Methods for assessing the efficacy of treatment protocols for neurodegenerative diseases

Abstract

The present invention relates to a method for assessing the efficacy of a treatment of a neurodegenerative disease in a subject, wherein the presence of said proteolytic peptides is assayed using mass spectrometry with reference to corresponding labeled and / or unlabeled reference proteolytic peptides. The present invention also relates to a method for stratifying a subject having a neurodegenerative disease, a method for diagnosing a neurodegenerative disease, a method for treating a subject having a neurodegenerative disease, and a kit for assessing the efficacy of a first line treatment of a neurodegenerative disease in a subject.

Description

[Technical field]

[0001] The present invention relates to a method for assessing the efficacy of treatment protocols for neurodegenerative diseases using a multi-targeted proteomics-based assay. [Background technology]

[0002] Neurodegenerative diseases are a heterogeneous group of conditions characterized by progressive degeneration of the function and structure of the peripheral or central nervous system (CNS). The prevalence of these diseases is increasing in many countries, making their effective treatment all the more important. One route to study the therapeutic course of these diseases is to examine biomarkers that are easily obtained in biological samples from patients (Heywood et al., Molecular Neurodegeneration, 10, 64 (2015)).

[0003] Currently, ELISA, SIMOA and other immunoassays are often used to measure a limited number of protein biomarkers in neurodegenerative diseases, and discovery proteomics is sometimes used to measure multiple proteins simultaneously, but all of these technology platforms have significant limitations. Summary of the Invention [Problem to be solved by the invention]

[0004] There are two main technical challenges to overcome. First, there is a lack of a comprehensive biomarker panel that measures multiple biomarkers associated with neurodegenerative diseases. Second, building a technology platform that measures these multiple biomarkers in a single test in an accurate and reproducible manner. Existing analytical technology platforms lack precision, reproducibility, and multiplexing capabilities.

[0005] Speed ​​of deployment is also an issue. Creating a new antibody can take six to nine months, in addition to the time needed to develop an immunoassay after the antibody is available. Targeted proteomics assays can be developed in three to four months, an acceptable lead time for most clinical trials.

[0006] A previous study by Heywood et al. (2015) showed that targeted proteomics in Parkinson's disease could be useful for identifying biomarkers for the disease. However, the paper did not address the concept of identifying biomarkers to evaluate the efficacy of treatment protocols for Parkinson's disease.

[0007] The present invention provides a method for evaluating the efficacy of a treatment protocol for neurodegenerative diseases using a multi-targeted proteomic assay. Advantageously, biomarkers are obtained from cerebrospinal fluid (CSF). Thus, the test provided by the present invention can monitor the therapeutic effect of new therapies currently under development for neurodegenerative diseases such as Parkinson's disease. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] According to a first aspect of the present invention there is provided a method of assessing the efficacy of a treatment for a neurodegenerative disease in a subject, the method comprising: (i) preparing a biological sample from a subject for the assay by incubating the sample with a protease to form a proteolytic digest of the proteins in the sample; and (ii) assaying the proteolytic digest of the protein of step (i) for the presence of proteolytic peptides of at least one protein selected from the group of proteins shown in Table 1, said group including matrix metalloprotease-9, chitinase-3-like protein 1, protein S100-A8, protein S100-A9, neutrophil collagenase (MMP8), complement C3, galectin-3 binding protein, catalase, extracellular superoxide dismutase, prosaposin, β-heterozygosyltransferase ... xosaminidase subunit β, cathepsin D, beclin-1, lysosomal-associated membrane glycoprotein 1, autophagy protein 13, E3 ubiquitin protein ligase RNF26, E3 ubiquitin protein ligase TRIM33, FAST kinase domain-containing protein 5, mitochondrial, apoptosis-inducing factor 1 mitochondrial, transmembrane protein 126A, neurofilament light polypeptide, chromogranin A, contactin-1, neurexin-1-β, protein MTSS Metastasis suppressor YGL-1, synaptotagmin-3, apolipoprotein E, apolipoprotein D, clusterin, flotillin-2, cadherin-2, neural cell adhesion molecule 2, amyloid-β precursor protein, amyloid-like protein 1, peptidyl prolyl cis-trans isomerase FKBP3, peptidyl prolyl cis-trans isomerase FKBP4, kallikrein-6, neutrophil gelatinase-associated lipocalin, protein disulfide isomerase A1 (PDIA1), limbic system-associated membrane protein (fragment), ceruloplasmin, serotransferrin, vacuolar protein sorting-associated protein 16 homolog (hVPS16), peptidyl prolyl cis-trans isomerase (PPIase; HEL-S-39), lymphocyte cytoplasmic protein 2, lactadherin, 72 kDa It consists of type IV collagenase, transforming growth factor-β-inducible protein IG-H3, neuronal proliferation and differentiation control protein 1, and ganglioside GM2 activator. wherein the presence of said proteolytic peptides is assayed using mass spectrometry with reference to corresponding labeled and / or unlabeled reference proteolytic peptides.

[0009] The treatment of neurodegenerative diseases can include the administration of a pharmaceutical composition comprising a pharma- ceutical active substance. The administration of the pharmaceutical composition can be part of a prescribed course of treatment or a clinical trial. The pharmaceutical composition can be administered once a day, twice a day, once a week, or at other frequencies according to the prescribed course of treatment or clinical trial protocol. The duration of the clinical trial can be one month, two months, six months, or twelve months.

[0010] The efficacy of treatment can be an indication of the efficacy of treatment with a generally suitable method for neurodegenerative disease. The efficacy can be expressed by the presence or absence of one or more clinical symptoms or one or more side effects of the pharmaceutical composition used to treat the subject. Thus, the efficacy of treatment can also be correlated with the change in protein biomarker concentration in a sample containing protein, as shown in Table 1. The change in the relevant biomarker concentration as defined herein can be an increase or decrease in the biomarker concentration compared to the baseline or control value of the biomarker.

[0011] Absolute peptide concentrations can be compared between a set of proteins in samples. Samples can be taken at different time intervals from the start of treatment (i.e., at week 0 ("week zero")). Subsequent samples can be taken from patients at about week 20 and / or week 45. The results of samples obtained at different time points can be normalized to baseline, and the results can be expressed as relative changes from baseline. For example, a 1.5-fold increase in peptide concentration or a 0.67-fold decrease in concentration from baseline in the drug-treated group, while no or less corresponding peptide change in the placebo group, would indicate drug treatment efficacy of the pharmaceutical composition comprising the drug under evaluation by the method of the present invention.

[0012] Subsequent samples may be taken from the patient at about weeks 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 or 30, and / or weeks 35, 40, 45, 50, 55 or 60.

[0013] Subsequent samples may be taken from the patient at about 1, 2, 3, 4, 5, 6, and / or 7, 8, 9, 10, 11, 12 months. Thereafter, samples may be taken from the patient at about six month intervals. Subsequent samples may be taken from the patient at about six and / or 12 months.

[0014] An increase in peptide concentration of at least 1.5-fold and / or a decrease in concentration of at least 0.67-fold from baseline in the drug treatment group, while there is no or little corresponding change in peptide in the placebo group, is considered to be indicative of drug treatment efficacy of the pharmaceutical composition comprising the drug under evaluation according to the methods of the present invention.

[0015] An increase in peptide concentration of at least 1.1 fold, at least 1.2 fold, at least 1.3 fold, at least 1.4 fold, at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1.8 fold, at least 1.9 fold or at least 2.0 fold, and / or a decrease in peptide concentration of at least 0.9 fold, at least 0.8 fold, at least 0.7 fold, at least 0.6 fold, at least 0.67 fold, at least 0.5 fold, at least 0.4 fold, at least 0.3 fold, at least 0.2 fold, or at least 0.1 fold in the drug treatment group, while there is no or less corresponding peptide change in the placebo group, is considered to be indicative of drug treatment efficacy of a pharmaceutical composition comprising the drug being evaluated according to the methods of the present invention.

[0016] The neurodegenerative disease may be selected from the group consisting of Parkinson's disease, Alzheimer's disease, Huntington's disease, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), Batten disease, or a transmissible spongiform encephalopathy (e.g., scrapie, bovine spongiform encephalopathy (BSE) or Creutzfeldt-Jakob disease (CJD), (including aberrant, variant, familial and sporadic CJD), a lysosomal neurodegenerative disease (such as Gaucher disease). The neurodegenerative disease may be Parkinson's disease.

[0017] As described herein, the method of the present invention can be applied to a wide range of neurodegenerative diseases, such as Alzheimer's disease, and other diseases with a neurodegenerative component, such as rare diseases / inborn errors of metabolism / lysosomal storage diseases, etc. Testing by the method of the present invention can also be applied to inflammatory diseases, such as Cystatin C in renal disease.

[0018] The sample may be a cerebrospinal fluid (CSF), blood (serum, whole blood (venous or peripheral), plasma, urine, tears, saliva, interstitial fluid, lymph, or tissue sample. The sample may be cerebrospinal fluid (CSF). The sample may be collected by any suitable means, including collection of spinal fluid by lumbar puncture, collection of venous blood by direct collection from a vein, or collection by finger prick blood collection device, blood spot card, or plasma spot card.

[0019] The same proteins can also be measured by different technology platforms. The same set of proteins can be measured using ELISA, SIMOA, Olink, Western blot, or other immunoassay platforms. Aptamers (oligonucleotide or peptide molecules that bind to specific target molecules) can be used in place of antibodies in similar assays.

[0020] A variety of sample types can be used for this test. Currently, the test has been validated with clear CSF samples, but different biofluid samples such as serum, plasma, etc. with various sample additives (anticoagulants) may be suitable. This includes K2 and K3 EDTA plasma tubes, heparin, potassium oxalate / sodium fluoride treated plasma tubes, etc.

[0021] Proteases include serine proteases (e.g., trypsin, chymotrypsin, thrombin, elastase, or subtilisin), cysteine ​​proteases (e.g., papain, caspase-1, adenine, pyroglutamic peptidase I, sortase A), hepatitis C virus peptidase 2, Sindbis virus type nsP2 peptidase, dipeptidyl peptidase VI, DeSI-1 peptidase, TEV protease, amidophosphoribosyltransferase precursor, gamma-glutamyl hydrolase, hedgehog protein , dmpA aminopeptidase), threonine protease (such as ornithine acetyltransferase), aspartic protease (such as pepsin, cathepsin D, cathepsin E, napsin A, nepenthesin, presenilin, renin (chymosin), etc.), glutamic protease (such as skitalid glutamic peptidase (aequorysin), aspergilloglutamic peptidase), metalloprotease (such as ADAM or matrix metalloprotease), or asparagine peptide lyase. The protease can be trypsin.

[0022] Thus, a proteolytic peptide is a peptide sequence derived from a protein, generated by the action of a protease cleaving the peptide bonds between amino acids in a protein sequence. Such a proteolytic peptide is thus an oligopeptide formed from a number of amino acids. A proteolytic peptide may be 5-30 amino acid residues in length, preferably 6-20 amino acids in length, 7-25 amino acids in length, or 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids in length.

[0023] Proteolytic peptides prepared by the action of the protease trypsin on proteins are sometimes called tryptic peptides.

[0024] The proteolytic peptides preferably have sequences as shown in Table 1.

[0025] The methods of the invention may include assaying for the presence of all up to 50 proteins in Table 1 for their proteolytic peptides. In some embodiments, the methods of the invention may include assaying for the presence of proteolytic peptides of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, or 50 proteins as shown in Table 1.

[0026] Thus, the method of the present invention includes measuring up to 118 proteolytic peptides from the 50 proteins shown in Table 1. In some embodiments, the method of the present invention may include assaying for at least 5 to 10, 5 to 15, 5 to 20, 5 to 25, 5 to 30, 5 to 35, 5 to 40, 5 to 45, or 5 to 50 proteolytic peptides from Table 1. Preferably, the number of proteolytic peptides assayed is at least 5, at least 15, at least 20, or at least 25 proteolytic peptides as shown in Table 1. The number of proteolytic peptides assayed may be 48 proteolytic peptides as shown in Table 1. When assaying the 48 proteolytic peptides shown in Table 1, the proteolytic peptide may be one proteolytic peptide from each protein in Table 1, except for protein MTSS 1 (Metastasis suppressor YGL-1) and lymphocyte cytoplasmic protein 2.

[0027] In case one or more proteins and / or proteolytic peptides are assayed, the method may comprise assaying a proteolytic digest of the protein of step (i) for the presence of a panel of proteolytic peptides of at least two proteins selected from the group of proteins set out in Table 1. Preferably, the method may comprise assaying a proteolytic digest of the protein of step (i) for the presence of a panel of proteolytic peptides of at least 48 proteins, wherein the 48 proteins are as follows: Matrix metalloproteinase-9 Chitinase-3-like protein 1 Protein S100-A8 Protein S100-A9 Neutrophil collagenase 8 (MMP8) Complement C3 Galectin-3 binding protein Catalase Extracellular superoxide dismutase Prosaposin β-hexosaminidase subunit β Cathepsin D Beclin-1 Lysosome-associated membrane glycoprotein 1 Autophagy Protein 13 E3 ubiquitin protein ligase RNF26 The E3 ubiquitin protein ligase TRIM33 FAST kinase domain-containing protein 5, mitochondrial Apoptosis-inducing factor 1, mitochondrial Transmembrane protein 126A Neurofilament Light Polypeptide Chromogranin A Contactin-1 Neurexin-1β Synaptotagmin-3 Apolipoprotein E Apolipoprotein D Clusterin Flotillin-2 Cadherin-2 Neural cell adhesion molecule 2 Amyloid β precursor protein Amyloid-like protein 1 Peptidylprolyl cis-trans isomerase FKBP3 Peptidylprolyl cis-trans isomerase FKBP4 Kallikrein-6 Neutrophil gelatinase-associated lipocalin Protein disulfide isomerase A1 (PDIA1) Limbic system-associated membrane protein (fragment) Ceruloplasmin Serotransferrin Vacuolar protein sorting-related protein 16 homologue (hVPS16) Peptidylprolyl cis-trans isomerase (PPIase; HEL-S-39) Lactadherin 72kDa type IV collagenase Transforming growth factor-β-induced protein IG-H3 Neural proliferation and differentiation control protein 1 and Ganglioside GM2 activator.

[0028] Table 1 lists 50 proteins differentially expressed in Parkinson's disease patients following drug treatment, along with the corresponding 118 proteolytic peptide sequences and the corresponding heavy isotope-labeled peptide internal standard sequences. Table 1 also lists hemoglobin subunit β as an optional analytical control protein, along with the corresponding proteolytic peptide sequences and the corresponding heavy isotope-labeled peptide internal standard sequences.

[0029] To analyze the expression of any protein according to the method of the present invention, an internal standard sequence may not be required. For example, a single protein internal standard such as yeast enolase can be used for the entire biomarker test without using heavy isotope-labeled internal standards. For quantification of peptides that do not have an internal standard, other heavy isotope-labeled internal standards with similar retention times can be used. There are examples of targeted proteomics methods that do not use any internal standards, such as the University College Dublin prostate cancer test.

[0030] In one embodiment, the method of the present invention may suitably comprise assaying for six proteolytic peptides in the following protein and corresponding proteolytic peptide groups: JPEG2024510734000001.jpg44170

[0031] The at least one protein may be selected from the group consisting of chromogranin-A, amyloid beta precursor protein, amyloid-like protein 1, transforming growth factor beta-induced protein IG-H3, prosaposin, and apolipoprotein E. The at least one protein may be selected from the group consisting of chromogranin-A, amyloid beta precursor protein, amyloid-like protein 1, transforming growth factor beta-induced protein IG-H3, and prosaposin.

[0032] The proteolytic peptides of the at least one protein may be selected from the group consisting of: SGELEQEEER (SEQ ID NO:52) QQLVETHMAR (SEQ ID NO:78) QMYPELQIAR (SEQ ID NO: 85) GDELADSALEIFK (SEQ ID NO: 114) QEILAALEK (SEQ ID NO:23) and AATVGSLAGQPLQER (sequence number 64).

[0033] The proteolytic peptides of the at least one protein may be selected from the group consisting of: SGELEQEEER (SEQ ID NO:52) QQLVETHMAR (SEQ ID NO:78) QMYPELQIAR (SEQ ID NO: 85) GDELADSALEIFK (SEQ ID NO: 114) and QEILAALEK (sequence number 23).

[0034] In one embodiment, the at least one protein is chromogranin A and the proteolytic peptide of the at least one protein is SGELEQEEER (SEQ ID NO:52).

[0035] In one embodiment, the at least one protein is an amyloid-β precursor protein and the proteolytic peptide of the at least one protein is QQLVETHMAR (SEQ ID NO: 78).

[0036] In one embodiment, the at least one protein is amyloid-like protein 1 and the proteolytic peptide of the at least one protein is QMYPELQIAR (SEQ ID NO: 85). The internal standard may be QMYPELQIAR(U-13C6,15N4)VEQAT (SEQ ID NO: 162).

[0037] In one embodiment, the at least one protein is transforming growth factor beta-induced protein IG-H3 and the proteolytic peptide of the at least one protein is GDELADSALEIFK (SEQ ID NO: 114).

[0038] In one embodiment, the at least one protein is prosaposin and the proteolytic peptide of the at least one protein is QEILAALEK (SEQ ID NO: 23). The internal standard may be QEILAALEK(U-13C6,15N2)GCSFL (SEQ ID NO: 132).

[0039] In one embodiment, the at least one protein is apolipoprotein E and the proteolytic peptide of the at least one protein is AATVGSLAGQPLQER (SEQ ID NO: 64). The internal standard may be AATVGSAGQPLQER(U-13C6,15N4)AQAWG (SEQ ID NO: 151).

[0040] In the method of the invention, mass spectrometry is used to identify the concentration of peptides, for example proteolytic peptides having sequences as shown in Table 1. In a mass spectrometry (MS) system for analyzing proteolytic peptides in a sample, the proteolytic peptides are injected into the MS system, where they are first detected as intact peptides and then fragmented into smaller pieces called peptide fragments. The method of the invention provides for the detection of up to 118 proteolytic peptides of up to 50 proteins, as shown in Table 1. The concentrations of the peptides can be used to assess the efficacy of treatments for neurodegenerative diseases and / or to stratify subjects with neurodegenerative diseases, with reference to a calibration curve of concentrations for known reference proteolytic peptides.

[0041] Thus, the concentration of proteolytic peptides in a sample is relevant for evaluating the efficacy of a treatment for a neurodegenerative disease and / or for stratifying subjects with a neurodegenerative disease. In general, a difference in the concentration of proteolytic peptides from a control (either an increase or decrease) indicates the efficacy of a treatment for a neurodegenerative disease and / or the stratification of subjects with a neurodegenerative disease.

[0042] The control may be a baseline control. The control may be a placebo group. The concentration of the proteolytic peptide may be normalized to the control. The concentration may be expressed as a relative fold change to the control, such as a fold change from baseline.

[0043] The method of the present invention can further describe whether proteolytic peptides are up-regulated or down-regulated herein. For example, in clinical trials, biomarker levels may differ between different patient groups; i.e., may be increased or decreased compared to other groups (such as control or placebo groups). Combinations of biomarkers can be used to separate patient groups using principal component analysis (PCA) or fold change differences based on biomarker concentrations. Fold change differences can be used to separate patient groups using thresholds or cut-off points, for example as shown in Figures 6 and 7.

[0044] The reference peptides can be used to pre-configure the mass spectrometer before use in the method of the present invention to detect and quantify the concentration of the peptides of Table 1 in a sample. The reference peptides also allow a calibration line to be constructed with each batch of samples tested to ensure reliable results. The heavy isotope-labeled peptides can be used as internal standards to control the analytical variability of each sample and can also provide a calibration curve.

[0045] According to the present invention, a mass spectrometry platform is used to measure the concentration of the analyte. For this test, a triple quadrupole mass spectrometry platform operating in MRM mode is preferred, however, other mass spectrometry platforms and other data acquisition modes can be used to design around this test. As an example, high resolution mass spectrometry platforms (such as Sciex 6600, Thermo Orbitrap, Waters QTOF type instruments) can use "pseudo" MRM or PRM modes for the measurements. Triple quadrupole instruments are more robust and provide more reproducible data, so other platforms may not achieve the same performance in all aspects of the test.

[0046] There are also a variety of chromatography systems available that can be coupled to mass spectrometry platforms. The test is designed to use a "standard flow" (ml / min) liquid chromatography system, but can also use low flow systems in the μl / min or nl / min range.

[0047] The peptide ionization interface / method prior to mass analysis may also vary: although this test is designed to use electrospray ionization (ESI), peptides can be ionized using matrix-assisted laser desorption ionization (MALDI), desorption electrospray ionization (DESI), atmospheric pressure chemical ionization (APCI), or other ionization methods.

[0048] Different internal standards can be used on the same targeted proteomics platform: although the test is designed to use heavy isotope-labeled internal standards for accurate measurements, different labeled or unlabeled peptide or protein internal standards can be used, which may or may not meet the performance of heavy isotope-labeled standards.

[0049] The method may include determining a relative concentration of the proteolytic peptide measured in the sample. The relative concentration of the peptide measured in the sample may refer to the relative concentration of the proteolytic peptide relative to a corresponding labeled and / or unlabeled reference proteolytic peptide.

[0050] All of the tests described herein can be multiplexed with another test that measures proteins in biological samples, such as CSF proteins, using the same technology platform. Thus, the test of the present invention can be part of a larger test. When other therapeutic efficacy tests emerge, the test of the present invention can be used to augment such other tests, improving overall performance.

[0051] The proteins from which the peptide sequences are derived are set out herein in Table 1. The method of the invention may be a multiplex assay.

[0052] As provided herein, different sets of peptides than those shown in Table 1 can be measured using the same targeted proteomics platform and from the same proteins as shown in Table 1 using different proteases. The examples of the invention described herein illustrate one embodiment of the methods of the invention, but different peptides from the same set of 50 proteins may be used, including peptides generated using the same protease (trypsin) as in this test or different proteases (LysC, GluC, etc.).

[0053] Typically, the mass spectrometric analysis of peptides according to the methods of the invention is liquid chromatography targeted mass spectrometry (LC-MS) using a triple quadrupole instrument operated in timed multiple reaction monitoring (MRM) mode.

[0054] Thus, the method of the present invention involves the use of targeted proteomics. This technique involves the quantification of specific, preselected proteins or proteolytic peptides from a given sample, and requires an existing understanding of disease biology to guide the selection of proteins. Thus, this technique differs from discovery proteomics, which seeks to gather information on all proteins or proteolytic peptides in a sample without any preexisting knowledge or hypotheses about disease biology. Discovery proteomics does not allow the use of internal standards or calibration lines for each protein or peptide of interest. Discovery proteomics is also performed using different instrument operation modes, such as SWATH and HDMSE, than MRM and PRM in targeted proteomics. Data processing also uses different approaches to signal normalization and quantification when absolute concentrations cannot be provided. Discovery proteomics platforms lack the robustness and reproducibility of targeted proteomics platforms. Targeted and discovery proteomics are different in that a team of scientists using a discovery proteomics platform would generally not be able to develop a targeted proteomic biomarker test without specific knowledge and experience in targeted proteomics, due to the differences in each step of the process, from the initial concept, sample preparation, data collection and processing, to the final test implementation at the clinical site.

[0055] The method of the invention is carried out using a proteolytic reference peptide, but can also be configured to use any suitable internal standard peptide on the same targeted proteomics platform. The method of the invention can be suitably used with heavy isotope-labeled internal standards for accurate measurements. Examples of internal standards as heavy isotope-labeled proteolytic peptides are given in Table 1 for the proteolytic peptides described therein. The internal standards can be added to the sample before proteolytic digestion of the proteins in the sample occurs in step (i) of the method of the invention. Alternatively, the internal standards can be added to the sample after proteolytic digestion of the proteins in the sample occurs in step (i) of the method of the invention. The mass spectrometer can be pre-configured with said internal standards. Suitable heavy isotope labels include: 13 C. 15 N, and / or 2 It's H.

[0056] In one embodiment, the method of the present invention can be used to evaluate the efficacy of treatment regimens for Parkinson's disease. The test measures the concentration of 118 peptides arising from 50 CSF proteins, as shown in Table 1. Several proteins described herein have not previously been associated with Parkinson's disease and have not been reported as biomarkers of drug treatment efficacy in Parkinson's disease (PD). The protein set covers all biochemical aspects of Parkinson's disease pathology and includes assays for: inflammation; oxidative stress; autophagy / lysosome; ubiquitin-proteasome system (UPS); mitochondria; axonal / neuronal degeneration; synaptic degeneration; lipoprotein metabolism; enhanced exosome release; endothelial dysfunction; amyloid processing and other proteins associated with PD. The test also includes hemoglobin as a control to monitor whether the CSF is not contaminated with blood.

[0057] The method of the invention comprises computer-implemented steps and may comprise a computer-controlled apparatus. The step of assaying the proteolytic digest of the protein for the presence of proteolytic peptides in step (i) may be performed by a computer-controlled apparatus.

[0058] The present invention also provides a computer-implemented method for assessing the effectiveness of a treatment for a neurodegenerative disease in a subject, the method comprising: receiving sample data representing a level of at least one proteolytic peptide in a sample obtained from the subject by a computer; running software on the computer for comparing the level of the at least one proteolytic peptide in the sample with a baseline control, wherein a difference between the level of the at least one proteolytic peptide and the baseline control indicates the effectiveness of the treatment for the neurodegenerative disease; and outputting efficacy data representing the effectiveness of the treatment for the neurodegenerative disease based on the comparison.

[0059] The present invention also provides a computer program comprising instructions which, when executed by a computer, cause the computer to carry out the computer-implemented method of the present invention.

[0060] It will be understood that the step of comparing the level of at least one proteolytic peptide in a sample with a baseline control may be performed on a computer different from the computer that originally received the data representing the at least one proteolytic peptide in the sample.

[0061] The present invention also provides a computer device for evaluating the effectiveness of a treatment for a neurodegenerative disease, the computer device comprising: a first device incorporating a computer; a second computer; and a communication channel for transmitting data between the first device and the second computer; wherein the first device is configured to receive sample data representative of a level of at least one proteolytic peptide in a sample obtained from a subject and transmit the sample data to the second computer via the communication channel; and the second computer is configured to execute software for comparing the level of at least one proteolytic peptide in the sample with a baseline control to determine the effectiveness of the treatment for the neurodegenerative disease, wherein a difference between the level of the at least one proteolytic peptide and the baseline control indicates the effectiveness of the treatment for the neurodegenerative disease, and outputting efficacy data representative of the effectiveness of the treatment for the neurodegenerative disease based on the comparison.

[0062] The second computer may be arranged to transmit the efficacy data over a communication path to the first device or to a third computer.

[0063] In some embodiments, the first device may incorporate a mass spectrometry device or instrument for measuring the level of at least one proteolytic peptide in a sample.

[0064] The test is built on the only mass spectrometry platform that meets existing regulatory standards, making it suitable for ongoing clinical testing. Uniquely, the test is able to simultaneously measure 118 peptides in the most robust manner possible. Every peptide measured has a dedicated calibration curve and internal standard, providing absolute concentrations and correcting for analytical signal variations. The CSF sample preparation protocol has also been experimentally optimized to detect this specific group of proteins.

[0065] This test allows for rapid iteration of protein sets that can include newly discovered molecules, which also allows the assay to be tailored to specific therapies, patient populations, and neurodegenerative disease (sub)types.

[0066] The present invention provides the first comprehensive targeted proteomic biomarker test for evaluating the therapeutic efficacy of novel therapies under development for neurodegenerative diseases such as Parkinson's disease.

[0067] The testing system provided by this invention can be commercialized in a standardized "assay kit" format, providing an end-to-end solution for customers to install in their own laboratories rather than simply sending samples to a centralized testing facility.

[0068] The methods of the present invention can be used as biofluid tests to evaluate the therapeutic efficacy of new therapies under development or existing / repurposed therapies for the treatment of neurodegenerative diseases, such as Parkinson's disease, including in clinical, preclinical development, and pharmacovigilance.

[0069] Figure 6 shows the concentration changes of 48 biomarkers. It shows that the biomarker signatures of the three patient groups (placebo, medium dose treatment group, and high dose treatment group) are different: most biomarkers decrease over time in the placebo group, whereas most biomarkers increase over time in both drug treatment groups. Therefore, any number of biomarkers from this 48 biomarker panel can be used to detect molecular level changes after drug treatment in a placebo-controlled randomized clinical trial, indicating that the present invention can be used as a tool to evaluate the efficacy of drug treatment.

[0070] The method of the present invention can be used as a biofluid test to stratify patient populations with neurodegenerative diseases, such as Parkinson's disease, according to their response to treatment. Such a method may serve as a companion or complementary diagnostic test, or as a tool for precision medicine in clinical trials, preclinical trials, or after therapeutic approval.

[0071] According to a second aspect of the present invention there is provided a method of stratifying a subject having a neurodegenerative disease, the method comprising: (i) preparing a biological sample from a subject for the assay by incubating the sample with a protease to form a proteolytic digest of the proteins in the sample; and (ii) assaying the proteolytic digest of the protein of step (i) for the presence of proteolytic peptides of at least one protein selected from the group of proteins shown in Table 1, said group including matrix metalloprotease-9, chitinase-3-like protein 1, protein S100-A8, protein S100-A9, neutrophil collagenase (MMP8), complement C3, galectin-3 binding protein, catalase, extracellular superoxide dismutase, prosaposin, β-heterozygosyltransferase ... xosaminidase subunit β, cathepsin D, beclin-1, lysosomal-associated membrane glycoprotein 1, autophagy protein 13, E3 ubiquitin protein ligase RNF26, E3 ubiquitin protein ligase TRIM33, FAST kinase domain-containing protein 5, mitochondrial, apoptosis-inducing factor 1 mitochondrial, transmembrane protein 126A, neurofilament light polypeptide, chromogranin A, contactin-1, neurexin-1-β, protein MTSS Metastasis suppressor YGL-1, synaptotagmin-3, apolipoprotein E, apolipoprotein D, clusterin, flotillin-2, cadherin-2, neural cell adhesion molecule 2, amyloid-β precursor protein, amyloid-like protein 1, peptidyl prolyl cis-trans isomerase FKBP3, peptidyl prolyl cis-trans isomerase FKBP4, kallikrein-6, neutrophil gelatinase-associated lipocalin, protein disulfide isomerase A1 (PDIA1), limbic system-associated membrane protein (fragment), ceruloplasmin, serotransferrin, vacuolar protein sorting-associated protein 16 homolog (hVPS16), peptidyl prolyl cis-trans isomerase (PPIase; HEL-S-39), lymphocyte cytoplasmic protein 2, lactadherin, 72 kDa It consists of type IV collagenase, transforming growth factor-β-inducible protein IG-H3, neuronal proliferation and differentiation control protein 1, and ganglioside GM2 activator. wherein the presence of said proteolytic peptides is assayed using mass spectrometry with reference to the corresponding labeled and / or contactin reference proteolytic peptides; and (b) stratifying the subjects according to the relative concentrations of the peptides measured in the samples.

[0072] To stratify patient groups following drug treatment, the trajectories of biomarkers (i.e., peptides) within drug-treated patient groups can be compared. All patients whose biomarker signal increased (i.e., the concentration of the assayed biomarker protein increased) can be stratified into a first subgroup, while all patients whose biomarker signal decreased or whose biomarker signal did not change significantly (i.e., the concentration of the assayed biomarker protein decreased or there was no change in concentration) can be classified into a second subgroup. The change in concentration (either increase or decrease) can be relative to a baseline control value or can be measured over a period of time (e.g., 20 weeks or 45 weeks).

[0073] The relative concentration of a peptide measured in a sample may refer to the relative concentration of the peptide relative to the corresponding labeled and / or unlabeled reference proteolytic peptides.

[0074] Stratifying subjects according to the relative concentrations of peptides measured in the samples may include separating subjects into two or more groups according to the relative concentrations of peptides measured in the samples. The relative concentrations used to stratify and / or group subjects may be any suitable time point, such as baseline, 20 weeks and / or 45 weeks after drug administration. Any suitable threshold value may be used to stratify and / or group subjects. For example, subjects may be stratified into two groups based on having a relative concentration of peptides that is higher or lower than the average relative concentration of peptides of all stratified subjects. The average value may be the mean, median, or mode. Alternatively, subjects may be stratified into two groups based on whether the relative concentration of peptides is higher or lower than a threshold value. The threshold value may be predetermined. The threshold value may be any suitable value, such as 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14 or 0.15.

[0075] Figure 7 shows the concentration changes over time of 48 biomarkers in five patients diagnosed with Parkinson's disease and administered a novel Parkinson's drug in clinical development. The figure shows the fold change from baseline in biomarker concentrations after 6 and 12 months. Patients 2 and 4 (most biomarkers decrease after treatment) respond differently than patients 3 and 5 (biomarkers increase after treatment). The current expert consensus in the Parkinson's disease field is that Parkinson's disease is a heterogeneous disease both clinically and at the molecular level, and therefore patients respond differently to drug treatment. Furthermore, this specific drug is expected to elicit different responses in different Parkinson's disease patients as a result of its multimodal mechanism of action. Thus, the biomarker results shown in this figure indicate that the present invention provides a practical tool (e.g., CSF biomarker testing) to identify these different patient groups and thus can be applied to patient stratification.

[0076] The method may comprise assaying the proteolytic digest of the protein of step (i) for the presence of a proteolytic peptide of at least one protein selected from the group consisting of E3 ubiquitin protein ligase TRIM33, fast kinase domain-containing protein 5, mitochondrial and contactin-1. The proteolytic peptide of E3 ubiquitin protein ligase TRIM33 may be LFCETCDR (SEQ ID NO: 40). The proteolytic peptide of FAST kinase domain-containing protein 5, mitochondrial may be LAVQFTNR (SEQ ID NO: 41). The proteolytic peptide of contactin-1 may be IVESYQIR (SEQ ID NO: 54).

[0077] The method may comprise assaying a proteolytic digest of the protein of step (i) for the presence of a proteolytic peptide of the contactin protein, wherein the protein is the E3 ubiquitin protein ligase TRIM33 and the proteolytic peptide may be LFCETCDR (SEQ ID NO: 40).

[0078] The method may comprise assaying a proteolytic digest of the protein of step (i) for the presence of at least one proteolytic peptide of the protein, wherein the protein is Fast kinase domain-containing protein 5, mitochondrial and the proteolytic peptide may be LAVQFTNR (SEQ ID NO: 41).

[0079] The method may comprise assaying a proteolytic digest of the protein of step (i) for the presence of at least one proteolytic peptide of the protein, wherein the protein is contactin-1 and the proteolytic peptide may be IVESYQIR (SEQ ID NO:54).

[0080] The methods of the invention can be used as biofluid tests to help evaluate the safety of new or existing treatments in development for neurodegenerative diseases, such as Parkinson's disease, including treatments in clinical or preclinical development.

[0081] The methods of the invention can be used as biofluid tests to compare the severity and other aspects of neurodegenerative diseases, e.g., Parkinson's disease, as a result of different etiologies (different genetic mutations, sporadic, early onset, different toxins, other diseases, e.g., Gaucher disease, etc.).

[0082] The methods of the present invention can be used as biofluid tests for population studies to determine which subgroups of the population will develop milder or more severe Parkinson's disease, including age, different geographic regions, diets, comorbidities, etc.

[0083] The method of the present invention can be used as a biofluid test to elucidate the mechanism of action of novel therapeutic agents under development.The method of the present invention can be used as a biofluid test as a reference method / benchmark for future trials for neurodegenerative diseases, such as Parkinson's disease, including cross-validation during the trial development stage.

[0084] Some of the tests described herein may be used in the future as diagnostic tests for neurodegenerative diseases, such as Parkinson's disease. Some of the tests described herein may be used in clinical practice to stratify Parkinson's disease patient populations according to disease stage / severity. Some of the tests described herein may be used as prognostic / predictive tools to determine an individual's risk of developing neurodegenerative diseases, such as Parkinson's disease, and more severe forms / stages of the disease.

[0085] According to a third aspect of the present invention there is provided a method for diagnosing a neurodegenerative disease, the method comprising: (i) preparing a biological sample from a subject for the assay by incubating the sample with a protease to form a proteolytic digest of the proteins in the sample; and (ii) assaying the proteolytic digest of the protein of step (i) for the presence of proteolytic peptides of at least one protein selected from the group of proteins shown in Table 1, said group including matrix metalloprotease-9, chitinase-3-like protein 1, protein S100-A8, protein S100-A9, neutrophil collagenase (MMP8), complement C3, galectin-3 binding protein, catalase, extracellular superoxide dismutase, prosaposin, β-heterozygosyltransferase ... xosaminidase subunit β, cathepsin D, beclin-1, lysosomal-associated membrane glycoprotein 1, autophagy protein 13, E3 ubiquitin protein ligase RNF26, E3 ubiquitin protein ligase TRIM33, FAST kinase domain-containing protein 5, mitochondrial, apoptosis-inducing factor 1 mitochondrial, transmembrane protein 126A, neurofilament light polypeptide, chromogranin A, contactin-1, neurexin-1-β, protein MTSS Metastasis suppressor YGL-1, synaptotagmin-3, apolipoprotein E, apolipoprotein D, clusterin, flotillin-2, cadherin-2, neural cell adhesion molecule 2, amyloid-β precursor protein, amyloid-like protein 1, peptidyl prolyl cis-trans isomerase FKBP3, peptidyl prolyl cis-trans isomerase FKBP4, kallikrein-6, neutrophil gelatinase-associated lipocalin, protein disulfide isomerase A1 (PDIA1), limbic system-associated membrane protein (fragment), ceruloplasmin, serotransferrin, vacuolar protein sorting-associated protein 16 homolog (hVPS16), peptidyl prolyl cis-trans isomerase (PPIase; HEL-S-39), lymphocyte cytoplasmic protein 2, lactadherin, 72 kDa type IV collagenase, transforming growth factor beta-induced protein IG-H3, neuronal proliferation and differentiation control protein 1, ganglioside GM2 activator, wherein the presence of said proteolytic peptides is assayed using mass spectrometry with reference to corresponding labeled and / or unlabeled reference proteolytic peptides; and (b) diagnosing a neurodegenerative disease in the subject according to the relative concentrations of the peptides measured in the sample.

[0086] According to a fourth aspect of the present invention there is provided a method for the treatment of a subject having a neurodegenerative disease, the method comprising: (a)(i) preparing a biological sample from a subject for an assay by incubating the sample with a protease to form a proteolytic digest of the proteins in the sample; and (ii) assaying the proteolytic digest of the protein of step (i) for the presence of proteolytic peptides of at least one protein selected from the group of proteins shown in Table 1, said group including matrix metalloprotease-9, chitinase-3-like protein 1, protein S100-A8, protein S100-A9, neutrophil collagenase (MMP8), complement C3, galectin-3 binding protein, catalase, extracellular superoxide dismutase, prosaposin, β-heterozygosyltransferase ... xosaminidase subunit β, cathepsin D, beclin-1, lysosomal-associated membrane glycoprotein 1, autophagy protein 13, E3 ubiquitin protein ligase RNF26, E3 ubiquitin protein ligase TRIM33, FAST kinase domain-containing protein 5, mitochondrial, apoptosis-inducing factor 1 mitochondrial, transmembrane protein 126A, neurofilament light polypeptide, chromogranin A, contactin-1, neurexin-1-β, protein MTSS Metastasis suppressor YGL-1, synaptotagmin-3, apolipoprotein E, apolipoprotein D, clusterin, flotillin-2, cadherin-2, neural cell adhesion molecule 2, amyloid-β precursor protein, amyloid-like protein 1, peptidyl prolyl cis-trans isomerase FKBP3, peptidyl prolyl cis-trans isomerase FKBP4, kallikrein-6, neutrophil gelatinase-associated lipocalin, protein disulfide isomerase A1 (PDIA1), limbic system-associated membrane protein (fragment), ceruloplasmin, serotransferrin, vacuolar protein sorting-associated protein 16 homolog (hVPS16), peptidyl prolyl cis-trans isomerase (PPIase; HEL-S-39), lymphocyte cytoplasmic protein 2, lactadherin, 72 kDa It consists of type IV collagenase, transforming growth factor-β-inducible protein IG-H3, neuronal proliferation and differentiation control protein 1, and ganglioside GM2 activator. wherein the presence of said proteolytic peptides is assayed using mass spectrometry with reference to corresponding labeled and / or unlabeled reference proteolytic peptides; (b) diagnosing a neurodegenerative disease in the subject according to the relative concentrations of the peptides measured in the sample; and (c) administering the pharmaceutical composition to a subject for the treatment of a neurodegenerative disease.

[0087] Pharmaceutical compositions suitable for the treatment of Parkinson's disease include, but are not limited to, bromocriptine, cabergoline, lazabemide, levodopa, pergolide, pramipexole, rasagiline, ropinirole, rotigotine and selegiline.

[0088] The pharmaceutical composition suitable for treating Alzheimer's disease includes, but is not limited to, cholinesterase inhibitor (CI) or NMDA receptor activity inhibitor.The examples of cholinesterase inhibitor (CI) include, but are not limited to, tacrine, donepezil, rivastigmine, galantamine, etc.The example of NMDA receptor activity inhibitor is memantine.

[0089] According to a fifth aspect of the present invention there is provided a method for second line treatment of a neurodegenerative disease in a subject, the method comprising: (a) assessing the efficacy of a first line treatment for a neurodegenerative disease in a subject, the method comprising: (i) preparing a biological sample from a subject for the assay by incubating the sample with a protease to form a proteolytic digest of the proteins in the sample; and (ii) assaying the proteolytic digest of the protein of step (i) for the presence of proteolytic peptides of at least one protein selected from the group of proteins shown in Table 1, said group including matrix metalloprotease-9, chitinase-3-like protein 1, protein S100-A8, protein S100-A9, neutrophil collagenase (MMP8), complement C3, galectin-3 binding protein, catalase, extracellular superoxide dismutase, prosaposin, β-heterozygosyltransferase ... xosaminidase subunit β, cathepsin D, beclin-1, lysosomal-associated membrane glycoprotein 1, autophagy protein 13, E3 ubiquitin protein ligase RNF26, E3 ubiquitin protein ligase TRIM33, FAST kinase domain-containing protein 5, mitochondrial, apoptosis-inducing factor 1 mitochondrial, transmembrane protein 126A, neurofilament light polypeptide, chromogranin A, contactin-1, neurexin-1-β, protein MTSS Metastasis suppressor YGL-1, synaptotagmin-3, apolipoprotein E, apolipoprotein D, clusterin, flotillin-2, cadherin-2, neural cell adhesion molecule 2, amyloid-β precursor protein, amyloid-like protein 1, peptidyl prolyl cis-trans isomerase FKBP3, peptidyl prolyl cis-trans isomerase FKBP4, kallikrein-6, neutrophil gelatinase-associated lipocalin, protein disulfide isomerase A1 (PDIA1), limbic system-associated membrane protein (fragment), ceruloplasmin, serotransferrin, vacuolar protein sorting-associated protein 16 homolog (hVPS16), peptidyl prolyl cis-trans isomerase (PPIase; HEL-S-39), lymphocyte cytoplasmic protein 2, lactadherin, 72 kDa It consists of type IV collagenase, transforming growth factor-β-inducible protein IG-H3, neuronal proliferation and differentiation control protein 1, and ganglioside GM2 activator. wherein the presence of said proteolytic peptides is assayed using mass spectrometry with reference to corresponding labeled and / or unlabeled reference proteolytic peptides; and (b) administering the pharmaceutical composition to a subject for second line treatment of a neurodegenerative disease.

[0090] A first line treatment for a neurodegenerative disease can be any appropriate therapeutic course prescribed. The first line and second line treatments can be the same or different in terms of pharmaceutical composition, dosage form and / or dosing regimen administered to the subject.

[0091] Pharmaceutical compositions suitable for the treatment of Parkinson's disease include, but are not limited to, bromocriptine, cabergoline, lazabemide, levodopa, pergolide, pramipexole, rasagiline, ropinirole, rotigotine and selegiline.

[0092] The pharmaceutical composition suitable for treating Alzheimer's disease includes, but is not limited to, cholinesterase inhibitor (CI) or NMDA receptor activity inhibitor.The examples of cholinesterase inhibitor (CI) include, but are not limited to, tacrine, donepezil, rivastigmine, galantamine, etc.The example of NMDA receptor activity inhibitor is memantine.

[0093] In addition to the above, the present invention can be commercialized in the form of an assay kit. Such kits can include a standardized set of consumables and be shipped to a customer site with instructions on how to perform the test in the customer's laboratory. Alternatively, the kits can be sold separately without the consumables and include instructions (standard operating procedures) detailing how to perform the test.

[0094] According to a sixth aspect of the present invention there is provided a kit for assessing the efficacy of a first line treatment for a neurodegenerative disease in a subject comprising: 2. A plurality of sample preparation media for analyzing a sample by mass spectrometry for the presence of proteolytic peptides of at least one protein selected from the group of proteins shown in Table 1, comprising: matrix metalloprotease-9, chitinase-3-like protein 1, protein S100-A8, protein S100-A9, neutrophil collagenase (MMP8), complement C3, galectin-3 binding protein, catalase, extracellular superoxide dismutase, prosaposin, β-hexosaminidase subunit β, cathepsin D, beclin-1, lysosomal associated membrane glycoprotein 1, autophagy protein 13, E3 ubiquitin protein ligase RNF26, E3 ubiquitin protein ligase TRIM33, FAST kinase domain-containing protein 5, mitochondria, apoptosis-inducing factor 1 mitochondrial, transmembrane protein 126A, neurofilament light polypeptide, chromogranin A, contactin-1, neurexin-1-β, protein MTSS Metastasis suppressor YGL-1, synaptotagmin-3, apolipoprotein E, apolipoprotein D, clusterin, flotillin-2, cadherin-2, neural cell adhesion molecule 2, amyloid-β precursor protein, amyloid-like protein 1, peptidyl prolyl cis-trans isomerase FKBP3, peptidyl prolyl cis-trans isomerase FKBP4, kallikrein-6, neutrophil gelatinase-associated lipocalin, protein disulfide isomerase A1 (PDIA1), limbic system-associated membrane protein (fragment), ceruloplasmin, serotransferrin, vacuolar protein sorting-associated protein 16 homolog (hVPS16), peptidyl prolyl cis-trans isomerase (PPIase; HEL-S-39), lymphocyte cytoplasmic protein 2, lactadherin, 72 kDa Multiple sample preparation media consisting of collagenase type IV, transforming growth factor beta-inducible protein IG-H3, neural proliferation and differentiation control protein 1, and ganglioside GM2 activator.

[0095] The sample preparation medium may include a protease. The protease is a serine protease, a cysteine ​​protease, a threonine protease, an aspartic acid protease, a glutamic acid protease, or a metalloprotease. The protease may be trypsin. Alternatively, the sample preparation medium may be suitable for use with a protease.

[0096] The sample preparation medium may include a buffer. The buffer may be suitable for use with a protease. Digestion of peptides may be performed in the sample preparation medium. Buffers suitable for proteases are known to those skilled in the art.

[0097] The sample preparation medium may include a peptide internal standard, such as an isotopically labeled peptide internal standard.

[0098] The present invention provides the first comprehensive targeted proteomic biomarker panel for neurodegeneration. It covers all aspects of Parkinson's disease pathology in a single robust assay. Each analyte has a calibration curve and a dedicated internal standard, so that every molecule in the panel is measured in the most accurate way possible. The panel provides absolute concentrations of each analyte, making it suitable for ongoing clinical testing. Overall, the test provides fundamentally rich and reliable biomarker data.

[0099] Preferred features of the second and subsequent aspects of the invention apply mutatis mutandis with respect to the first aspect.

[0100] The present invention will now be described with reference to the following examples, which are presented for illustrative purposes only and are not to be construed as limiting the invention. Please also refer to the following drawings: [Brief description of the drawings]

[0101] [Figure 1]Figure 1 shows the results of differential protein expression of chromogranin A in Parkinson's disease patients. Bars represent median, error bars represent interquartile range (IQR), dots represent individual samples. W0: baseline, W20: patients after 20 weeks of drug / placebo, W45: patients after 45 weeks of drug / placebo. [Diagram 2] Figure 2 shows the results of differential expression of amyloid beta precursor protein in Parkinson's disease patients. Bars represent median, error bars represent interquartile range (IQR), dots represent individual samples. W0: baseline, W20: patients after 20 weeks of drug / placebo treatment, W45: patients after 45 weeks of drug / placebo treatment. [Diagram 3] Figure 3 shows the results of differential protein expression of amyloid-like protein 1 in Parkinson's disease patients. Bars represent median, error bars represent interquartile range (IQR), dots represent individual samples. W0: baseline, W20: patients after 20 weeks of drug / placebo, W45: patients after 45 weeks of drug / placebo. [Figure 4] Figure 4 shows the results of differential protein expression of the transforming growth factor beta-induced protein IG-H3 in Parkinson's disease patients. Bars represent median, error bars represent interquartile range (IQR), dots represent individual samples. W0: baseline, W20: patients after 20 weeks of drug / placebo, W45: patients after 45 weeks of drug / placebo. [Diagram 5] Figure 5 shows the results of differential protein expression of prosaposin in Parkinson's disease patients. Bars represent median, error bars represent interquartile range (IQR), dots represent individual samples. W0: baseline, W20: patients after 20 weeks of drug / placebo, W45: patients after 45 weeks of drug / placebo. [Figure 6] Figure 6 shows a summary of differential protein expression of all proteins in the biomarker study. Forward hatching represents fold change increase compared to baseline, backward hatching represents fold change decrease compared to baseline. W0 - baseline, W20 - patients after 20 weeks of drug / placebo, W45 - patients after 45 weeks of drug / placebo. FC - fold change. [Figure 7]Figure 7 shows a summary of differential protein expression for all proteins within the drug-treated patient group. Different biomarker trajectories indicate different patient responses to treatment and this biomarker testing can be used for patient stratification. Forward hatching represents fold-change increase compared to baseline, backward hatching represents fold-change decrease compared to baseline. W0-baseline, W20-patients after 20 weeks of drug / placebo, W45-patients after 45 weeks of drug / placebo. FC-fold change, MO-months. [Figure 8] Figure 8 shows the results of differential protein expression of the E3 ubiquitin protein ligase TRIM33 in Parkinson's disease patients treated with medication. Different biomarker trajectories indicate that different patients respond differently to treatment, and this biomarker test can be used for patient stratification. Dots represent individual samples. W0 - baseline, W20 - patients after 20 weeks of medication, W45 - patients after 45 weeks of medication. [Figure 9] Figure 9 shows the results of differential expression of FAST kinase domain-containing protein 5, mitochondrial protein, in Parkinson's disease patients treated with medication. Different biomarker trajectories indicate that different patients respond differently to treatment, and this biomarker test can be used for patient stratification. Dots represent individual samples. W0 - baseline, W20 - patients after 20 weeks of medication, W45 - patients after 45 weeks of medication. [Figure 10] Figure 10 shows the results of differential protein expression of Contactin-1 in Parkinson's disease patients treated with medication. Different biomarker trajectories indicate that different patients respond differently to treatment, and this biomarker test can be used for patient stratification. Dots represent individual samples. W0 - baseline, W20 - patients after 20 weeks of medication, W45 - patients after 45 weeks of medication.

[0102] The protein biomarker names shown in Figures 6 and 7 are referred to as "biomarker 1" through "biomarker 48," corresponding to 48 of the 50 proteins in Table 1. Two proteins included in Table 1 but omitted in Figures 6 and 7 are the proteins MTSS 1 (metastasis suppressor YGL-1) and lymphocyte cytoplasmic protein 2.

[0103] The method may include detecting an increase and / or decrease in any one or more proteolytic peptides, and Figure 6 and / or Figure 7 show an increase and / or decrease for the corresponding biomarkers. The increase and / or decrease may be detected in samples obtained at any time points shown in Figure 6 (e.g., week 20 or week 45) and / or Figure 7 (e.g., month 6 or month 12). With reference to Figure 6, the biomarker may be a biomarker that decreases in placebo at week 20 and / or week 45. With reference to Figure 6, the biomarker may be a biomarker that increases in one or more drug treatment groups at week 20 and / or week 45. With reference to Figure 6, the biomarker may be a biomarker that decreases in placebo at week 20 and / or week 45 and increases in one or more drug treatment groups at week 20 and / or week 45. For example, the biomarker may be biomarker 23 that decreases in placebo at week 20 and increases in both drug treatment groups at week 20. For example, the biomarker can be biomarker 33, which decreases in the placebo group at week 20 and increases in both drug groups at week 20. For example, the biomarker can be biomarker 3, which decreases in the placebo group at week 20 and increases in the medium drug group at week 20. For example, the biomarker can be biomarker 41, which decreases in the placebo group at weeks 20 and 45, increases in the medium drug group at week 20, and increases in the high drug group at weeks 20 and 45. The increase or decrease can be the fold change relative to placebo for any biomarker in FIG. 6. The biomarker can be any biomarker for which FIG. 7 shows a difference between any two patients at any time point, for example, at 6 months and / or 12 months. For example, the biomarker can be biomarker 48, which increases in patient 3 at 6 and 12 months and decreases in patients 2 and 4 at 3 and 6 months. Thus, biomarker 48 is an example of a biomarker that can be used to stratify patients for treatment response according to the relative concentrations of peptides measured in their samples.The biomarker may be any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 or 48 of the following proteins: Matrix metalloproteinase-9 Chitinase-3-like protein 1 Protein S100-A8 Protein S100-A9 Neutrophil collagenase 8 (MMP8) Complement C3 Galectin-3 binding protein Catalase Extracellular superoxide dismutase Prosaposin β-hexosaminidase subunit β Cathepsin D Beclin-1 Lysosome-associated membrane glycoprotein 1 Autophagy Protein 13 E3 ubiquitin protein ligase RNF26 The E3 ubiquitin protein ligase TRIM33 FAST kinase domain-containing protein 5, mitochondrial Apoptosis-inducing factor 1, mitochondrial Transmembrane protein 126A Neurofilament Light Polypeptide Chromogranin A Contactin-1 Neurexin-1β Synaptotagmin-3 Apolipoprotein E Apolipoprotein D Clusterin Flotillin-2 Cadherin-2 Neural cell adhesion molecule 2 Amyloid β precursor protein Amyloid-like protein 1 Peptidylprolyl cis-trans isomerase FKBP3 Peptidylprolyl cis-trans isomerase FKBP4 Kallikrein-6 Neutrophil gelatinase-associated lipocalin Protein disulfide isomerase A1 (PDIA1) Limbic system-associated membrane protein (fragment) Ceruloplasmin Serotransferrin Vacuolar protein sorting-related protein 16 homologue (hVPS16) Peptidylprolyl cis-trans isomerase (PPIase; HEL-S-39) Lactadherin 72kDa type IV collagenase Transforming growth factor-β-induced protein IG-H3 Neural proliferation and differentiation control protein 1 and Ganglioside GM2 activator.

[0104] Thus, the at least one protein used in the method or kit of the present invention may be any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 or 48 of the above 48 proteins. Preferably, the at least one protein may be all 48 of the above proteins. Thus, the method may comprise assaying the proteolytic digest of the protein of step (i) for the presence of all 48 proteolytic peptides of the above proteins.

[0105] Further advantages of the present invention are: ●We demonstrate that it is possible to develop highly multiplexed biomarker assays that cover all aspects of Parkinson's disease molecular pathology in a single test. Table 1 details these disease processes, selected proteins, and corresponding tryptic peptides. We believe this to be the world's first comprehensive targeted proteomic biomarker panel for Parkinson's disease / neurodegeneration. • This demonstrates that this test can be applied to stratify patient populations and evaluate the efficacy of drug treatment (Figures 6 and 7). A person skilled in the art can refer to Tables 1 and 2 and this specification to set up this biomarker test on a particular analytical device. Table 2 discloses specific instrument settings to a level of detail that can be directly copied to set up this biomarker test. - We identified proteins not previously associated with Parkinson's disease and demonstrated that these proteins may be part of a way to stratify Parkinson's disease patient populations and evaluate the effectiveness of drug treatment. Demonstrating that this comprehensive biomarker panel can be established on a specific analytical device, allowing more accurate and robust analytical measurements of all molecules in the panel compared to other analytical techniques, and that the assay can be validated against existing regulatory standards in the clinical setting. • Overall, this biomarker test provides fundamentally richer and more reliable biomarker data compared to other technologies in the field. EXAMPLES

[0106] Example 1 Proteomic analysis of CSF samples from Parkinson's disease (PD) patients undergoing clinical trials Sample collection CSF samples were collected by lumbar puncture from Parkinson's disease patients in clinical trials at baseline, 20 weeks, and 45 weeks after treatment with placebo or an investigational PD drug.

[0107] Sample preparation Equal amounts of CSF were lyophilized and isotopically labeled peptide internal standards were added prior to protein digestion.CSF proteins were enzymatically digested into peptides using trypsin, with prior reduction and alkylation.After digestion, samples were purified using a C18-based solid-phase extraction microplate before injection into the LC-MS / MS system.

[0108] LC-MS / MS setup Tryptic peptides were then quantified by liquid chromatography-tandem mass spectrometry system (LC-MS / MS) operated in timed multiple reaction monitoring mode (MRM). A Thermo Scientific TSQ Altis triple quadrupole mass spectrometer was used for quantification. For each protein biomarker, at least two unique tryptic peptides were quantified using the corresponding internal standards shown in Table 1 and peptide calibration lines in assay buffer and CSF. A minimum of two transitions were used for positive identification of each peptide, as detailed in Tables 2 and 3. Each patient sample was injected onto a Kinetex reversed-phase C18-based liquid chromatography column and peptides were separated with a water-acetonitrile gradient at a flow rate of 0.5 ml / min. Samples were blinded and in random order. Chromatograms were analyzed using Xcalibur v4.1 software and absolute concentrations were determined from standard curves constructed with peptides shown in Table 1. Standard curves for each peptide were analyzed at the beginning and end of the run for quantification and performance normalization.

[0109] Statistical analysis / interpretation of results A linear regression model was fitted to the assay buffer standard curve data set to determine absolute peptide concentrations. The standard curves were plotted on a log-log scatter plot and expressed as 1 / x 2Weighting was applied. Lower limits of quantification, intraday and interday coefficients of variation (%CV) were also established for each peptide at multiple concentrations. Absolute peptide concentrations were then compared across sample sets. Week 45 and week 20 results were normalized to baseline and expressed as fold change from baseline. A 1.5-fold increase or 0.67-fold decrease in peptide concentration from baseline in the drug-treated groups, with no or small corresponding peptide change in the placebo group, was considered to indicate efficacy of drug treatment.

[0110] Example 2: Patient Stratification To stratify patient groups after drug treatment, we compared the trajectories of biomarkers (i.e., peptides) within the drug-treated patient group. All patients with increased biomarker signals were stratified into subgroup 1, and all patients with decreased biomarker signals or no significant change in biomarker signals were classified into subgroup 2.

[0111] This application also refers to the following tables: Table 1 shows 50 proteins differentially expressed in Parkinson's disease patients and the corresponding 118 proteolytic peptide sequences and the corresponding heavy isotope-labeled peptide internal standard sequences. [Table 1] JPEG2024510734000003.jpg245170JPEG2024510734000004.jpg243170JPEG20245107340 00005.jpg247170JPEG2024510734000006.jpg247170JPEG2024510734000007.jpg246170

[0112] Table 2 shows the experimentally optimized target LC-MS / MS conditions for monitoring the native peptides shown in Table 1. The data in Table 2 are based on ThermoScientific's triple quadrupole LC-MS / MS platform TSQ Altis, but are equally applicable to platforms from other manufacturers. The following abbreviations are used in Table 2: RF - radio frequency; [Table 2] JPEG2024510734000009.jpg219170JPEG2024510734000010.jpg216170JPEG2024510734000011.jpg219170 JPEG2024510734000012.jpg216170JPEG2024510734000013.jpg221170JPEG2024510734000014.jpg213170 JPEG2024510734000015.jpg224170JPEG2024510734000016.jpg221170JPEG2024510734000017.jpg223170 JPEG2024510734000018.jpg228170JPEG2024510734000019.jpg218170JPEG2024510734000020.jpg236170

[0113] Table 3 shows the experimentally optimized target LC-MS / MS conditions for monitoring the heavy isotope-labeled peptide internal standards shown in Table 1, based on the triple quadrupole LC-MS / MS platform TSQ Altis from ThermoScientific. The following abbreviations are used in Table 3: RF - radio frequency; [Table 3] JPEG2024510734000022.jpg232170JPEG2024510734000023.jpg226170JPEG2024510734000024.jpg223170JPEG2024510734000025.jpg222170JPEG2024510734000026.jpg224170

Claims

1. A method for assessing the efficacy of a therapy for a neurodegenerative disease in a subject, the method comprising: (i) preparing a biological sample from a subject for the assay by incubating the sample with a protease to form a proteolytic digest of the proteins in the sample; and (ii) assaying the proteolytic digests of the proteins of step (i) for the presence of proteolytic peptides of contactin-1 and proteolytic peptides of the E3 ubiquitin protein ligase TRIM33, and optionally of at least one protein selected from the group of proteins shown in Table 1, said group of proteins shown in Table 1 being matrix metalloprotease-9, chitinase-3-like protein 1, protein S100-A8, protein S100-A9, neutrophil collagenase (MMP8), , complement C3, galectin-3 binding protein, catalase, extracellular superoxide dismutase, prosaposin, β-hexosaminidase subunit β, cathepsin D, beclin-1, lysosomal-associated membrane glycoprotein 1, autophagy protein 13, E3 ubiquitin protein ligase RNF26, FAST kinase domain-containing protein 5, mitochondria, apoptosis-inducing factor 1 mitochondrial, transmembrane protein 126A, neurofilament light polypeptide, chromogranin A, neurexin-1-β, protein MTSS 1 (metastasis suppressor YGL-1), synaptotagmin-3, apolipoprotein E, apolipoprotein D, clusterin, flotillin-2, cadherin-2, neural cell adhesion molecule 2, amyloid beta precursor protein, amyloid-like protein 1, peptidyl prolyl cis-trans isomerase FKBP3, peptidyl prolyl cis-trans isomerase FKBP4, kallikrein-6, neutrophil gelatinase-associated lipocalin, protein disulfide isomerase A1 (PDIA1), limbic system-associated membrane protein (fragment), ceruloplasmin, serotransferrin, vacuolar protein sorting-associated protein 16 homolog (hVPS16), peptidyl prolyl cis-trans isomerase (PPIase; HEL-S-39), lymphocyte cytoplasmic protein 2, lactadherin, 72 kDa It is composed of type IV collagenase, transforming growth factor β-inducible protein IG-H3, neural proliferation and differentiation control protein 1, and ganglioside GM2 activator, wherein the presence of said proteolytic peptides is assayed using mass spectrometry with reference to corresponding labeled and / or unlabeled reference proteolytic peptides. A method comprising:

2. 1. A method of stratifying a subject having a neurodegenerative disease, the method comprising: (i) preparing a biological sample from a subject for the assay by incubating the sample with a protease to form a proteolytic digest of the proteins in the sample; and (ii) assaying the proteolytic digests of the proteins of step (i) for the presence of proteolytic peptides of contactin-1 and proteolytic peptides of the E3 ubiquitin protein ligase TRIM33, and optionally of at least one protein selected from the group of proteins shown in Table 1, said group comprising: matrix metalloprotease-9, chitinase-3-like protein 1, protein S100-A8, protein S100-A9, neutrophil collagenase (MMP8), complement C3, Galectin-3 binding protein, catalase, extracellular superoxide dismutase, prosaposin, β-hexosaminidase subunit β, cathepsin D, beclin-1, lysosomal-associated membrane glycoprotein 1, autophagy protein 13, E3 ubiquitin protein ligase RNF26, FAST kinase domain-containing protein 5, mitochondria, apoptosis-inducing factor 1 mitochondrial, transmembrane protein 126A, neurofilament light polypeptide, chromogranin A, neurexin-1-β, protein MTSS 1 (metastasis suppressor YGL-1), synaptotagmin-3, apolipoprotein E, apolipoprotein D, clusterin, flotillin-2, cadherin-2, neural cell adhesion molecule 2, amyloid beta precursor protein, amyloid-like protein 1, peptidyl prolyl cis-trans isomerase FKBP3, peptidyl prolyl cis-trans isomerase FKBP4, kallikrein-6, neutrophil gelatinase-associated lipocalin, protein disulfide isomerase A1 (PDIA1), limbic system-associated membrane protein (fragment), ceruloplasmin, serotransferrin, vacuolar protein sorting-associated protein 16 homolog (hVPS16), peptidyl prolyl cis-trans isomerase (PPIase; HEL-S-39), lymphocyte cytoplasmic protein 2, lactadherin, 72 kDa It is composed of type IV collagenase, transforming growth factor β-inducible protein IG-H3, neural proliferation and differentiation control protein 1, and ganglioside GM2 activator, wherein the presence of said proteolytic peptides is assayed using mass spectrometry with reference to corresponding labeled and / or unlabeled reference proteolytic peptides; and (b) stratifying the subjects according to the relative concentrations of the peptides measured in the samples. A method comprising:

3. 1. A method for assaying a proteolytic digest to aid in the diagnosis of a neurodegenerative disease, the method comprising: (i) preparing a biological sample from a subject for the assay by incubating the sample with a protease to form a proteolytic digest of the proteins in the sample; and (ii) assaying the proteolytic digests of the proteins of step (i) for the presence of proteolytic peptides of contactin-1 and proteolytic peptides of the E3 ubiquitin protein ligase TRIM33, and optionally of at least one protein selected from the group of proteins shown in Table 1, said group comprising: matrix metalloprotease-9, chitinase-3-like protein 1, protein S100-A8, protein S100-A9, neutrophil collagenase (MMP8), complement C3, Galectin-3 binding protein, catalase, extracellular superoxide dismutase, prosaposin, β-hexosaminidase subunit β, cathepsin D, beclin-1, lysosomal-associated membrane glycoprotein 1, autophagy protein 13, E3 ubiquitin protein ligase RNF26, FAST kinase domain-containing protein 5, mitochondria, apoptosis-inducing factor 1 mitochondrial, transmembrane protein 126A, neurofilament light polypeptide, chromogranin A, neurexin-1-β, protein MTSS 1 (metastasis suppressor YGL-1), synaptotagmin-3, apolipoprotein E, apolipoprotein D, clusterin, flotillin-2, cadherin-2, neural cell adhesion molecule 2, amyloid beta precursor protein, amyloid-like protein 1, peptidyl prolyl cis-trans isomerase FKBP3, peptidyl prolyl cis-trans isomerase FKBP4, kallikrein-6, neutrophil gelatinase-associated lipocalin, protein disulfide isomerase A1 (PDIA1), limbic system-associated membrane protein (fragment), ceruloplasmin, serotransferrin, vacuolar protein sorting-associated protein 16 homolog (hVPS16), peptidyl prolyl cis-trans isomerase (PPIase; HEL-S-39), lymphocyte cytoplasmic protein 2, lactadherin, 72 kDa It is composed of type IV collagenase, transforming growth factor β-inducible protein IG-H3, neural proliferation and differentiation control protein 1, and ganglioside GM2 activator, wherein the presence of said proteolytic peptides is assayed using mass spectrometry with reference to corresponding labeled and / or unlabeled reference proteolytic peptides; and (b) determining the neurodegenerative disease of the subject according to the relative concentrations of the peptides measured in the sample; A method comprising:

4. 1. A method for assaying a proteolytic digest to aid in the treatment of a subject with a neurodegenerative disease, the method comprising: (a)(i) preparing a biological sample from a subject for an assay by incubating the sample with a protease to form a proteolytic digest of the proteins in the sample; and (ii) assaying the proteolytic digests of the proteins of step (i) for the presence of proteolytic peptides of contactin-1 and proteolytic peptides of the E3 ubiquitin protein ligase TRIM33, and optionally of at least one protein selected from the group of proteins shown in Table 1, said group comprising: matrix metalloprotease-9, chitinase-3-like protein 1, protein S100-A8, protein S100-A9, neutrophil collagenase (MMP8), complement C3, Galectin-3 binding protein, catalase, extracellular superoxide dismutase, prosaposin, β-hexosaminidase subunit β, cathepsin D, beclin-1, lysosomal-associated membrane glycoprotein 1, autophagy protein 13, E3 ubiquitin protein ligase RNF26, FAST kinase domain-containing protein 5, mitochondria, apoptosis-inducing factor 1 mitochondrial, transmembrane protein 126A, neurofilament light polypeptide, chromogranin A, neurexin-1-β, protein MTSS 1 (metastasis suppressor YGL-1), synaptotagmin-3, apolipoprotein E, apolipoprotein D, clusterin, flotillin-2, cadherin-2, neural cell adhesion molecule 2, amyloid beta precursor protein, amyloid-like protein 1, peptidyl prolyl cis-trans isomerase FKBP3, peptidyl prolyl cis-trans isomerase FKBP4, kallikrein-6, neutrophil gelatinase-associated lipocalin, protein disulfide isomerase A1 (PDIA1), limbic system-associated membrane protein (fragment), ceruloplasmin, serotransferrin, vacuolar protein sorting-associated protein 16 homolog (hVPS16), peptidyl prolyl cis-trans isomerase (PPIase; HEL-S-39), lymphocyte cytoplasmic protein 2, lactadherin, 72 kDa It is composed of type IV collagenase, transforming growth factor β-inducible protein IG-H3, neural proliferation and differentiation control protein 1, and ganglioside GM2 activator, wherein the presence of said proteolytic peptides is assayed using mass spectrometry with reference to corresponding labeled and / or unlabeled reference proteolytic peptides; and (b) determining the neurodegenerative disease of the subject according to the relative concentrations of the peptides measured in the sample; A method comprising:

5. A method for assaying a proteolytic digest to aid in second line treatment of a neurodegenerative disease in a subject, the method comprising: (a) assessing the efficacy of a first-line treatment for a neurodegenerative disease in a subject, the method comprising: (i) preparing a biological sample from a subject for the assay by incubating the sample with a protease to form a proteolytic digest of the proteins in the sample; and (ii) assaying the proteolytic digests of the proteins of step (i) for the presence of proteolytic peptides of contactin-1 and proteolytic peptides of the E3 ubiquitin protein ligase TRIM33, and optionally of at least one protein selected from the group of proteins shown in Table 1, said group comprising: matrix metalloprotease-9, chitinase-3-like protein 1, protein S100-A8, protein S100-A9, neutrophil collagenase (MMP8), complement C3, Galectin-3 binding protein, catalase, extracellular superoxide dismutase, prosaposin, β-hexosaminidase subunit β, cathepsin D, beclin-1, lysosomal-associated membrane glycoprotein 1, autophagy protein 13, E3 ubiquitin protein ligase RNF26, FAST kinase domain-containing protein 5, mitochondria, apoptosis-inducing factor 1 mitochondrial, transmembrane protein 126A, neurofilament light polypeptide, chromogranin A, neurexin-1-β, protein MTSS 1 (metastasis suppressor YGL-1), synaptotagmin-3, apolipoprotein E, apolipoprotein D, clusterin, flotillin-2, cadherin-2, neural cell adhesion molecule 2, amyloid beta precursor protein, amyloid-like protein 1, peptidyl prolyl cis-trans isomerase FKBP3, peptidyl prolyl cis-trans isomerase FKBP4, kallikrein-6, neutrophil gelatinase-associated lipocalin, protein disulfide isomerase A1 (PDIA1), limbic system-associated membrane protein (fragment), ceruloplasmin, serotransferrin, vacuolar protein sorting-associated protein 16 homolog (hVPS16), peptidyl prolyl cis-trans isomerase (PPIase; HEL-S-39), lymphocyte cytoplasmic protein 2, lactadherin, 72 kDa It is composed of type IV collagenase, transforming growth factor β-inducible protein IG-H3, neural proliferation and differentiation control protein 1, and ganglioside GM2 activator, wherein the presence of said proteolytic peptides comprises assaying using mass spectrometry with reference to corresponding labeled and / or unlabeled reference proteolytic peptides; (b) determining a pharmaceutical composition for second line treatment of a neurodegenerative disease; A method comprising:

6. The method according to any one of claims 1 to 5, wherein the protease is a serine protease, a cysteine ​​protease, a threonine protease, an aspartic acid protease, a glutamic acid protease or a metalloprotease.

7. The method according to any one of claims 1 to 6, wherein the protease is trypsin.

8. 1. A kit for assessing the efficacy of a first-line treatment for a neurodegenerative disease in a subject, comprising: A plurality of sample preparation media for analyzing a sample by mass spectrometry for the presence of proteolytic peptides of contactin-1 and proteolytic peptides of E3 ubiquitin protein ligase TRIM33, and optionally at least one proteolytic peptide selected from the group of proteins shown in Table 1, said group including matrix metalloprotease-9, chitinase-3-like protein 1, protein S100-A8, protein S100-A9, neutrophil collagenase (MMP8), complement C3, galactamylase (GAMMA), phosphodiesterase (PHOS ... cytopenic acid-3 binding protein, catalase, extracellular superoxide dismutase, prosaposin, β-hexosaminidase subunit β, cathepsin D, beclin-1, lysosomal-associated membrane glycoprotein 1, autophagy protein 13, E3 ubiquitin protein ligase RNF26, FAST kinase domain-containing protein 5, mitochondria, apoptosis-inducing factor 1 mitochondrial, transmembrane protein 126A, neurofilament light polypeptide, chromogranin A, neurexin-1-β, protein MTSS 1 (metastasis suppressor YGL-1), synaptotagmin-3, apolipoprotein E, apolipoprotein D, clusterin, flotillin-2, cadherin-2, neural cell adhesion molecule 2, amyloid beta precursor protein, amyloid-like protein 1, peptidyl prolyl cis-trans isomerase FKBP3, peptidyl prolyl cis-trans isomerase FKBP4, kallikrein-6, neutrophil gelatinase-associated lipocalin, protein disulfide isomerase A1 (PDIA1), limbic system-associated membrane protein (fragment), ceruloplasmin, serotransferrin, vacuolar protein sorting-associated protein 16 homolog (hVPS16), peptidyl prolyl cis-trans isomerase (PPIase; HEL-S-39), lymphocyte cytoplasmic protein 2, lactadherin, 72 kDa A kit comprising a sample preparation medium consisting of type IV collagenase, transforming growth factor beta-inducible protein IG-H3, neural proliferation and differentiation control protein 1, and ganglioside GM2 activator.

9. 9. The kit of claim 8, wherein the sample preparation medium comprises a protease, optionally wherein the protease is a serine protease, a cysteine ​​protease, a threonine protease, an aspartic acid protease, a glutamic acid protease, or a metalloprotease.

10. The kit of claim 9 , wherein the protease is trypsin.

11. The method or kit according to any one of claims 1 to 10, wherein the neurodegenerative disease is selected from the group consisting of Parkinson's disease, Alzheimer's disease, Huntington's disease, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), Batten disease, or a transmissible spongiform encephalopathy (e.g. scrapie, bovine spongiform encephalopathy (BSE) or Creutzfeldt-Jakob disease (CJD), including aberrant, variant, familial and sporadic CJD, and a lysosomal neurodegenerative disease (such as Gaucher disease).

12. The method or kit according to any one of claims 1 to 11, wherein the neurodegenerative disease is Parkinson's disease.

13. 13. The method or kit according to any one of claims 1 to 12, wherein the sample is selected from the group consisting of cerebrospinal fluid (CSF), blood (serum, whole blood (venous or peripheral), plasma, urine, tears, saliva, interstitial fluid, lymphatic fluid or a tissue sample.

14. The method or kit according to any one of claims 1 to 13, wherein the sample is cerebrospinal fluid (CSF).

15. 15. The method or kit according to any one of claims 1 to 14, wherein the proteolytic peptide of at least one protein is selected from the group consisting of: VNVDEVGGEALGR (SEQ ID NO: 1) AFALWSAVTPLTFTR (SEQ ID NO: 2) QLSLPETGELDSATLK (SEQ ID NO: 3) LVMGIPTFGR (SEQ ID NO: 4) TLLSVGGWNFGSQR (SEQ ID NO:5) FSNTDYAVGYMLR (SEQ ID NO: 6) ALNSIIDDVYHK (SEQ ID NO: 7) GNFHAVYR (SEQ ID NO: 8) VIEHIMEDLDTNADK (SEQ ID NO: 9) LTWASHEK (SEQ ID NO: 10) YYAFDLIAQR (SEQ ID NO: 11) VDAVFQQEHFFHVFSGPR (SEQ ID NO: 12) VLLDGVQNPR (SEQ ID NO: 13) NTLIIYLDK (SEQ ID NO: 14) ASHEEVEGLVEK (SEQ ID NO: 15) SQLVYQSR (SEQ ID NO: 16) IYTSPTWSAFVTDSSWSAR (SEQ ID NO: 17) FSTVAGESGSADTVR (SEQ ID NO: 18) GAGAFGYFEVTHDITK (SEQ ID NO: 19) AGLAASLAGPHSIVGR (SEQ ID NO: 20) AVVVHAGEDDLGR (SEQ ID NO: 21) EILDAFDK (SEQ ID NO:22) QEILAALEK (SEQ ID NO:23) VEPLDFGGTQK (SEQ ID NO:24) VLDIIATINK (SEQ ID NO:25) LSPEDYTLK (SEQ ID NO:26) VSTLPAITLK (SEQ ID NO:27) WGLAWVSSQFYNK (SEQ ID NO: 28) TSNNSTMQVSFVCQR (SEQ ID NO: 29) ELALEEER (SEQ ID NO:30) ENTSDPSLVIAFGR (SEQ ID NO: 31) TVESITDIR (SEQ ID NO:32) ALQATVGNSYK (SEQ ID NO: 33) DDILPMDLGTFYR (SEQ ID NO: 34) TVQVIVQAR (SEQ ID NO: 35) LLGHLASHGALR (SEQ ID NO: 36) QDTLPEAGR (SEQ ID NO:37) SLQLASWPNR (SEQ ID NO: 38) QIDLVDNYFVK (SEQ ID NO: 39) LFCETCDR (SEQ ID NO: 40) LAVQFTNR (SEQ ID NO:41) ASTLQLGSPR (SEQ ID NO: 42) GVIFYLR (SEQ ID NO:43) ELWFSDDPNVTK (SEQ ID NO:44) YQSALLPHK (SEQ ID NO:45) ENITIVDISR (SEQ ID NO:46) YEEEVLSR (SEQ ID NO:47) IDSLMDEISFLK (SEQ ID NO: 48) VLEAELLVLR (SEQ ID NO:49) FTVLTESAAK (SEQ ID NO:50) YPGPQAEGDSEGLSQGLVDR (SEQ ID NO:51) SGELEQEEER (SEQ ID NO:52) FIPLIPIPER (SEQ ID NO:53) IVESYQIR (SEQ ID NO:54) SDLYIGGVAK (SEQ ID NO:55) ITTQITAGAR (SEQ ID NO:56) TTVVAAAAAFLDAFQK (SEQ ID NO:57) SLTMDPHK (SEQ ID NO:58) YLYGSDQLVVR (SEQ ID NO:59) ILQALDLPAK (SEQ ID NO:60) KPVEHWHQLVEVSR (SEQ ID NO:61) GPTPTPDK (SEQ ID NO:62) HPC (carbamidomethyl) TLGPFIHATNALHVR (SEQ ID NO: 63) AATVGSLAGQPLQER (SEQ ID NO: 64) GEVQAMLGQSTEELR (SEQ ID NO:65) LGPLVEQGR (SEQ ID NO:66) IPTTFENGR (SEQ ID NO:67) VLNQELR (SEQ ID NO:68) VTTVASHTSDSDVPSGVTEVVVK (SEQ ID NO: 69) ASSIIDELFQDR (SEQ ID NO:70) NVVLQTLEGHLR (SEQ ID NO:71) EGHLRSILGTLVEQIYQDRDQFAK (SEQ ID NO:72) TAEAQLAYELQGAR (SEQ ID NO:73) GPFPQELVR (SEQ ID NO:74) DVHEGQPLLNVK (SEQ ID NO:75) EVVSPQEFK (SEQ ID NO:76) ALLQVTISLSK (SEQ ID NO:77) QQLVETHMAR (SEQ ID NO:78) GAIIGLMVGGVV (SEQ ID NO:79) GAIIGLMVGGVVIA (SEQ ID NO: 80) LVFFAEDVGSNK (SEQ ID NO: 81) WYFDVTEGK (SEQ ID NO:82) DDTPMTLPK (SEQ ID NO:83) HGYENPTYR (SEQ ID NO:84) QMYPELQIAR (SEQ ID NO:85) AWTVEQLR (SEQ ID NO: 86) SEETLDEGPPK (SEQ ID NO:87) ESWEMNSEEK (SEQ ID NO:88) ALELDSNNEK (SEQ ID NO:89) LSELIQPLPLER (SEQ ID NO: 90) YTNWIQK (SEQ ID NO:91) MYATIYELK (SEQ ID NO:92) VPLQQNFQDNQFQGK (SEQ ID NO:93) MDSTANEVEAVK (SEQ ID NO:94) ALAPEYAK (SEQ ID NO:95) VTVNYPPTITESK (SEQ ID NO:96) SGIIFAGHDK (SEQ ID NO:97) MYYSAVEPTK (SEQ ID NO:98) IYHSHIDAPK (SEQ ID NO:99) DSAHGFLK (SEQ ID NO: 100) DGAGDVAFVK (SEQ ID NO: 101) LGDTPGVSYSDIAAR (SEQ ID NO: 102) ALLLVGDVAQAADVAIEHR (SEQ ID NO: 103) TVDNFVALATGEK (SEQ ID NO: 104) VYFDLR (SEQ ID NO: 105) FLNLTENDIQK (SEQ ID NO: 106) LPAPSIDR (SEQ ID NO: 107) GKEDFLSVSDIIDYFR (SEQ ID NO: 108) VTFLGLQHWVPELAR (SEQ ID NO: 109) LASHEYLK (SEQ ID NO: 110) AFQVWSDVTPLR (SEQ ID NO: 111) FFGLPQTGDLDQNTIETMR (SEQ ID NO: 112) LTLLAPLNSVFK (SEQ ID NO: 113) GDELADSALEIFK (SEQ ID NO: 114) LPEPATLGFSA (SEQ ID NO: 115) NPLFDHAALSAPLPAPSPPALP (SEQ ID NO: 116) SEFVVPDLELPSWLTTGNYR (SEQ ID NO: 117) and EGTYSLPK (sequence number 118).

16. 16. The method or kit of any one of claims 1 to 15, wherein the at least one protein is selected from the group consisting of chromogranin A, amyloid beta precursor protein, amyloid-like protein 1, transforming growth factor beta-induced protein IG-H3, prosaposin and apolipoprotein E.

17. 17. The method or kit according to any one of claims 1 to 16, wherein the proteolytic peptide of at least one protein is selected from the group consisting of: SGELEQEEER (SEQ ID NO:52) QQLVETHMAR (SEQ ID NO:78) QMYPELQIAR (SEQ ID NO:85) GDELADSALEIFK (SEQ ID NO: 114) QEILAALEK (SEQ ID NO:23) and AATVGSLAGQPLQER (sequence number 64).

18. 16. The method or kit of any one of claims 1 to 15, wherein at least one protein is selected from the group consisting of: Matrix metalloproteinase-9 Chitinase-3-like protein 1 Protein S100-A8 Protein S100-A9 Neutrophil collagenase 8 (MMP8) Complement C3 Galectin-3 binding protein Catalase Extracellular superoxide dismutase Prosaposin β-hexosaminidase subunit β Cathepsin D Beclin-1 Lysosome-associated membrane glycoprotein 1 Autophagy Protein 13 E3 ubiquitin protein ligase RNF26 FAST kinase domain-containing protein 5, mitochondrial Apoptosis-inducing factor 1, mitochondrial Transmembrane protein 126A Neurofilament Light Polypeptide Chromogranin A Neurexin-1β Synaptotagmin-3 Apolipoprotein E Apolipoprotein D Clusterin Flotillin-2 Cadherin-2 Neural cell adhesion molecule 2 Amyloid β precursor protein Amyloid-like protein 1 Peptidylprolyl cis-trans isomerase FKBP3 Peptidylprolyl cis-trans isomerase FKBP4 Kallikrein-6 Neutrophil gelatinase-associated lipocalin Protein disulfide isomerase A1 (PDIA1) Limbic system-associated membrane protein (fragment) Ceruloplasmin Serotransferrin Vacuolar protein sorting-related protein 16 homologue (hVPS16) Peptidylprolyl cis-trans isomerase (PPIase; HEL-S-39) Lactadherin 72 kDa type IV collagenase Transforming growth factor beta-induced protein IG-H3 Neural proliferation and differentiation control protein 1 and Ganglioside GM2 activator.

19. 20. The method or kit of claim 18, wherein at least one protein is all 46 of the proteins of claim 18.

20. The method or kit of any one of claims 1 to 15, wherein the at least one protein is selected from the group consisting of FAST kinase domain-containing protein 5 and mitochondria.

21. The method or kit of claim 20, wherein the proteolytic peptide of E3 ubiquitin protein ligase TRIM33 is LFCETCDR (SEQ ID NO: 40), the proteolytic peptide of fast kinase domain-containing protein 5, mitochondrial is LAVQFTNR (SEQ ID NO: 41); and / or the proteolytic peptide of contactin-1 is IVESYQIR (SEQ ID NO: 54).

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