Methods for detecting synuclein peptides

Abstract

A method is provided for detecting various isoforms of synuclein protein by detecting the amino acid sequences of synuclein peptides generated by proteolysis of synuclein present in a sample from a subject suspected of suffering from a neurodegenerative disease.

Description

[Technical Field]

[0001] The present invention relates to a method for detecting synuclein peptides, and more particularly to a method for detecting various isoforms of synuclein protein by detecting the amino acid sequences of synuclein peptides generated by proteolysis of synuclein present in a sample from a subject suspected of suffering from a neurodegenerative disease. [Background technology]

[0002] Synucleins ("syn") are a family of proteins found in the presynaptic terminals of dopaminergic neurons. Three distinct proteins, α-synuclein, β-synuclein, and γ-synuclein, are known, and at least α-syn has been reported to be present in several neurodegenerative conditions, including Parkinson's disease ("PD"), dementia with Lewy bodies ("LBD"), Alzheimer's disease ("AD"), multiple system atrophy ("MSA"), and Creutzfeldt-Jakob disease ("CJD") (Candelise et al., 2019). These conditions caused by abnormalities in synuclein are called synucleinopathies.

[0003] To date, the differentiation and diagnosis of various forms of synucleinopathy rely on the evaluation of clinical symptoms. For example, the diagnosis of idiopathic PD consists of three clinical evaluation steps. The first step detects signs of bradykinesia and motor abnormalities such as rigidity, resting tremor, or postural instability. If a patient exhibits bradykinesia and any one of the above-mentioned motor conditions, the patient is then further evaluated in the second step to ensure the absence of atypical symptoms (e.g., eye rotation seizures, delayed remission of disease symptoms, unilateral onset of symptoms 3 years after symptom evolution, supranuclear gaze palsy, cerebellar morphological changes, early severe autonomic neuropathy, early severe dementia, language impairment, memory impairment, and apraxia, lack of response to sufficient L-dopa, a history of stroke with a stepwise worsening course, multiple other cases of familial PD in close relatives, etc.). The third step involves detecting additional symptoms (e.g., unilateral symptom development, resting tremor, progressive deterioration, favorable response to dopaminergic therapy, initial persistent symptom asymmetry on the affected side indicated by a relatively high sensitivity to dopaminergic therapy, clinical evolution, or worsening of symptoms over a period of 10 years or more). To distinguish between PD and LBD, clinicians must also monitor patients for cognitive impairment in addition to hallucinations (e.g., visual hallucinations) in the absence of dopaminergic medications, as well as fluctuations in cognitive symptoms and vigilance, neuroleptic hypersensitivity, delusions, and psychosis (Krolak-Salmon, 2019 Elsevier Masson).

[0004] Synucleins, particularly α-synuclein ("α-syn"), have been shown to be major components of Lewy bodies ("LBs") and Lewy neurites ("LNs"), toxic inclusions found in the neurodegenerative conditions described above. LBs and LNs are found in the cytoplasm of dopaminergic neurons in PD and LBD and in oligodendrocytes in MSA. Therefore, α-synuclein and the synuclein family have been quantified in biological samples as potential biomarkers for diagnosing synucleinopathies. To date, synuclein detection has primarily been achieved using antibody-based assays such as ELISA. However, antibody-based detection techniques have proven suboptimal due to the presence of multiple protein or proteoforms, nonspecific detection, variable affinity, and limited antibody selection. There are also observations that α-synuclein concentrations measured differently in a single cerebrospinal fluid ("CSF") sample across multiple different laboratories (Mollenhauer et al., 2018) (Viode et al., 2019; Mollenhauer et al., 2018; Yang et al., 2017).

[0005] Clinical mass spectrometry ("MS") has been a promising method for identifying proteins indicative of pathological conditions over the past few decades and has been used to quantify α-syn. This technique, which uses the mass-to-charge ratio after ionization and fragmentation of target proteins, is specific, sensitive, accurate, and easy to use for multiple proteins and proteoforms.

[0006] Several studies have been conducted on the analysis of synuclein (most specifically α-synuclein) from CSF, but no significant differences were found between the various types of synucleinopathies. Summary of the Invention [Problem to be solved by the invention]

[0007] To utilize synuclein proteins as markers for neurodegenerative conditions, the present disclosure provides methods for detecting, in a sample of a subject, multiple synuclein peptides, each having a specific amino acid sequence, each of which may have a sequence that can be used to identify the exact identity of the synuclein protein, and which allow for quantification of each synuclein protein in the sample. [Means for solving the problem]

[0008] In one aspect, the disclosure relates to a method for detecting a combination of synuclein peptides in a subject, comprising detecting in a sample from the subject at least one combination selected from the group consisting of MDVFMK (SEQ ID NO: 4), EGVVAAAEK (SEQ ID NO: 5), QGVAEAAGK (SEQ ID NO: 6), EGVLYVGSK (SEQ ID NO: 7), EGVVHGVATVAEK (SEQ ID NO: 8), EQVTNVGGAVVTGVTAVAQK (SEQ ID NO: 9), and TVEGAGSIAAATGFVK (SEQ ID NO: 10).

[0009] In one aspect, the present disclosure relates to a method for detecting a combination of synuclein peptides in a subject, the method comprising detecting, in a sample from the subject, at least one combination selected from the group consisting of: (i) an α-synuclein peptide comprising QGVAEAAGK (SEQ ID NO: 6), an α-synuclein peptide comprising EQVTNVGGAVVTGVTAVAQK (SEQ ID NO: 9), an α / β-synuclein peptide comprising EGVVAAAEK (SEQ ID NO: 5), and an α / β-synuclein peptide comprising EGVLYVGSK (SEQ ID NO: 7); (ii) the α-synuclein peptide comprising QGVAEAAGK (SEQ ID NO: 6) and the α / β-synuclein peptide comprising EGVLYVGSK (SEQ ID NO: 7); and (iii) the α-synuclein peptide comprising EQVTNVGGAVVTGVTAVAQK (SEQ ID NO: 9) and the α / β-synuclein peptide comprising EGVLYVGSK (SEQ ID NO: 7). In some embodiments, the at least one combination of peptides each has a length of 20 amino acids or less. In some embodiments, the method includes quantifying the at least one combination of peptides. In some embodiments, the method includes comparing the amount of each of the at least one combination of peptides in the sample with a control, the control being a predetermined corresponding value obtained from a subject without PD. In some embodiments, the method further distinguishes between PD and dementia with Lewy bodies (LBD) or multiple system atrophy (MSA). In some embodiments, the detecting step is performed with a mass spectrometer. In some embodiments, the subject is human and the sample is blood or plasma, excluding cerebrospinal fluid. In some embodiments, the method includes detecting PD by the method described above and treating the subject.

[0010] In one aspect, the present disclosure relates to a method for diagnosing Parkinson's disease (PD) in a subject, comprising quantifying at least one peptide selected from the group consisting of an α-synuclein peptide comprising QGVAEAAGK (SEQ ID NO: 6), an α-synuclein peptide comprising EQVTNVGGAVVTGVTAVAQK (SEQ ID NO: 9), an α / β-synuclein peptide comprising EGVVAAAEK (SEQ ID NO: 5), and an α / β-synuclein peptide comprising EGVLYVGSK (SEQ ID NO: 7). In some embodiments, the at least one peptide comprises the α-synuclein peptide and / or the α / β-synuclein peptide. In some embodiments, the at least one peptide has a length of 30 amino acids, 20 amino acids, or less than 20 amino acids. In some embodiments, the detecting step comprises quantifying the at least one peptide. In some embodiments, the method further comprises comparing the amount of each of the at least one peptide in the sample to a control, which may be a predetermined corresponding value obtained from a subject without PD or a predetermined corresponding value obtained from a subject with PD. In some embodiments, the method distinguishes PD from other types of synucleinopathies, including LBD and MSA. In some embodiments, the method further distinguishes PD from dementia with Lewy bodies (LBD) or multiple system atrophy (MSA). In some embodiments, the detecting step is performed with a mass spectrometer. In some embodiments, the subject is human and the sample is blood or plasma, excluding cerebrospinal fluid. In some embodiments, the method comprises detecting PD by the method described above and treating the subject.

[0011] These and other embodiments, features, and advantages of the present disclosure may be more readily understood by those of ordinary skill in the art upon reading the following detailed description. [Brief explanation of the drawings]

[0012] [Figure 1] 1 shows the procedure for LC-MRM analysis of α-synuclein peptides. [Figure 2] 1 shows a data comparison graph comparing synuclein peptide levels in disease and control patients. [Figure 3] The results of the clinical performance of the α / β-syn (EGVLYVGSK (SEQ ID NO: 7)) peptide are shown in the ROC curve. [Figure 4A] A comparison of the PD group, other synucleinopathies (MSA and LBD), and control group using an established model is shown (α-syn[EQVTNVGGAVVTGVTAVAQK (SEQ ID NO: 9)]*-0.011268+α / β-syn[EGVLYVGSK (SEQ ID NO: 7)]*0.0096545). [Figure 4B] A comparison between the PD group and the MSA group using an established peptide combination is shown (α-syn[EQVTNVGGAVVTGVTAVAQK (SEQ ID NO: 9)]*-0.011268+α / β-syn[EGVLYVGSK (SEQ ID NO: 7)]*0.0096545). [Figure 5] A comparison between PD and MSA groups using an established peptide ratio is shown (α / β-syn EGVLYVGSK (SEQ ID NO: 7) / α-syn EQVTNVGGAVVTGVTAVAQK (SEQ ID NO: 9)). [Figure 6] Receiver operating characteristic curves monitoring the clinical performance (sensitivity and specificity) of peptide combinations in comparing PD and LBD patients are shown. [Figure 7] 1 shows ROC curves monitoring the clinical performance (sensitivity and specificity) of peptide combinations when comparing PD and MSA groups. [Figure 8] 1 shows a graph comparing data between diseases using a highly sensitive immunochemiluminescent assay. DETAILED DESCRIPTION OF THE INVENTION

[0013] In the context of this description, all publications, patent applications, patents, and other prior art documents referred to herein, unless otherwise indicated, are expressly incorporated herein by reference in their entirety for all purposes as if fully set forth herein.

[0014] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In case of conflict, the present specification, including definitions, will control.

[0015] Unless otherwise specified, trademarks are shown in superscript. Unless otherwise specified, all percentages, parts, ratios, etc. are by weight.

[0016] When an amount, concentration, or other value or parameter is expressed as a range, or when its upper and lower limits are listed, it is understood that all ranges formed from any pair of any of the upper limits of the range and any of the lower limits of the range are specifically disclosed, regardless of whether a range is separately disclosed. When a range of numerical values ​​is described herein, unless otherwise specified, the range is intended to include both ends of the range, and all integers and decimals within the range. It is not intended that the scope of the present disclosure be limited to the specific values ​​listed when defining a range.

[0017] When the term "about" is used, it is used to mean a particular action or result that can be obtained within a certain tolerance, and one of ordinary skill in the art knows how to obtain that tolerance. When the term "about" is used in describing a value within a range or an end value of a range, it should be understood that the disclosure includes the specific value or end value referred to.

[0018] As used herein, the terms "comprises," "comprising," "includes," "including," "has," "having," or any variations thereof, are intended to cover a non-exclusive inclusion. For example, a process, method, article, or apparatus comprising multiple elements is not necessarily limited to only those elements and may include other elements not expressly listed or inherent in the process, method, article, or apparatus.

[0019] The transitional phrase "consisting of" excludes any element, step, or ingredient not expressly stated in the claim, and excludes from the claim the inclusion of materials other than those recited, except for impurities ordinarily associated therewith. When the phrase "consists of" appears in a clause reciting the characterizing portion of a claim, rather than immediately following the preamble, it limits only the elements recited in that clause, and does not exclude other elements from the claim as a whole.

[0020] The transitional phrase "consisting essentially of" limits the scope of a claim to those materials or steps explicitly recited and which do not materially affect the basic and novel characteristics of the invention. Claims recited with "consisting essentially of" fall between closed claims recited in the "consisting of" format and fully open claims recited in the "comprising" format. Optional additives (at levels appropriate for such additives) and minor amounts of impurities, as defined herein, are not excluded from compositions recited with the term "consisting essentially of."

[0021] Furthermore, unless expressly stated otherwise, "or" and "and / or" refer to an inclusive condition and not to an exclusive condition. For example, a condition A or B, or A and / or B, is satisfied by any one of the following: A is true (or exists) and B is not true (or does not exist); A is not true (or does not exist) and B is true (or exists); or both A and B are true (or exist).

[0022] The use of the singular forms "a" or "an" to describe various elements and components herein is merely used for convenience and to give the general gist of the disclosure. The description should be read as including one or at least one, and such singular forms also include the plural unless it is clear that otherwise is intended.

[0023] The terms "substantial portion" or "substantially," as used herein and unless otherwise defined, mean all, nearly all, or the majority as would be understood by one of ordinary skill in the art in that context. It is intended to allow for some reasonable deviation from 100% that may normally occur in an industrial or commercial scale situation.

[0024] The terms "depleted" or "reduced" are synonymous with less than was originally present. For example, removing a significant portion of a material from a stream results in a "material-deficient stream" that is substantially depleted of that material. Conversely, the terms "enriched" or "increased" are synonymous with more than was originally present.

[0025] For convenience, many elements in this disclosure may be described separately, alternative lists may be provided, and numerical values ​​may be given in range form; however, for purposes of this disclosure, any claim combining any of these separate elements, lists, or ranges should not be considered as limiting the scope or support of this disclosure. Unless otherwise specified, each and every combination possible in this disclosure should be considered expressly disclosed for all purposes.

[0026] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described herein. Therefore, the materials, methods, and examples herein are illustrative only and, unless otherwise specified, are not intended to be limiting.

[0027] Synuclein Peptides The above three synuclein proteins have the following amino acid sequences represented by SEQ ID NOs: 1 to 3, as outlined in Table 1 below.

[0028] [Table 1]

[0029] The present disclosure enables the use of specific peptide segments of these synuclein proteins to identify and quantify synuclein proteoforms present in samples obtained from patients suspected of suffering from neurodegenerative conditions. Each of these synuclein proteins has distinct molecular behaviors, and identifying and quantifying their presence in a subject may provide a molecular / biochemical basis useful for diagnosing and distinguishing between various types of neurodegenerative conditions and synucleinopathies.

[0030] The sample from the subject subjected to detection of the peptide segment may be obtained from a variety of sources, including blood, plasma, cerebrospinal fluid, biopsy, saliva, nasal swab, oral swab, and any other biological sample that can be obtained invasively or non-invasively from the subject. In certain embodiments, the sample used in the detection may be blood, plasma, and / or cerebrospinal fluid. In other embodiments, the sample is blood or plasma. In yet another embodiment, the sample is plasma. In some embodiments, the sample excludes cerebrospinal fluid.

[0031] The synuclein protein contained in the sample may be proteolyzed using one or more proteases prior to detection. In some embodiments, the protease may be one or more of trypsin, LysC, LysN, and Glu-C. For example, the protease may be a combination of trypsin and Lys-C. In another example, the protease may be a combination of trypsin and LysN.

[0032] To facilitate the protease reaction, the sample from the subject may be cleaned up to remove non-protein molecules prior to the proteolysis step. For example, proteins in the sample may be precipitated and isolated from the remaining supernatant, and further purified using affinity columns, purification cartridges, and other means for separating non-protein materials from the sample.

[0033] By using different proteases or different protease combinations, different peptide segments can be generated from the synuclein protein. The amino acid sequences of these proteins (some of which may be unique to one particular synuclein proteoform or may be common to two or more proteoforms) can be used to identify and quantify the synuclein protein present in the sample tested. For example, digesting α-synuclein with a combination of trypsin and LysC can generate peptide segments with the amino acid sequences MDVFMK (SEQ ID NO: 4), EGVVAAAEK (SEQ ID NO: 5), QGVAEAAGK (SEQ ID NO: 6), EGVLYVGSK (SEQ ID NO: 7), EGVVHGVATVAEK (SEQ ID NO: 8), EQVTNVGGAVVTGVTAVAQK (SEQ ID NO: 9), and TVEGAGSIAAATGFVK (SEQ ID NO: 10). The resulting peptides can have 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, or fewer amino acids. Exemplary peptide segments generated by proteolysis of the three synuclein proteins with a combination of trypsin and LysC are outlined below in Table 2.

[0034] [Table 2]

[0035] Peptides generated by proteolysis can be detected and / or analyzed by a variety of methods, including mass spectrometry, immunoassays, gel electrophoresis and immunoblotting, and chromatography. In one embodiment, the peptides are detected, sequenced, and / or quantified by mass spectrometry.

[0036] Suitable methods for detecting and / or measuring the peptides described herein in a sample may include, for example, liquid chromatography (LC) and liquid chromatography-mass spectrometry (LC-MS). Examples include LC-MS, immunoassay, and electrochemiluminescence (ECL). LC methods use various detectors, such as an absorption detector (e.g., an ultraviolet-visible absorption detector) and an emission detector (e.g., a fluorescence detector), to quantify the abundance or abundance ratio from the peak area of ​​a chromatogram. Examples of LC-MS methods include selected ion monitoring (SIM), which uses one mass spectrometry (MS) detector, and multiple reaction monitoring (MRM), which uses a tandem mass spectrometry (MS / MS) detector. Examples include multiple reaction monitoring (MRM) and selected reaction monitoring (SRM). Among these, MRM (and SRM) are preferred from the viewpoint of high-sensitivity analysis.

[0037] Examples of immunological analysis methods include ELISA (enzyme-linked immunosorbent assay), FLISA (fluorescence-linked immunosorbent assay), and RIA (radioimmunoassay). Among these, ELISA is considered preferable from the viewpoint of safety, etc.

[0038] The peptides thus detected, sequenced and quantified can be further analyzed to determine whether a certain neurological condition exists in the subject from whom test sample is collected.For example, a certain peptide or peptide combination may be increased or decreased in a certain neurological condition.Therefore, in one embodiment, sample is analyzed to detect at least one combination selected from the group consisting of MDVFMK (SEQ ID NO: 4), EGVVAAAEK (SEQ ID NO: 5), QGVAEAAGK (SEQ ID NO: 6), EGVLYVGSK (SEQ ID NO: 7), EGVVHGVATVAEK (SEQ ID NO: 8), EQVTNVGGAVVTGVTAVAQK (SEQ ID NO: 9) and TVEGAGSIAAATGFVK (SEQ ID NO: 10). In some embodiments, the sample is analyzed to detect at least one combination selected from the group consisting of: (i) an alpha-synuclein peptide comprising QGVAEAAGK (SEQ ID NO: 6), an alpha-synuclein peptide comprising EQVTNVGGAVVTGVTAVAQK (SEQ ID NO: 9), an alpha / beta-synuclein peptide comprising EGVVAAAEK (SEQ ID NO: 5), and an alpha / beta-synuclein peptide comprising EGVLYVGSK (SEQ ID NO: 7); (ii) an alpha-synuclein peptide comprising QGVAEAAGK (SEQ ID NO: 6) and an alpha / beta-synuclein peptide comprising EGVLYVGSK (SEQ ID NO: 7); and (iii) an alpha-synuclein peptide comprising EQVTNVGGAVVTGVTAVAQK (SEQ ID NO: 9) and an alpha / beta-synuclein peptide comprising EGVLYVGSK (SEQ ID NO: 7). In another embodiment, the sample is analyzed to detect a combination comprising an α-synuclein peptide comprising QGVAEAAGK (SEQ ID NO: 6), an α-synuclein peptide comprising EQVTNVGGAVVTGVTAVAQK (SEQ ID NO: 9), an α / β-synuclein peptide comprising EGVVAAAEK (SEQ ID NO: 5), and an α / β-synuclein peptide comprising EGVLYVGSK (SEQ ID NO: 7). In one embodiment, the sample is analyzed to detect a combination comprising an α-synuclein peptide comprising QGVAEAAGK (SEQ ID NO: 6) and an α / β-synuclein peptide comprising EGVLYVGSK (SEQ ID NO: 7).In one embodiment, the sample is analyzed to detect a combination comprising an α-synuclein peptide comprising EQVTNVGGAVVTGVTAVAQK (SEQ ID NO: 9) and an α / β-synuclein peptide comprising EGVLYVGSK (SEQ ID NO: 7). In one embodiment, the peptide combination described above is quantified. In some embodiments, the present disclosure relates to a method of diagnosing a neurological condition in a subject by analyzing, detecting, sequencing, or qualifying the peptide combinations described herein.

[0039] In one embodiment, the neurological condition detected or diagnosed based on the above-described peptide detection method may be Parkinson's disease (PD). In such a case, the method may include detecting at least one combination as described above. The method may further include quantifying each peptide and comparing the quantified amount of each peptide of the at least one combination in a sample with a control. In some embodiments, the control in such an embodiment may be a predetermined corresponding value obtained from a subject without PD or a predetermined corresponding value obtained from a subject with PD. For example, the predetermined corresponding value may be the amount of an α-synuclein peptide comprising QGVAEAAGK (SEQ ID NO: 6) in a sample from a subject without PD, and such predetermined corresponding value is compared with the measured amount of an α-synuclein peptide comprising QGVAEAAGK (SEQ ID NO: 6) in a sample from the subject to determine whether the subject has PD. If there is a statistically significant difference between the predetermined corresponding value and the measured amount, the subject may be determined to have PD. If there is no statistically significant difference between the predetermined corresponding value and the measured amount, it can be determined that the subject does not have PD. Alternatively, the predetermined corresponding value can be the amount of an α-synuclein peptide comprising QGVAEAAGK (SEQ ID NO: 6) in a sample from a subject with PD, and such predetermined corresponding value is compared with the measured amount of an α-synuclein peptide comprising QGVAEAAGK (SEQ ID NO: 6) in a sample from the subject to determine whether the subject has PD. If there is a statistically significant difference between the predetermined corresponding value and the measured amount, it can be determined that the subject does not have PD. If there is no statistically significant difference between the predetermined corresponding value and the measured amount, it can be determined that the subject has PD. In some embodiments, the method can further include quantifying each peptide and comparing the ratio of the quantified amounts of each peptide in the sample to a control.

[0040] The methods described above can be used to diagnose or distinguish between PD and other types of synucleinopathies. For example, in one embodiment, the method can distinguish between PD and LBD. In such an example, the method can include detecting an α-synuclein peptide comprising QGVAEAAGK (SEQ ID NO: 6). The method can include detecting an α / β-synuclein peptide comprising EGVLYVGSK (SEQ ID NO: 7). In a further embodiment, the method for distinguishing between PD and LBD includes comparing the amount of each peptide of at least one combination in a sample to a control. The control in such an embodiment can be a predetermined corresponding value obtained from a subject with LBD or a predetermined corresponding value obtained from a subject with PD.

[0041] In one embodiment, the method can diagnose or distinguish between PD and multiple system atrophy ("MSA"). In such an example, the method can include detecting an α-synuclein peptide comprising EQVTNVGGAVVTGVTAVAQK (SEQ ID NO: 9). The method can include detecting an α / β-synuclein peptide comprising EGVLYVGSK (SEQ ID NO: 7). The method can include detecting an α-synuclein peptide comprising EQVTNVGGAVVTGVTAVAQK (SEQ ID NO: 9) and an α / β-synuclein peptide comprising EGVLYVGSK (SEQ ID NO: 7). In a further embodiment, the method for distinguishing between PD and MSA includes comparing the amount of each peptide of at least one combination in a sample to a control. The control in such an embodiment can be a predetermined corresponding value obtained from a subject with MSA or a predetermined corresponding value obtained from a subject with PD. Similarly, the method can be used to distinguish between PD and any one synucleinopathy or control.

[0042] In a further embodiment, the method for distinguishing between PD and other synucleinopathies may further comprise applying the amount of at least one peptide to a computational model adapted to distinguish between PD and other types of synucleinopathies, wherein the synucleinopathy for which the computational model is adapted may be LBD, MSA, or any other synucleinopathy.

[0043] The above-described method can be used to detect synuclein peptides in any subject suspected of suffering from a neurodegenerative condition. For example, the subject may be a subject who has not previously been diagnosed with a neurodegenerative condition. In another embodiment, the subject may be a subject who has not previously been diagnosed with a synucleinopathy. Furthermore, the subject may be a subject who has not previously been diagnosed with PD, MSA, or LBD. The subjects for which the above-described method can be used are not particularly limited and include human subjects, non-human mammalian subjects, and other vertebrates.

[0044] In one aspect, the methods described herein may relate to methods of treating a subject with PD. In some embodiments, the methods may include detecting or diagnosing PD as described above. In some embodiments, the treatment may include administering a pharmaceutical composition to increase or replace dopamine. Such a pharmaceutical composition may include at least one selected from the group consisting of a dopamine promoter, an antidepressant, a cognitive enhancer, or an antitremor agent. In some embodiments, the treatment may include deep brain stimulation. In some embodiments, the treatment may include implantation of an electrical pulse generator. [Example]

[0045] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0046] [Example 1] Reagents and internal standards Recombinant full-length α-synuclein and nitrogen-15 uniformly labeled ("U-15N") recombinant full-length alpha-synuclein proteins were obtained from LGC (Teddington, UK). U-15N human recombinant β- and γ-synuclein proteins were purchased from Promise advanced proteomics (Grenoble, France). Human recombinant light forms of beta and gamma were obtained from Anaspec (Fremont, California, USA) and Interchim (Montlucon, France), respectively. Standard concentrations of light protein were assayed for α-synuclein by immunochemiluminescence using a MesoScale Discovery (Rockville, Maryland, USA) system. MS-grade trypsin / Lys-C, rLys-C, glu-C, and Lys-N were purchased from Promega (Walldorf, Germany). Ammonium bicarbonate (ABC), 70% perchloric acid, trifluoroacetic acid (TFA), and human serum albumin (HSA) were purchased from Sigma-Aldrich (Saint Quentin Fallavier, France). RPW cartridge tips were purchased from Agilent Technologies (Santa Clara, California, USA). All liquid chromatography (LC) solvents, including acetonitrile, water, and formic acid, were LC-MS grade and purchased from Biosolve (Dieuze, France). 99% pure AQUA (absolute quantitation) peptides for α-syn-126 / 98, α-syn-112 / 98, and α-syn-41 were purchased from Thermo Fisher Scientific (Lincoln, Massachusetts, USA). Alternative α-syn and its variant specific peptides (95% pure) were also purchased from Thermo Fisher Scientific (Lincoln, Massachusetts, USA). Goat serum (Capra hircus) was ordered from Clinisciences (Nanterre, France).An immunoassay kit named U-PLEX Human α-Synuclein Kit was purchased from MesoScale Discovery.

[0047] Stock solutions, calibration standards, and quality control ("QC") samples Protein and peptide standards were dissolved in 50 mM ammonium bicarbonate to a final concentration of 1 microgram / microliter, aliquoted into low protein binding tubes (1.5 or 0.5 mL), and stored at -20°C or -80°C.

[0048] Method development was performed on a pool of plasma samples at one level, which were prepared as follows: samples from suspected AD patients were mixed in a 50 ml Falcon tube, aliquoted into 1.5 ml Eppendorf LoBind tubes (150 microliters), and stored at -80°C.

[0049] For method validation, plasma quality control (QC) samples containing three levels of α-syn were prepared by immunoassay (MSD measurement) of 42 plasma patient samples. These patients all had different α-synuclein levels, with the highest (25%) α-syn concentration pooled in a high pool, the lowest (25%) in a low pool, and the remaining patient samples in an intermediate pool, prepared as previously described.

[0050] For the clinical validation part, one level of control QC was established for plasma, consisting of a pool of samples previously tested for synuclein concentration using the MesoScale Discovery ("MSD") immunoassay and taken as the median mean value (52.16 ng / mL plasma).

[0051] calibration Nine calibration points for plasma were prepared by diluting recombinant light α-, β-, and γ-synuclein standards with goat (Capra hircus) serum. The calibration ranges in plasma (detailed in Table 3) were [0–5571] ng / mL for α-synuclein, [0–410] ng / mL for β-synuclein, and [0–1401] ng / mL for γ-synuclein. N-terminal peptide concentrations, α-synuclein isoform peptide concentrations, and synuclein alternative protein heavy-labeled peptide concentrations were as listed in Table 4.

[0052] [Table 3]

[0053] [Table 4]

[0054] Sample preparation Ninety-five microliters of plasma sample was thawed on ice for 1 hour, and then 855 microliters of deionized water was added to the plasma sample. 5.7 microliters of 50 mM ammonium bicarbonate containing U-15N-labeled recombinant α-, β-, and γ-synuclein internal standards (10 ng / microliter) was added to the sample and QC. The sample was mixed and protein was precipitated by placing it on ice for 15 minutes with 142.5 microliters of 70% perchloric acid. The sample was then centrifuged at 16,000 g for 15 minutes at 4°C, and the supernatant was collected in a new low-binding tube. 95 microliters of 1% trifluoroacetic acid (TFA) was added. After reducing the volume by vacuum drying (Speedvac, Labconco) at room temperature, the supernatant was cleaned up using an AssayMap Bravo (Agilent Technologies) in a RPW cartridge and placed in a LoBind 96-well (deep-well) plate. The cartridge was washed with water at a suction rate of 10 microliters / min, and then the sample was loaded onto the cartridge at a rate of 5 microliters / min. Proteins were washed using a reverse phase W (RP-W) tip with 10% ACN and 0.1% formic acid at 10 microliters / min and eluted with 45% ACN and 0.1% formic acid at 5 microliters / min. The sample was evaporated to dryness under vacuum at room temperature using a SpeedVac for 1.5 hours. The sample was reconstituted with 20 microliters of 50 mM ABC. Finally, 7 microliters of 1 microgram / microliter trypsin / Lys-C were added. The plasma sample was incubated at 37°C for 4 hours with gentle agitation (450 rpm). After incubation, 0.5 microliters of formic acid was added to the sample.

[0055] The overall procedure for LC-MRM analysis of α-synuclein peptides is shown in Figure 1. LC-MRM analysis Samples were analyzed using a Shimadzu LC (Mikros) and a Shimadzu triple quadrupole 8060 mass spectrometer (Duisburg, Germany) in positive ionization mode. Fifteen microliters of sample were injected onto an Agilent Technologies ZORBAX SB-Aq (1 × 150 mm, 3.5 micrometer) column at 35 °C. Mobile phase A consisted of water and 0.1% formic acid (FA), and mobile phase B consisted of ACN and 0.1% formic acid. The solvent gradient used was a gradual increase of organic solvent (phase B) from 0% to 30% over 30 min. The total run time for the LC-MRM analysis was 40 min at a flow rate of 50 microliters / min. The LC gradient is listed in Table 5. The minimum dwell time used was 20 milliseconds ("msec") per peptide (with a range of 22 to 111 msec). The ion source parameters were nebulizer gas flow rate of 3 L / min, heating gas flow rate of 10 L / min, interface temperature of 300 °C, desolvation line temperature of 250 °C, heat block temperature of 400 °C, and drying gas flow rate of 10 L / min. These parameters were optimized in advance for peptide analysis. Three to six transitions were obtained for each peptide in the biological sample. The peptide positions, selections, and transitions of synuclein are listed in Table 6. The concentrations of α-syn and β-syn peptides were investigated by comparing unlabeled and labeled peptides using multiple reaction monitoring (MRM) analysis.

[0056] [Table 5]

[0057] [Table 6-1]

[0058] [Table 6-2]

[0059] [Table 6-3]

[0060] Analytical Method Validation Intra- and inter-assay precision was determined by analyzing one QC sample (in triplicate, plasma samples, three different α-syn levels: high 230 ng / ml, medium 87 ng / ml, and low 45 ng / ml) daily for four days. For CSF samples, one QC (in triplicate, one level) was determined by analyzing one QC (in triplicate, one level) daily for four days.

[0061] Sample stability, including the effect of thawing samples on ice or at room temperature, was tested as follows: one goat serum was thawed in triplicate on ice for 0, 2, 4, and 6 hours or at room temperature, and then spiked with light and heavy recombinant α-syn, β-syn, and γ-syn standards (n=3). Additionally, QC samples (CSF at one level and plasma at three levels: high, medium, and low) were tested after storage in the autosampler at 4°C for 0, 12, 24, 36, and 48 hours (n=3). Parallelism and dilution effects were investigated by spiking goat serum with α-syn, β-syn, and γ-syn at high concentrations (5 ng / ml for α-syn and γ-syn, 10 ng / ml for β-syn) and then serially diluting 2-, 3-, and 4-fold, respectively, in 50 mM ABC or normal goat serum (spiked with the same high concentrations) (n=3).

[0062] Precision and linearity were examined on each of the four days using three duplicate calibration standards, while intra- and inter-assay precision was examined.

[0063] The lower limit of quantification ("LLOQ") was determined as the lowest concentration at which the coefficient of variation was greater than 20% (n=3) and the signal-to-noise ratio (S / N) was at least greater than 3. For this purpose, α-syn and γ-syn peptides were spiked into goat serum at four different concentrations (6.45, 12.90, 19.35, and 32.25 ng / ml; these are close to the LLOQs of the peptides). Details of the LLOQs are shown in Table XO for α-syn peptide. β-syn peptide was spiked into goat serum at three concentrations (80.7, 96.9, and 113.1 ng / ml). The same method was applied to AQUA peptides containing N-terminal α / β-syn peptides, alternative splicing, and alternative peptides at three concentrations: 0.155, 0.306, and 0.613 ng / ml (N-ter), 0.0134, 0.0674, and 0.134 ng / ml (α-syn-41), 0.02, 0.101, and 0.202 ng / ml (α-syn-112 / 98), 0.019, 0.093, and 0.186 ng / ml (α-syn-126 / 98), and 0.008, 0.015, and 0.03 ng / ml (α-syn-alt).

[0064] Matrix effects and recovery rates were investigated by comparing MS signals between normal goat serum added at the beginning of the experiment, normal goat serum added at the same concentration after digestion, and solvent added at the same concentration after digestion (n=3).

[0065] After the highest concentration calibration standard, blank samples were analyzed in the same way as the tested samples to check for carryover effects (n=4). All these parameters are shown in the results column in Tables 7 and 8.

[0066] [Table 7]

[0067] [Table 8-1]

[0068] [Table 8-2]

[0069] Immunoassays Plasma samples were analyzed by immunochemiluminescence assay using MesoScale Discovery ("MSD"). Plasma samples were diluted 200-fold to determine total α-synuclein concentration. All measurements were performed according to the manufacturer's instructions. For antibodies (AB), a capture rabbit monoclonal antibody targets the C-terminal portion of α-syn (residues 110-125), and a monoclonal mouse antibody targets residues 15-125.

[0070] Patients and fluid collection All participants provided informed consent for enrollment in the study, which was approved by the local ethics committee of the Montpellier University Hospital. The general characteristics of the 143 patients are shown in Table 9. The mean age of participants was 71 (±8.2) years, and 65% were male. Patients were divided into three disease groups: PD (n = 82), LBD (n = 32), and MSA (n = 8). The control group (n = 21) had various non-neurodegenerative disorders, including neuropathic changes (33.3%), vascular changes (29.3%), immune changes (20.8%), and hydrocephalus (23.1%). The exclusion criterion for the control group was an abnormal Alzheimer's biomarker profile (i.e., a low amyloid beta ratio (Ab 40 / 42) and elevated Tau / pTau protein levels). PD patients had been diagnosed with the condition an average of 11 (±7.4) years prior to the time of analysis. Of these, 41.9% showed cognitive impairment, 50.8% showed anosmia, and 69.7% showed non-motor signs. The mean Hoehn and Yahr scores and Unified Parkin's Disease Rating Scale (UPDRS) II and III scores were 2.4 (±0.7), 19.7 (±11.2), and 36 (±18.9), respectively, corresponding to mild motor impairment. Intravenous plasma samples were stored at the Montpellier Neurobank (#DC-2008-417, certified NFS 96-900 CHU resource center BB-0033-00031). Approval for the handling of personal data was obtained from the French Data Protection Authority (CNIL). Upon reception, 0.5 mL of plasma was aliquoted into 1.5 mL polypropylene LoBind tubes and stored at -80°C until further analysis.

[0071] [Table 9]

[0072] Data Reprocessing Statistical analysis was performed using MedCalc (version 19.0.3). The Shapiro-Wilk test was used to check the normality of the dataset, and the Mann-Whitney-Wilcoxon test was used to test the significance of the results. ROC curves (sensitivity, corresponding to a true-positive result, as a function of specificity minus 100, representing a false-positive result) were used to obtain clinical performance. The statistical model for determining the results was examined by logistic regression using backward and forward regression (due to the small number of variables and the large number of samples). For selection, the smallest variable and a p-value of <0.05, the same as the coefficient obtained by logistic regression, were used. The coefficients (shown below) were applied to the quantification of alpha and beta syn peptides: α-syn [EQVTNVGGAVVTGVTAVAQK (SEQ ID NO: 9)] * -0.011268 + α / β-syn [EGVLYVGSK (SEQ ID NO: 7)] * 0.0096545).

[0073] Chromatogram extraction and the area of ​​synuclein peptides for quantification were obtained using Skyline software (version 20.1.0) and LabSolutions Insight Browser. LC-MRM analysis was performed using LabSolutions software (version 5.99). Optimization of MS parameters, such as collision energy and voltage applied to the quadrupole, was performed using the "optimization for method" function available from LabSolutions software; these parameters and the peptides included in the method are listed in Table 5.

[0074] result Method development For α-synuclein, all proteotypic peptides were selected except for the C-terminal peptide, which accounts for approximately 70% of the protein sequence. All peptides and transitions were selected based on the sensitivity and selectivity observed in biological samples compared with added internally labeled standards. The C-terminal peptide has a long residue chain (40 amino acids long) and is composed of many acidic residues, resulting in low sensitivity in MS analysis. In biological fluids such as CSF and plasma, this C-terminal peptide is always below the LLOQ. The same was true for β-syn and γ-syn. All synuclein species and sequences monitored by the above method are listed in Table 7.

[0075] Analytical Method Validation Both β-syn and γ-syn were consistently below the LOQ in all three levels of plasma QC (high, medium, and low α-syn) and in patient samples. Similar results were obtained for alternative splicing isoforms and alternative α-syn peptides. Only α-syn peptides and α / β-syn consensus peptides were detected and quantified in all QC and patient samples. (ac) Two peptides with the sequences MDVFMK (SEQ ID NO: 1) and EGVVHGVATVAEK (SEQ ID NO: 8) were excluded from analytical (and clinical) validation due to instability and nonlinear response in MS calibration.

[0076] The intra-assay precision results for α-syn peptide were 5-13% for low-level QC samples (mean α-syn peptide 46.11 ng / ml, mean α / β-syn peptide 195.95 ng / ml), 3-8% for medium-level QC samples (mean α-syn peptide 74.70 ng / ml, mean α / β-syn peptide 270.82 ng / ml), and 3-5% for high-level QC samples (mean α-syn peptide 140.24 ng / ml, mean α / β-syn peptide 427.09 ng / ml). The intra-assay precision for α / β-syn peptide was 7-8% for low-level QC samples, 4-5% for medium-level QC samples, and 2-6% for high-level QC samples.

[0077] Inter-assay precision for α-synuclein peptides in low-level QC samples was within the range of 12-19% (except for the TVEGAGSIAAATGFVK (SEQ ID NO: 10) peptide, which was 22% in the low-level QC sample). The coefficient of variation (CV) for α-syn peptides was 13-15% in medium-level QC samples and 10-13% in high-level QC samples. Inter-assay precision for common α / β-syn peptides was 15-17% in low-level QC samples, 13-16% in medium-level QC samples, and 12-14% in high-level QC samples.

[0078] For the α-syn peptide, the LOQ and linearity ranged from 6.45 to 19.35 ng / ml and from 0 to 5571 ng / ml, respectively. For the α / β-syn consensus peptide, the LOQ was similar to that of the α-syn peptide, with a linearity range of 0 to 5981 ng / ml.

[0079] The LOQs were 113.1 ng / ml for the β-syn peptide, greater than 32.25 ng / ml for the β / γ-syn QGVTEAAEK peptide, and ranged from 6.45 to 32.25 ng / ml for the γ-syn peptide. The linearity ranged from 0 to 410 ng / ml for the β-syn peptide, 0 to 1811.5 ng / ml for the common β / γ-syn, and 0 to 1401.2 ng / ml for the γ-syn peptide.

[0080] The LLOQs for AQUA peptides were >0.613 ng / ml for the N-terminal peptide, >0.134 ng / ml for the α-syn-41 peptide, 0.101 ng / ml for the α-syn-112 / 98 peptide, 0.093 ng / ml for the α-syn-126 / 98 peptide, and 0.03 ng / ml for the α-syn-alt peptide. The mutant α-syn-alt peptide was not detected.

[0081] After high-concentration synuclein peptide analysis, no peptides were detected in the blanks and the carryover was less than 1% (except for α-syn EQVTNVGGAVVTGVTAVAQK (SEQ ID NO: 9) which was 1.18%). All these parameters are shown in Tables 7 and 8.

[0082] Regarding sample stability, the % accuracy for the effect of dilution ranged from 98 to 113% (over 120% accuracy) for the α-syn peptide TVEGAGSIAAATGFVK (SEQ ID NO: 10), which remained stable up to 2-fold dilution, and for the α / β-syn peptide EGVLYVGSK (SEQ ID NO: 7), which remained stable up to 3-fold dilution, ranged from 87 to 117%.

[0083] The percent parallelism precision ranged from 86-100% for the α-syn peptides (>120% precision), except for TVEGAGSIAAATGFVK (SEQ ID NO: 10), which remained stable up to 2-fold dilution, and EQVTNVGGAVVTGVTAVAQK, which remained stable up to 3-fold dilution. For the α / β-syn consensus peptides, this precision ranged from 89-100%.

[0084] For samples thawed on ice, the percent precision ranged from 88-103% for α-syn peptides and 95-100% for α / β-syn peptides after 6 hours of thawing. For samples thawed at room temperature, the stability remained stable for up to 4 hours for all synuclein peptides, with precision ranging from 99-106% for α-syn peptides and 100-106% for α / β-syn peptides.

[0085] For stability in the autosampler at 4°C, the % precision was 95-121% for α-syn peptides, 93-103% for α / β-syn peptides at low levels, 80-111% for α-syn peptides, and 96-105% for α / β-syn peptides at medium levels (except for the TVEGAGSIAAATGFVK (SEQ ID NO: 10) peptide, which was below 80%). For high-level QC samples, the precision for stability was 97-115% for α-syn peptides and 96-108% for α / β-syn peptides.

[0086] The matrix effect and recovery were investigated simultaneously for all peptides (methods are described in "Materials and Methods"). The matrix effect ranged from -51% to 9%, meaning a maximum 51% loss and a maximum 9% gain in signal due to interference, and a maximum 93% loss in recovery (-93% to -41% for all peptides). Details of these parameters are listed in Table 8.

[0087] The levels of α-syn peptide and α / β-syn peptide at the three levels of QC were determined according to the Westgard rules (Westgard et al., 1981, Clin Chem).

[0088] Clinical Validation A total of 143 plasma samples were obtained from PD (n=82), MSA (n=8), LBD (n=32), and control (n=21), all obtained by MS and immunoassay. Details of the patients who participated in the study are shown in Table 9.

[0089] MS quantitative In clinical validation, only peptides derived from α-syn and consensus α / β-syn were detected in plasma patient samples. β-synuclein and γ-synuclein were below the LOQ in patient samples. Similar results were obtained for alternative splicing isoforms and alternative α-syn peptides.

[0090] The clinical performance of the developed MS method was evaluated by examining the area under the receiver operating characteristic curve (AUC), sensitivity, and specificity, as well as the p-value for distinguishing between disease groups. The clinical performance results of α-syn peptides in plasma patient samples are summarized in Table 10.

[0091] [Table 10]

[0092] Specifically, the α-syn TVEGAGSIAAATGFVK (SEQ ID NO: 10) peptide showed no significant difference between groups (minimum p-value = 0.2134). The α-syn EQVTNVGGAVVTGVTAVAQK (SEQ ID NO: 9) peptide and the α / β-syn EGVVAAAEK (SEQ ID NO: 5) peptide were able to distinguish between PD and control patients (p-values ​​of 0.0205 and 0.0277, respectively), with sensitivity and specificity of 0.87 and 0.67, respectively. The area under the receiver operating characteristic curve (AUC) was 0.664 and 0.656, respectively. Two peptides, α / β-syn EGVLYVGSK (SEQ ID NO: 7) and α-syn QGVAEAAGK (SEQ ID NO: 6), were able to distinguish between PD and control and LBD patients. The p-values ​​for α / β-syn EGVLYVGSK (SEQ ID NO: 7) differentiation from controls and LBD were 0.0001 and 0.0014, respectively, and for α-syn QGVAEAAGK (SEQ ID NO: 6) differentiation from controls and LBD were 0.0014 and 0.0331, respectively. The sensitivity, specificity, and AUC evaluated for PD patients compared with control patients were 0.84, 0.76, and 0.787, respectively, for α / β-syn EGVLYVGSK (SEQ ID NO: 7). These values ​​were also examined for the same peptides for differentiation between PD and LBD groups, yielding values ​​of 0.7, 0.69, and 0.694, respectively. The sensitivity, specificity, and AUC of α-syn QGVAEAAGK (SEQ ID NO: 6) for differentiation between PD and control patients were 0.83, 0.76, and 0.726. Only the MSA group could not be distinguished from PD patients.

[0093] A graph comparing data between disease groups and control patients for the α / β-syn EGVLYVGSK (SEQ ID NO: 7) peptide is shown in Figure 2. The best clinical performance in distinguishing between PD and control patients was obtained with the α / β-syn EGVLYVGSK (SEQ ID NO: 7) peptide. The sensitivity and specificity for PD versus controls were 0.84 and 0.76, respectively. The clinical performance results for the α / β-syn EGVLYVGSK (SEQ ID NO: 7) peptide are shown in the form of a receiver operating characteristic curve in Figure 3.

[0094] The combination of these two peptides effectively discriminated between PD and MSA patients (p-value = 0.0021). Because this combination and its coefficient signature were similar to the Ab40 / 42 ratio, the ratio of α-syn peptide was tested, and the results are shown in Figure 5 in the form of a group comparison graph.

[0095] Although this peptide combination has a slightly smaller p-value for distinguishing PD from MSA than the ratio, the ratio is easier to apply because it does not require the use of coefficients, as is the case with logistic regression.

[0096] The best clinical performance in distinguishing between PD and DLB groups was obtained from the peptide combination. In fact, the obtained sensitivity and specificity values ​​were 0.7 and 0.68, respectively. The ROC curve showing the clinical performance of the peptide combination in PD versus LBD is shown in Figure 6.

[0097] The best clinical performance in distinguishing between PD and MSA groups was obtained with the peptide combination. The sensitivity, specificity, and AUC of the peptide combination in distinguishing between PD patients and MSA were 0.84, 0.85, and 0.831, respectively. The ROC curves showing the clinical performance of the peptide combination in PD versus LBD are shown in Figure 7.

[0098] Immunoassay quantification The same plasma cohort was also quantified by a highly sensitive immunochemiluminescence assay. A graph comparing the data between the diseases is shown in Figure 8. No significant differences were observed in α-syn levels quantified by immunochemiluminescence assay. The α-syn levels in the control group were slightly lower than those in the PD group.

[0099] Correlation The same plasma cohort was also quantified by a highly sensitive immunochemiluminescence assay. A graph comparing the data between the diseases is shown in Figure 8. No significant differences were observed in α-syn levels quantified by immunochemiluminescence assay. The α-syn levels in the control group were slightly lower than those in the PD group.

[0100] A strong correlation was observed between α-syn QGVAEAAGK (SEQ ID NO: 6) and α / β-syn EGVLYVGSK (SEQ ID NO: 7) in patient plasma samples (r=0.827). Moderate correlations were observed between the α / β-syn EGVLYVGSK (SEQ ID NO: 7) peptide and peptide combinations and ratios (r=0.568 and r=0.66, respectively). No correlation was observed between peptide and immunoassay quantification (r<0.277). There was no correlation between gender, age, and sample collection date and α-syn peptide, combinations containing α-syn peptide, or ratios (Pearson coefficients averaged r<-0.124). Concentrations of α / β-syn EGVLYVGSK (SEQ ID NO: 7) peptide correlated with Ab40 concentrations in CSF (r = 0.765) and inversely correlated with the Ab42 / 40 ratio (r = -0.74). There was also a slight, inverse correlation between α-syn QGVAEAAGK (SEQ ID NO: 6) peptide and the Ab42 / 40 ratio (r = -0.52). Plasma peptide ratios and the Ab42 / 40 ratio in CSF were approximately correlated (r = 0.444). α-syn peptides did not correlate with any of the UPDRS measures used to monitor PD disease severity (r < -0.206).

[0101] The results of LC-MRM methods for quantification in patient samples are of interest in terms of diagnostics or treatment development, as multiple peptides are observed at different levels in plasma.

[0102] Consideration During method development and validation, the absence of cysteine ​​residues in the synuclein sequence obviates the need for denaturation and alkylation steps typically performed on proteins.

[0103] The β-syn, γ-syn, α-syn, and alternative α-syn splicing isoforms were always below the LOQ in plasma QC and patient samples because of their very low concentrations in biological fluids (likely below the pg / ml range).

[0104] At the RNA level, multiple alternative splicing isoforms were detected at different expression levels in multiple different synucleinopathies, and we aimed to target these α-syn species to gain the same insight at the protein level, since, to the best of our knowledge, such differences at the protein level have not been described in the literature.

[0105] As a proof of concept, two proteotypic peptides derived from alternative α-syn were monitored in an attempt to detect these specific species in plasma QC or patient samples.

[0106] In clinical validation of MS, all α-syn peptides, and all but the α-syn TVEGAGSIAAATGFVK (SEQ ID NO: 10) peptide, tended to be significantly higher in the plasma of PD patients than in controls. Considering the gut-brain hypothesis in PD, this implies that α-syn species may originate from the peripheral enteric plexus or erythrocytes in the early stages of PD, which may not be the case in other synucleinopathies, especially MSA (Chang et al., 2020 (Frontiers in neurology)). Although all synuclein peptides correlated with each other (minimum r value < 0.727), none of them correlated with disease severity (UPDRS II, III, and V; r < -0.206). This may be explained by the low values ​​of the various UPDRS scores in PD patients due to mild or moderate progression (see Patient Body Fluid Collection).

[0107] Peptides detected in the same patients were quantified by a highly sensitive immunoassay, revealing no significant differences between disease groups. The main difference between these two proteomic approaches is the use of antibodies by immunoassays. In this case, the antibodies used target the C-terminal portion of the protein, which has undergone considerable post-translational modifications and truncations (described in a previous study (Pons et al., 2022, Frontiers in aging neuroscience)). These have a significant impact on the total quantification of the soluble form of α-syn. Furthermore, immunoassay techniques, unlike MS, cannot simultaneously quantify proteoforms or truncated segments.

[0108] Previous literature highlights the fact that α-syn species / conformations (the content of B-sheet conformation in the overall conformation of α-syn) differ among various synucleinopathies, depending on the subcellular localization of α-syn (e.g., oligodendrocytes in MSA and dopaminergic neurons in PD / DLB (Mehra et al., 2019; Mou et al., and Porro et al.)). This has been demonstrated by Real-Time Quaking-Induced Conversion (RT-QuIC) and Protein Modification Cyclic Amplification (PMCA) techniques in brain homogenates or CSF samples (Shahnawaz et al., 2018 and Groveman et al., 2018 (Acta neuropathologica communications)). This means that, depending on the pathology, distinct α-synuclein species may be expressed in the insoluble fraction (LB or aggregated forms) and the soluble fraction (monomers, dimers, oligomers). Here, we used LC-MRM to analyze the soluble fraction of α-syn in the plasma of participating patients.

[0109] The goal was to distinguish the PD group from all other synucleinopathies and controls. To this end, peptide combinations and ratios of α-syn peptides were investigated. The peptide combinations and ratios were based on the same principle as the amyloid beta ratio (Ab40 / Ab42). In fact, the ratio Ab40 / Ab42 is obtained by dividing the concentration of the peptide with a lower aggregation tendency by the concentration of the peptide with a higher aggregation potential and longer size. For AD, the Ab40 / Ab42 ratio is more robust and has better clinical performance than two separate peptides (Hansson et al., 2019, Alzheimer's Research & Therapy).

[0110] The peptides in the α-syn peptide combination are not derived from the same part of the protein. In fact, the α / β-syn EGVLYVGSK (SEQ ID NO: 7) peptide (positions 35-43 in the sequence) is part of the N-terminal region.

[0111] McGlinchey et al. (2021) reported that 36–40 residues are important for the replication of the full-length structure and the formation of various fibril structures. This suggests that the N-terminal portion may alter the intermolecular interactions in the amyloidogenesis mechanism and fibril assembly. The authors noted that C-terminal truncation resulted in less conformational changes than N-terminal truncation. Deletion of the C- and / or N-terminal portions typically enhances the interaction of the hydrophobic NAC portion with other proteins or other α-syn fragments / species (Sorrentino et al., 2020 (JBC)).

[0112] The α-syn EQVTNVGGAVVTGVTAVAQK (SEQ ID NO: 9) peptide (positions 61-80 in the full-length sequence) is incorporated into the NAC moiety that constitutes the fibril core of the protein and is involved in the aggregation process of α-syn (Sorrentino et al., 2020 (JBC) and (McGlinchey et al., 2021)).

[0113] Similar to what was done with Ab40 / 42 ratios for AD, peptide combinations and / or alpha-syn peptide ratios were established with the aim of improving clinical performance for PD, especially in distinguishing it from other synucleinopathies.

[0114] Currently, neurologists have difficulty distinguishing between various synucleinopathies (especially MSA and PD) because the core symptoms are common, especially in the prodromal stage. The combinations or ratios described above allow biomarkers to distinguish between these diseases with good sensitivity and specificity (similar to that of Ab40 / 42) (Hansson et al., 2019, Alzheimer's Research & Therapy).

Claims

1. 1. A method for detecting a combination of synuclein peptides in a subject, comprising: A method comprising detecting at least one combination selected from the group consisting of MDVFMK (SEQ ID NO: 4), EGVVAAAEK (SEQ ID NO: 5), QGVAEAAGK (SEQ ID NO: 6), EGVLYVGSK (SEQ ID NO: 7), EGVVHGVATVAEK (SEQ ID NO: 8), EQVTNVGGAVVTGVTAVAQK (SEQ ID NO: 9), and TVEGAGSIAAATGFVK (SEQ ID NO: 10) in a sample from the subject.

2. The at least one combination is (i) an α-synuclein peptide comprising QGVAEAAGK (SEQ ID NO: 6), an α-synuclein peptide comprising EQVTNVGGAVVTGVTAVAQK (SEQ ID NO: 9), an α / β-synuclein peptide comprising EGVVAAAEK (SEQ ID NO: 5), and an α / β-synuclein peptide comprising EGVLYVGSK (SEQ ID NO: 7); (ii) an α-synuclein peptide comprising QGVAEAAGK (SEQ ID NO: 6) and an α / β-synuclein peptide comprising EGVLYVGSK (SEQ ID NO: 7); or (iii) an α-synuclein peptide comprising the EQVTNVGGAVVTGVTAVAQK (SEQ ID NO: 9) and an α / β-synuclein peptide comprising the EGVLYVGSK (SEQ ID NO: 7) The method of claim 1, wherein

3. 10. The method of any one of the preceding claims, wherein the at least one combination comprises (i) an α-synuclein peptide comprising QGVAEAAGK (SEQ ID NO: 6), an α-synuclein peptide comprising EQVTNVGGAVVTGVTAVAQK (SEQ ID NO: 9), an α / β-synuclein peptide comprising EGVVAAAEK (SEQ ID NO: 5), and an α / β-synuclein peptide comprising EGVLYVGSK (SEQ ID NO: 7).

4. 10. The method of any one of the preceding claims, wherein the at least one combination comprises (ii) the alpha-synuclein peptide comprising QGVAEAAGK (SEQ ID NO: 6) and the alpha / beta-synuclein peptide comprising EGVLYVGSK (SEQ ID NO: 7).

5. 10. The method of any one of the preceding claims, wherein the at least one combination comprises (iii) the α-synuclein peptide comprising EQVTNVGGAVVTGVTAVAQK (SEQ ID NO: 9) and the α / β-synuclein peptide comprising EGVLYVGSK (SEQ ID NO: 7).

6. 10. The method of any one of the preceding claims, wherein the peptides of the at least one combination each have a length of 20 amino acids or less.

7. 10. The method of any one of the preceding claims, wherein said detecting step comprises quantifying said at least one combination of peptides.

8. 10. A method for detecting Parkinson's disease (PD) in a subject, comprising detecting said at least one combination by the method of any one of the preceding claims.

9. 9. The method of claim 8, further comprising the step of comparing the amount of each peptide of said at least one combination in said sample with a control.

10. 10. The method of claim 9, wherein the control is a predetermined matched value obtained from a subject without PD.

11. 11. The method of claim 9 or 10, wherein the control is a predetermined matched value obtained from a subject with PD.

12. 12. The method of any one of claims 8 to 11, wherein the method further distinguishes between PD and Lewy body dementia (LBD), and the at least one combination comprises (ii) an α-synuclein peptide comprising QGVAEAAGK (SEQ ID NO: 6) and an α / β-synuclein peptide comprising EGVLYVGSK (SEQ ID NO: 7).

13. 13. The method of claim 12, further comprising comparing the amount of each peptide of said at least one combination in said sample with a control.

14. 14. The method of claim 13, wherein the control is a predetermined matched value obtained from a subject with LBD.

15. 15. The method of claim 13 or 14, wherein the control is a predetermined matched value obtained from a subject with PD.

16. 16. The method of any one of claims 9 to 15, wherein the method further distinguishes between PD and multiple system atrophy (MSA), and the at least one combination comprises (iii) an α-synuclein peptide comprising EQVTNVGGAVVTGVTAVAQK (SEQ ID NO: 9) and an α / β-synuclein peptide comprising EGVLYVGSK (SEQ ID NO: 7).

17. 17. The method of claim 16, further comprising the step of comparing the ratio of the amount of the α-synuclein peptide comprising EQVTNVGGAVVTGVTAVAQK (SEQ ID NO: 9) to the amount of the α / β-synuclein peptide comprising EGVLYVGSK (SEQ ID NO: 7) in the sample with a control.

18. 18. The method of claim 17, wherein the control is a predetermined corresponding value obtained from a subject with MSA.

19. 19. The method of claim 17 or 18, wherein the control is a predetermined matched value obtained from a subject with PD.

20. The method of any one of claims 8 to 19, further comprising applying the amounts of the at least one combination of peptides to a computational model adapted to distinguish between PD and other forms of synucleinopathy.

21. 21. The method of claim 20, wherein the other forms of synucleinopathy include MSA, and the computational model is adapted to distinguish between PD and MSA.

22. 10. The method of any one of the preceding claims, wherein the subject has not been previously diagnosed with PD.

23. 10. The method of any one of the preceding claims, wherein the detecting step is performed in a mass spectrometer.

24. 10. The method of any one of the preceding claims, wherein the detecting step excludes an immunoassay.

25. 10. The method of any one of the preceding claims, wherein the subject is a human.

26. 10. The method of any one of the preceding claims, wherein the sample is blood or plasma.

27. 10. The method of any one of the preceding claims, wherein the sample excludes cerebrospinal fluid.

28. 28. A method of treating a subject having PD, comprising detecting PD by the method of any one of claims 8 to 27, and treating the subject.

29. 1. A method for diagnosing Parkinson's disease (PD) in a subject, comprising quantifying at least one peptide selected from the group consisting of an α-synuclein peptide comprising QGVAEAAGK (SEQ ID NO: 6), an α-synuclein peptide comprising EQVTNVGGAVVTGVTAVAQK (SEQ ID NO: 9), an α / β-synuclein peptide comprising EGVVAAAEK (SEQ ID NO: 5), and an α / β-synuclein peptide comprising EGVLYVGSK (SEQ ID NO: 7).

30. 30. The method of claim 29, wherein the at least one peptide comprises the alpha-synuclein peptide.

31. 31. The method of claim 29 or 30, wherein the at least one peptide comprises the α / β-synuclein peptide.

32. 32. The method of any one of claims 29 to 31, wherein the at least one peptide has a length of 30 amino acids or less.

33. 33. The method of any one of claims 29 to 32, wherein the at least one peptide has a length of 20 amino acids or less.

34. 34. The method of any one of claims 29 to 33, wherein the at least one peptide comprises at least one alpha-synuclein peptide and at least one alpha / beta-synuclein peptide.

35. The method of any one of claims 29 to 34, wherein the at least one peptide comprises an alpha-synuclein peptide comprising QGVAEAAGK (SEQ ID NO: 6).

36. 36. The method of any one of claims 29 to 35, wherein the at least one peptide comprises an alpha-synuclein peptide comprising EQVTNVGGAVVTGVTAVAQK (SEQ ID NO: 9).

37. 37. The method of any one of claims 29 to 36, wherein the at least one peptide comprises an α / β-synuclein peptide comprising EGVVAAAEK (SEQ ID NO: 5).

38. 38. The method of any one of claims 29 to 37, wherein the at least one peptide comprises an α / β-synuclein peptide comprising the EGVLYVGSK (SEQ ID NO: 7).

39. 39. The method of any one of claims 29 to 38, wherein the at least one peptide is selected from the group consisting of a peptide consisting of QGVAEAAGK (SEQ ID NO: 6), a peptide consisting of EQVTNVGGAVVTGVTAVAQK (SEQ ID NO: 9), a peptide consisting of EGVVAAAEK (SEQ ID NO: 5), and a peptide consisting of EGVLYVGSK (SEQ ID NO: 7).

40. The method of any one of claims 29 to 39, wherein the detecting step comprises quantifying the at least one peptide.

41. The method of any one of claims 29 to 40, further comprising the step of comparing the amount of each of said at least one peptide in said sample with a control.

42. 42. The method of claim 41, wherein the control is a predetermined matched value obtained from a subject without PD.

43. 42. The method of claim 41, wherein the control is a predetermined corresponding value obtained from a subject with PD.

44. 44. The method of any one of claims 29 to 43, wherein the method distinguishes between PD and other forms of synucleinopathy.

45. 45. The method of any one of claims 29 to 44, further comprising applying the amount of each of the at least one peptide to a computational model adapted to distinguish between Parkinson's disease and other forms of synucleinopathy.

46. 46. ​​The method of claim 44 or 45, wherein the other type of synucleinopathy comprises multiple system atrophy (MSA).

47. 47. The method of claim 46, wherein the computational model is tuned to distinguish between Parkinson's disease and multiple system atrophy.

48. 48. The method of any one of claims 29 to 47, wherein the method further distinguishes between PD and LBD, and the at least one peptide comprises an α-synuclein peptide comprising QGVAEAAGK (SEQ ID NO: 6) and / or an α / β-synuclein peptide comprising EGVLYVGSK (SEQ ID NO: 7).

49. 49. The method of claim 48, further comprising comparing the amount of each of the at least one peptide in the sample with a control.

50. 50. The method of claim 49, wherein the control is a predetermined matched value obtained from a subject with LBD.

51. 50. The method of claim 49, wherein the control is a predetermined matched value obtained from a subject with PD.

52. 52. The method of any one of claims 29 to 51, wherein the method further distinguishes between PD and multiple system atrophy (MSA), and the at least one peptide comprises an α-synuclein peptide comprising EQVTNVGGAVVTGVTAVAQK (SEQ ID NO: 9) and an α / β-synuclein peptide comprising EGVLYVGSK (SEQ ID NO: 7).

53. 53. The method of claim 52, further comprising comparing the ratio of the amount of α-synuclein peptide comprising EQVTNVGGAVVTGVTAVAQK (SEQ ID NO: 9) to the amount of α / β-synuclein peptide comprising EGVLYVGSK (SEQ ID NO: 7) in the sample with a control.

54. 54. The method of claim 53, wherein the control is a predetermined corresponding value obtained from a subject with MSA.

55. 54. The method of claim 53, wherein the control is a predetermined corresponding value obtained from a subject with PD.

56. 56. The method of any one of claims 29 to 55, wherein the detecting step is carried out in a mass spectrometer.

57. 57. The method of any one of claims 29 to 56, wherein the detecting step excludes an immunoassay.

58. The method of any one of claims 29 to 57, wherein the subject is a human.

59. The method of any one of claims 29 to 58, wherein the sample is blood or plasma.

60. 60. The method of any one of claims 29 to 59, wherein the sample excludes cerebrospinal fluid.

61. 61. A method of treating a subject having PD, comprising diagnosing PD by the method of any one of claims 29 to 60, and treating the subject diagnosed with PD.

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