invention was made with government support under

R01 NS065667 awarded by the National Institutes of Health. The government has certain rights in the invention.

CROSS REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the priority of US provisional application number 61 /804,731 , filed March 24, 2013, which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0003] The present invention encompasses methods for preparing a sample comprising amyloid beta for analysis. When the method is used to prepare a sample for analysis by mass spectrometry, the unique combination of steps comprising the invention results in several improvements over previous methods, including (1 ) an increase in signal, (2) significantly decreased background, (3) reduced signal suppression, (4) increased reproducibility, and (5) improved throughput.

BACKGROUND OF THE INVENTION

[0004] Sensitive, accurate and reproducible methods for analyzing amyloid beta in biological samples is an unmet need in the study of neurodegenerative diseases. To some degree, the analytical methods are constrained by the sensitivity of the detection devices used in applications like mass spectrometry. An alternative approach, however, may be to increase the sensitivity and accuracy of the existing methods by improving the process by which samples are prepared for analysis by downstream applications.

SUMMARY OF THE INVENTION

[0005] In one aspect, the present disclosure provides a method for purifying amyloid beta from a liquid sample. The method comprises: (a) contacting the sample with a complete protease inhibitor cocktail comprising about 0.2% polysorbate-20 and about 1 .2M urea; (b) contacting the sample from (a) with an epitope binding agent conjugated to a solid support for about 90 minutes to about 120 minutes at room temperature, wherein the epitope binding agent binds to an epitope on amyloid beta and forms an epitope binding agent:amyloid beta complex that is conjugated to the solid support; (c) performing a first wash step with a solution comprising about 1 .2M urea and about 0.2% polysorbate 20 in a phosphate buffered saline solution, and a second and third wash step with about a 25mM ammonium bicarbonate solution or about a 50mM triethyl ammonium bicarbonate solution, wherein the first was step is optionally repeated and wherein the solution is removed from the solid support and discarded at the end of each wash step; (d) contacting the epitope binding agent:amyloid beta complex conjugated to the solid support with about a 50mM triethyl ammonium bicarbonate solution at a pH of about 8.5 comprising about 2.5 ng to about 5 ng of a protease for about 16 hours at about 37°C; and (e) performing solid phase extraction on the solution from (d) to purify the amyloid beta peptides, whereby the extraction comprises a wash step with about 10% acetonitrile / about 10% trifluoroacetic acid solution or about a 0.05% trifluoroacetic acid solution and an elution step with about 60% acetonitrile / about 10% trifluoroacetic acid solution.

[0006] In another aspect, the present disclosure provides a method for purifying amyloid beta from a liquid sample. The method comprises: (a) contacting the sample with a complete protease inhibitor cocktail comprising about 0.2% polysorbate- 20 and about 1 .2M urea; (b) contacting the sample from (a) with an epitope binding agent conjugated to a solid support for about 90 minutes to about 120 minutes at room temperature, wherein the epitope binding agent binds to an epitope on amyloid beta and forms an epitope binding agent:amyloid beta complex that is conjugated to the solid support; (c) performing a first wash step with a solution comprising about 1 .2M urea and about 0.2% polysorbate 20 in a phosphate buffered saline solution, and a second and third wash step with about a 25mM ammonium bicarbonate solution or about a 50mM triethyl ammonium bicarbonate solution, wherein the first was step is optionally repeated and wherein the solution is removed from the solid support and discarded at the end of each wash step; (d) contacting the epitope binding agent:amyloid beta complex conjugated to the solid support with about a 50mM triethyl ammonium bicarbonate solution at a pH of about 8.5 comprising about 2.5 ng to about 5 ng of a protease for about 16 hours at about 37°C; and (e) performing solid phase extraction on the solution from (d) to purify the amyloid beta peptides, whereby the extraction comprises a wash step with about 10% acetonitrile / about 10% trifluoroacetic acid solution or about a 0.05% trifluoroacetic acid solution and an elution step with about 60% acetonitrile / about 10% trifluoroacetic acid solution; and (f) drying the eluate from (e) and

resuspending the dried sample in a volume of buffer suitable for mass spectrometry.

[0007] In still another aspect, the present disclosure provides a method for purifying amyloid beta from a liquid sample. The method comprises: (a) contacting the sample with a complete protease inhibitor cocktail comprising about 0.2% polysorbate- 20 and about 1 .2M urea; (b) contacting the sample from (a) with an epitope binding agent conjugated to a solid support for about 90 minutes at room temperature, wherein the epitope binding agent binds to an epitope on amyloid beta and forms an epitope binding agent:amyloid beta complex that is conjugated to the solid support; (c) performing a first and second wash step with a solution comprising about 1 .2M urea and about 0.2% polysorbate 20 in a phosphate buffered saline solution, and a third and fourth wash step with about a 50mM triethyl ammonium bicarbonate solution, wherein the solution is removed from the solid support and discarded at the end of each wash step; (d) contacting the epitope binding agent:amyloid beta complex conjugated to the solid support with about a 50mM triethyl ammonium bicarbonate solution at a pH of about 8.5 comprising about 2.5 ng of a protease for about 16 hours at about 37°C; and (e) performing solid phase extraction on the solution from (d) to purify the amyloid beta peptides, whereby the extraction comprises a wash step with about 10% acetonitrile / about 10% trifluoroacetic acid solution and an elution step with about 60% acetonitrile / about 10% trifluoroacetic acid solution.

BRIEF DESCRIPTION OF THE FIGURES

[0008] The application file contains at least one drawing executed in color. Copies of this patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [0009] FIG. 1 depicts a graph illustration of human plasma Αβ isoforms C ¹³ - leu labeling curves. Subjects were labeled by IV infusion for 60 min with 800 mg of C ¹³ - leucine. Plasma samples were prepared by IP/digestion protocol, then analyzed by LC/MS for C ¹³ -leu incorporation into Αβ isoforms.

DETAILED DESCRIPTION OF THE INVENTION

[0010] The present invention encompasses a method for preparing a sample comprising amyloid beta for analysis. Generally speaking, the method encompasses inhibiting proteases that may be present in the sample, purifying amyloid beta from other components in the sample, digesting the purified amyloid beta into discrete fragments, and separating purified amyloid beta fragments from the digestion reaction. Thus, in an aspect, the present invention encompasses a method for purifying amyloid beta fragments from a sample. The purified amyloid beta fragments may then be analyzed or used in any downstream application. When the method is used to prepare a sample for analysis by mass spectrometry, the Applicants have found that the unique combination of steps comprising the invention results in several improvements over previous methods, including (1 ) an increase in signal, (2) significantly decreased background, (3) reduced signal suppression, (4) increased reproducibility, and (5) improved throughput. Each aspect of the invention is described in further detail below.

A. Sample comprising amyloid beta

[001 1 ] Amyloid beta (also referred to as Αβ or Abeta) is a peptide that is processed from the amyloid precursor protein (APP). As used herein, the term "amyloid beta" refers to total amyloid beta (Αβ) protein, Αβ40, Αβ42, Αβ38, or another Αβ isoform. In some embodiments, a sample may comprise total amyloid beta. In other embodiments, a sample may comprise Αβ40. In still other embodiments, a sample may comprise Αβ42. In still other embodiments, a sample may comprise Αβ38. In yet other embodiments, a sample may comprise another Αβ isoform. In different embodiments, a sample may comprise at least two members of the group consisting of total amyloid beta (Αβ) protein, Αβ40, Αβ42, Αβ38, and another Αβ isoform. In alternative

embodiments, a sample may comprise at least three members of the group consisting of total amyloid beta (Αβ) protein, Αβ40, Αβ42, Αβ38, and another Αβ isoform. In other embodiments, a sample may comprise at least three members of the group consisting of total amyloid beta (Αβ) protein, Αβ40, Αβ42, Αβ38, and another Αβ isoform. In other embodiments, a sample may comprise at least four members of the group consisting of total amyloid beta (Αβ) protein, Αβ40, Αβ42, Αβ38, and another Αβ isoform. In other embodiments, a sample may comprise total amyloid beta (Αβ) protein, Αβ40, Αβ42, Αβ38, and another Αβ isoform.

[0012] A sample comprising amyloid beta may or may not be a biological sample. In some embodiments, a sample is not a biological sample. For example, the sample may be the end-product of a synthesis reaction performed in vitro to produce synthetic amyloid beta. Methods for synthesizing peptides are known in the art, and may include, but are not limited to, liquid phase synthesis and solid phase synthesis. In other embodiments, a sample is a biological sample.

[0013] As used herein, "biological sample" refers to a sample derived from a subject. Suitable subjects may include a human, a livestock animal, a companion animal, a lab animal, or a zoological animal. In one embodiment, a subject may be a rodent, e.g. a mouse, a rat, a guinea pig, etc. In another embodiment, a subject may be a livestock animal. Non-limiting examples of suitable livestock animals may include pigs, cows, horses, goats, sheep, llamas and alpacas. In yet another embodiment, a subject may be a companion animal. Non-limiting examples of companion animals may include pets such as dogs, cats, rabbits, and birds. In yet another embodiment, a subject may be a zoological animal. As used herein, a "zoological animal" refers to an animal that may be found in a zoo. Such animals may include non-human primates, large cats, wolves, and bears. In a preferred embodiment, a subject is human.

[0014] A method of the invention may be used with any of the numerous types of biological samples known in the art. Non-limiting examples may include tissue samples or bodily fluids. In some embodiments, a biological sample is a tissue sample such as a tissue biopsy. The tissue biopsy may be a brain biopsy, a spinal cord biopsy or a CNS microvascular biopsy. A sample may also be primary and/or transformed cell cultures derived from tissue from a subject. In embodiments where a biological sample is a tissue sample, or cells derived therefrom, the tissue may be processed into a cell extract using techniques that are well known to those with skill in the art, whereby the cell extract will comprise amyloid beta. In other embodiments, a sample may be a bodily fluid. Non-limiting examples of bodily fluids include cerebrospinal fluid, interstitial fluid, blood, serum, plasma, saliva, sputum, semen, tears, and urine. The fluid may be used "as is", the cellular components may be isolated from the fluid, or a protein faction may be isolated from the fluid using standard techniques. For example, a sample of cerebrospinal fluid may be fractionated into individual cellular components using techniques that are well known to those with skill in the art. In preferred embodiments, a sample may be cerebrospinal fluid, blood, plasma or serum. In some embodiments, a sample may be pre-processed. For instance, a sample may be concentrated, diluted, or extracted. Suitable extraction techniques may include detergents, acids, bases, organic solvents, or other methods known in the art.

[0015] As will be appreciated by a skilled artisan, the method of collecting a biological sample can and will vary depending upon the nature of the biological sample. Any of a variety of methods generally known in the art may be utilized to collect a biological sample. Generally speaking, the method preferably maintains the integrity of proteins comprising the sample, specifically amyloid beta, such that it can be accurately measured according to the method of the invention.

B. Method for preparing a sample

[0016] In an aspect, methods described herein may be used to prepare a sample comprising amyloid beta for analysis. Generally speaking, the method encompasses inhibiting proteases that may be present in the sample, purifying amyloid beta from other components in the sample, digesting purified amyloid beta into discrete fragments, and separating purified amyloid beta fragments from the digestion reaction. Thus, in another aspect, methods of the invention also provide means to purify amyloid beta fragments from a sample.

(i) Inhibiting proteases for analysis

[0017] Methods for inhibiting proteases that may be present in the sample comprise contacting the sample with a complete protease inhibitor cocktail. The phrase "complete protease inhibitor cocktail" is a term of art, and refers to a composition that inhibits a broad range of proteases. Complete protease inhibitor cocktails are available commercially or mixtures of proteases inhibitors may be prepared according to methods known in the art. A sample is contacted with a complete protease inhibitor cocktail under conditions suitable for complete protease inhibitor cocktail function. Conditions for complete protease inhibitor cocktail function, which may include, but are not limited to, duration, temperature, and amount are known to those of skill in the art. In preferred embodiments, the complete protease inhibitor cocktail comprises about 0.20%

polysorbate 20 and about 1 .20M urea. For example, the complete protease inhibitor cocktail may comprise about 0.05% to about 0.40% polysorbate 20 and about 1 .0M to about 1 .40M urea. In other examples, the complete protease inhibitor cocktail may comprise about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.10%, about 0.1 1 %, about 0.12%, about 0.13%, about 0.14%, about 0.15%, about 0.16%, about 0.17%, about 0.18%, about 0.19%, about 0.20%, about 0.21 %, about 0.22%, about 0.23%, about 0.24%, about 0.25%, about 0.26%, about 0.27%, about 0.28%, about 0.29%, about 0.30%, about 0.31 %, about 0.32%, about 0.33%, about 0.34%, about 0.35%, about 0.36%, about 0.37%, about 0.38%, about 0.39%, and about 0.40% polysorbate 20. In other examples, the complete protease inhibitor cocktail may comprise about 1 .00M, about 1 .01 M, about 1 .02M, about 1 .03M, about 1 .04M, about 1 .05M, about 1 .06M, about 1 .07M, about 1 .08M, about 1 .09M, about 1 .10M, about 1 .1 1 M, about 1 .12M, about 1 .13M, about 1 .14M, about 1 .15M, about 1 .16M, about 1 .17M, about 1 .18M, about 1 .19M, about 1 .20M, about 1 .21 M, about 1 .22 M, about 1 .23M, about 1 .24M, about 1 .25M, about 1 .26M, about 1 .27M, about 1 .28M, about 1 .29M, about 1 .30M, about 1 .31 M, about 1 .32M, about 1 .33M, about 1 .34M, about 1 .35M, about 1 .36M, about 1 .37M, about 1 .38M, about 1 .39M, or about 1 .40M urea.

(ii) Purifying amyloid beta from other components

[0018] Methods for purifying amyloid beta from other components in the sample are typically epitope binding agent-based methods. As used herein, the term "epitope binding agent" refers to an antibody, an aptamer, a nucleic acid, an

oligonucleic acid, an amino acid, a peptide, a polypeptide, a protein, a lipid, a

metabolite, a small molecule, or a fragment thereof that recognizes and is capable of binding to an epitope of amyloid beta. Suitable epitopes of amyloid beta include amino acids 1 -42 of amyloid beta. For example, an epitope of amyloid beta may comprise at least amino acids 1 -5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, or 35-42. In some embodiments, an epitope of amyloid beta may comprise amino acids on either side of the alpha-secretase cleavage site, thereby resulting in preferential purification of amyloid beta that has not been cleaved by alpha-secretase. In other embodiments, an epitope of amyloid beta is selected to simplify clean-up following proteolytic digestion as described below. In an exemplary embodiment, an epitope of amyloid beta may comprise SEQ ID NO:1 (HHQKLVFF; amino acids 13-20 of amyloid beta). In another exemplary embodiment, an epitope of amyloid beta may comprise SEQ ID NO:2

(HHQKLVFFAEDV; amino acids 13-24 of amyloid beta). In another exemplary embodiment, an epitope of amyloid beta may comprise SEQ ID NO:3

(HHQKLVFFAEDVGSNK; amino acids 13-28 of amyloid beta). In a preferred

embodiment, an epitope binding agent is an anti-amyloid beta antibody. Non-limiting examples of suitable antibodies include the HJ5.1 monoclonal antibody (Cirrito et al., Neuron 2008; 58(1 ):42-51 ). In another preferred embodiment, an epitope binding agent is an aptamer.

[0019] As used herein, the term "antibody" generally means a polypeptide or protein that recognizes and can bind to an epitope of an antigen. An antibody, as used herein, may be a complete antibody as understood in the art, i.e., consisting of two heavy chains and two light chains, or may be any antibody-like molecule that has an antigen binding region, and includes, but is not limited to, antibody fragments such as Fab', Fab, F(ab')2, single domain antibodies, Fv, and single chain Fv. The term antibody also refers to a polyclonal antibody, a monoclonal antibody, a chimeric antibody and a humanized antibody. The techniques for preparing and using various antibody-based constructs and fragments are well known in the art. Means for preparing and

characterizing antibodies are also well known in the art (See, e.g. Antibodies: A

Laboratory Manual, Cold Spring Harbor Laboratory, 1 988; herein incorporated by reference in its entirety).

[0020] As used herein, the term "aptamer" refers to a polynucleotide, generally a RNA or DNA that has a useful biological activity in terms of biochemical activity, molecular recognition or binding attributes. Usually, an aptamer has a molecular activity such as binging to a target molecule at a specific epitope (region). It is generally accepted that an aptamer, which is specific in it binding to a polypeptide, may be synthesized and/or identified by in vitro evolution methods. Means for preparing and characterizing aptamers, including by in vitro evolution methods, are well know in the art (See, e.g. US 7,939,313; herein incorporated by reference in its entirety).

[0021 ] In general, an epitope binding agent-based method for purifying amyloid beta from other components in the sample comprises contacting a sample comprising amyloid beta with an epitope binding agent under conditions effective to allow for formation of a complex between the epitope binding agent and amyloid beta. Epitope binding agent-based methods may occur in solution, or the epitope binding agent or sample may be immobilized on a solid surface. In a preferred embodiment, the epitope binding agent is immobilized on a solid surface. Non-limiting examples of suitable surfaces include microtitre plates, test tubes, beads, resins, and other polymers.

[0022] An epitope binding agent may be attached to the substrate in a wide variety of ways, as will be appreciated by those in the art. The epitope binding agent may either be synthesized first, with subsequent attachment to the substrate, or may be directly synthesized on the substrate. The substrate and the epitope binding agent may be derivatized with chemical functional groups for subsequent attachment of the two. For example, the substrate may be derivatized with a chemical functional group including, but not limited to, amino groups, carboxyl groups, oxo groups or thiol groups. Using these functional groups, the epitope binding agent may be attached directly using the functional groups or indirectly using linkers.

[0023] The epitope binding agent may also be attached to the substrate non- covalently. For example, a biotinylated epitope binding agent may be prepared, which may bind to surfaces covalently coated with streptavidin, resulting in attachment.

Alternatively, an epitope binding agent may be synthesized on the surface using techniques such as photopolymerization and photolithography. Additional methods of attaching epitope binding agents to solid surfaces and methods of synthesizing biomolecules on substrates are well known in the art, i.e. VLSI PS technology from Affymetrix (e.g., see U.S. Pat. No. 6,566,495, and Rockett and Dix, Xenobiotica

30(2):155-177, both of which are hereby incorporated by reference in their entirety).

[0024] Contacting the sample with an epitope binding agent under effective conditions for a period of time sufficient to allow formation of a complex generally involves adding the sample to the epitope binding agent composition and incubating the mixture for a period of time long enough for the epitope binding agent to bind to amyloid beta present in the sample and form a complex. In some embodiments, the sample and epitope binding agent are incubated for at least 30 minutes. For example, the sample and epitope binding agent may be incubated for at least 30 minutes, at least 60 minutes, at least 90 minutes, or at least 120 minutes. In a preferred embodiment, the sample and epitope binding agent may be incubated from about 90 minutes to about 120 minutes. In an exemplary embodiment, the sample and epitope binding agent are incubated for 90 minutes. In another exemplary embodiment, the sample and epitope binding agent are incubated for 120 minutes. Incubation may occur at room temperature (e.g. 20 to 25 °C) or higher. Generally, incubation times decrease at temperatures greater than room temperature.

[0025] After incubating the sample and epitope binding agent to form an epitope binding agent:amyloid beta complex, the complex is washed one or more times with a solution to remove unbound components of the sample. Each wash step is performed with fresh solution (i.e. after each wash step, the solution used to wash the complex is not re-used in the next wash step) comprising the same or different components as the previous wash step. In a preferred embodiment, a first wash step is performed with a solution comprising about 1 .2M urea and about 0.2% polysorbate-20 in a phosphate buffered saline solution, and a second and a third wash step is performed with about a 25mM ammonium bicarbonate (AmBiC) solution or about a 50mM triethyl ammonium bicarbonate (TEABC) solution, wherein the first step is optionally repeated. In another preferred embodiment, a first and a second wash step is performed with a solution comprising about 1 .2M urea and about 0.2% polysorbate-20 in a phosphate buffered saline solution, and a third and a fourth wash step is performed with about a 50mM triethyl ammonium bicarbonate (TEABC) solution. [0026] In embodiments where the first wash step is performed with a solution comprising about 0.20% polysorbate 20 and about 1 .20M urea, the solution may comprise about 0.05% to about 0.40% polysorbate 20 and about 1 .00M to about 1 .40M urea. For example, the first wash solution may comprise about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.10%, about 0.1 1 %, about 0.12%, about 0.13%, about 0.14%, about 0.15%, about 0.16%, about 0.17%, about 0.18%, about 0.19%, about 0.20%, about 0.21 %, about 0.22%, about 0.23%, about 0.24%, about 0.25%, about 0.26%, about 0.27%, about 0.28%, about 0.29%, about 0.30%, about 0.31 %, about 0.32%, about 0.33%, about 0.34%, about 0.35%, about 0.36%, about 0.37%, about 0.38%, about 0.39% , and about 0.40% polysorbate 20. In other examples, the first wash solution may comprise about 1 .00M, about 1 .01 M, about 1 .02M, about 1 .03M, about 1 .04M, about 1 .05M, about 1 .06M, about 1 .07M, about 1 .08M, about 1 .09M, about 1 .10M, about 1 .1 1 M, about 1 .12M, about 1 .13M, about 1 .14M, about 1 .15M, about 1 .16M, about 1 .17M, about 1 .18M, about 1 .19M, about 1 .20M, about 1 .21 M, about 1 .22M, about 1 .23M, about 1 .24M, about 1 .25M, about 1 .26M, about 1 .27M, about 1 .28M, about 1 .29M, about 1 .30M, about 1 .31 M, about 1 .32M, about 1 .33M, about 1 .34M, about 1 .35M, about 1 .36M, about 1 .37M, about 1 .38M, about 1 .39M, or about 1 .40M urea.

[0027] In embodiments where the second and third wash steps are performed with a solution comprising about 25mM AmBiC, the solution may comprise about 5mM to about 45 AmBiC. For example, the second and third wash solution may comprise about 5mM, about 6mM, about 7mM, about 8mM, about 9mM, about 10mM, about 1 1 mM, about 12mM, about 13mM, about 14mM, about 15mM, about 16mM, about 17mM, about 18mM, about 19mM, about 20mM, about 21 mM, about 22mM, about 23mM, about 24mM, about 25mM, about 26mM, about 27mM, about 28mM, about 29mM, about 30mM, about 31 mM, about 32mM, about 33mM, about 34mM, about 35mM, about 36mM, about 37mM, about 38mM, about 39mM , about 40mM, and about 45mM AmBiC. Outside of the above exemplary embodiments, a skilled artisan would appreciate that there may be other equivalents, that while functional, are not preferred.

[0028] In embodiments where the second and third wash steps are performed with a solution comprising about 50mM TEABC, the solution may comprise about 30mM to about 70 TEABC. For example, the second and third wash solution may comprise about 30mM, about 31 mM, about 32mM, about 33mM, about 34mM, about 35mM, about 36mM, about 37mM, about 38mM, about 39mM, about 40mM, about 41 mM, about 42mM, about 43mM, about 44mM, about 45mM, about 46mM, about 47mM, about 48mM, about 49mM, about 50mM, about 51 mM, about 52mM, about 53mM, about 54mM, about 55mM, about 56mM, about 57mM, about 58mM, about 59mM, about 60mM, about 61 mM, about 62mM, about 63mM, about 64mM, about 65mM, and about 70mM AmBiC. Outside of the above exemplary embodiments, a skilled artisan would appreciate that there may be other equivalents, that while functional, are not preferred.

[0029] In embodiments where the first and second wash step is performed with a solution comprising about 0.20% polysorbate 20 and about 1 .20M urea, the solution may comprise about 0.05% to about 0.40% polysorbate 20 and about 1 .00M to about 1 .40M urea. For example, the first and second wash solution may comprise about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.10%, about 0.1 1 %, about 0.12%, about 0.13%, about 0.14%, about 0.15%, about 0.16%, about 0.17%, about 0.18%, about 0.19%, about 0.20%, about 0.21 %, about 0.22%, about 0.23%, about 0.24%, about 0.25%, about 0.26%, about 0.27%, about 0.28%, about 0.29%, about 0.30%, about 0.31 %, about 0.32%, about 0.33%, about 0.34%, about 0.35%, about 0.36%, about 0.37%, about 0.38%, about 0.39% , and about 0.40% polysorbate 20. In other examples, the first and second wash solution may comprise about 1 .00M, about 1 .01 M, about 1 .02M, about 1 .03M, about 1 .04M, about 1 .05M, about 1 .06M, about 1 .07M, about 1 .08M, about 1 .09M, about 1 .10M, about 1 .1 1 M, about 1 .12M, about 1 .13M, about 1 .14M, about 1 .15M, about 1 .16M, about 1 .17M, about 1 .18M, about 1 .19M, about 1 .20M, about 1 .21 M, about 1 .22M, about 1 .23M, about 1 .24M, about 1 .25M, about 1 .26M, about 1 .27M, about 1 .28M, about 1 .29M, about 1 .30M, about 1 .31 M, about 1 .32M, about 1 .33M, about 1 .34M, about 1 .35M, about 1 .36M, about 1 .37M, about 1 .38M, about 1 .39M, or about 1 .40M urea.

[0030] In embodiments where the third and fourth wash steps are performed with a solution comprising about 50mM TEABC, the solution may comprise about 30mM to about 70 TEABC. For example, the third and fourth wash solution may comprise about 30mM, about 31 mM, about 32mM, about 33mM, about 34mM, about 35mM, about 36mM, about 37mM, about 38mM, about 39mM, about 40mM, about 41 mM, about 42mM, about 43mM, about 44mM, about 45mM, about 46mM, about 47mM, about 48mM, about 49mM, about 50mM, about 51 mM, about 52mM, about 53mM, about 54mM, about 55mM, about 56mM, about 57mM, about 58mM, about 59mM, about 60mM, about 61 mM, about 62mM, about 63mM, about 64mM, about 65mM, and about 70mM AmBiC. Outside of the above exemplary embodiments, a skilled artisan would appreciate that there may be other equivalents, that while functional, are not preferred.

[0031 ] After the wash steps, purified amyloid beta may be eluted from the complex using a suitable eluent. After elution, the amyloid beta may be digested, or, in some alternative embodiments, digestion of amyloid beta may occur while amyloid beta is bound to an epitope binding agent.

(Hi) Digesting amyloid beta

[0032] Digesting amyloid beta into discrete fragments is useful in many downstream applications, particularly in mass spectrometry based analyses. For example, when amyloid beta is labeled with ¹³ C-leucine, such as in stable isotope labeling kinetic (SILK) assays, a C-terminal amyloid beta fragment can be used to discriminate between amyloid beta isoforms (for further details, see Bateman RJ et al J Am Soc Mass Spectrom (2007) 18(6): 997-1006). As used herein, the term "amyloid beta fragments" refers to the peptide fragments of amyloid beta produced by in vitro proteolytic digestion according to a method of the invention. Amyloid beta fragments are distinct from amyloid beta isoforms, though an amyloid beta fragment may comprise the same number and sequence of amino acids as an amyloid beta isoform.

[0033] Methods for digesting amyloid beta are known in the art. In brief, a solution comprising amyloid beta is contacted with a protease at a defined temperature for a specified amount of time, wherein the protease is selected from the group consisting of a serine protease, a threonine protease, a cysteine protease, an aspartate protease, a glutamic protease, and a metalloprotease. Typically a protease is selected based on the intended downstream application. For example, when a method of the invention is used to prepare a sample for analysis by mass spectrometry, non-limiting examples of suitable proteases may include trypsin, pepsin, and LysN. Other suitable proteases would result in the same or similar fragments as those listed above. In some embodiments, the protease is trypsin. In other embodiments, the protease is LysN.

[0034] As noted above, amyloid beta purified according to a method of the invention may be eluted from the epitope binding agent:amyloid beta complex and digested, or digestion of amyloid beta may occur while amyloid beta is bound to the epitope binding agent. Thus, a solution comprising amyloid beta to be contacted with a protease may comprise purified amyloid beta or purified epitope binding agent:amyloid beta complex suspended in suitable buffer. A non-limiting example of a suitable buffer includes 50mM triethyl ammonium bicarbonate buffer at pH 8.5 (TEABC, pH 8.5).

Another non-limiting example of a suitable buffer includes phosphate buffered saline (pH 7.2 to 7.4). In preferred embodiments the pH range of a buffer is between 7 and 9. Stated another way, in preferred embodiments, the pH range of the buffer is about 7.0, 7.1 , 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1 , 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.9, or 9.0.

[0035] In a preferred embodiment, amyloid beta is digested while bound to an epitope binding agent immobilized to a solid support. Digestion of amyloid beta while bound to an epitope binding agent immobilized to a solid support can save considerable time and decrease loss of material provided the correct epitope binding agent and protease are selected. Solid support digestion produced higher Αβ42 C ¹² -signal compared to digestion in solution. Following LysN cleavage, Αβ 28-38/40/42 forms may be detected by MS. LysN produces peptides one amino acid (lysine) longer than trypsin in this system. The lysine present on the longer Αβ peptide fragments results in better MS signal. In one embodiment, Αβ 16-27 is used to detect Αβ total signal. Other Αβ peptide fragments in the solution should not interfere with LC/MS detection of the Αβ isoforms of interest.

[0036] A skilled artisan will appreciate that temperature, time and protease concentration are three variables that may be manipulated in order to change the efficiency of digestion.

[0037] The amount of enzyme may be increased or decreased proportionally to the amount of amyloid beta to be digested. In embodiments where the protease is LysN, a suitable amount of LysN may be about 0.5ng to about 50 ng of LysN per reaction. For example, a suitable amount of LysN may be about 0.5, about 1 .0, about 1 .5, about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, about 8.5, about 9.0, about 9.5, about 10.0, about 10.5, about 1 1 .0, about 1 1 .5, about 12.0, about 12.5, about 13.0, about 13.5, about 14.0, about 14.5, about 15.0, about 15.5, about 16.0, about 16.5, about 17.0, about 17.5, about 18.0, about 18.5, about 19.0, about 19.5, about 20.0, about 20.5, about 21 .0, about 21 .5, about 22.0, about 22.5, about 23.0, about 23.5, about 24.0, about 24.5, about 25.0, about 25.5, about 26.0, about 26.5, about 27.0, about 27.5, about 28.0, about 29.5, about 30.0, about 30.5, about 31 .0, about 31 .5, about 32.0, about 32.5, about 33.0, about 33.5, about 34.0, about 34.5, about 35.0, about 35.5, about 36.0, about 36.5, about 37.0, about 37.5, about 38.0, about 39.5, or about 40.0 ng of LysN per reaction. In preferred embodiments, the amount of LysN used is the amount which produces the highest MS Αβ42 C ¹² -signal. In an exemplary embodiment, a suitable amount of LysN may be about 0.5ng to about 5ng. In another exemplary embodiment, a suitable amount of LysN may be about 1 to about 10ng. In yet another exemplary embodiment, a suitable amount of LysN may be about 2.5 ng to about 5 ng of LysN. In still yet another exemplary embodiment, a suitable amount of LysN may be about 2.5 ng of LysN.

[0038] Temperature and time are generally inversely related, such that as the temperature increases, the amount of time required to achieve satisfactory digestion decreases. In an exemplary embodiment, digestion may occur for about 10 to about 20 hours at 37 °C. For example, digestion may occur for about 10, about 1 1 , about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 hours at 37 °C. In another exemplary embodiment, digestion may occur for about 10 to about 15 hours at 37 °C. In an exemplary embodiment, digestion may occur for about 15 to about 20 hours at 37 °C. In an exemplary embodiment, digestion may occur for about 13 to about 18 hours at 37 °C.

(iv) Separating amyloid beta fragments from the digestion reaction [0039] Typically, amyloid beta fragments are separated from other components of the digestion reaction by solid phase extraction. Given that the methods of the invention are typically performed on a small scale, microscale solid phase extraction processes known in the art are particularly useful. Briefly, solid phase extraction uses the affinity of components dissolved or suspended in a liquid (known as the mobile phase) for a solid through which the sample is passed (known as the solid phase) to separate a mixture into desired and undesired components. Solid phase extraction cartridges and tips are commercially available, or may be prepared according to methods known in the art. In some embodiments, the solid phase is C18. In other embodiments, the solid phase may be C6, C8, carbon, SPE, or other suitable resins. In these embodiments, the portion retained on the solid phase includes the desired amyloid beta fragments, and the portion that passes through the solid phase is discarded. In certain embodiments, following digestion, the samples are centrifuged and the supernatant fluid above the epitope binding agent immobilized to a solid support may be removed and placed onto the top of the solid phase resin.

[0040] The amyloid beta fragments retained on the solid phase may be optionally washed one or more times to remove additional contaminants prior to removal from the solid phase. In a preferred embodiment, the wash solution is about 0.05% trifluoroacetic acid (TFA). In another preferred embodiment, the wash solution is about 10% trifluoroacetic acid (TFA) / about 10% acetonitrile. However, about 0.025% to about 15% TFA may be sufficient. For instance, about 0.05, 0.1 , 0.5, 1 .0, 1 .5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 1 1 , 1 1 .5, 12, 12.5, 13, 13.5, 14, 14.5, or 15% may be used. In addition, the wash solution may comprise other components, such as acetonitrile. In some embodiments, the wash solution may comprise between about 0.05% and 15% acetonitrile. For instance, about 0.05, 0.1 , 0.5, 1 .0, 1 .5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 1 1 , 1 1 .5, 12, 12.5, 13, 13.5, 14, 14.5, or 15% may be used.

[0041 ] A final rinse of the solid phase with a suitable eluent will then remove the amyloid beta fragments from the solid phase. Such eluents are known in the art. In a preferred embodiment, the eluent is a solution of about 60% acetonitrile / about 10% TFA. [0042] The eluate comprising the purified amyloid beta fragments may then be dried and resuspended in a buffer suitable for downstream applications. Suitable drying methods are known in the art and may include, but are not limited to, evaporation in a centrifugal vacuum concentrator (e.g. Thermo SpeedVac, Genevac) and

lyophilization.

[0043] In some embodiments, the downstream application is mass

spectrometry. Suitable solvent solutions for liquid chromatography/mass spectrometry are known in the art. Non-limiting examples may include 10% formic acid/20 % methanol. In other embodiments, the downstream application may be an antibody based detection method such as ELISA, AlphaScreen, xMAP or similar methodologies.

(B) Preferred embodiments

[0044] In a preferred embodiment, a method for purifying amyloid beta from a liquid sample comprises: (a) contacting the sample with a complete protease inhibitor cocktail comprising about 0.2% polysorbate-20 and about 1 .2M urea; (b) contacting the sample from (a) with an epitope binding agent conjugated to a solid support for about 90 minutes to about 120 minutes at room temperature, wherein the epitope binding agent binds to an epitope on amyloid beta and forms an epitope binding agent:amyloid beta complex that is conjugated to the solid support; (c) performing a first wash step with a solution comprising about 1 .2M urea and about 0.2% polysorbate 20 in a phosphate buffered saline solution, and a second and third wash step with about a 25mM ammonium bicarbonate solution or about a 50mM triethyl ammonium bicarbonate solution, wherein the first was step is optionally repeated and wherein the solution is removed from the solid support and discarded at the end of each wash step; (d) contacting the epitope binding agent:amyloid beta complex conjugated to the solid support with about a 50mM triethyl ammonium bicarbonate solution at a pH of about 8.5 comprising about 2.5 ng to about 5 ng of a protease for about 16 hours at about 37°C; and (e) performing solid phase extraction on the solution from (d) to purify the amyloid beta peptides, whereby the extraction comprises a wash step with about 10%

acetonitrile / about 10% trifluoroacetic acid solution or about a 0.05% trifluoroacetic acid solution and an elution step with about 60% acetonitrile / about 10% trifluoroacetic acid solution.

[0045] In another preferred embodiment, a method for purifying amyloid beta from a liquid sample comprises: (a) contacting the sample with a complete protease inhibitor cocktail comprising about 0.2% polysorbate-20 and about 1 .2M urea; (b) contacting the sample from (a) with an epitope binding agent conjugated to a solid support for about 90 minutes to about 120 minutes at room temperature, wherein the epitope binding agent binds to an epitope on amyloid beta and forms an epitope binding agent:amyloid beta complex that is conjugated to the solid support; (c) performing a first wash step with a solution comprising about 1 .2M urea and about 0.2% polysorbate 20 in a phosphate buffered saline solution, and a second and third wash step with about a 25mM ammonium bicarbonate solution or about a 50mM triethyl ammonium

bicarbonate solution, wherein the first was step is optionally repeated and wherein the solution is removed from the solid support and discarded at the end of each wash step; (d) contacting the epitope binding agent:amyloid beta complex conjugated to the solid support with about a 50mM triethyl ammonium bicarbonate solution at a pH of about 8.5 comprising about 2.5 ng to about 5 ng of a protease for about 16 hours at about 37°C; and (e) performing solid phase extraction on the solution from (d) to purify the amyloid beta peptides, whereby the extraction comprises a wash step with about 10%

acetonitrile / about 10% trifluoroacetic acid solution or about a 0.05% trifluoroacetic acid solution and an elution step with about 60% acetonitrile / about 10% trifluoroacetic acid solution; and (f) drying the eluate from (e) and resuspending the dried sample in a volume of buffer suitable for mass spectrometry.

[0046] In still another preferred embodiment, a method for purifying amyloid beta from a liquid sample comprises: (a) contacting the sample with a complete protease inhibitor cocktail comprising about 0.2% polysorbate-20 and about 1 .2M urea; (b) contacting the sample from (a) with an epitope binding agent conjugated to a solid support for about 90 minutes at room temperature, wherein the epitope binding agent binds to an epitope on amyloid beta and forms an epitope binding agent:amyloid beta complex that is conjugated to the solid support; (c) performing a first and second wash step with a solution comprising about 1 .2M urea and about 0.2% polysorbate 20 in a phosphate buffered saline solution, and a third and fourth wash step with about a 50mM triethyl ammonium bicarbonate solution, wherein the solution is removed from the solid support and discarded at the end of each wash step; (d) contacting the epitope binding agent:amyloid beta complex conjugated to the solid support with about a 50mM triethyl ammonium bicarbonate solution at a pH of about 8.5 comprising about 2.5 ng of a protease for about 16 hours at about 37°C; and (e) performing solid phase extraction on the solution from (d) to purify the amyloid beta peptides, whereby the extraction comprises a wash step with about 10% acetonitrile / about 10% trifluoroacetic acid solution and an elution step with about 60% acetonitrile / about 10% trifluoroacetic acid solution.

[0047] In still yet another preferred embodiment, a method for purifying amyloid beta from a liquid sample comprises: (a) contacting the sample with a complete protease inhibitor cocktail comprising about 0.2% polysorbate-20 and about 1 .2M urea; (b) contacting the sample from (a) with an epitope binding agent conjugated to a solid support for about 90 minutes at room temperature, wherein the epitope binding agent binds to an epitope on amyloid beta and forms an epitope binding agent:amyloid beta complex that is conjugated to the solid support; (c) performing a first and second wash step with a solution comprising about 1 .2M urea and about 0.2% polysorbate 20 in a phosphate buffered saline solution, and a third and fourth wash step with about a 50mM triethyl ammonium bicarbonate solution, wherein the solution is removed from the solid support and discarded at the end of each wash step; (d) contacting the epitope binding agent:amyloid beta complex conjugated to the solid support with about a 50mM triethyl ammonium bicarbonate solution at a pH of about 8.5 comprising about 2.5 ng of a protease for about 16 hours at about 37°C; and (e) performing solid phase extraction on the solution from (d) to purify the amyloid beta peptides, whereby the extraction comprises a wash step with about 10% acetonitrile / about 10% trifluoroacetic acid solution and an elution step with about 60% acetonitrile / about 10% trifluoroacetic acid solution; and (f) drying the eluate from (e) and resuspending the dried sample in a volume of buffer suitable for mass spectrometry. EXAMPLES

[0048] The following examples illustrate various iterations of the invention.

Example 1. Sample processing for LC/MS.

[0049] Blood and CSF samples obtained from human subjects were processed as follows:

[0050] 1 . Pre-treatment of sample with complete protease inhibitor cocktail (Roche) plus 0.2% Tween-20 and 1 .2M urea.

[0051 ] 2. Immunoprecipitation with HJ5.1 (recognizes amyloid beta amino acids 13-28) (30 μΙ slurry) - 2 hours at room temperature. HJ5.1 antibody was conjugated to GE Healthcare CNBr-activated Sepharose 4B beads according to the manufacturer's protocol. Following conjugation, the beads were washed with phosphate buffered saline plus 0.02% sodium azide to produce a bead slurry of equal parts packed beads and buffer. For use, 30 μΙ of the bead slurry was added to each tube for the IP.

[0052] 3. Wash: 1 x with 1 .2M urea/0.2% Tween-20 in phosphate buffered saline and 2x with 25mM AmBiC.

[0053] 4. Add: 20 μΙ of 50 mM triethyl ammonium bicarbonate buffer (TEABC, pH 8.5).

[0054] 5. Digest: 5 ng of LysN (in 10 μΙ of TEABC) at 37°C for 16 hours.

[0055] 6. Cleanup: TopTip200 - 0.05% trifluoroacetic acid (TFA) to wash, 60% acetonitrile / TFA to elute

[0056] 7. Dry and resuspend: 10% formic acid/ 20% methanol (20 μΙ)

[0057] 8. Separation /detection: nano-liquid chromatography / mass spectrometer (lc/ms); Waters Acquity /Xevo TQ-S - inject 5 μΙ.

Example 2. Sample processing for LC/MS.

[0058] Blood and CSF samples obtained from human subjects were processed as follows:

[0059] 1 . Pre-treatment of sample with complete protease inhibitor cocktail (Roche) plus 0.2% Tween-20 and 1 .2M urea.

[0060] 2. Immunoprecipitation with HJ5.1 (recognizes amyloid beta amino acids 13-28) (30 μΙ slurry) - 90 minutes at room temperature. HJ5.1 antibody was conjugated to GE Healthcare CNBr-activated Sepharose 4B beads according to the manufacturer's protocol. Following conjugation, the beads were washed with phosphate buffered saline plus 0.02% sodium azide to produce a bead slurry of equal parts packed beads and buffer. For use, 30 μΙ of the bead slurry was added to each tube for the IP.

[0061 ] 3. Wash: 2x with 1 .2M urea/0.2% Tween-20 in Pierce 37573 (Protein- Free T20 (PBS) Blocking Buffer) and 2x with 50mM triethyl ammonium bicarbonate buffer (TEABC).

[0062] 4. Add: 20 μΙ of 50 mM triethyl ammonium bicarbonate buffer (TEABC, pH 8.5).

[0063] 5. Digest: 2.5 ng of LysN (in 20 μΙ of TEABC) at 37°C for 16 hours.

[0064] 6. Cleanup: TopTip200 - 60% acetonitrile / 0.05% trifluoroacetic acid (TFA) to wet; 0% acetonitrile / 0.05% TFA to equilibrate; load sample; 10% acetonitrile / 10% TFA to wash; 60% acetonitrile / 10% TFA to elute

[0065] 7. Dry and resuspend: 10% acetonitrile / 5% methanol / 0.1 % formic acid (20 μΙ) at 37°C for 10 minutes.

[0066] 8. Centrifuge 25,000 xg for 10 minutes and transfer to mass spec vials.

[0067] 9. Separation / detection: nano-liquid chromatography / mass spectrometery (nano-lc/ms); Waters Acquity / Xevo TQ-S - inject 5 μΙ into HSS T3 column.

Example 3. Protocol optimization

[0068] The protocol of Example 1 and Example 2 works equally well for all biological fluids tested to date including: CSF, plasma, media and buffered solutions of Αβ (surrogate/artificial CSF and plasma). The same increase in Αβ42-Ο ¹² signal occurred.

[0069] The development of the protocol was driven by Αβ42-Ο ¹² signal. This isoform has the lowest concentration of the four isoforms studied by the inventors.

[0070] Total Αβ concentration is 50-fold less in human plasma (200 pg/ml) than CSF (10,000 pg/ml). Total protein (all proteins) is 60-fold greater in plasma (60 mg/ml) versus CSF (1 mg/ml). On top of that, Αβ42 is 10% of the total or 20 pg/ml in plasma. Thus, plasma Αβ as a fraction of total protein is 3000-fold lower than that in CSF. As a result, purification followed by detection of plasma Αβ42 is a significant challenge.

[0071 ] Improvements are shown in Table 1 below:

[0072] Before the optimization was started, Αβ isoforms could not be detected from the plasma IP samples because the samples would clog the nano-lc columns. Once TopTip cleanup was added to the protocol, Αβ isoform signal was detected. That was the starting point for the plasma Αβ protocol development.

[0073] With the Αβ42-ΰ ¹² signal of 108,000, we can clearly detect C ¹³ labeling at <1 %. 1 % label would be a calculated Αβ42-ΰ ¹³ signal of 1 ,080 which was 30-fold above the background of 35.

[0074] The signal/noise ratio was increase by >1000 fold through a

combination of both significantly increased signal plus lower background.

[0075] See Fig. 1 for a full labeling curve of human plasma using the current protocol.

[0076] Table 2 outlines the conditions and combinations that were explored.

IP Dilution of HJ5.1 beads

Time and temperature

Washes Chaotropic agents: urea, guanidine, guanidine isothicyanate at multiple concentrations

Detergents: Tween20 and SDS at multiple concentrations

Multiple combinations of chaotropic agents and detergents

Phosphate buffered saline (pH 7.2)

Acidic and basic buffers - pH range 6 to 9

Organic solvents: methanol, ethanol, acetonitrile at multiple concentrations

Digestion On/Bead versus Off/Bead

Amount of enzyme (LysN)

Time and temperature

Combination of LysN concentration, time and temperature

Ammonium bicarbonate solution

TEABC buffer

Phosphate buffered saline (pH 7.4)

Cleanup TopTip200 C18

Acetonitrile at multiple concentrations

Acids: formic and trifluoroacetic at multiple concentrations

Organic solvents: methanol and ethanol at multiple concentrations

Multiple combinations of acetonitrile concentrations, acids and organic solvents

Alternative cleanup tips: at least twelve were tested

Solid phase extraction (SPE) with varied conditions

Re-suspension Acetonitrile at multiple concentrations

solvent

Acids: formic and trifluoracetic at multiple concentrations

Organic solvents: methanol and ethanol at multiple concentrations

Multiple combinations of acetonitrile concentrations, acids and organic solvents

LC/MS C18 - multiple types

MS parameters