Methods for measuring concentrations of biomolecules in biofluids

Abstract

The present invention provides methods for measuring the absolute concentration of Tau, and other protein, peptide fragments and proteoforms in CSF and plasma samples collected from a subject. Such biomolecules may be implicated in one or more neurological and neurodegenerative diseases or disorders. Also provided is a method for determining whether a therapeutic agent affects the CSF or plasma concentration of a central nervous system derived biomolecule. Also provided are kits for performing the methods of the invention.

Claims

1. A method comprising: a) isolating unlabeled Tau protein from a sample; b) obtaining unlabeled Tau protein fragments from the unlabeled Tau protein; and c) measuring the amount of at least two unlabeled Tau protein fragments by relating the ratio of a stable isotope-labeled Tau protein fragments quantitation internal standard to each of the at least two unlabeled Tau protein fragments, wherein: i) one of the at least two unlabeled Tau protein fragments is selected from the group consisting of SEQ ID NOs: 54, 74, 92 and 102; and ii) another of the at least two unlabeled Tau protein fragments is selected from the group consisting of SEQ ID NOs: 49-53, 55-91, 93-101 and 103-106.

2. The method of claim 1, wherein the sample is a cerebrospinal fluid (CSF), blood or plasma sample.

3. The method of claim 1, wherein the isotope is selected from the group consisting of 2H, 13C, 15N, 170, 180, 33S, 34S, and 36S.

4. The method of claim 1, wherein the isotope labeled Tau protein is 15N labeled Tau.

5. The method of claim 1, wherein measuring comprises analysis via mass spectroscopy.

6. The method of claim 5, wherein obtaining the at least two unlabeled Tau protein fragments comprises digesting the unlabeled Tau protein in the sample with a digestion enzyme.

7. The method of claim 6, wherein the digestion enzyme is trypsin.

8. The method of claim 6, wherein the digesting generates the at least two unlabeled Tau protein fragment having an amino acid sequence selected from the group consisting of SEQ ID NOs: 49-102.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1A shows an overview of how .sup.15N-Tau-441 is used as the Quantitation Internal Standard, added to the biological sample at a known concentration, the Quantitation Internal Standard and endogenous proteins are digested with trypsin, or another proteolytic enzyme, to form peptide fragments that are specific to the protein from which they derive. The mass, amino acid sequence, and relative abundance of the peptides derived from endogenous proteins and .sup.15N-Tau-441 protein are determined using a mass spectrometer. The concentration of each peptide derived from endogenous Tau is determined by the mass spectrometer signal intensity for each peptide expressed as a ratio to the signal intensity for the corresponding .sup.15N-labeled peptide derived from .sup.15N-Tau-441. A series of standards that contain different but known quantities of endogenous Tau and .sup.15N-Tau-441 and that are processed exactly the same as the biological samples are used to generate a quantitation standard curve. To those persons skilled in the art, FIG. 1B details the mass fragmentation pattern obtained from a select endogenous Tau peptide, the mass fragmentation pattern obtained from the corresponding peptide present in the .sup.15N-Tau-441 Quantitation Internal Standard, and the relative abundance (Gaussian peaks or signals) obtained by mass spectrometry for several endogenous and .sup.15N-labeled fragments (y- and a-ions). In this aspect, the mass fragmentation pattern specifically identifies the amino acid sequence for this Tau peptide, and the relative abundance signals for the known concentration of the .sup.15N-labeled fragments are used to calculate the concentration of the endogenous .sup.14N-Tau peptide.

(2) FIG. 2 shows the entire sample and standard preparation, processing, and analysis workflow.

DETAILED DESCRIPTION OF THE INVENTION

(3) The present invention is based, in part, on the principle that stable isotope labeled proteins and peptides have a known, slightly greater molecular weight than their corresponding endogenous proteins and peptides, but they have identical physical or chemical properties, behave the same way in a mass spectrometer, except for their greater mass, which makes them ideal quantitative internal standards. Using the techniques provided herein, endogenous proteins, peptide fragments, and proteoforms are quantified and can be used to diagnose and/or treat a subject having or at risk of developing a neurological or neurodegenerative disorder. Accordingly, the present invention provides methods and kits useful for calculating the concentration of one or more proteins, peptide fragments, and proteoforms of interest in a subject.

(4) The invention also provides a method to assess whether a therapeutic intervention affects the concentration of proteins, peptide fragments, and proteoforms in the subject, where the biomolecules are relevant to neurological or neurodegenerative diseases. Accordingly, the method may be used to determine the optimal doses and/or optimal dosing regimens of the therapeutic intervention. Additionally, the method may be used to determine which subjects respond better to a particular therapeutic intervention. For example, subjects with high protein, peptide fragment, proteoform concentrations may respond better to one therapeutic agent, whereas subjects with normal concentrations may be at lower risk for developing a neurodegenerative disorder and are not eligible to enroll in clinical trials of experimental therapeutic agents or interventions. Alternatively, subjects with one particular genotype or proteotype may respond better to a particular therapeutic agent than those with a different genotype or proteotype. Finally, by allowing isoform specific quantitation, the method may be used to determine whether a therapeutic agent can modulate the relative concentration of one isoform to another isoform of the same protein.

(5) As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, references to “the method” includes one or more methods, and/or steps of the type described herein which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.

(6) Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, the preferred methods and materials are now described.

(7) The term “subject” as used herein refers to any individual or patient to which the subject methods are performed. Generally, the subject is human, although as will be appreciated by those in the art, the subject may be an animal. Thus, other animals, including mammals such as rodents (including mice, rats, hamsters and guinea pigs), cats, dogs, rabbits, farm animals including cows, horses, goats, sheep, pigs, etc., and primates (including monkeys, chimpanzees, orangutans and gorillas) are included within the definition of subject. In addition, the term “subject” may refer to a culture of cells, where the methods of the invention are performed in vitro to assess, for example, efficacy of a therapeutic agent.

(8) As used herein, the terms “sample” and “biological sample” refer to any sample suitable for the methods provided by the present invention. A sample of cells used in the present method can be obtained from tissue samples or bodily fluid from a subject, or tissue obtained by a biopsy procedure (e.g., a needle biopsy) or a surgical procedure. In certain embodiments, the biological sample of the present invention is a sample of bodily fluid, e.g., cerebral spinal fluid (CSF), blood, plasma, urine, saliva, and tears.

(9) As disclosed herein, stable isotope labeled Quantitation Internal Standards have a slightly higher molecular weight than their endogenous counterparts, but does not alter the physical or chemical properties of the proteins, peptide fragments, and proteoforms. Thus, these biomolecules and their stable isotope labeled counterparts will bind to antibodies and elute off a liquid chromatography column in an identical fashion. Sensitive instruments, such as mass spectrometers, provide the ability to measure small differences in mass between labeled and unlabeled biomolecules.

(10) Accordingly, in one aspect, the invention provides a method of calculating the concentration of a biomolecule in a subject. In one embodiment, the method includes contacting a sample from the subject with a Quantitation Internal Standard. As used herein, a “Quantitation Internal Standard” refers to a known concentration of a stable isotope labeled biomolecule, which has a distinct molecular weight from other labeled or unlabeled biomolecules that may exist in the sample. Thereafter, a sensitive measuring device, such as a mass spectrometer, a tandem mass spectrometer, or a combination of both, is used to measure the ratio of labeled to unlabeled biomolecules. Since the physical properties of the labeled and unlabeled biomolecules are identical, the ratio measured by the mass spectrometer is identical to the ratio in the original sample. Thus, by adding a known amount of one or more biomolecules, each labeled with a unique isotopic label, the invention provides the ability to quantitate the amount of those biomolecules that have different isotopic composition.

(11) As used herein, the term “biomolecule” refers to any organic molecule in a living organism. Exemplary biomolecules include, but are not limited to proteins, peptides, proteoforms. In one embodiment, the biomolecule is a peptide, such as a protein, that is synthesized in the central nervous system (CNS) of the subject. Exemplary proteins that can be measured by the methods of the invention include, but are not limited to, Tau and post-translationally modified such as phospho-Tau (associated with Alzheimer's Disease). In one embodiment, the protein whose in vivo concentration is measured may be Tau or its variants or isoforms. Exemplary isoforms of Tau whose concentrations may be measured include, but are not limited to, the following phosphorylated or unphosphorylated isoforms of Tau: Tau-4R2N, Tau-4R1N, Tau-4R0N, Tau-3R2N, Tau-3R1N, Tau-3R0N. The following shows a multiple sequence alignment of the 6 different isoforms of Tau.

(12) TABLE-US-00001 SEQ ID Nos. 1 441 aa MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTEDGSEEPG 60 4R/2N 2 412 aa MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTEDGSEEPG 60 4R/1N 3 383 aa MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLK 44 4R/0N 4 410 aa MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTEDGSEEPG 60 3R/2N 5 381 aa MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTEDGSEEPG 60 3R/1N 6 352 aa MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLK 44 3R/0N               ******************************************** 7 441 aa SETSDAKSTPTAEDVTAPLVDEGAPGKQAAAQPHTEIPEGTTAEEAGIGDTPSLEDEAAG 120 8 412 aa SETSDAKSTPTAE-----------------------------AEEAGIGDTPSLEDEAAG 91 9 383 aa ------------------------------------------AEEAGIGDTPSLEDEAAG 62 10 410 aa SETSDAKSTPTAEDVTAPLVDEGAPGKQAAAQPHTEIPEGTTAEEAGIGDTPSLEDEAAG 120 11 381 aa SETSDAKSTPTAE-----------------------------AEEAGIGDTPSLEDEAAG 91 12 352 aa ------------------------------------------AEEAGIGDTPSLEDEAAG 62                         ****************** 13 441 aa HVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPK 180 14 412 aa HVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPK 151 15 383 aa HVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPK 122 16 410 aa HVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPK 180 17 381 aa HVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPK 151 18 352 aa HVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPK 122 ************************************************************ 19 441 aa TPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPKSPSSAK 240 20 412 aa TPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPKSPSSAK 211 21 383 aa TPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPKSPSSAK 182 22 410 aa TPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPKSPSSAK 240 23 381 aa TPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPKSPSSAK 211 24 352 aa TPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPKSPSSAK 182 ************************************************************ 25 441 aa SRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHV 300 26 412 aa SRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHV 271 27 383 aa SRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHV 242 28 410 aa SRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGGK 274 29 381 aa SRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGGK 245 30 352 aa SRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGGK 216                  ********************************** 31 441 aa PGGGSVQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNI 360 32 412 aa PGGGSVQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNI 331 33 383 aa PGGGSVQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNI 302 34 410 aa VQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNI 329 35 381 aa VQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNI 300 36 352 aa VQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNI 271    ******************************************************* 37 441 aa THVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMV 420 38 412 aa THVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMV 391 39 383 aa THVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMV 362 40 410 aa THVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMV 389 41 381 aa THVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMV 360 42 352 aa THVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMV 331 ************************************************************ 43 441 aa DSPQLATLADEVSASLAKQGL 441 44 412 aa DSPQLATLADEVSASLAKQGL 412 45 383 aa DSPQLATLADEVSASLAKQGL 383 46 410 aa DSPQLATLADEVSASLAKQGL 410 47 381 aa DSPQLATLADEVSASLAKQGL 381 48 352 aa DSPQLATLADEVSASLAKQGL 352                        *********************

(13) By way of example and not limitation, it is noted that several unique isoforms of Tau exist in CSF and plasma, and that these isoforms can be post-translationally modified in several ways including phosphorylation. Trypsin digestion of Tau yields several peptides which may or may not be unique to each isoform, see Table 1. Thus, quantitation of some of these peptides allows for calculation of the concentration of these isoforms in the original biological fluid.

(14) TABLE-US-00002 TABLE 1 Includes Tau 441 Residues Plasma SEQ With CSF CSF LLOQ Plasma ID Cleavage (ng/ Curve (ng/ Std NO. Peptide Sequence Residues Isoforms mL) R.sup.2 mL) Curve R.sup.2 49 R.QEFEVMEDHAGTYGLGDR.K  6-23 Total 0.08 0.9987 nd — 50 R.KDQGGYTMHQDQEGDTDAGLK.E 24-44 Total 0.08 0.9999 nd — 51 K.ESPLQTPTEDGSEEPGSETSDAK.S 45-67 1N/2N 0.03 0.9932 0.05 0.9932 52 K.STPTAEDVTAPLVDEGAPGK.Q 68-87 2N 0.03 0.9956 0.05 0.9956 53 K.QAAAQPHTEIPEGTTAEEAGIGDTPSLEDEAAGHVTQAR.M  88-126 2N 0.03 0.9929 0.05 0.9397 54 K.IATPR.G 151-155 Total 0.03 0.9979 2.5 0.8603 55 R.GAAPPGQK.G 156-163 Total nd — 2.5 0.9937 56 K.TPPAPK.T 175-180 Total 0.25 0.9982 nd — 57 K.TPPSSGEPPK.S 181-190 Total 0.03 0.9948 nd — 58 R.SGYSSPGSPGTPGSR.S 195-209 Total 0.003 0.9974 0.05 0.9937 59 R.TPSLPTPPTR.E 212-221 Total 0.003 0.9994 0.05 0.9994 60 R.TPSLPTPPTREPK.K 212-224 Total 0.5 0.9988 2.5 0.9941 61 K.IGSTENLK.H 260-267 Total 0.03 0.9886 0.05 0.9988 62 K.VQIINK.K 275-280 4R 0.01 0.9831 0.5 0.9967 63 K.VQIVYKPVDLSK.V 275-286 3R 10 nd 10 nd 64 K.LDLSNVQSK.C 282-290 4R 0.05 0.9910 0.05 0.9969 65 K.HVPGGGSVQIVYKPVDLSK.V 299-317 4R 0.05 0.9999 0.05 0.9883 66 K.IGSLDNITHVPGGGNK.K 354-369 Total 0.05 0.999 0.05 0.9977 67 K.TDHGAEIVYK.S 386-395 Total 0.1 0.9985 0.05 0.9710 68 K.SPVVSGDTSPR.H 396-406 Total 0.05 0.9952 0.5 0.9819

(15) As such, the methods provide the ability to measure concentrations of various isoforms of Tau, such as fragments produced after digestion with an endoprotease (e.g., trypsin, LysN, or V8 protease). Exemplary fragments of Tau isoforms include but are not limited to regions of Tau that are different between the different isoforms and their boundaries, such as the N-terminal region (2N/1N/0N) and the C-terminal repeat region (4R/3R).

(16) As used herein, the term “nucleic acid” refers to DNA, RNA, single-stranded, double-stranded or triple stranded and any chemical modifications thereof. Virtually any modification of the nucleic acid is contemplated. A “nucleic acid molecule” can be of almost any length, from 10, 20, 30, 40, 50, 60, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 6000, 7000, 8000, 9000, 10,000, 15,000, 20,000, 30,000, 40,000, 50,000, 75,000, 100,000, 150,000, 200,000, 500,000, 1,000,000, 1,500,000, 2,000,000, 5,000,000 or even more bases in length, up to a full-length chromosomal DNA molecule.

(17) The terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to two or more amino acid residues joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers, those containing modified residues, and non-naturally occurring amino acid polymer. “Polypeptide” refers to both short chains, commonly referred to as peptides, oligopeptides or oligomers, and to longer chains, generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids. Likewise, “protein” refers to at least two covalently attached amino acids, which includes proteins, polypeptides, oligopeptides and peptides. A protein may be made up of naturally occurring amino acids and peptide bonds, or synthetic peptidomimetic structures. Thus “amino acid”, or “peptide residue”, as used herein means both naturally occurring and synthetic amino acids. For example, homo-phenylalanine, citrulline and noreleucine are considered amino acids for the purposes of the invention. “Amino acid” also includes imino acid residues such as proline and hydroxyproline. The side chains may be in either the (R) or the (S) configuration.

(18) Several different moieties may be used to label the biomolecule of interest. Generally speaking, the two types of labeling moieties utilized in the method of the invention are radioactive isotopes and non-radioactive (stable) isotopes. In one embodiment, non-radioactive isotopes may be used and measured by mass spectrometry. Preferred stable isotopes include deuterium (.sup.2H), .sup.13C, .sup.15N, .sup.17 or 18O, and .sup.33, 34, or 36S, but it is recognized that a number of other stable isotopes that change the mass of an atom by more or less neutrons than is seen in the prevalent native form would also be effective. A suitable label generally will change the mass of the biomolecule under study such that it can be detected in a mass spectrometer. In one embodiment, the biomolecule to be measured may be a peptide or protein, and the labeled moiety may be an amino acid comprising a non-radioactive isotope (e.g., .sup.13C). In another embodiment, the biomolecule to be measured may be a nucleic acid, and the labeled moiety may be a nucleoside triphosphate comprising a non-radioactive isotope (e.g., .sup.15N). Alternatively, a radioactive isotope may be used, and the labeled biomolecules may be measured with a scintillation counter (or via nuclear scintigraphy) as well as by a mass spectrometer. One or more labeled moieties may be used simultaneously or in sequence.

(19) Thus, in one embodiment, when the method is employed to measure the concentration of proteins, the labeled moiety typically will be an amino acid. Those of skill in the art will appreciate that several amino acids may be used to provide the label of biomolecules. Generally, the choice of amino acid is based on a variety of factors such as: (1) The amino acid generally is present in at least one residue of the protein or peptide of interest. (2) The amino acid is generally able to reach the site of protein production and rapidly equilibrate tissue or cellular barriers. And (3) commercial availability of the desired amino acid (i.e., some amino acids are much more expensive or harder to manufacture than others).

(20) In one embodiment, the amino acid is an essential amino acid (not produced by the body), so that a higher percent of labeling may be achieved. In another embodiment, the amino acid is a non-essential amino acid. Exemplary amino acids include, but are not limited to, leucine, isoleucine, and phenylalanine. As such, in one embodiment, the labeled amino acid is one or more of a .sup.15N-labeled amino acid, a .sup.13C.sub.x-labeled phenylalanine, where x=1 to 9, a .sup.13C.sub.x-labeled isoleucine, where x=1 to 6. For example, .sup.13C.sub.6-phenylalanine, which contains six .sup.13C atoms, may be used to label a biomolecule of interest (e.g., a CNS derived protein). In another embodiment, .sup.13C.sub.6-leucine may be used to label a biomolecule of interest (e.g., a CNS derived protein). In yet another embodiment, .sup.13C.sub.6-leucine is used to label amyloid-beta (Aβ).

(21) There are numerous commercial sources of labeled amino acids, both non-radioactive isotopes and radioactive isotopes. Generally, the labeled amino acids may be produced either biologically or synthetically. Biologically produced amino acids may be obtained from an organism (e.g., kelp/seaweed) grown in an enriched mixture of .sup.13C, .sup.15N, or another isotope that is incorporated into amino acids as the organism produces proteins. The amino acids are then separated and purified. Alternatively, amino acids may be made with known synthetic chemical processes.

(22) Once disease is established and a treatment protocol is initiated, the methods of the invention may be repeated on a regular basis to monitor the concentration(s) of biomolecule(s) of interest in the subject. The results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to months. Accordingly, another aspect of the invention is directed to methods for monitoring a therapeutic regimen for treating a subject having a neurological or neurodegenerative disorder. A comparison of the concentration(s) of biomolecule(s) of interest prior to and during therapy will be indicative of the efficacy of the therapy. Therefore, one skilled in the art will be able to recognize and adjust the therapeutic approach as needed.

(23) The method of the invention provides that a sample be obtained from the subject such that the in vivo concentration of one or more biomolecules of interest can be determined. In one embodiment, the sample is a body fluid. Suitable body fluids include, but are not limited to, cerebral spinal fluid (CSF), blood plasma, blood serum, urine, saliva, perspiration, and tears. It should be understood that biological fluids typically contain a multitude of quantifiable biomolecules. For example, where the sample is CSF, exemplary biomolecules that can be quantified include, but are not limited to, Tau, variants of Tau, amyloid-beta protein, variants of amyloid-beta protein (Aβ), digestion products of amyloid-beta protein, amyloid precursor protein (APP), apolipoprotein E, apolipoprotein J, alpha-synuclein, or any combination thereof. In another embodiment, the sample is a tissue sample, such as a sample of tissue from the central nervous system (CNS). The sample generally will be collected using standard procedures well known to those of skill in the art.

(24) In one embodiment, the sample is a CNS sample, which includes, but is not limited to, tissue from the central nervous system, which comprises brain tissue and spinal cord tissue. In one embodiment of the invention, the CNS sample may be taken from brain tissue, including, but not limited to, tissue from the forebrain (e.g., cerebral cortex, basal ganglia, hippocampus), the interbrain (e.g., thalamus, hypothalamus, subthalamus), the midbrain (e.g., tectum, tegmentum), or the hindbrain (e.g., pons, cerebellum, medulla oblongata). In another embodiment, the CNS sample may be collected from spinal cord tissue. In still other embodiments, CNS samples from more than one CNS region may be taken. Accordingly, the concentration of a biomolecule of interest may be measured in different CNS samples, e.g., in the cortex and the hippocampus, simultaneously.

(25) CNS samples may be obtained by known techniques. For instance, brain tissue or spinal cord tissue may be obtained via dissection or resection. Alternatively, CNS samples may be obtained using laser microdissection. The subject may or may not have to be sacrificed to obtain the sample, depending on the CNS sample desired and the subject utilized.

(26) In general, when the biomolecule under study is a peptide or protein, the invention provides that a first sample may be taken from a subject prior to administration of the therapeutic agent to provide a baseline concentration. After administration of the therapeutic agent, one or more samples are obtained from the subject. As will be appreciated by those of skill in the art, the number of samples and when the samples are taken generally will depend upon a number of factors such as: the type of analysis, type of administration, the protein of interest, the rate of metabolism, the type of detection, and the type of subject.

(27) It should be understood that if samples at different time-points are desired, more than one subject may be used. For instance, one subject may be used for a baseline sample, another subject for a time-point of one-hour post administration of the therapeutic agent, another subject for a time-point six hours post administration of the therapeutic agent.

(28) Accordingly, the present invention provides that detection of the amount of labeled biomolecule and the amount of unlabeled biomolecule in the sample may be used to determine the ratio of labeled biomolecule to unlabeled biomolecule, which in turn, may be used to calculate the concentration of the biomolecule of interest in the subject. In one embodiment, the ratio is determined by means of detecting changes in mass of the labeled biomolecule (e.g., peptide or protein) with respect to the unlabeled biomolecule. Exemplary means for detecting differences in mass between the labeled and unlabeled biomolecules include, but are not limited to, liquid chromatography mass spectrometry, gas chromatography mass spectrometry, MALDI-TOF mass spectrometry, and tandem mass spectrometry.

(29) However, prior to detecting the ratio of labeled biomolecule to unlabeled biomolecule, it may be desirable to isolate and/or separate the biomolecule of interest from other biomolecules in the sample. Thus, in one embodiment, immunoprecipitation may be used to isolate and purify the biomolecule (e.g., peptide or protein) of interest before it is analyzed. In another embodiment, the biomolecule of interest may be isolated or purified by affinity chromatography or immunoaffinity chromatography. Alternatively, mass spectrometers having chromatography setups may be used to separate biomolecules without immunoprecipitation, and then the biomolecule of interest may be measured directly. In an exemplary embodiment, the protein of interest may be immunoprecipitated and then analyzed by a liquid chromatography system interfaced with a tandem MS unit equipped with an electrospray ionization source (LC-ESI-tandem MS). One example of using 3 different antibodies to immunoprecipitate Tau protein from 3 aliquots of the same human CSF sample, followed by digestion of Tau protein, and quantitation of the relative abundance of different Tau peptides is shown in Table 2. This aspect includes the observation that different antibodies have different affinity for different portions of the Tau protein, and the selection of immunoprecipitation antibody can be used to focus the quantitative analysis on different portions or proteoforms of the Tau biomolecule.

(30) TABLE-US-00003 TABLE 2 SEQ ID Nos. Peptide Sequence (Residue) HJ8.5 HJ8.7 Tau12 69 QEFEVMEDHAGTYHLHDR (6-23) 0.97 0.68 1.00 70 KDQGGYTMHQDQEGDTDAGLK (24-44) 1.00 0.84 0.15 71 ESPLQTPTEDFSEEPGSETSDAK 0.74 1.00 0.92 (45-67) 72 STPTAEDVTAPLVDEGAPGK (68-87) 0.47 1.00 0.47 73 QAAAQPHTEIPEGTTAEEAGIGDTPSL 0.36 1.00 0.59 EDEAAGHVTQAR (88-126) 74 IATPR (151-155) 0.55 1.00 0.87 75 TPPAPK (175-180) 0.97 1.00 0.99 76 TPPSSGEPPK (181-190) 0.52 1.00 0.46 77 SGYSSPGSPGTPGSR (195-209) 0.83 0.80 1.00 78 TPSLPTPPTR (212-221) 0.49 0.86 1.00 79 TPSLPTPPTREPK (212-224) 0.94 0.94 1.00 80 IGSTENLK (260-267) 0.37 1.00 0.86 81 VQIINK (275-280) 0.36 1.00 0.31 82 LDLSNVWSK (282-290) 0.08 0.04 1.00 83 HVPGGGSVQICYKPVDLSK (299-316) 0.40 1.00 0.82 84 IGSLDNITHVPGGGNK (354-369) 0.61 1.00 0.86 85 TDHGAEIVYK (386-395) 0.44 1.00 0.68 86 SPVVSGDTSPR (396-406) 1.00 0.39 0.42 Peptide Intensity *Values are reported as a fraction of the abundance for the immuno-precipitation antibody that provided the highest abundance (1.0) for that specific Tau peptide

(31) In another aspect, the invention provides that multiple biomolecules in the same sample may be measured simultaneously. That is, both the amount of unlabeled and labeled biomolecule may be detected and measured separately or at the same time for multiple biomolecules. As such, the invention provides a useful method for screening changes in concentration, of one or more biomolecules on a large scale (i.e., proteomics/metabolomics) and provides a sensitive means to detect and measure biomolecules involved in the underlying pathophysiology. In this aspect, the invention also provides a means to measure multiple types of biomolecules. In this context, for example, a protein and a lipid may be measured simultaneously or sequentially.

(32) Once the amount of labeled and unlabeled biomolecule has been detected in a sample, the ratio or percent of labeled biomolecule to unlabeled biomolecule may be determined. Thereafter, the concentration of the unlabeled biomolecule in the sample can be determined. In other words, since a known amount of labeled biomolecule is added to an unknown amount of biomolecules and the ratio of labeled to unlabeled is measured, the concentration of the unlabeled biomolecules can be calculated from the ratio as follows:
Concentration of unlabeled=(ratio of unlabeled to labeled)×(concentration of labeled).  (i)
The equation may be simplified as:
Concentration of unlabeled=(ratio of unlabeled:Quantitation Internal Standard)×(concentration of Quantitation Internal Standard).  (ii)

(33) Conversely, if a known amount of unlabeled is added to an unknown amount labeled the concentration of the labeled can be calculated as follows:
Concentration of labeled=(ratio of labeled to unlabeled)×(concentration of unlabeled).  (iii)

(34) In addition, if a known amount of biomolecule 1, labeled with label 1, is added to an unknown amount of biomolecule 2, labeled with label 2, the concentration of the biomolecule 2 can be calculated as follows:
Concentration of label 2=(ratio of label 2 to label 1)×(concentration of label 1).  (iv)

(35) Similarly, if a known amount of biomolecule 1, labeled with label 1, is added to an unknown amount of biomolecule 2, labeled with label 2, and biomolecule 3, labeled with label 3, the concentration of the biomolecule 2 and biomolecule 3 can be calculated as follows:
Concentration of label 2=(ratio of label 2 to label 1)×(concentration of label 1)  (v)
Concentration of label 3=(ratio of label 3 to label 1)×(concentration of label 1).  (vi)

(36) Finally, if a known amount of biomolecule 1, labeled with label 1, is added to an unknown amount of biomolecule 2, labeled with label 2, and an unknown amount of unlabeled biomolecule 3, the concentration of the biomolecule 2 and unlabeled biomolecule can be calculated as follows:
Concentration of label 2=(ratio of label 2 to label 1)×(concentration of label 1)  (vii)
Concentration of unlabeled=(ratio of unlabeled to label 1)×(concentration of label 1).  (viii)

(37) In another embodiment, the methods further include the step of normalizing the calculated concentration to a standard curve based on the curve fitting equation generated by the standard curve. The standard curve used herein is generated by determining two or more ratios of unlabeled biomolecules to their respective Quantitation Internal Standards, where the concentration of the unlabeled biomolecule of interest is known.

(38) In another aspect, the invention allows measurement of the labeled and unlabeled protein at the same time, so that the ratio of labeled to unlabeled protein, as well as other calculations, may be made.

(39) In one aspect, Tau is isolated from the biologic samples by immunoprecipitation using an antibody that recognizes Tau. In this embodiment, the isolated peptides are eluted from the antibody, for example by using formic acid and then digested with trypsin or another protease. This invention measures the concentration of Tau peptides.

(40) The term “antibody” as used in this invention is meant to include intact molecules of polyclonal or monoclonal antibodies, as well as fragments thereof, such as Fab and F(ab′).sub.2, Fv and SCA fragments which are capable of binding an epitopic determinant. The term “specifically binds” or “specifically interacts,” when used in reference to an antibody means that an interaction of the antibody and a particular epitope has a dissociation constant of at least about 1×10.sup.−6, generally at least about 1×10.sup.−7, usually at least about 1×10.sup.−8, and particularly at least about 1×10.sup.−9 or 1×10.sup.−10 or less.

(41) The method of the invention may be used to diagnose or monitor the progression of a neurological or neurodegenerative disease by measuring the in vivo concentration of one or more biomolecules of interest in a subject. Additionally, the methods of the invention may be used to monitor the treatment of a neurological or neurodegenerative disease by measuring the in vivo concentration of a biomolecule of interest in a subject. The concentration of the biomolecule may be linked to a neurological or neurodegenerative disease such that any increase or decrease may be indicative of the presence or progression of the disease. Thus, the calculated concentration of one or more biomolecules of interest may be compared to the concentration of the same biomolecules in a corresponding normal sample, to the concentration of the same biomolecules in a subject of known neurological or neurodegenerative disease state, to the concentration of the same biomolecules from the same subject determined at an earlier time, or any combination thereof.

(42) In addition, such methods may help identify an individual as having a predisposition for the development of the disease or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms. A more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the disease.

(43) As used herein a “corresponding normal sample” refers to a sample from the same organ and/or of the same type as the sample being examined In one aspect, the corresponding normal sample comprises a sample of cells obtained from a healthy individual. Such a corresponding normal sample can, but need not be, from an individual that is age-matched and/or of the same sex as the individual providing the sample being examined In another aspect, the corresponding normal sample comprises a sample of cells obtained from an otherwise healthy portion of tissue of the subject from which the sample being tested is obtained.

(44) Reference to the concentration of biomolecules in a subject of known neurological or neurodegenerative disease state includes a predetermined concentration of a biomolecule linked to a neurological or neurodegenerative disease. Thus, the concentration may be compared to a known concentration of biomolecules obtained from a sample of a single individual or may be from an established cell line of the same type as that of the subject. In one aspect, the established cell line can be one of a panel of such cell lines, wherein the panel can include different cell lines of the same type of disease and/or different cell lines of different diseases associated with the same biomolecule. Such a panel of cell lines can be useful, for example, to practice the present method when only a small number of cells can be obtained from the subject to be treated, thus providing a surrogate sample of the subject's cells, and also can be useful to include as control samples in practicing the present methods.

(45) Exemplary neurological or neurodegenerative diseases that may be linked to the concentration ranges of biomolecules of interest include, but are not limited to, Alzheimer's Disease, Pick's Disease, Parkinson's Disease, stroke, frontal temporal dementias (FTDs), Huntington's Disease, progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), aging-related disorders and dementias, Multiple Sclerosis, Prion Diseases (e.g., Creutzfeldt-Jakob Disease, bovine spongiform encephalopathy or Mad Cow Disease, and scrapie), Lewy Body Disease, schizophrenia, Amyotrophic Lateral Sclerosis (ALS or Lou Gehrig's Disease) or other motor neuron diseases, restless legs syndrome, epilepsy or other seizure disorders, tremors, depression, mania, anxiety disorders, brain trauma or injury, narcolepsy, insomnia or other sleep disorders, autism, normal pressure hydrocephalus, pain disorders or syndromes, migraines, cluster headaches or other forms of headache, spinocerebellar disorders, muscular dystrophies, myasthenia gravis, retinitis pigmentosa or other forms of retinal degeneration. It is also envisioned that the method of the invention may be used to study the normal physiology, metabolism, and function of the CNS.

(46) In another aspect, the present invention provides a method for assessing whether a therapeutic agent used to treat a neurological or neurodegenerative disease affects the concentration of a biomolecule of interest in the subject. For example, the concentration of the biomolecule may be measured to determine if a given therapeutic agent results in an increase, or a decrease in the concentration of the biomolecule. In one embodiment, the method is performed in vivo, as herein described. In another embodiment, the method is performed in vitro utilizing a culture of cells, where the culture of cells is the “subject” in the methods described herein. Accordingly, use of the methods provided herein will allow those of skill in the art to accurately determine the degree of change in the concentration of the biomolecule of interest, and correlate these measurements with the clinical outcome of the disease modifying treatment. Results from this aspect of the invention, therefore, may help determine the optimal doses and frequency of doses of a therapeutic agent, may assist in the decision-making regarding the design of clinical trials, and may ultimately accelerate validation of effective therapeutic agents for the treatment of neurological or neurodegenerative diseases.

(47) Thus, the method of the invention may be used to predict which subjects will respond to a particular therapeutic agent. For example, subjects with increased concentrations of a particular biomolecule may respond to a particular therapeutic agent differently than subjects with decreased concentrations of the biomolecule. In particular, results from the method may be used to select the appropriate treatment (e.g., an agent that blocks the production of the biomolecule or an agent that increases the clearance of the biomolecule) for a particular subject. Similarly, results from the method may be used to select the appropriate treatment for a subject having a particular genotype.

(48) Those of skill in the art will appreciate that the therapeutic agent can and will vary depending upon the neurological or neurodegenerative disease or disorder to be treated and/or the biomolecule whose metabolism is being analyzed. In embodiments in which the biomolecule is Tau, non-limiting examples of suitable therapeutic agents include Tau metabolism modulators, Tau kinase inhibitors, cathepsin D inhibitors, Tau autophagy activators, and Tau aggregation inhibitors. Other suitable AD therapeutic agents include hormones, neuroprotective agents, and cell death inhibitors. Many of the above mentioned therapeutic agents may also affect the in vivo metabolism of other proteins implicated in neurodegenerative disorders. Furthermore, therapeutic agents that may affect the in vivo metabolism of synuclein include sirtuin 2 inhibitors, synuclein aggregation inhibitors, proteasome inhibitors, etc.

(49) The therapeutic agent may be administered to the subject in accordance with known methods. Typically, the therapeutic agent will be administered orally, but other routes of administration such as parenteral or topical may also be used. The amount of therapeutic agent that is administered to the subject can and will vary depending upon the type of agent, the subject, and the particular mode of administration. Those skilled in the art will appreciate that dosages may be determined with guidance from Goodman & Goldman's The Pharmacological Basis of Therapeutics, Tenth Edition (2001), Appendix II, pp. 475-493, and the Physicians' Desk Reference.

(50) It should be understood that the methods of the invention described herein can be adapted to a high throughput format, thus allowing the examination of a plurality (i.e., 2, 3, 4, or more) of samples and/or biomolecules, which independently can be the same or different, in parallel. A high throughput format provides numerous advantages. For example, a high throughput format allows for the examination/quantitation of two, three, four, etc., different biomolecules, alone or in combination, of a subject. Finally, a high throughput format allows, for example, control samples (positive controls and or negative controls) to be run in parallel with test samples. In addition, a high throughput method may allow immunoprecipitation of multiple proteins at the same time using multiple antibodies.

(51) In another aspect, the invention provides a kit for performing the methods of the invention. In one embodiment, a kit is provided for diagnosing and/or monitoring the progression or treatment of a neurological or neurodegenerative disease in a subject. The kit may further include an appropriate Quantitation Internal Standard and means for obtaining a biological sample at regular time intervals from the subject. In certain embodiments, the kit will also include instructions for detecting and determining the ratio of labeled to unlabeled biomolecules of interest over time and for calculating the concentration of the endogenous unlabeled biomolecule. In one embodiment, the instructions will disclose methods for comparing the calculated concentration to certain standards and/or controls as disclosed herein.

(52) In another embodiment, the kit of the invention provides a compartmentalized carrier including one or more containers containing the Quantitation Internal Standard and the various means for performing the methods of the invention.

(53) The following examples are provided to further illustrate the advantages and features of the present invention but are not intended to limit the scope of the invention. While they are typical of those that might be used, other procedures, methodologies, or techniques known to those skilled in the art may alternatively be used.

EXAMPLE

Quantitation of Tau by SISAQ

(54) .sup.15N labeled Tau was used as Quantitation Internal Standard and spiked into a standard curve of samples containing concentrations of Tau ranging from 0.05 pg/mL to 100 pg/mL. In addition, the Quantitation Internal Standard was spiked into CSF and plasma from two different individuals. Tau was isolated from the samples using immunoprecipitation and then digested with trypsin and analyzed by mass spectrometry. The ratio of unlabeled endogenous Tau to Quantitation Internal Standard was calculated for all samples and a standard curve generated. The standard curve was linear in the range tested (0.05 pg/mL to 100 pg/mL) and was used to calculate the concentration of Tau in the CSF and plasma samples. The concentration of Tau was approximately 5 pg/mL in the CSF samples and around 0.1 pg/mL in plasma, and the intra- and inter-assay CV for triplicate measures of CSF and plasma Tau concentrations was less than 25%. Our data show that regardless of the concentration, Tau can be reliably and reproducibly measured using the invention methods. Examples of the intra-assay and inter-assay reliability (% CV) for quantifying several Tau peptides present in human CSF are shown in Tables 3 & 4. The intra-assay reliability (Table 3) was calculated by triplicate analysis of a single pooled human CSF sample where these injection replicate analyses were dispersed among many other standard curve samples and unknown samples included in the same analytical run. The inter-assay reliability (Table 4) was calculated by triplicate analysis of a single human CSF pool that was prepared and analyzed on 3 separate days (process replicates) in different analytical runs that included many other samples and standards.

(55) TABLE-US-00004 TABLE 3 SEQ. Average ID. Inter-Assay Analysis Calculated Concentration (ng/mL) Concentration Standard Nos. Peptide Sequence (Residue #s) Replicate 1 Replicate 2 Replicate 3 (ng/mL) Deviation % CV 87 QEFEVMEDHAGTYHLHDR (6-23) 3.174 3.220 3.111 3.168 0.055 1.7 88 KDQGGYTMHQDQEGDTDAGLK (24-44) 3.516 3.653 3.845 3.672 0.165 4.5 89 ESPLQTPTEDFSEEPGSETSDAK (45-67) 1.738 1.885 1.852 1.825 0.078 4.2 90 STPTAEDVTAPLVDEGAPGK (68-87) 0.223 0.212 0.215 0.216 0.006 2.6 91 QAAAQPHTEIPEGTTAEEAGIGDTPSLE 0.174 0.158 0.199 0.177 0.020 11.5 DEAAGHVTQAR (88-126) 92 IATPR (151-155) 2.074 2.190 1.315 1.860 0.475 25.6 93 TPPAPK (175-180) 1.317 1.499 1.598 1.471 0.142 9.7 94 TPPSSGEPPK (181-190) 1.418 1.551 1.733 1.567 0.158 10.1 95 SGYSSPGSPGTPGSR (195-209) 1.240 1.289 1.047 1.192 0.128 10.7 96 TPSLPTPPTR (212-221) 1.191 1.259 1.230 1.227 0.034 2.8 SEQ. Average ID. Inter-Assay Analysis Calculated Concentration (ng/mL) Concentration Standard Nos. Peptide Sequence (Residue #s) Day 1 Day 2 Day 3 (ng/mL) Deviation % CV 97 QEFEVMEDHAGTYHLHDR (6-23) 2.935 3.898 3.174 3.336 0.501 15.0 98 KDQGGYTMHQDQEGDTDAGLK (24- 2.419 2.870 3.516 2.935 0.552 18.8 44) 99 ESPLQTPTEDFSEEPGSETSDAK (45-67) 1.488 2.003 1.738 1.743 0.258 14.8 100 STPTAEDVTAPLVDEGAPGK (68-87) 0.217 0.274 0.223 0.238 0.031 13.2 101 QAAAQPHTEIPEGTTAEEAGIGDTPSLE 0.184 0.218 0.174 0.192 0.023 12.2 DEAAGHVTQAR (88-126) 102 IATPR (151-155) 1.302 1.587 2.074 1.654 0.390 23.6 103 TPPAPK (175-180) 1.423 1.310 1.317 1.350 0.063 4.7 104 TPPSSGEPPK (181-190) 1.177 1.686 1.418 1.427 0.255 17.9 105 SGYSSPGSPGTPGSR (195-209) 1.195 1.435 1.240 1.290 0.128 9.9 106 TPSLPTPPTR (212-221) 1.004 1.129 1.191 1.108 0.095 8.6

(56) This illustrates the feasibility of using stable isotope labeled Tau as a quantitation internal standard and relating the ratio of unlabeled to labeled Tau to a standard curve to allow for measurement of concentrations of Tau in unknown samples.

(57) Although the invention has been described with reference to the above example, it will be understood that modifications and variations are encompassed within the spirit and scope of the invention. Accordingly, the invention is limited only by the following claims.