Mass spectrometry method for post-translational modification and / or fragmentation of tau protein in biological fluid

Abstract

Provided is a mass spectrometry method for post-translational modification and / or fragmentation of tau protein in a biological fluid, the method enabling stable detection of both modified endogenous tau fragments that include post-translational modification sites such as phosphorylation, and unmodified endogenous tau fragments, without requiring the performance of additional fragmentation processing on a group of endogenous tau fragments in the biological fluid. The method includes: a step of extracting endogenous tau fragments derived from tau protein in a biological fluid sample to obtain a group of endogenous tau fragments; a step of performing mass spectrometry on the group of endogenous tau fragments to obtain mass-to-charge ratio (m / z) and intensity information for each of the endogenous tau fragments, and thereby conducting mass profiling of the group of endogenous tau fragments; and a step of analyzing at least one of the difference in fragmentation of tau protein and the difference in post-translational modifications of tau protein between samples by comparing the mass profiling results of the groups of endogenous tau fragments between samples.

Description

Mass spectrometry of post-translational modifications and / or fragmentation of tau protein in biological fluids

[0001] The present invention relates to mass spectrometric methods for post-translational modifications and / or fragmentation of tau protein in biological fluids.

[0002] Neurofibrillary tangles (NFTs) are aggregates of abnormal tau protein (hereafter simply referred to as "tau") that accumulate within neurons. Tauopathies (tau-related neurodegenerative diseases) are a group of neurodegenerative disorders that result from these NFTs. Representative tauopathies include Alzheimer's disease (AD), progressive supranuclear palsy, corticobasal degeneration (CBD), and Pick's disease. Tau protein is localized in neuronal axons and is involved in stabilizing the microtubule cytoskeleton and axonal transport. In the brains of AD patients, hyperphosphorylated tau accumulates abnormally within neurons, and some of the phosphorylated tau protein is fragmented and detected in cerebrospinal fluid (CSF) and blood.

[0003] It is believed that tau protein phosphorylation and fragmentation progress as the pathology of tau-related neurodegenerative diseases progresses, and therefore analysis of these proteins in biological fluids is expected to be useful for early detection of disease and assessment of treatment effectiveness (see, for example, Anniina Snellman et al., "N-terminal and mid-region tau fragments as fluid biomarkers in neurological diseases," BRAIN 2022:145;2834-2848 (Non-Patent Document 1)).

[0004] In recent years, phosphorylation of threonine residues at positions 181, 205, 217, and 231 (threonine residues at each position from the N-terminus of the 2N4R isoform of tau protein, which consists of a full-length of 441 amino acids (the amino acid sequence is shown in SEQ ID NO: 1 in the Sequence Listing)) has attracted attention as an indicator for detecting early-stage AD. Analytical techniques for quantifying these residues, such as immunoassay ELISA (enzyme-linked immunosorbent assay) and LC-MS / MS (QQQ, Q-TOF, Orbitrap), have been actively developed, and the quantitative values ​​of phosphorylated tau protein obtained by these methods and the phosphorylation rate of each site (the proportion of protein phosphorylated at a specific site) are being evaluated as biomarkers.

[0005] When analyzing phosphorylated tau using immunoassays, quantification is performed using antibodies (anti-phosphorylation antibodies) that recognize the target phosphorylation site. To achieve highly sensitive and specific measurements, two different recognition sites for the target component are generally required, requiring two types of antibodies: a capture antibody and a detection antibody. The capture antibody is an anti-tau antibody that recognizes the specific phosphorylation to be analyzed. Therefore, the phosphorylated tau protein that can be captured and detected by this method is limited to fragments of a specific length surrounded by a pair of antibodies. Because tau protein in biological fluids is highly fragmented by endogenous proteases, the quantitative value measured by this method may be underestimated. Furthermore, the capture antibody does not bind or only binds very weakly to components whose recognition site is not phosphorylated. Therefore, it is not possible to determine the phosphorylation rate.

[0006] On the other hand, when analyzing phosphorylated tau using the LC-MS / MS method, as shown in an example of the analysis flow in Figure 7, the captured tau protein (a mixture of full-length tau and tau fragments that have undergone fragmentation or post-translational modification) is subjected to enzymatic digestion (trypsin digestion) to convert it into a uniform peptide mixture. Next, the sample is subjected to LC-MRM (Liquid Chromatography-Multiple Reaction Monitoring) to measure peptides containing the phosphorylation site to be analyzed, thereby determining a quantitative value of phosphorylation (see, for example, Nicolas R. Barthelemy et al., "Tau Phosphorylation Rates Measured by Mass Spectrometry Differ in the Intracellular Brain vs. Extracellular Cerebrospinal Fluid Compartments and Are Differentially Affected by Alzheimer's Disease", Frontiers in Aging Neuroscience, (2019), Volume 11, Article 121, pp. 1-18 (Non-Patent Document 2); Laia Montoliu-Gaya et al., "Mass spectrometric simultaneous quantification of tau species in plasma shows differential associations with amyloid and tau pathologies", Nature Aging, Volume 3, (2023), pp. 661-669 (Non-Patent Document 3)). The phosphorylation rate can also be determined from the ratio of the ion intensity (peak area) of a non-phosphorylated peptide containing the phosphorylation site to be analyzed to that of a phosphorylated peptide (Non-Patent Document 2).

[0007] Regarding tau enrichment as a pretreatment for LC-MS / MS, it is not necessary to use anti-phosphorylation antibodies. Rather, immunoprecipitation (IP) using anti-tau antibodies (or a combination of antibodies) that do not recognize specific phosphorylation sites can be performed to more efficiently recover and simultaneously analyze endogenous tau fragments with various fragmentation and post-translational modifications present in biological fluids. This method improves the depth of analytical measurements of phosphorylation and fragmentation. It is also effective to combine this with a non-specific phosphorylation enrichment method (antibody-free), such as IMAC (immobilized metal affinity chromatography), which can similarly efficiently capture multiple phosphorylation sites.

[0008] In previously reported analytical methods for phosphorylated tau, such as those described in Non-Patent Documents 2 and 3, trypsin digestion is performed to reduce the captured endogenous tau fragments to a size that is easy to detect by mass spectrometry. This decomposes tau protein into endogenous tau fragments of uniform size, allowing efficient detection of the target endogenous tau fragments with and without post-translational modifications by mass spectrometry.

[0009] On the other hand, there are also the following disadvantages. The reproducibility of the digestion reaction is greatly affected by the amino acid sequence and post-translational modifications of the target protein, as well as the digestion conditions. Figure 8 is a schematic diagram showing the fragmentation of tau protein by trypsin digestion, as described in Non-Patent Documents 2 and 3. As shown in Figure 8, due to the influence of phosphorylation, a tryptic peptide containing T181 (threonine at position 181) (181-190, miss cleavage 0) is incompletely cleaved when T181 is phosphorylated (175-190, miss cleavage 1).

[0010] Due to the influence of the charge and three-dimensional structure of amino acid residues, there is a tendency for undigested fragments to be generated. For tau, for example, it has been confirmed that the reaction efficiency is low for trypsin digestion fragments 45-67 and 212-221. Furthermore, tau protein in biological fluids is not only highly fragmented, but is further fragmented by trypsin digestion, resulting in the generation of semi-tryptic fragments.

[0011] As a result, the peptides to be analyzed are diverse and complex. For example, to analyze the peptidic T231 (pT231), Non-Patent Document 2 targets 226-234 / 226-230, while Non-Patent Document 3 targets 225-240, as shown in Figure 8.

[0012] Tau in biological fluids not only exists in six isoforms (proteins with different molecular weights of 352-441 amino acid residues), but also has a highly complex structure due to numerous phosphorylations (along with other known post-translational modifications such as glycosylation and acetylation) and fragmentation. Therefore, the conventional methods described above have problems in that the digestion process is difficult to reproducibly, and the peptides containing specific phosphorylation sites become more diverse, resulting in reduced analytical sensitivity and difficulty in accurate quantification.

[0013] For detection, for example, tau protein is present in extremely small amounts in cerebrospinal fluid (reference concentration: approximately 100 pg / mL), and highly sensitive detection is required after highly separating the complex tau fragments. The LC-MS / MS methods described in Non-Patent Documents 2 and 3 use high-performance equipment (nanoLC, Orbitrap) with high sensitivity and selectivity. Furthermore, for digests of tau protein with multiple phosphorylation sites, quantifying the phosphorylation rate and phosphate groups at each site requires the setting of MRM transitions corresponding to each phosphorylation site, making this analysis cumbersome.

[0014] Furthermore, for example, Claudia Cicognola1 et al., "Novel tau fragments in cerebrospinal fluid: relation to tangle pathology and cognitive decline in Alzheimer's disease," Acta Neuropathologica (2019) 137:279-296 (Non-Patent Document 4), describes the identification of endogenous tau fragments extracted by immunoprecipitation from a few milliliters of cerebrospinal fluid using LC-MS / MS. There is a need for a method that can detect endogenous tau fragments with high sensitivity and accuracy using smaller sample volumes. The establishment of such a detection method is expected to play an important role in early diagnosis and monitoring of treatment effectiveness.

[0015] Anniina Snellman et al., “N-terminal and mid-region tau fragments as fluid biomarkers in neurological diseases”, BRAIN 2022:145;2834-2848Nicolas R. Barthelemy et al., “Tau Phosphorylation Rates Measured by Mass Spectrometry Differ in the Intracellular Brain vs. Extracellular Cerebrospinal Fluid Compartments and Are Differentially Affected by "Alzheimer's Disease", Frontiers in Aging Neuroscience, (2019), Volume 11, Article 121, p1-18Laia Montoliu-Gaya et al., "Mass spectrometric simultaneous quantification of tau species in plasma shows differential associations with amyloid and tau pathologies", Nature Aging, Volume 3, (2023), p661-669Claudia Cicognola1 et al., “Novel tau fragments in cerebrospinal fluid: relation to tangle pathology and cognitive decline in Alzheimer's disease”, Acta Neuropathologica (2019) 137:279-296

[0016] The present invention has been proposed to solve the above-mentioned problems, and its purpose is to provide a mass spectrometry method that can stably detect modified and unmodified endogenous tau fragments containing phosphorylation and other post-translational modification sites without performing an additional fragmentation process on endogenous tau fragments in biological fluids.

[0017] A first aspect of the present invention relates to a mass spectrometry method for post-translational modification and / or fragmentation of tau protein in a biological fluid, and comprises the following steps: 1. extracting endogenous tau fragments derived from tau protein from a biological fluid sample to obtain a group of endogenous tau fragments; 2. subjecting the group of endogenous tau fragments to mass analysis to obtain mass-to-charge ratio (m / z) and intensity information derived from each endogenous tau fragment, and performing mass profiling of the group of endogenous tau fragments; and 3. comparing the mass profiling results of the group of endogenous tau fragments between samples to analyze differences in at least one of tau protein fragmentation and post-translational modification of tau protein between samples.

[0018] According to the present invention, a mass spectrometry method can be provided that can stably detect modified and unmodified endogenous tau fragments containing post-translational modification sites without performing additional fragmentation processing on endogenous tau fragments in biological fluids.

[0019] 1 is a flow chart showing an example of the method of the present invention. 2 is a diagram showing a schematic diagram of tau protein isoforms and antibody epitope sequences. 3 is a diagram showing various examples of endogenous tau fragments according to Experimental Example 1, with the start and end positions and [M+H] + 1 is a diagram showing the m / z values ​​(theoretical average mass) of tau protein fragments in Experimental Example 1. FIG. 2 is a diagram showing the m / z values ​​(theoretical average mass) of tau protein fragments in Experimental Example 1. FIG. 3 is a diagram showing the m / z values ​​(theoretical average mass) of tau protein fragments in Experimental Example 1. FIG. 4 is a diagram showing the m / z values ​​(theoretical average mass) of tau protein fragments in Experimental Example 1. FIG. 5 is a diagram showing the m / z values ​​(theoretical average mass) of tau protein fragments in Experimental Example 1. FIG. 6 is a diagram showing the m / z values ​​(theoretical average mass) of tau protein fragments in Experimental Example 1. FIG. 7 is a diagram showing the m / z values ​​(theoretical average mass) of tau protein fragments in Experimental Example 1. FIG. 8 is a diagram showing the m / z values ​​(theoretical average mass) of tau protein fragments in Experimental Example 1. FIG. 9 is a diagram showing the m / z values ​​(theoretical average mass) of tau protein fragments in Experimental Example 1.

[0020] The method of the present invention is a mass spectrometry method for detecting post-translational modifications and / or fragmentation of tau protein in a biological fluid, comprising the steps of extracting endogenous tau fragments derived from tau protein from a biological fluid sample to obtain a group of endogenous tau fragments, mass profiling the group of endogenous tau fragments by mass spectrometry to obtain mass-to-charge ratio (m / z) and intensity information derived from each endogenous tau fragment, and comparing the mass profiling results of the group of endogenous tau fragments between samples to analyze differences in at least one of tau protein fragmentation and tau protein post-translational modifications between samples. This method provides a mass spectrometry method that can stably detect modified and unmodified endogenous tau fragments containing post-translational modification sites without subjecting the endogenous tau fragment group in a biological fluid to additional fragmentation treatment. The method of the present invention is considered useful for detecting post-translational modifications (e.g., phosphorylation) and / or fragmentation of tau protein in tauopathies. Conventional methods generally involve absolute / relative quantification using stable isotope-labeled proteins / peptides, but the method of the present invention utilizes the relative ratios of endogenous tau fragments, enabling more efficient and economical analysis.

[0021] FIG. 1 is a flow chart showing an example of the method of the present invention. In the method of the present invention, first, endogenous tau fragments derived from tau protein are extracted from a biological fluid as a sample. In the present invention, the "biological fluid" refers to a liquid collected as a sample and can be selected from blood, cerebrospinal fluid (CSF), urine, bodily secretions, feces, saliva, sputum, etc. Blood includes whole blood, plasma, serum, etc. Blood can be prepared by appropriately treating collected whole blood. The biological fluid may be used directly for measuring the concentration of a component, or may be subjected to appropriate pretreatment as necessary before use for measuring the concentration of a component.

[0022] Extraction of endogenous tau fragments from biological fluids can be carried out by immunoprecipitation (IP) using an anti-tau antibody, which is a conventionally known technique.

[0023] The anti-tau antibody used in IP can be appropriately selected depending on the target to be detected (fragmentation of tau protein, post-translational modification of tau protein) and its region.

[0024] Objective I: Targeting tau protein fragmentation. Any antibody can be selected that has as its epitope a specific sequence within the region of tau protein that is to be detected (the target region). In this case, an antibody that does not contain as an epitope an amino acid sequence that may be subject to post-translational modification is preferred. Focusing on the type of fragmentation occurring in the target region (the antibodies used and the data obtained overlap with those in Objective II, Mode A (described below), but the analysis method is different), the fragmentation profile of endogenous tau fragments in a specific region can be calculated.

[0025] Objective II: When post-translational modifications of tau protein are the subject of analysis (Aspect A) Antibodies that do not contain amino acids that may be subject to post-translational modifications in their epitope sequences We focus on what kind of post-translational modifications have occurred in the target region (the antibodies used and the data obtained overlap with those in Objective I (described above), but the analytical method is different), and can calculate the "phosphorylation profile" of endogenous tau fragments that have been post-translationally modified in a specific region, for example, phosphorylated endogenous tau fragments (phosphorylated tau fragments).

[0026] (Aspect B) Antibodies Comprising Post-translationally Modified Amino Acids at Specific Sites as Epitope Sequences When such antibodies are used, a group of endogenous tau fragments in which the specific amino acids recognized by the antibody have been post-translationally modified can be recovered. Examples of such antibodies include known anti-phosphorylation antibodies (antibodies that recognize phosphorylation at specific sites (e.g., antibody PT3 that recognizes pT217 of tau protein)). In this case, the ability to recognize phosphorylation at specific sites on tau protein and detect it as multiple phosphorylated tau fragments can be utilized to calculate a "fragmentation profile" of tau fragments phosphorylated at specific sites on tau protein.

[0027] The focus is on what kind of fragmentation has occurred in a target region that has post-translational modifications at a specific site. By limiting the analysis target to phosphorylated tau fragments at a specific site (for example, in the above specific example, detection of various endogenous tau fragments including pT217 is expected), they can be analyzed with high sensitivity.

[0028] In the above-described embodiment, information on endogenous tau fragments is separately assigned, and the fragmentation rate and / or phosphorylation rate can be calculated from the peak intensity ratio between them. These fragmentation rates and / or phosphorylation rates are considered to reflect changes in the process of tau detachment from microtubules.

[0029] Here, the above-mentioned Objective I and Aspect A of Objective II will be explained with reference to FIG. 2. FIG. 2 is a diagram schematically showing tau protein isoforms and the epitope sequences of each anti-tau antibody clone. As shown in the upper part of FIG. 2, six isoforms of tau protein are known to exist: 2N4R (the amino acid sequence is shown in SEQ ID NO: 1 in the Sequence Listing. The amino acid sequences of each endogenous tau fragment described below are determined by counting the N-terminus of the amino acid sequence of SEQ ID NO: 1 as 1, and the position and amino acid sequence will be understood by those skilled in the art), 0N3R (the amino acid sequence is shown in SEQ ID NO: 2 in the Sequence Listing), 1N3R (the amino acid sequence is shown in SEQ ID NO: 3 in the Sequence Listing), 2N3R (the amino acid sequence is shown in SEQ ID NO: 4 in the Sequence Listing), 0N4R (the amino acid sequence is shown in SEQ ID NO: 5 in the Sequence Listing), and 1N4R (the amino acid sequence is shown in SEQ ID NO: 6 in the Sequence Listing). The amino acid sequences of each isoform are shown in Tables 1 and 2 below. Figure 2 also shows an enlarged view of the Mid region of tau protein, showing the positions of amino acids S (serine), T (threonine), and Y (tyrosine), which may be phosphorylated during post-translational modifications, as described below, and the positions of amino acids K (lysine) and R (arginine).

[0030]

[0031]

[0032] Suitable antibody clones (hereinafter sometimes simply referred to as "antibodies") include, for example, HT7, which has an epitope sequence of 159-163; BT2, which has an epitope sequence of 194-198; 77G7(R1), which has an epitope sequence of 268-271; 77G7(R2), which has an epitope sequence of 299-302; 77G7(R3), which has an epitope sequence of 330-333; and 77G7(R4), which has an epitope sequence of 362-365. These antibodies are known, and commercially available products can be used without particular restrictions. Here, an antibody is selected that has an epitope at a specific sequence within the region to be detected. HT7 is an antibody that does not contain an amino acid sequence that may be subject to post-translational modification as an epitope, and therefore can be used in both Objective I and Objective II, Aspect A. However, the BT2 epitope contains amino acids that can be phosphorylated, and the phosphorylation state of these sites may affect the binding properties of the antibody, so the target tau fragments that can be analyzed using BT2 are relatively limited.

[0033] Immunoprecipitation can be performed using conventionally known techniques. For example, after preparing antibody beads using magnetic beads and an antibody, the biological fluid as a sample is mixed with an equal volume of an IP reaction solution (composition: 0.04% DDM, 300 mM NaCl, 100 mM Tris-HCl (pH 7.4)), which is then mixed with the antibody beads and reacted by, for example, mixing by inversion at 4°C for 1 hour. Any conventionally known appropriate antibody beads can be used without any particular limitation, and specifically, magnetic beads such as Dynabeads （商標） A suitable example is M-270 Epoxy (manufactured by Thermo Fisher Science). After the immune reaction, this is mixed with the eluate to release the endogenous tau peptides bound to the antibody beads. The endogenous tau peptides in the eluate from which the endogenous tau peptides have been released are used as extracted endogenous tau fragments.

[0034] In the method of the present invention, the extracted endogenous tau fragments are then subjected to mass spectrometry and observed. In the method of the present invention, the extracted endogenous tau fragments are subjected to mass spectrometry directly, without additional fragmentation (such as trypsin digestion) as in conventional methods. The mass spectrometry method is not particularly limited, but MALDI (Matrix Assisted Laser Desorption / Ionization)-MS (quadrupole ion trap-TOF (Time-of-flight) type, TOF / TOF type) and LC-MS (quadrupole-TOF type, triple quadrupole type) can be suitably used. When combined with LC, the highly complex mixture of tau fragments can be selectively separated and concentrated, improving detectability. Because a certain tau peptide fragment and its corresponding phosphorylated peptide fragment have similar physicochemical properties, it is preferable to select LC conditions that allow them to be detected in the same fraction without separating them.

[0035] It is preferable to separate and remove antibodies and non-specifically adsorbed salts while leaving them in the extracted endogenous tau fragments under conditions that do not separate the endogenous tau fragments to be analyzed in the present invention, extracted in the IP step. Examples of the endogenous tau fragments to be analyzed include endogenous tau fragments with zero phosphorylation, one phosphorylation, and two phosphorylation (Aspect A of Objective II) and endogenous tau fragments of different lengths, including pT217 (Aspect B1 of Objective II). However, if internal standards for each endogenous tau fragment are prepared and added to correct for intensity, improved detection sensitivity and quantitative accuracy can be expected, and therefore the endogenous tau fragments may be separated by LC.

[0036] Next, the endogenous tau fragments are subjected to mass profiling to obtain the mass-to-charge ratio (m / z) and intensity information for each endogenous tau fragment. "Mass profiling" refers to a technique that uses a mass spectrometer to acquire the mass spectral pattern of molecules in a sample and analyze the resulting profile (characteristic pattern). Mass profiling primarily provides information on the mass-to-charge ratio (m / z) of molecules present in a sample and the relative abundance (peak intensity) of each molecule. When attempting to detect subtle differences between different samples, mass profiling allows for (i) comparison of the intensities of specific peaks, (ii) visual comparison of peak patterns, and (iii) quantitative comparison using statistical methods (principal component analysis, cluster analysis, etc.). Mass profiling can quantify protein states through relatively simple relative quantification. For example, comparing healthy and diseased groups may potentially reveal disease-related protein changes. Specifically, the signal intensity ratio of characteristic peaks can be calculated, and this can be used to determine the phosphorylation rate and / or fragmentation rate. From this perspective, "mass profiling" may be rephrased as "phosphorylation profiling," "fragmentation profiling," or "phosphorylation-fragmentation profiling," and the above-mentioned "profile" may be rephrased as "phosphorylation profile," "fragmentation profile," or "phosphorylation-fragmentation profile." Mass profiling may be affected by differences in ionization efficiency and fluctuations in measurement conditions, but more accurate quantification can be achieved by introducing an internal standard using a stable isotope label.

[0037] Mass profiling in the method of the present invention provides the following information: - Endogenous tau fragments containing the epitope sequence of the antibody used; - These fragments are a group of fragments of different lengths that share a partial sequence due to nonspecific cleavage (the observed peaks are the difference in amino acid mass); - Some of these fragments are endogenous tau fragments containing post-translational modifications. Information on the type and number of post-translational modifications and their partial locations can be obtained, and the type (unmodified, monophosphorylated, diphosphorylated, etc.) and their relative amounts can also be quantified. Note that, because this is a "relative amount," a known amount of internal standard may be added to the sample, and the peak intensities of each component may be corrected based on the peak intensity information derived from the internal standard. The signal intensity ratio of a specific phosphorylation cluster peak (unmodified, monophosphorylated, diphosphorylated, etc.) or a specific endogenous tau fragment peak can be calculated, and the state of the endogenous tau fragments can be quantified as the phosphorylation rate or fragmentation rate, which can be used to compare differences between samples (the signal intensity ratio of the above peaks may also be calculated after adding an internal standard and correcting for intensity). Here, the components are endogenous tau fragments and their post-translation modification-related ions, and the endogenous tau fragments contain the epitope sequence of the antibody used, undergo non-specific cleavage, and are a group of fragments of different lengths that share a partial sequence.

[0038] In the method of the present invention, the mass profiling results of the endogenous tau fragments are compared between samples to analyze differences in at least one of tau protein fragmentation and tau protein post-translational modification between samples. Here, "post-translational modification" includes at least one of tau protein phosphorylation, glycosylation, acetylation, methylation, dimethylation, ubiquitination, sumoylation, and oxidation, with phosphorylation being preferred. This is because tau phosphorylation is considered a promising diagnostic marker and has been widely reported. Furthermore, it is preferable that endogenous tau fragments contain one post-translational modification or a combination of two or more post-translational modifications. The analysis of differences may be performed between samples collected from the same subject at different time points, or between samples collected from different subjects. Analysis of such differences can be used as an indicator for estimating the pathological progression of tau-related neurodegenerative diseases. "Tau-related neurodegenerative diseases" that are used as indicators to estimate the progression of the disease include Alzheimer's disease (AD), progressive supranuclear palsy (PSP), corticobasal syndrome (CBS), and Pick's disease.

[0039] The process and mechanisms by which tau protein undergoes post-translational modification and fragmentation are not well understood. It is thought that tau protein phosphorylation and fragmentation progress as the disease progresses, but the detailed mechanism and causal relationship with the disease remain largely unknown. The relationship between phosphorylation and fragmentation is also poorly understood. How endogenous tau fragments are fragmented, which positions on endogenous tau fragments have post-translational modifications, and how many positions there are vary depending on the sample. Mass profiling obtains ion intensity information for endogenous tau fragments with slightly different lengths of N- or C-terminus containing epitope sequences, as well as the associated peaks derived from their phosphorylation.

[0040] Hereinafter, as specific embodiments, analysis of post-translational modifications of tau protein and analysis of fragmentation of tau protein will be described.

[0041] (Analysis of Post-translational Modification of Tau Protein) In one embodiment of the method of the present invention, the phosphorylation state of tau protein is quantitatively evaluated. Specifically, the procedure involves the following steps: 1. Using mass spectrometry, obtain mass-to-charge ratios (m / z) and intensity information for endogenous tau fragments derived from tau protein recovered by the IP method described in the above-mentioned aspect A of Objective II; 2. From the obtained mass spectrum, identify peaks corresponding to non-phosphorylated tau fragments and phosphorylated tau fragments in each phosphorylation state (monophosphorylation, diphosphorylation, etc.); 3. Using the intensity of each peak, calculate the phosphorylation rate using the following formula: Phosphorylation rate = phosphorylated peptide intensity / (non-phosphorylated peptide intensity + phosphorylated peptide intensity) 4. When multiple phosphorylation states exist, calculate the relative proportion of each state to create a phosphorylation profile. For example, when six peaks, from non-phosphorylated peptide to pentaphosphorylated peptide, are detected, and the intensities of each peak are I0, I1, I2, I3, I4, and I5, the formula is as follows: - non-phosphorylated rate = I0 / (I0 + I1 + I2 + I3 + I4 + I5) - monophosphorylated rate = I1 / A phosphorylation profile can be created as follows: (I0 + I1 + I2 + I3 + I4 + I5) - 2 phosphorylation rate = I2 / (I0 + I1 + I2 + I3 + I4 + I5) - 3 phosphorylation rate = I3 / (I0 + I1 + I2 + I3 + I4 + I5) - 4 phosphorylation rate = I4 / (I0 + I1 + I2 + I3 + I4 + I5) - 5 phosphorylation rate = I5 / (I0 + I1 + I2 + I3 + I4 + I5). This phosphorylation profile can be used to quantitatively evaluate changes in the phosphorylation pattern of tau protein between different samples.

[0042] (Tau Protein Fragmentation Analysis) In another embodiment of the present invention, the fragmentation pattern of tau protein is quantitatively evaluated. Specifically, the following steps are performed: 1. Pretreatment by immunoprecipitation is performed using the methods described in the above-mentioned Objective I and Objective II Aspect B, and endogenous tau fragments are directly analyzed by mass spectrometry without trypsin digestion; 2. Peaks corresponding to endogenous tau fragments of slightly different lengths that contain a common epitope are identified from the obtained mass spectrum. For example, multiple endogenous tau fragments containing a certain epitope sequence are detected; 3. The intensity of each endogenous tau fragment is used to calculate the relative fragmentation rate using the following formula: relative fragmentation rate = intensity of a specific endogenous tau fragment / total intensity of all detected endogenous tau fragments containing the same epitope; 4. If multiple endogenous tau fragments are present, the relative proportion of each endogenous tau fragment is calculated to create a fragmentation profile. For example, if eight endogenous tau fragments containing a certain epitope sequence are detected, the following can be calculated: Fragment 1 ratio = I1 / (I1 + I2 + I3 + I4 + I5 + I6 + I7 + I8) Fragment 2 ratio = I2 / (I1 + I2 + I3 + I4 + I5 + I6 + I7 + I8) Fragment 3 ratio = I3 / (I1 + I2 + I3 + I4 + I5 + I6 + I7 + I8) Fragment 4 ratio = I4 / (I1 + I2 + I3 + I4 + I5 + I6 + I7 + I8) Fragment 5 ratio = I5 / (I1 + I2 + I3 + I4 + I5 + I6 + I7 + I8) Fragment 6 ratio = I6 / (I1 + I2 + I3 + I4 + I5 + I6 + I7 + I8) Fragment 7 ratio = I7 / (I1 + I2 + I3 + A fragmentation profile can be generated as follows: (I4 + I5 + I6 + I7 + I8) - Fragment 8 ratio = I8 / (I1 + I2 + I3 + I4 + I5 + I6 + I7 + I8), where In (n is an integer between 1 and 8) represents the intensity of each endogenous tau fragment. This method allows for quantitative evaluation of subtle changes in the fragmentation pattern of tau protein. In particular, it can capture changes in the distribution of fragment lengths around each epitope.

[0043] Furthermore, by combining multiple antibodies that recognize specific sequences within the target region as epitopes, differences in fragmentation between those target regions can be assessed. For example, by combining antibodies that recognize the MID and MTBR regions, respectively (e.g., HT7 and 77G7), it is possible to compare endogenous tau fragments containing two different epitopes (HT7-MID and 77G7-MTBR) using the following formula (antibody clone 77G7 recognizes the repeat region within R1, R2, R3, and R4 of the 2N4R isoform of tau protein (see Figure 2)):

[0044] MID / MTBR fragmentation ratio = (total intensity of all fragments containing IMID region epitopes) / (total intensity of all fragments containing IMMTBR ​​region epitopes). These phosphorylation and / or fragmentation profiles enable the following analyses: Tracking changes in tau protein phosphorylation and / or fragmentation patterns as the disease progresses; Evaluating changes in tau protein phosphorylation and / or fragmentation patterns due to the effects of therapeutic drugs; and Comparing tau protein phosphorylation and / or fragmentation patterns between different patient groups. This method effectively analyzes endogenous tau fragments even when full-length tau protein is difficult to detect, enabling more sensitive phosphorylation and / or fragmentation analysis. Furthermore, by targeting only endogenous tau fragments containing common epitopes, highly specific analysis can be achieved.

[0045] Compared to conventional methods using isotope-labeled proteins / peptides, the method of the present invention offers the following advantages: 1. Cost-effectiveness: Since expensive stable isotope-labeled peptides are not required, analytical costs are significantly reduced. 2. Rapidity: Since the preparation of standard materials is not required, analytical preparation time is reduced. 3. Comprehensive analysis: Since multiple phosphorylation states and fragmentation patterns can be evaluated simultaneously, it is easier to understand the overall picture of post-translational modifications and fragmentation of tau protein. 4. High sensitivity: Since the relative ratios of endogenous tau fragments are used, analysis is possible even with small amounts of sample. 5. Flexibility: Even if new phosphorylation or fragmentation sites are discovered, new information can be obtained by reanalyzing existing data. These features make the method of the present invention a more effective and efficient approach than conventional methods for studying post-translational modifications and / or fragmentation of tau protein and for discovering biomarkers for neurodegenerative diseases. A stepwise approach, in which candidates are first narrowed down using relative quantification and then verified using absolute quantification, is also effective. It is believed that the method of the present invention is likely to be useful in the initial screening stage.

[0046] Here, FIG. 3 shows various examples of endogenous tau fragments according to Experimental Example 1 described below, with their start and end positions and [M+H] + The masses are shown by m / z values ​​(theoretical average masses). Figure 4 shows a specific example of a mass spectrum obtained as a result of mass analysis of the endogenous tau fragment shown in Figure 3 in Experimental Example 1 described later. Figure 3 shows the mass spectrum of 156-168 (the amino acid sequence of which is shown in SEQ ID NO: 7 in the Sequence Listing) ([M+H] + 1,167.3), 158-171 (amino acid sequence shown in SEQ ID NO: 8 in the sequence listing) ([M+H] + 1,409.6), 158-172 (amino acid sequence shown in SEQ ID NO: 9 in the Sequence Listing) ([M+H] + 1,506.7), 158-173 (amino acid sequence shown in SEQ ID NO: 10 in the sequence listing) ([M+H] + 1,577.8), 156-172 (amino acid sequence shown in SEQ ID NO: 11 in the sequence listing) ([M+H] +1,634.8), 157-173 (amino acid sequence shown in SEQ ID NO: 12 in the sequence listing) ([M+H] + 1,648.9), 156-173 (amino acid sequence shown in SEQ ID NO: 13 in the sequence listing) ([M+H] + 1,705.9), 157-180 (amino acid sequence shown in SEQ ID NO: 14 in the sequence listing) ([M+H] + 2,368.7), 156-180 (amino acid sequence shown in SEQ ID NO: 15 in the sequence listing) ([M+H] + 2,425.8), 156-189 (amino acid sequence shown in SEQ ID NO: 16 in the sequence listing) / 158-190 (amino acid sequence shown in SEQ ID NO: 17 in the sequence listing) ([M+H] + 3,275.7), 156-190 (amino acid sequence shown in SEQ ID NO: 18 in the sequence listing) ([M+H] + 3,403.9), 134-172 (amino acid sequence shown in SEQ ID NO: 19 in the sequence listing) / 155-193 (amino acid sequence shown in SEQ ID NO: 20 in the sequence listing) / 156-194 (amino acid sequence shown in SEQ ID NO: 21 in the sequence listing) ([M+H] + 3,819.3), 156-196 (amino acid sequence shown in SEQ ID NO: 22 in the sequence listing) ([M+H] + 3,963.4), 156-208 (amino acid sequence shown in SEQ ID NO: 23 in the sequence listing) ([M+H] + 5,038.5), 156-210 (amino acid sequence shown in SEQ ID NO: 24 in the Sequence Listing) / 146-198 (amino acid sequence shown in SEQ ID NO: 25 in the Sequence Listing) ([M+H] + 5,281.8), 153-221 (amino acid sequence shown in SEQ ID NO: 26 in the sequence listing) / 156-224 (amino acid sequence shown in SEQ ID NO: 27 in the sequence listing) / 158-225 (amino acid sequence shown in SEQ ID NO: 28 in the sequence listing) ([M+H] + 6,840.6), 122-202 (amino acid sequence shown in SEQ ID NO: 29 in the sequence listing) / 135-216 (amino acid sequence shown in SEQ ID NO: 30 in the sequence listing) / 136-217 (amino acid sequence shown in SEQ ID NO: 31 in the sequence listing) / 158-237 (amino acid sequence shown in SEQ ID NO: 32 in the sequence listing) ([M+H] +8,060.1), 157-190 (amino acid sequence shown in SEQ ID NO: 33 in the sequence listing) ([M+H] + 3,346.8), 158-190 (amino acid sequence shown in SEQ ID NO: 36 in the sequence listing) ([M+H] + The endogenous tau fragments (134-172 / 155-193 / 156-194) are shown. Here, the notation "134-172 / 155-193 / 156-194" means "134-172" or "155-193" or "156-194", but all of them are [M+H] + This indicates that the fragment is a tau fragment with m / z=3,819.3. In Figure 3, descriptions using " / " such as "134-172 / 155-193 / 156-194" indicate the position of an example fragment among the fragment candidates (representatively, candidates starting at 156 or 158). The amino acid sequences of each endogenous tau fragment shown in Figure 3 are shown in Tables 3 and 4 below.

[0047]

[0048]

[0049] In Figures 3 and 4, +1p indicates one phosphorylation site, +2p indicates two phosphorylations, +3p indicates three phosphorylations, +4p indicates four phosphorylations, and +5p indicates five phosphorylations (each phosphorylation (S / T / Y) increases the peak by 80 Da. In addition, post-translational modifications other than phosphorylation increase the peak by 42 Da for acetylation (K), 14 Da for methylation (K), and 28 Da for dimethylation (K). For example, comparing the 156-208 endogenous tau fragment with the 156-210 / 146-198 endogenous tau fragment, which differ by only two residues on the C-terminal side, results show that the phosphorylation states are different. In addition, the endogenous tau fragments 156-210 / 146-198 and 153-221 / 156-224 / 158-225 were found to have 0 to 4 and 0 to 2 phosphorylated S / T / Y residues, respectively, although it is unclear which S / T / Y residues were phosphorylated.In addition, the endogenous tau fragments 122-202 / 135-216 / 136-217 / 158-237 were found to have 0 to 5 phosphorylated S / T / Y residues, although it is unclear which S / T / Y residues were phosphorylated.

[0050] Figures 4(a), 4(b), and 5 show mass spectra obtained as a result of mass analysis of endogenous tau fragments according to Experimental Example 1. Figure 5 confirms triplet peaks at m / z 5281, 5361, and 5441, spaced 80 Da apart. These peaks are likely to represent phosphate-added endogenous tau fragments (156-210 / 146-198). Among the amino acid sequences shown in Figure 5, the amino acid sequence of 155-191, which is not shown in Figures 3 and 4, is shown in SEQ ID NO: 34, and the amino acid sequence of 156-193, which is not shown in Figures 3 and 4, is shown in SEQ ID NO: 35. The amino acid sequences of these endogenous tau fragments are also shown in Table 5 below.

[0051]

[0052] According to the method of the present invention, information on post-translational modifications (preferably phosphorylation) of each endogenous tau fragment can be obtained. Because trypsin digestion is not performed after collection of the biological fluid, no information is lost during trypsin digestion. Therefore, when comparing tau fragments that differ only by a few residues, results showing different phosphorylation states can be obtained. Furthermore, when it is believed that diverse phosphorylation and fragmentation are not occurring, it is possible to narrow down the phosphorylation sites and number from the differential information obtained by comparing endogenous tau fragments. Furthermore, the relative phosphorylation rate of unidentified sites may be used as an indicator. The relative phosphorylation rate can be determined, for example, by calculating the ion intensity (peak area) of the phosphorylated fragment relative to the sum of the ion intensities (peak areas) of the unphosphorylated and phosphorylated fragments.

[0053] The method of the present invention has the advantage of simplifying the pretreatment process and reducing the complexity of the analysis target. The method of the present invention can simplify the pretreatment process by subjecting endogenous tau fragments in biological fluids to analysis while retaining information on phosphorylation and fragmentation observed in the fragments. Furthermore, since the complexity of the analysis target is not increased, the method is not affected by the low reproducibility of the reaction in the enzymatic digestion process.

[0054] The method of the present invention also has the advantage of allowing for the simple measurement of the phosphorylation rate of a specific phosphorylation site, eliminating the need to design and synthesize a stable isotope-labeled peptide in advance, and eliminating the need to set site-specific detection conditions even when there are multiple phosphorylation sites.

[0055] In the method of the present invention, the endogenous tau fragments are extracted using immunoprecipitation, and the method may include a step of designing and selecting an epitope of an antibody to be used for immunoprecipitation, and a step of assigning peaks derived from the target endogenous tau fragment contained in the sample using the detection of internal fragment peaks as an index in mass profiling of the endogenous tau fragment group obtained by immunoprecipitation using the antibody.

[0056] Conventional immunoprecipitation-based specific extraction methods for biomolecules enable the detection of trace components in biological samples by combining highly specific antibodies with highly sensitive detection methods. However, in many cases, contaminants, such as nonspecific binding components, direct and indirect binding components, the antibody itself, and magnetic beads, are extracted in addition to the target component. These contaminants can potentially produce false-positive or false-negative results, reducing the accuracy and reproducibility of measurement results. Detection methods such as those combining mass spectrometry with LC can improve the detectability of target components by separating the extracted component mixture based on its physicochemical properties and mass-to-charge ratio. However, this requires the time-consuming task of attributing the peaks of the diverse mixture of components detected to contaminants and the target component, respectively.

[0057] The method includes the steps of designing and selecting an epitope for an antibody used in immunoprecipitation, and assigning peaks derived from the target endogenous tau fragment contained in a sample using the detection of internal fragment peaks as an indicator in mass profiling of endogenous tau fragments obtained by immunoprecipitation using the antibody, thereby achieving the following effects: - Reliable assignment of peaks derived from the target endogenous tau fragments: By designing and selecting an epitope for the antibody, peaks derived from the target endogenous tau fragments can be reliably assigned using internal fragment peaks as an indicator. In conventional methods, peak assignment was sometimes difficult due to the influence of contaminants. - Reduction of analysis time: By using internal fragment peaks from a mass spectrometer as an indicator, analysis using a database search or the like is no longer necessary, thereby reducing analysis time. In conventional methods, these analyses required a lot of time. - Reduction of analysis costs: The aforementioned reduction in analysis time allows for reduced analysis costs.

[0058] The following sequences are likely to produce internal fragments: those containing proline (P), those containing aspartic acid (D) and glutamic acid (E), and those containing lysine (K) and arginine (R). The N-terminal side of proline (P) is near the C-terminal side of aspartic acid (D) and glutamic acid (E) to (nearly equal) the vicinity of lysine (K) and arginine (R) > other amino acids. These sequences either have low peptide bond energy or have properties that promote peptide bond cleavage, making them more likely to produce internal fragments. However, these are general trends, and the actual ease of cleavage varies depending on the peptide sequence, experimental conditions, mass spectrometer, and fragmentation method used.

[0059] In the process of designing and selecting an epitope for an antibody to be used in immunoprecipitation, the design and selection are based on the likelihood of it being generated as an internal fragment in mass spectrometry. Specifically, when designing and selecting an epitope for an antibody, it is preferable to consider the following factors: - The structure and properties of the target component contained in the sample - The dissociation method and conditions for mass spectrometry. This allows for more sophisticated antibody epitope design and selection, and by designing and selecting an epitope that takes into account the structure and properties of the target endogenous tau fragment and the dissociation method and conditions for mass spectrometry, more reliable peak assignment and higher analytical sensitivity can be achieved.

[0060] In mass profiling of the endogenous tau fragment group obtained by immunoprecipitation using the antibody, a technique for improving the detection sensitivity of the internal fragment peaks (such as isotope labeling or chemical modification) may be used in the step of assigning peaks derived from the target endogenous tau fragments contained in the sample using the detection of internal fragment peaks as an index, thereby improving the detection sensitivity of the internal fragment peaks and enabling the detection of minute amounts of the target endogenous tau fragments.

[0061] Furthermore, in the mass profiling of the endogenous tau fragment group obtained by immunoprecipitation using the antibody, in the step of assigning peaks derived from the target endogenous tau fragment contained in the sample using the detection of internal fragment peaks as an indicator, a technique (such as a database search) for more reliably assigning peaks derived from the target endogenous tau fragment may be used, thereby further improving the reliability of peak assignment.

[0062] The present invention will be explained in more detail below by way of experimental examples, but the present invention is not limited to these examples.

[0063] Experimental Example 1: Endogenous tau fragments were extracted from 17 CSF (cerebrospinal fluid) samples (0.5 mL / sample) by IP treatment (using HT7 or BT2 as antibody clones), and mass spectrometry data was obtained by MALDI-MS. In the IP treatment, magnetic beads (Dynabeads) were used instead of antibody beads. （商標）The IP assay was prepared using M-270 Epoxy (Thermo Fisher Science) and each antibody clone. First, 0.5 mL of CSF was mixed with an equal volume of IP reaction solution (composition: 0.04% DDM, 300 mM NaCl, 100 mM Tris-HCl (pH 7.4)), and then mixed with antibody beads. The mixture was incubated at 4°C for 1 hour by inversion. After washing the antibody beads, the eluate was applied to a MALDI plate (2,5-Dihydrobenzoic acid (DHB) matrix). MALDI-MS was performed using ion-trap TOF-MS data for peak assignment and prediction, followed by high-sensitivity detection using linear TOF-MS data. A schematic diagram of the resulting list of representative endogenous tau fragments, showing the identified / predicted start and end amino acid sequence numbers, post-translational modifications, and their positional relationship to the epitope sequence of each antibody clone, is shown in Figure 3. Fragments are indicated by a / in the diagram. A / in the start and end amino acid sequence numbers indicates multiple candidates. The fragments 158-173, 156-173, 156-180, 156-189, 158-190, 156-190, 155-193, and 156-196 are tau fragments reported in Non-Patent Document 4. On the other hand, fragments 156-168, 158-171, 158-172, 156-172, 157-173, 134-172, 156-194, 156-208, 156-210, 146-198, 153-221, 156-224, 158-225, 122-202, 135-216, 136-217, and 158-237 represent unreported or phosphorylated fragments detected by this method. The mass spectra of some of these fragments are shown in Figures 4 and 5. Figure 4(a) shows a typical mass spectrum (m / z 1,000 - m / z 2,400) obtained by IP-MS of one of the samples. Figure 4(b) shows a mass spectrum (around m / z 8,200) obtained by IP-MS of one of the samples. This may represent endogenous tau fragments (122-202 / 135-216 / 136-217 / 158-237) that are non-phosphorylated and have one to five phosphate groups.Figure 5 shows the mass spectrum obtained by IP-MS of one of the samples, revealing triplet peaks at m / z 5281, 5361, and 5441, spaced 80 Da apart. These peaks may represent endogenous tau fragments (156-210 / 146-198) that are non-phosphorylated and mono- or diphosphate-added. Because there are many S / T / Y positions where phosphorylation can occur within each fragment, the positions of phosphorylation within each fragment have not been identified.

[0064] The assigned amino acid sequences were analyzed in a chain reaction, and many novel endogenous tau fragments (shown in Figures 3 to 5) were identified, including 156-168 ([M+H] + 1,167.3), 158-171 ([M+H] + 1,409.6), 158-172 ([M+H] + 1,506.7), 156-172 ([M+H] + 1,634.8), 157-173 ([M+H] + 1,648.9), 157-180 ([M+H] + 2,368.7), 134-172 / 155-193 / 156-194 ([M+H] + 3,819.3), 156-208 ([M+H] + 5,038.5), 156-210 ([M+H] + 5,281.8), 146-198 ([M+H] + 5,281.8), 153-221 ([M+H] + 6,840.6), 156-224 ([M+H] + 6,840.6), 158-225 ([M+H] + 6,840.6), 122-202 ([M+H] + 8,060.1), 135-216 ([M+H] + 8,060.1), 136-217 ([M+H] + 8,060.1), 158-237 ([M+H] + The amino acid sequence of the gene encoding the nucleotide sequence of ...

[0065] Experimental Example 2: As in Experimental Example 1, 0.5 mL of CSF was subjected to IP treatment (HT7 was designed and selected as the antibody clone) to extract tau fragments, and mass spectrometry data was acquired by MALDI-MS. Two of the resulting MS peaks, m / z 1,505.82 (158-172, SEQ ID NO: 9) and m / z 1,166.59 (156-168, SEQ ID NO: 7), were subjected to CID-MS / MS analysis. Figure 6(a) shows the results of CID-MS / MS analysis for m / z 1,505.82, and Figure 6(b) shows the results of CID-MS / MS analysis for m / z 1,166.59. Examination of the peaks obtained in each MS / MS spectrum confirmed that peaks derived from internal fragments (internal fragment peaks) corresponding to the epitope sequence (PPGQK) of antibody clone HT7 were preferentially detected. Proline (P) is a cyclic amino acid, and lysine (K) has a positive charge on the side chain. These amino acids are known to affect the strain and electronic state of the preceding and following peptide bonds, leading to their susceptibility to cleavage and internal fragmentation. Therefore, these (m / z 1505.82, m / z 1166.59) were determined to be endogenous tau fragments containing PPGQK in the internal sequence (presumably captured by antibody clone HT7). This easily confirmed that these were not contaminants, such as nonspecific binding components, direct or indirect binding components, the antibody itself, or magnetic beads, which may be contaminated during the immunoprecipitation process. Because the HT7 epitope sequence is readily observed as an internal fragment peak, it can be used as a screening indicator for endogenous tau fragments.

[0066] Aspects It will be understood by those skilled in the art that the exemplary embodiments and experimental examples described above are examples of the following aspects.

[0067] (Item 1) A method according to one embodiment is a mass spectrometry method for post-translational modification and / or fragmentation of tau protein in a biological fluid, comprising the steps of extracting endogenous tau fragments derived from tau protein from a biological fluid sample to obtain a group of endogenous tau fragments, mass analyzing the group of endogenous tau fragments to obtain the mass-to-charge ratio (m / z) and intensity information derived from each endogenous tau fragment, and mass profiling the group of endogenous tau fragments, and comparing the mass profiling results of the group of endogenous tau fragments between samples to analyze differences in at least one of the fragmentation of tau protein and the post-translational modification of tau protein between samples.

[0068] According to the method described in paragraph 1, modified and unmodified endogenous tau fragments containing post-translational modification sites can be stably detected without performing an additional fragmentation process on the endogenous tau fragments in a biological fluid.

[0069] (Item 2) The method according to item 1, wherein the endogenous tau fragments are extracted using immunoprecipitation, comprises the steps of designing and selecting an epitope for an antibody to be used for immunoprecipitation, and assigning peaks derived from the target endogenous tau fragment contained in the sample using the detection of internal fragment peaks as an indicator in mass profiling of the endogenous tau fragment group obtained by immunoprecipitation using the antibody.

[0070] According to the method described in the second paragraph, it is possible to reliably assign the peak of the target endogenous tau fragment, and it is also possible to shorten the analysis time and reduce the analysis cost.

[0071] (Item 3) In the method according to item 1, the post-translational modification is phosphorylation of tau protein.

[0072] According to the method described in Section 3, although the location of phosphorylation is unknown, the differences between fragments or the phosphorylation profile of specific fragments are expected to be effective biomarkers for determining the progression of tau-related neurodegenerative diseases.

[0073] (Item 4) In the method according to item 1, the endogenous tau fragment comprises one post-translational modification or a combination of two or more post-translational modifications.

[0074] According to the method described in item 4, the post-translational modification rate of a post-translational modification site can be easily measured, and even when there are multiple post-translational modification sites, it is not necessary to set site-specific detection conditions.

Claims

1. A mass spectrometry method for analyzing post-translational modification and / or fragmentation of tau protein in a biological fluid, comprising the steps of: extracting endogenous tau fragments derived from tau protein from a biological fluid sample to obtain a group of endogenous tau fragments; mass analyzing the group of endogenous tau fragments to obtain the mass-to-charge ratio (m / z) and intensity information derived from each endogenous tau fragment, and performing mass profiling of the group of endogenous tau fragments; and comparing the mass profiling results of the group of endogenous tau fragments between samples to analyze differences in at least one of tau protein fragmentation and post-translational modification of tau protein between samples.

2. The method of claim 1, wherein the endogenous tau fragments are extracted using immunoprecipitation, and the method comprises the steps of: designing and selecting an epitope for an antibody to be used in the immunoprecipitation; and assigning peaks derived from the target endogenous tau fragment contained in the sample using the detection of internal fragment peaks as an indicator in mass profiling of the endogenous tau fragment group obtained by immunoprecipitation using the antibody.

3. The method of claim 1, wherein the post-translational modification is phosphorylation of tau protein.

4. The method of claim 1, wherein the endogenous tau fragment comprises one post-translational modification or a combination of two or more post-translational modifications.

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