WO2025183053A1 - Mass spectrometry method for post-translational modification and/or fragmentation of tau protein in biological fluid - Google Patents

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

The present invention relates to a mass spectrometry method for post-translational modifications and/or fragmentation of tau protein in biological fluids.

Aggregates of abnormal tau protein (hereafter simply referred to as "tau") that accumulate within neurons are called neurofibrillary tangles (NFTs), and the group of neurodegenerative diseases that result from these NFTs are called tauopathies (tau-related neurodegenerative diseases). Typical tauopathies include Alzheimer's disease (AD), progressive supranuclear palsy, corticobasal degeneration, and Pick's disease. Tau protein is localized in the axons of neurons 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.

It is believed that as the pathology of tau-related neurodegenerative diseases progresses, tau protein phosphorylation and fragmentation progress. Therefore, analysis of these proteins in biological fluids is expected to be useful for early detection of the disease and assessing the effectiveness of treatment (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)).

In recent years, attention has been focused on phosphorylation of threonine residues at positions 181, 205, 217, and 231 of the amino acid sequence (threonine residues at each position from the N-terminus of the 2N4R isoform of tau protein, which has a full length of 441 amino acids (the amino acid sequence is shown in SEQ ID NO: 1 in the Sequence Listing)) 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), are being actively developed, and quantitative values of phosphorylated tau protein obtained using these methods, as well as the phosphorylation rate of each site (referring to the proportion of protein phosphorylated at a specific site), are being evaluated as biomarkers.

When analyzing phosphorylated tau using an immunoassay, quantification is performed using an antibody (anti-phosphorylation antibody) that recognizes the phosphorylation site being analyzed. 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 being analyzed. Therefore, the phosphorylated tau protein that can be captured and detected using 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 using 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. As a result, it is not possible to determine the phosphorylation rate.

On the other hand, when analyzing phosphorylated tau using LC-MS/MS, as shown in an example of the analysis flow in Figure 7, the captured tau protein (a mixture of full-length tau and fragmented or post-translationally modified tau fragments) is enzymatically digested (trypsin digested) to create a uniform peptide mixture. Next, the mixture is subjected to LC-MRM (Liquid Chromatography-Multiple Reaction Monitoring) to measure peptides containing the phosphorylation site being analyzed, thereby obtaining a quantitative value of phosphorylation (see, for example, Nicolas R. Barthelemy et al., "Tau Phosphorylation Rates Measured by Mass Spectrometry: Difference in the Intracellular Brain vs. Extracellular Cerebrospinal Fluid Compartments and Are Differentially Affected by See "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 calculated from the ion intensity (peak area) ratio of the non-phosphorylated peptide containing the phosphorylation site being analyzed to the phosphorylated peptide (Non-Patent Document 2).

When it comes to tau enrichment as a pretreatment for LC-MS/MS, it is not necessarily necessary to use anti-phosphorylation antibodies. Rather, immunoprecipitation (IP) using anti-tau antibodies (or a combination of multiple types) that do not recognize specific phosphorylation can be performed to more efficiently recover endogenous tau fragments that have undergone various fragmentation and post-translational modifications present in biological fluids and analyze them all at once. This method improves the depth of analytical measurement of phosphorylation and fragmentation. It is also effective to combine it with a site-nonspecific phosphorylation enrichment method (which does not use antibodies), such as IMAC (Immobilized Metal Affinity Chromatography), which can similarly efficiently capture many phosphorylation sites.

In previously reported analytical methods for phosphorylated tau, such as those 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 breaks down tau protein into endogenous tau fragments of uniform size, allowing for efficient detection of endogenous tau fragments containing the target post-translational modifications and endogenous tau fragments without post-translational modifications by mass spectrometry.

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 effects of phosphorylation, a tryptic peptide containing T181 (threonine at position 181) (181-190, miss cleavage 0) will undergo incomplete cleavage when T181 is phosphorylated (175-190, miss cleavage 1).

Due to the influence of the charge and three-dimensional structure of amino acid residues, there are cases where undigested fragments are relatively more likely to be produced. In the case of 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 production of semi-tryptic fragments.

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

Tau in biological fluids not only exists in six different 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, with the conventional methods described above, it is difficult to achieve reproducibility of the digestion process, and peptides containing specific phosphorylation sites become more diverse, resulting in reduced analytical sensitivity and making accurate quantification difficult.

In terms of detection, for example, tau protein is present in extremely small amounts in cerebrospinal fluid (reference concentration: approximately 100 pg/mL), and it is necessary to highly separate the complex tau fragments and then detect them with high sensitivity. 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, it is necessary to set MRM transitions corresponding to each phosphorylation site in order to quantify the phosphorylation rate and phosphate groups at each site, making this analysis cumbersome.

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

Anniina Snellman et al., “N-terminal and mid-region tau fragments as fluid biomarkers in neurological diseases”, BRAIN 2022:145;2834-2848 Nicolas R. Barthelemy et al., “Tau Phosphorylation Rates Measured by Mass Spectrometry Differ in the Intracellular Brain vs. Extracellular Cerebrospi nal Fluid Compartments and Are Differently Affected by Alzheimer’s Disease”, Frontiers in Aging Neuroscience, (2019), Volume 11, Article 121, p1-18 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), p661-669 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

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 additional fragmentation processes on endogenous tau fragments in biological fluids.

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

The present invention provides a mass spectrometry method that can stably detect modified and unmodified endogenous tau fragments containing post-translational modification sites without performing additional fragmentation treatment on endogenous tau fragments in biological fluids.

1 is a flow diagram illustrating an example of a method of the present invention. FIG. 1 is a diagram showing a schematic diagram of tau protein isoforms and antibody epitope sequences. FIG. 1 shows various examples of endogenous tau fragments according to Experimental Example 1, with their start and end positions and m/z values of [M+H] ⁺ (theoretical average mass). 4 shows a specific example of a mass spectrum obtained as a result of mass analysis of the endogenous tau fragment of FIG. 3 according to Experimental Example 1. 4 shows a specific example of a mass spectrum obtained as a result of mass analysis of the endogenous tau fragment of FIG. 3 according to Experimental Example 1. CID (Collision Induced Dissociation)-MS/MS analysis results for Experimental Example 2. This is an example of an analysis flow using the conventional IP/LC-MS method. FIG. 1 is a diagram schematically showing fragmentation of tau protein by trypsin digestion as described in Non-Patent Documents 2 and 3.

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 the mass-to-charge ratio (m/z) and intensity information derived from each endogenous tau fragment; and analyzing differences in at least one of tau protein fragmentation and post-translational modifications between samples by comparing the mass profiling results of the group of endogenous tau fragments. This method provides a mass spectrometry method capable of stably detecting modified and unmodified endogenous tau fragments containing post-translational modification sites without subjecting the endogenous tau fragments in a biological fluid to additional fragmentation treatment. The method of the present invention is believed to be 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.

Here, Figure 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 sample. In the present invention, a "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 processing collected whole blood. The biological fluid may be used directly to measure the concentration of a component, or may be pretreated as necessary before use to measure the concentration of a component.

Endogenous tau fragments can be extracted from biological fluids by immunoprecipitation (IP) using anti-tau antibodies, a conventionally known method.

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

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.

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).

(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.

The focus is on what type of fragmentation occurs in target regions that have post-translational modifications at specific sites. By limiting the analysis to phosphorylated tau fragments at specific sites (for example, in the above specific example, detection of a variety of endogenous tau fragments, including pT217, is anticipated), they can be analyzed with high sensitivity.

In the above-described embodiment, information on endogenous tau fragments is assigned separately, and the fragmentation rate and/or phosphorylation rate can be calculated from their peak intensity ratio. These fragmentation rates and/or phosphorylation rates are thought to reflect changes in the process of tau detaching from microtubules.

Here, aspect A of Objective I and Objective II will be explained using Figure 2. Figure 2 is a diagram schematically illustrating tau protein isoforms and the epitope sequences of each anti-tau antibody clone. As shown at the top of Figure 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 assuming that the N-terminus in the amino acid sequence of SEQ ID NO: 1 is 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 expanded 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).

Suitable antibody clones (hereinafter sometimes referred to simply as "antibodies") include, for example, HT7, whose epitope sequence is 159-163; BT2, whose epitope sequence is 194-198; 77G7(R1), whose epitope sequence is 268-271; 77G7(R2), whose epitope sequence is 299-302; 77G7(R3), whose epitope sequence is 330-333; and 77G7(R4), whose epitope sequence is 362-365. These antibodies are publicly known, and commercially available products can be used without particular restrictions. Here, an antibody is selected whose epitope is 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. On the other hand, the BT2 epitope contains amino acids that can be phosphorylated, and the phosphorylation state of this site may affect the binding properties of the antibody. Therefore, the target tau fragments that can be analyzed using BT2 are relatively limited.

Immunoprecipitation can be performed using conventional techniques. For example, antibody beads are prepared using magnetic beads and an antibody, and then the biological fluid 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. The mixture is then reacted, for example, by inversion at 4°C for 1 hour. Any conventionally known antibody beads can be used without particular limitation. A specific example of a suitable antibody bead is the magnetic beads Dynabeads ^™ M-270 Epoxy (manufactured by Thermo Fisher Science). After the immune reaction, the beads are mixed with an eluate to release 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.

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 directly to mass spectrometry without additional fragmentation (trypsin digestion, etc.) as in conventional methods. There are no particular limitations on the mass spectrometry method, 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, highly complex mixtures 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.

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 that are the subject of the present invention and that are extracted in the IP step. Examples of the endogenous tau fragments that are the subject of the present invention include endogenous tau fragments that are zero-phosphorylated, mono-phosphorylated, and di-phosphorylated (Aspect A of Objective II), and endogenous tau fragments of different lengths that contain 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 improved quantification accuracy can be expected, and therefore the endogenous tau fragments may be separated by LC.

Next, the endogenous tau fragments are subjected to mass analysis to obtain the mass-to-charge ratio (m/z) and intensity information from each endogenous tau fragment, thereby performing mass profiling of the endogenous tau fragments. "Mass profiling" refers to a technique that uses a mass spectrometer to obtain the mass spectral pattern of molecules in a sample and analyze that 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 enables (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 the state of proteins through relatively simple relative quantification, potentially identifying disease-related protein changes by comparing healthy and diseased groups, for example. Specifically, the signal intensity ratio of multiple characteristic peaks can be calculated, and this can be determined as the phosphorylation rate and/or fragmentation rate. From this perspective, "mass profiling" can be rephrased as "phosphorylation profiling," "fragmentation profiling," or "phosphorylation-fragmentation profiling," and the above-mentioned "profile" can be rephrased as "phosphorylation profile," "fragmentation profile," or "phosphorylation-fragmentation profile." Mass profiling can 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.

Mass profiling in the method of the present invention provides the following information:
- an endogenous tau fragment containing the epitope sequence of the antibody used;
- They are fragments of different lengths that share a partial sequence due to non-specific cleavage (the observed peaks are due to differences in the mass of amino acids),
Some of them are endogenous tau fragments containing post-translational modifications.
The present invention provides a method for quantifying the type and number of post-translational modifications and their relative positions. This method also provides information on the type (e.g., unmodified, monophosphorylated, diphosphorylated) and their relative amounts. Because this method involves "relative amounts," 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 between specific phosphorylation cluster peaks (e.g., unmodified, monophosphorylated, diphosphorylated) or specific endogenous tau fragment peaks is calculated, and this is used to quantify the state of endogenous tau fragments 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). The components are endogenous tau fragments and their post-translational modification-related ions. The endogenous tau fragments contain the epitope sequence of the antibody used, undergo nonspecific cleavage, and are a group of fragments of different lengths that share a partial sequence.

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 modifications between samples. Here, "post-translational modifications" include 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. Differences may be analyzed 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.

The process and mechanism by which tau protein undergoes post-translational modification and fragmentation are not well understood. It is thought that as the disease progresses, tau protein phosphorylation and fragmentation progress, but the detailed mechanism and causal relationship with the disease remain largely unknown. The relationship between phosphorylation and fragmentation is also not well understood. How endogenous tau fragments are fragmented, which parts of endogenous tau fragments have post-translational modifications, and how many parts have post-translational modifications vary depending on the sample. Mass profiling obtains ion intensity information for groups of 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.

Below, specific embodiments will be described, including analysis of post-translational modifications of tau protein and analysis of tau protein fragmentation.

(Post-translational modification analysis of tau protein)
In one embodiment of the method of the present invention, the phosphorylation state of tau protein is quantitatively evaluated. Specifically, the method comprises the following steps:
1. Obtaining mass-to-charge ratio (m/z) and intensity information of endogenous tau fragments derived from tau protein recovered by the IP method described in the above-mentioned Objective II, Mode A by mass spectrometry;
2. Identifying peaks corresponding to non-phosphorylated tau fragments and phosphorylated tau fragments of each phosphorylation state (monophosphorylation, diphosphorylation, etc.) from the obtained mass spectrum;
3. Using the intensity of each peak, calculate the phosphorylation rate using the following formula:
Phosphorylation rate = intensity of phosphopeptide/(intensity of non-phosphorylated peptide + intensity of phosphopeptide)
4. If multiple phosphorylation states exist, calculate the relative proportion of each state and generate a phosphorylation profile.
For example, when six peaks from a non-phosphorylated peptide to a five-phosphorylated peptide are detected, and the intensities of the peaks are I0, I1, I2, I3, I4, and I5, the following is obtained:
- Unphosphorylated ratio = I0 / (I0 + I1 + I2 + I3 + I4 + I5)
- 1 Phosphorylation rate = I1 / (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)
Using this phosphorylation profile, it is possible to quantitatively evaluate changes in the phosphorylation pattern of tau protein between different samples.

(Tau protein fragmentation analysis)
In another embodiment of the present invention, the fragmentation pattern of tau protein is quantitatively evaluated, specifically by the following steps:
1. Pretreatment by immunoprecipitation using the methods described in the above-mentioned Objective I and Objective II, Mode B, and direct analysis of endogenous tau fragments by mass spectrometry without trypsin digestion;
2. From the obtained mass spectrum, peaks corresponding to endogenous tau fragments of slightly different lengths containing a common epitope are identified. For example, multiple endogenous tau fragments containing a certain epitope sequence are detected.
3. Using the intensity of each endogenous tau fragment, calculate the relative fragmentation rate using the following formula:
Relative fragmentation rate = intensity of a specific endogenous tau fragment / total intensity of all endogenous tau fragments containing the same epitope detected 4. If multiple endogenous tau fragments are present, calculate the relative proportion of each endogenous tau fragment to create a fragmentation profile. For example, if eight endogenous tau fragments containing a certain epitope sequence are detected, the following will be calculated:
- Fragment 1 rate = I1 / (I1 + I2 + I3 + I4 + I5 + I6 + I7 + I8)
- Fragment 2 rate = I2 / (I1 + I2 + I3 + I4 + I5 + I6 + I7 + I8)
- Fragment 3 rate = I3 / (I1 + I2 + I3 + I4 + I5 + I6 + I7 + I8)
- Fragment 4 rate = I4 / (I1 + I2 + I3 + I4 + I5 + I6 + I7 + I8)
- Fragment 5 rate = I5 / (I1 + I2 + I3 + I4 + I5 + I6 + I7 + I8)
- Fragment 6 rate = I6 / (I1 + I2 + I3 + I4 + I5 + I6 + I7 + I8)
- Fragment 7 rate = I7 / (I1 + I2 + I3 + I4 + I5 + I6 + I7 + I8)
- Fragment 8 rate = I8 / (I1 + I2 + I3 + I4 + I5 + I6 + I7 + I8)
A fragmentation profile can be created as follows: 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.

Furthermore, by combining multiple antibodies that recognize specific sequences within the region of interest (target region) as epitopes, it is possible to evaluate differences in fragmentation between those target regions. 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 fragment groups containing two different epitopes (HT7-MID and 77G7-MTBR) using the following formula (the recognition sequence of antibody clone 77G7 is the repeat region within R1, R2, R3, and R4 of the tau protein 2N4R isoform (see Figure 2)).

MID/MTBR fragmentation ratio = (total intensity of all fragments containing IMID region epitopes)/(total intensity of all fragments containing IMTB region epitopes)
These phosphorylation and/or fragmentation profiles can be used to analyze:
This method enables the following: 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 analysis of phosphorylation and/or fragmentation. Furthermore, by targeting only endogenous tau fragments containing common epitopes, highly specific analysis can be achieved.

The method of the present invention has the following advantages over conventional methods using isotope-labeled proteins/peptides:
1. Cost-effectiveness: The absence of expensive stable isotope-labeled peptides significantly reduces analytical costs. 2. Rapidity: The absence of the need for standard preparation reduces analytical preparation time. 3. Comprehensive Analysis: The ability to simultaneously evaluate multiple phosphorylation states and fragmentation patterns facilitates understanding the overall picture of tau protein post-translational modifications and fragmentation. 4. High Sensitivity: By utilizing the relative ratios of endogenous tau fragments, analysis is possible even with minute 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 enable the method of the present invention to provide a more effective and efficient approach than conventional methods for studying tau protein post-translational modifications and/or fragmentation 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 in biomarker discovery. It is believed that the method of the present invention is likely to be useful in the initial screening stage.

Here, Fig. 3 shows various examples of endogenous tau fragments by their start and end positions and m/z values (theoretical average mass) of [M+H] ⁺ according to Experimental Example 1, which will be described later. Fig. 4 shows specific examples of mass spectra obtained as a result of mass analysis of the endogenous tau fragments of Fig. 3 according to Experimental Example 1, which will be described later. 3 shows the amino acid sequence of 156-168 (the amino acid sequence is shown in SEQ ID NO:7 in the Sequence Listing) ([M+H] ⁺ 1,167.3), 158-171 (the amino acid sequence is shown in SEQ ID NO:8 in the Sequence Listing) ([M+H] ⁺ 1,409.6), 158-172 (the amino acid sequence is shown in SEQ ID NO:9 in the Sequence Listing) ([M+H] ⁺ 1,506.7), 158-173 (the amino acid sequence is shown in SEQ ID NO:10 in the Sequence Listing) ([M+H] ⁺ 1,577.8), 156-172 (the amino acid sequence is shown in SEQ ID NO:11 in the Sequence Listing) ([M+H] ⁺ 1,634.8), 157-173 (the amino acid sequence is shown in SEQ ID NO:12 in the Sequence Listing) ([M+H] ⁺ 1,648.9), and 156-173 (the amino acid sequence is 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 (the amino acid sequence is shown in SEQ ID NO:22 in the Sequence Listing) ([M+H] ⁺ 3,963.4), 156-208 (the amino acid sequence is shown in SEQ ID NO:23 in the Sequence Listing) ([M+H] ⁺ 5,038.5), 156-210 (the amino acid sequence is shown in SEQ ID NO:24 in the Sequence Listing) / 146-198 (the amino acid sequence is shown in SEQ ID NO:25 in the Sequence Listing) ([M+H] ⁺ 5,281.8), 153-221 (the amino acid sequence is shown in SEQ ID NO:26 in the Sequence Listing) / 156-224 (the amino acid sequence is shown in SEQ ID NO:27 in the Sequence Listing) / 158-225 (the amino acid sequence is shown in SEQ ID NO:28 in the Sequence Listing) ([M+H] ⁺ The endogenous tau fragments shown are 122-202 (the amino acid sequence is shown in SEQ ID NO:29 in the Sequence Listing)/135-216 (the amino acid sequence is shown in SEQ ID NO:30 in the Sequence Listing)/136-217 (the amino acid sequence is shown in SEQ ID NO:31 in the Sequence Listing)/158-237 (the amino acid sequence is shown in SEQ ID NO:32 in the Sequence Listing) ([M+H] ⁺ 8,060.1), 157-190 (the amino acid sequence is shown in SEQ ID NO:33 in the Sequence Listing) ([M+H] ⁺ 3,346.8), and 158-190 (the amino acid sequence is shown in SEQ ID NO:36 in the Sequence Listing) ([M+H] ⁺ 3,275.7). Here, the notation "134-172/155-193/156-194" refers to "134-172,""155-193," or "156-194," all of which indicate a tau fragment with [M+H] ⁺ m/z = 3,819.3. In Figure 3, notations 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.

Furthermore, 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 (the peak increases by 80 Da for each phosphorylation (S/T/Y). In addition to phosphorylation, 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, the fragments differ by only two residues on the C-terminus, but the results show different phosphorylation states. Furthermore, although it is unclear which S/T/Y residues are phosphorylated in the endogenous tau fragments 156-210/146-198 and 153-221/156-224/158-225, results showed that they were phosphorylated at 0 to 4 and 0 to 2 sites, respectively. Furthermore, although it is unclear which S/T/Y residues are phosphorylated in the endogenous tau fragments 122-202/135-216/136-217/158-237, results showed that they were phosphorylated at 0 to 5 sites.

Figures 4(a), 4(b), and 5 show mass spectra obtained as a result of mass analysis of endogenous tau fragments in Experimental Example 1. Figure 5 confirms triplet peaks at m/z 5281, 5361, and 5441, spaced 80 Da apart. These are likely to be phosphate-added endogenous tau fragments (156-210/146-198). Of 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 shown in Table 5 below.

The method of the present invention makes it possible to obtain information on post-translational modifications (preferably phosphorylation) for each endogenous tau fragment. Because trypsin digestion is not performed after collecting the biological fluid, no information is lost during trypsin digestion, and when comparing tau fragments that differ only by a few residues, results showing different phosphorylation states can be obtained. Furthermore, if 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 non-phosphorylated and phosphorylated fragments.

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 simplifies the pretreatment process by preserving information on phosphorylation and fragmentation observed in endogenous tau fragments in biological fluids and subjecting them to analysis. Furthermore, because it does not increase the complexity of the analysis target, it is not affected by the low reaction reproducibility of the enzymatic digestion process.

Furthermore, the method of the present invention has the advantage of being able to easily measure the phosphorylation rate of a phosphorylation site. There is no need to design or synthesize a stable isotope-labeled peptide in advance to determine the phosphorylation rate of a specific phosphorylation site, and even when there are multiple phosphorylation sites, there is no need to set site-specific detection conditions.

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 for an antibody to be used in the 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.

Traditional 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, in addition to the target component, contaminant components such as non-specific binding components, direct and indirect binding components, the antibody used itself, and magnetic beads are also extracted. These contaminant components can produce false positive and false negative results, reducing the accuracy and reproducibility of measurement results. Detection methods that combine mass spectrometry with LC, for example, can improve the detectability of target components by separating the extracted component mixture based on physicochemical properties and mass-to-charge ratio. However, the peak groups of the detected diverse mixture components must be assigned to contaminant components and the target component, respectively, which is a time-consuming process.

The method includes a step of designing and selecting an epitope of an antibody used in immunoprecipitation, and a step of assigning peaks derived from the target endogenous tau fragment contained in a sample using detection of an internal fragment peak as an index in mass profiling of an endogenous tau fragment group obtained by immunoprecipitation using the antibody, thereby achieving the following:
- Reliable assignment of peaks derived from the target endogenous tau fragments By designing and selecting the epitope of the antibody, peaks derived from the target endogenous tau fragments can be reliably assigned using internal fragment peaks as indicators. With conventional methods, peak assignment was sometimes difficult due to the influence of contaminant components. - Reduced analysis time By using the internal fragment peaks of the mass spectrometer as indicators, analysis using database searches is no longer necessary, shortening analysis time. With conventional methods, these analyses took a long time. - Reduced analysis costs The aforementioned shortened analysis time has the effect of reducing analysis costs.

An array like this:
・Sequences containing proline (P) ・Sequences containing aspartic acid (D) and glutamic acid (E) ・Sequences containing lysine (K) and arginine (R) N-terminal side of proline (P) > C-terminal side of aspartic acid (D) and glutamic acid (E) to (nearly equal) around lysine (K) and arginine (R) > Other amino acids are thought to be more likely to produce internal fragments because they have low peptide bond energy or properties that promote peptide bond cleavage. However, these are general trends, and the actual ease of cleavage varies depending on the entire peptide sequence, experimental conditions, mass spectrometer used, fragmentation method, etc.

In the process of designing and selecting an epitope of an antibody to be used for immunoprecipitation, the design and selection are based on the criterion that the epitope is likely to be generated as an internal fragment in mass spectrometry. Specifically, when designing and selecting an epitope of an antibody, the following factors are taken into consideration:
It is preferable to take into consideration the structure and properties of the target components contained in the sample, and the dissociation method and conditions for mass spectrometry. This will enable more sophisticated antibody epitope design and selection, and epitope design and selection that takes into account the structure and properties of the target endogenous tau fragment and the dissociation method and conditions for mass spectrometry will enable more reliable peak assignment and higher analytical sensitivity.

In mass profiling of endogenous tau fragments obtained by immunoprecipitation using the antibody, techniques for improving the detection sensitivity of internal fragment peaks (isotope labeling, chemical modification, etc.) may be used in the process of assigning peaks derived from the target endogenous tau fragments contained in the sample using the detection of internal fragment peaks as an indicator. This improves the detection sensitivity of internal fragment peaks, making it possible to detect minute amounts of the target endogenous tau fragments.

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) may be used to more reliably assign peaks derived from the target endogenous tau fragment. This can further improve the reliability of peak assignment.

The present invention will be explained in more detail below using experimental examples, but the present invention is not limited to these.

<Experimental Example 1>
Endogenous tau fragments were extracted from 17 CSF samples (0.5 mL per sample) using IP (antibody clone HT7 or BT2) and mass spectrometry (MALDI-MS) data were acquired. For IP, antibody beads were prepared using magnetic beads (Dynabeads ^™ 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 the antibody beads were mixed and reacted at 4°C for 1 hour by inversion. After washing the antibody beads, the eluate was applied to a MALDI plate (matrix: DHB (2,5-Dihydrobenzoic acid)). MALDI-MS was performed by prior peak assignment and estimation using ion trap TOF-MS data, followed by high-sensitivity detection using linear TOF-MS data. A schematic diagram showing the identified/estimated start and end amino acid sequence numbers, post-translational modifications, and the positional relationship between each antibody clone's epitope sequence and the representative endogenous tau fragments obtained is shown in Figure 3. Each fragment is indicated by a / in the diagram. The / symbol in the start and end amino acid sequence numbers indicates that there are multiple candidates. 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. 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 the mass spectrum (around m/z 8,200) obtained by IP-MS of one of the samples. This may represent endogenous tau fragments with unphosphorylated and one to five phosphate groups (122-202/135-216/136-217/158-237). Figure 5 shows the mass spectrum obtained by IP-MS of one of the samples. Triplet peaks at m/z 5281, 5361, and 5441, spaced 80 Da apart, were identified. This may represent endogenous tau fragments with unphosphorylated and one to two phosphate groups (156-210/146-198). Because there are many possible S/T/Y phosphorylations within each fragment, the positions of phosphorylation within each fragment have not been identified.

The assigned amino acid sequences were analyzed in a chain reaction, revealing numerous novel endogenous tau fragments (shown in Figures 3 to 5), 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 ^... The amino acid sequences of 122-202 ([M+H] ⁺ 8,060.1), ^135-216 ([M+H] ⁺ 8,060.1), 136-217 ([M+H] ⁺ 8,060.1), and 158-237 ( ^[ M+H] ⁺ 8,060.1) ^were identified ^and predicted.

<Experimental Example 2>
As in Experiment 1, 0.5 mL of CSF was subjected to IP (antibody clone HT7 was designed and selected) to extract tau fragments, and mass spectrometry data were 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 analyzed by CID-MS/MS. Figure 6(a) shows the results of CID-MS/MS analysis for m/z 1,505.82, and Figure 6(b) shows the results for m/z 1,166.59. Examination of the peaks obtained in each MS/MS spectrum confirmed that peaks derived from internal fragments corresponding to the epitope sequence (PPGQK) of antibody clone HT7 (internal fragment peaks) 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.

[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.

(Section 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, and includes 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 the fragmentation of tau protein and the post-translational modification of tau protein between samples.

The method described in paragraph 1 makes it possible to stably detect modified and unmodified endogenous tau fragments containing post-translational modification sites without subjecting endogenous tau fragments in biological fluids to additional fragmentation treatment.

(Section 2)
The method according to paragraph 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 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 index in mass profiling of the endogenous tau fragment group obtained by immunoprecipitation using the antibody.

The method described in Section 2 enables reliable assignment of peaks to the target endogenous tau fragments, and also shortens analysis time and reduces analysis costs.

(Section 3)
Item 1. The method according to item 1, wherein the post-translational modification is phosphorylation of tau protein.

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

(Section 4)
2. 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.

The method described in paragraph 4 allows for easy measurement of the post-translational modification rate at a post-translational modification site, and even when there are multiple post-translational modification sites, there is no need to set site-specific detection conditions.

Claims (4)

extracting endogenous tau fragments derived from tau protein from a biological fluid sample to obtain a group of endogenous tau fragments;
performing mass profiling of the endogenous tau fragment group by subjecting the endogenous tau fragment group to mass analysis to obtain mass-to-charge ratio (m/z) and intensity information from each endogenous tau fragment;
and a step of analyzing differences in at least one of tau protein fragmentation and tau protein post-translational modification between samples by comparing the mass profiling results of the endogenous tau fragment group between samples. The extraction of endogenous tau fragments is carried out using immunoprecipitation,
Designing and selecting epitopes for antibodies used in immunoprecipitation;
The method of claim 1, further comprising a step of assigning peaks derived from the target endogenous tau fragment contained in the sample using detection of internal fragment peaks as an indicator in mass profiling of the endogenous tau fragment group obtained by immunoprecipitation using the antibody. The method of claim 1, wherein the post-translational modification is phosphorylation of tau protein. The method of claim 1, wherein the endogenous tau fragment contains one post-translational modification or a combination of two or more post-translational modifications.