EP4639172A1 - Method for detecting synuclein peptides - Google Patents

Abstract

Provided are methods of detecting different isoforms of the synuclein protein by detecting the amino acid sequences of synuclein peptides generated by proteolysis of synucleins present in a sample from a subject suspected of having a neurodegenerative disease.

Description

Description

Title of Invention: METHOD FOR DETECTING SYNUCLEIN PEPTIDES

Technical Field

[0001] The present invention relates to methods of detecting synuclein peptides. More specifically, the present invention relates to methods of detecting different isoforms of the synuclein protein by detecting the amino acid sequences of synuclein peptides generated by proteolysis of synucleins present in a sample from a subject suspected of having a neurodegenerative disease.

Background Art

[0002] Synuclein ("syn") is a family of proteins that is found in the pre-synaptic terminals in dopaminergic neurons. Three different proteins-a, [3, and y-of synuclein are known, of which at least the a- syn is reported to be indicated in several neurodegenerative conditions such as Parkinson’s disease ("PD"), Lew Body dementia ("LBD"), Alzheimer’s disease ("AD"), Multi system atrophy ("MSA"), and Creutzfeldt-Jakob disease ("CJD") (Candelise et al, 2019). These conditions caused by synuclein abnormalities are termed synucleinopathies.

[0003] Distinction and diagnosis of different forms of synucleinopathies have thus far relied on evaluation of clinical symptoms. For example, diagnosis of idiopathic PD involves three steps of clinical evaluation. The first step involves detecting signs of bradykinesia in combination with presence of motor abnormalities such as rigidity, resting tremors, or postural instability. If a patient demonstrates bradykinesia and any one of the aforementioned motor conditions, then the patient is further evaluated in the second step for absence of atypical symptoms such as oculogyric crises, prolonged remission from the symptoms of the disease, unilateral appearance of the symptoms after 3 years of evolution, supra-nuclear gaze palsy, morphological changes in the cerebellum, early severe dysautonomia, early severe dementia and language, memory and apraxia disorders, negative response to sufficient dosage of L-dopa, history of stroke with stair-step progression, existence of more than one other case of familial PD in the immediate family. The third step consists of detecting additional symptoms such as onset of unilateral symptoms, resting tremor, progressive worsening, positive response to dopaminergic therapy, persistent asymmetry of symptoms on the affected side at the beginning showing a greater sensitivity to dopaminergic therapy, clinical evolution, or worsening of symptoms over the time span of 10 years and more. Moreover, to distinguish PD from LBD, a clinician has to observe a patient for a cognitive disorder in addition of hallucinations (visual and others) in the absence of dopaminergic agonist, fluctuation of cognitive symptoms and vigilance, hypersensitivity to neuroleptics, delusions and psychosis (Krolak-Salmon, 2019 Elsevier Masson).

[0004] Synucleins, in particular a-synuclein ("a-syn"), are found to be a major constituent of Lewy bodies ("LB") and Lewy neurites ("LN"), toxic inclusion bodies that are found in the aforementioned neurodegenerative conditions. LBs and LNs are found in the cytoplasm of dopaminergic neurons in PD and LBD and in oligodendrocyte for MSA. As such, a-synuclein and the synuclein family have been quantified in biological samples because of their potential as biomarkers in diagnosing synucle- inopathies. Thus far, detection of synucleins was mainly achieved through antibodybased assay such as ELISA. Antibody-based detection techniques technique, however, have proven to be less than optimal due to occurrence of multiplex proteins or pro- teoforms, unspecific detection, affinity variability, and limited choice of available antibodies. It has also been observed that a single cerebrospinal fluid ("CSE") sample yielded different a-synuclein concentrations when measured in different laboratories (Mollenhauer et al, 2018) (Viode et al, 2019; Mollenhauer et al, 2018; Yang et al, 2017).

[0005] Clinical mass spectrometry ("MS") has emerged during last decades as a promising method to identify proteins indicative of pathologic conditions and has been use for a- syn quantitation. By using mass on charge ratio after ionization and fragmentation of the targeted proteins, this technic is highly specific, sensitive, accurate and easy to multiplex for proteins and proteoforms.

[0006] Several studies on the analysis of synuclein (most specifically the a-synuclein) from CSE were developed but no significant differences were observed between different types of synucleinopathies.

Summary of Invention Technical Problem

[0007] In order to utilize the synuclein proteins as markers for neurodegenerative conditions, the present disclosure provides a method of detecting synuclein peptides of certain amino acid sequences in a sample of a subject. The peptides may each have a sequence that can be used to identify the precise identity of the synuclein protein, and the peptides also may allow quantification of each synuclein protein in the sample. Solution to Problem

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

[0009] In one aspect, the present disclosure relates to a method of detecting a combination of synuclein peptides in a subject, comprising detecting, in a sample of the subject, at least one combination selected from the group consisting of: (i) an a- synuclein peptide comprising QGVAEAAGK (SEQ ID NO: 6), an a-synuclein peptide comprising EQVTNVGGAVVTGVTAVAQK (SEQ ID NO: 9), an a/[3- synuclein peptide comprising EGVVAAAEK (SEQ ID NO: 5), and an a/[3- synuclein peptide comprising EGVLYVGSK (SEQ ID NO: 7); (ii) the a-synuclein peptide comprising QGVAEAAGK (SEQ ID NO: 6) and the a/[3- synuclein peptide comprising EGVLYVGSK (SEQ ID NO: 7); and (iii) the a-synuclein peptide comprising EQVTNVGGAVVTGVTAVAQK (SEQ ID NO: 9) and the a/[3- synuclein peptide comprising EGVLYVGSK (SEQ ID NO: 7). In some embodiments, the peptides of said at least 20 amino acids, or less than 20 amino acids. In some embodiments the method comprises quantifying peptides of the at least one combination. In some embodiment, the method comprises comparing an amount of each peptide of the at least one combination in the sample with a control, which is a predetermined corresponding value obtained from a subject without PD. In some embodiments, the method further discriminates PD from Lewy Body Dementia (LBD) or multiple system atrophy (MSA). In some embodiments, the detecting is performed by a mass spectrometer. In some embodiments, the subject is human and the sample is blood or plasma and excludes cerebrospinal fluid. In some embodiments, the method comprises detecting PD according to the method of mentioned above and treating the subject.

[0010] In one aspect, the present disclosure relates a method of diagnosing Parkinson disease (PD) in a subject, comprising quantifying at least one peptide selected from the group consisting of an a-synuclein peptide comprising QGVAEAAGK (SEQ ID NO: 6), an a-synuclein peptide comprising EQVTNVGGAVVTGVTAVAQK (SEQ ID NO: 9), an a/[3- synuclein peptide comprising EGVVAAAEK (SEQ ID NO: 5), and an a/[3- synuclein peptide comprising EGVLYVGSK (SEQ ID NO: 7). In some embodiments, said at least one peptide comprises the a-synuclein peptide and/or the a/[>- synuclein peptide. In some embodiment, said at least one peptide has a length of 30 amino acids, 20 amino acids, or less than 20 amino acids. In some embodiments, said detecting comprises quantifying the at least one peptide. In some embodiments, the method further comprises comparing an amount of each of the at least one peptide in the sample with 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 discriminates PD from Lewy Body Dementia (LBD) or multiple system atrophy (MSA). In some embodiments, the detecting is performed by a mass spectrometer. In some embodiments, the subject is human and the sample is blood or plasma and excludes cerebrospinal fluid. In some embodiments, the method comprises detecting PD according to the method of mentioned above and treating the subject.

[0011] These and other embodiments, features and advantages of the present disclosure will be more readily understood by those of ordinary skill in the art from a reading of the following detailed description.

Brief Description of Drawings

[0012] [Fig.l]FIG. 1 shows workflow for the LC-MRM analysis of the a-synuclein peptides.

[Fig.2]FIG. 2 shows data comparison graphs of synuclein peptide concentrations in diseased patient groups and control patients.

[Fig.3]FIG. 3 shows clinical performance results for a/[3-syn EGVLYVGSK (SEQ ID NO: 7) peptide illustrated via a ROC curve.

[Fig.4A]FIG. 4A shows a comparison of the PD group with the other synucle- inopathies (MSA and LBD) and the control group with the established Model (a-syn [EQVTNVGGAVVTGVTAVAQK (SEQ ID NO: 9)]*-0.011268 + a/[3-syn [EGVLYVGSK (SEQ ID NO: 7)] * 0.0096545).

[Fig.4B]Fig. 4B shows comparison of the PD with MSA groups with the established peptide combination (a-syn [EQVTNVGGAVVTGVTAVAQK (SEQ ID NO: 9)]*-0.011268 + a/[3-syn [EGVLYVGSK (SEQ ID NO: 7)] * 0.0096545).

[Fig.5]FIG. 5 shows a comparison of the PD with MSA group with the established ratio of peptides (a/[3-syn EGVLYVGSK (SEQ ID NO: 7)/ a-syn EQVTNVGGAVVTGVTAVAQK (SEQ ID NO: 9)).

[Fig.6]Fig. 6 shows a ROC curve monitoring the clinical performance (sensitivity and specificity) for the peptide combination when PD is compared to LBD patients.

[Fig.7]Fig. 7 shows a ROC curve monitoring the clinical performance (sensitivity and specificity) for the peptide combination when PD is compared to MSA group.

[Fig.8]Fig. 8 shows a data comparison graph between diseases using sensitive immune-chemiluminescence assay.

Description of Embodiments

[0013] In the context of the present description, all publications, patent applications, patents and other references mentioned herein, if not otherwise indicated, are explicitly incorporated by reference herein in their entirety for all purposes as if fully set forth.

[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] Except where expressly noted, trademarks are shown in upper case.

[0016] Unless stated otherwise, all percentages, parts, ratios, etc, are by weight.

[0017] When an amount, concentration, or other value or parameter is given as a range, or a list of upper and lower values, this is to be understood as specifically disclosing all ranges formed from any pair of any upper and lower range limits, regardless of whether ranges are separately disclosed. Where a range of numerical values is recited herein, unless otherwise stated, the range is intended to include the endpoints thereof, and all integers and fractions within the range. It is not intended that the scope of the present disclosure be limited to the specific values recited when defining a range.

[0018] When the term "about" is used, it is used to mean a certain effect or result can be obtained within a certain tolerance, and the skilled person knows how to obtain the tolerance. When the term "about" is used in describing a value or an end-point of a range, the disclosure should be understood to include the specific value or end-point referred to.

[0019] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having" or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but can include other elements not expressly listed or inherent to such process, method, article, or apparatus.

[0020] The transitional phrase "consisting of" excludes any element, step, or ingredient not specified in the claim, closing the claim to the inclusion of materials other than those recited except for impurities ordinarily associated therewith. When the phrase "consists of" appears in a clause of the body of a claim, rather than immediately following the preamble, it limits only the element set forth in that clause; other elements are not excluded from the claim as a whole.

[0021] The transitional phrase "consisting essentially of" limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed invention. A "consisting essentially of" claim occupies a middle ground between closed claims that are written in a "consisting of" format and fully open claims that are drafted in a "comprising" format. Optional additives as defined herein, at a level that is appropriate for such additives, and minor impurities are not excluded from a composition by the term "consisting essentially of".

[0022] Further, unless expressly stated to the contrary, "or" and "and/or" refers to an inclusive and not to an exclusive. For example, a condition A or B, or A and/or B, is satisfied by any one of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present). [0023] The use of "a" or "an" to describe the various elements and components herein is merely for convenience and to give a general sense of the disclosure. This description should be read to include one or at least one and the singular also includes the plural unless it is obvious that it is meant otherwise.

[0024] The term "substantial portion" or "substantially", as used herein, unless otherwise defined, means all or almost all or the vast majority, as would be understood by the person of ordinary skill in the context used. It is intended to take into account some reasonable variance from 100% that would ordinarily occur in industrial- scale or commercial- scale situations.

[0025] The term "depleted" or "reduced" is synonymous with reduced from originally present. For example, removing a substantial portion of a material from a stream would produce a material-depleted stream that is substantially depleted of that material. Conversely, the term "enriched" or "increased" is synonymous with greater than originally present.

[0026] For convenience, many elements of the present disclosure are discussed separately, lists of options may be provided and numerical values may be in ranges; however, for the purposes of the present disclosure, that should not be considered as a limitation on the scope of the disclosure or support of the present disclosure for any claim of any combination of any such separate components, list items or ranges. Unless stated otherwise, each and every combination possible with the present disclosure should be considered as explicitly disclosed for all purposes.

[0027] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described herein. The materials, methods, and examples herein are thus illustrative only and, except as specifically stated, are not intended to be limiting.

Svnuclein Peptides

[0028] The three synuclein proteins have the following amino acid sequences shown in SEQ ID NO: 1-3 summarized in Table 1 below.

[0029]

[Table 1]

Table 1 : amino acid sequences of the symiclem proteins.

[0030] The present disclosure provides that certain peptide segments of the synuclein proteins can be used to identify and quantify the synuclein proteoforms present in the samples obtained from patients suspected of having a neurodegenerative condition. Each of the synuclein proteins has different molecular behaviors and identifying and quantifying their presence in subject may provide a useful molecular/biochemical basis to diagnose and distinguish different types of neurodegenerative conditions and synu- cleinopathies.

[0031] Samples from a subject in which the peptide segments are to be detected may come from a variety of sources, including blood, blood plasma, cerebrospinal fluid, biopsy, saliva, nasal swab, oral swab, and any other biological samples that can be obtained in- vasively or noninvasively from a subject. In certain embodiments, the sample used in the detection may be blood, blood plasma, and/or cerebrospinal fluid. In other embodiments, the sample is blood or blood plasma. In yet another embodiment the sample is blood plasma. In some embodiments, the sample excludes cerebrospinal fluid.

[0032] The synuclein proteins contained in the sample may be proteolyzed using one or more proteases prior to detection. In some embodiment, the protease may be one or more of trypsin, Lys C, Lys N, and Glu-C. For example, the protease may be a combination of trypsin and Lys-C. In a different example, the protease may be a combination of trypsin and Lys N.

[0033] In order to facilitate the protease reaction, the sample from a subject may be cleaned up to remove non-proteinaceous molecules prior to the proteolysis step. For example, the proteins in the sample may be precipitated and isolated from the remaining supernatant, and further purified using affinity columns, purification cartridges, and other means of separating non-proteinaceous matters from the sample.

[0034] Use of a different protease or a different combination of proteases may yield different peptide segments from the synuclein proteins. The amino acid sequences of these proteins, some of which may be unique to a particular synuclein proteoform or may be shared between two or more proteoforms, may be used to identify and quantify the synuclein proteins present in the sample being tested. For example, from the digestion of a-synuclein by the combination of trypsin and Lys C, peptide segments having 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), may be generated. The peptides generated may 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 amino acids or less. Exemplary peptide segments generated by proteolysis of the three synuclein proteins by the combination of trypsin and Lys C are summarized in Table 2 below.

[0035]

[Table 2]

Table 2: amino acid sequences of syimclein peptides generated by proteolysis using trypsin and

Lys C proteases.

The reference sequence for SEQ ID NO 23 is following SEQ ID NO 24: sp P69905 HBA_HUMAN OS=Homo sapiens OX—9606 GN=HBA1 PE=1 SV=2

MVLSPADKTN YKAAWGKVGA HAGEYGAEAL ERMFL SFPTT KTYFPHFDLS

HGSAQXXGHG KKVADALTXA VAHXDDMPNA LSALSDLHAH KLRYDPVNFK

LLSHCLLVTL AAHLPAEFTP AVHASLDKFL ASVSTVLTSK YR

[0036] The peptide generated by proteolysis may be detected and/or analyzed using various methods, including mass spectrometry, immunoassays, gel electrophoresis and im- munoblotting, and chromatography. In one embodiment, the peptides are detected, sequenced, and/or quantified using mass spectrometry.

[0037] Suitable methods for detecting and/or measuring peptides described herein in a sample may also include, for example, LC (Liquid Chromatography), LC-MS (Liquid Chromatography - Mass Spectrometry:). LC-MS, immunological analysis, ECL (Electrochemiluminescence) can be mentioned. As the LC method, for example, various detectors such as an absorption detector such as an ultraviolet-visible absorption detector and a light emission detector such as a fluorescence detector are used to quantify the abundance amount or abundance ratio from the peak area of the chromatogram. Examples of the LC-MS method include a SIM method (Selected Ion Monitoring) using a single mass spectrometry (MS) detector and an MRM method (Multiple Reaction Monitoring) using a tandem mass spectrometry (MS / MS) detector. Multiple reaction monitoring method) or SRM method (Selected Reaction Monitoring) can be mentioned. Of these, the MRM method (and SRM method) is preferable from the viewpoint of high- sensitivity analysis.

[0038] Examples of the immunological analysis method include an ELISA method (Enzyme-Linked Immunosorbent Assay), a FLISA method (Fluorescense Linked Im- munososorbent Assay), and an RIA method (Radioimmunoassay). Of these, the ELISA method may be preferable from the viewpoint of safety and the like.

[0039] The peptide thus detected, sequenced, and quantified may be further analyzed to determine whether a certain neurological condition is present in the subject from whom the sample being tested is obtained. For example, a certain peptide or a combination of peptides may be elevated or lowered in a particular neurological condition. As such, in one embodiment, the 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 a-synuclein peptide comprising QGVAEAAGK (SEQ ID NO:6), an a-synuclein peptide comprising EQVTNVGGAVVTGVTAVAQK (SEQ ID NO: 9), an a/[3- synuclein peptide comprising EGVVAAAEK (SEQ ID NO: 5), and an a/[3- synuclein peptide comprising EGVLYVGSK (SEQ ID NO: 7); (ii) the a-synuclein peptide comprising QGVAEAAGK (SEQ ID NO:6) and the a/[>- synuclein peptide comprising EGVLYVGSK (SEQ ID NO: 7); and (iii) the a-synuclein peptide comprising EQVTNVGGAVVTGVTAVAQK (SEQ ID NO: 9) and the a/[3- synuclein peptide comprising EGVLYVGSK (SEQ ID NO: 7). In another embodiment, the sample is analyzed to detect a combination comprising the a-synuclein peptide comprising QGVAEAAGK (SEQ ID NO:6), the a-synuclein peptide comprising EQVTNVGGAVVTGVTAVAQK (SEQ ID NO: 9), the a/[3- synuclein peptide comprising EGVVAAAEK (SEQ ID NO: 5), and the a/[3- synuclein peptide comprising EGVLYVGSK (SEQ ID NO: 7). In one embodiment, the sample is analyzed to detect a combination comprising the a-synuclein peptide comprising QGVAEAAGK (SEQ ID NO:6) and the a/[3- synuclein peptide comprising EGVLYVGSK (SEQ ID NO: 7). In one embodiment, the sample is analyzed to detect a combination comprising the a-synuclein peptide comprising EQVTNVGGAVVTGVTAVAQK (SEQ ID NO: 9) and the a/[3- synuclein peptide comprising EGVLYVGSK (SEQ ID NO: 7). In one embodiment, the aforementioned combinations of peptides are quantified. In some embodiments, the disclosure relates to a method of diagnosing a neurological condition in a subject by analyzing, detecting, sequencing or qualifying the combination of peptides described herein.

[0040] In one embodiment, the neurological condition that is detected or diagnosed based on the method of detecting the peptides discussed above may be Parkinson disease (PD). The method in such a case may comprise detecting the at least one combination as discussed above. The method may further comprise quantifying each peptide and comparing the quantified amount of each peptide of the at least one combination in the 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, a predetermined corresponding value may be an amount of an a-synuclein peptide comprising QGVAEAAGK (SEQ ID NO: 6) in a sample from a subject without PD, and such a predetermined corresponding value is compared to a measured amount of an a-synuclein peptide comprising QGVAEAAGK (SEQ ID NO: 6) in a sample from a subject at issue to determine whether the subject at issue has PD or not. If the predetermined corresponding value and the measured amount have a statistically significant difference, the subject at issue may be determined to have PD. If the predetermined corresponding value and the measured amount have a statistically insignificant difference, the subject at issue may be determined not to have PD. On the other hand, a predetermined corresponding value may be an amount of an a-synuclein peptide comprising QGVAEAAGK (SEQ ID NO: 6) in a sample from a subject with PD, and such a predetermined corresponding value is compared to a measured amount of an a- synuclein peptide comprising QGVAEAAGK (SEQ ID NO: 6) in a sample from a subject at issue to determine whether the subject at issue has PD or not. If the predetermined corresponding value and the measured amount have a statistically significant difference, the subject at issue may be determined not to have PD. If the predetermined corresponding value and the measured amount have a statistically insignificant difference, the subject at issue may be determined to have PD. In some embodiments, the method may further comprise quantifying each peptide and comparing a ratio of the quantified amounts of peptides in the sample with a control.

[0041] The method described above may be used to diagnose or discriminate PD from other types of synucleinopathies. For example, in one embodiment, the method may discriminate PD from LBD. The method in such an example may comprise detecting the a-synuclein peptides comprising QGVAEAAGK (SEQ ID NO:6). The method may comprise detecting the a/[3- synuclein peptide comprising EGVLYVGSK (SEQ ID NO: 7). In a further embodiment, the method that discriminates PD from LBD comprises comparing an amount of each peptide of the at least one combination in the sample with a control. The control in such an embodiment may be a predetermined corresponding value obtained from a subject with LBD or a predetermined corresponding value obtained from a subject with PD.

[0042] In one embodiment, the method may diagnose or discriminate PD from multiple system atrophy ("MSA"). The method in such an example may comprise detecting the a-synuclein peptide comprising EQVTNVGGAVVTGVTAVAQK (SEQ ID NO: 9). The method may comprise detecting the a/[3-synuclein peptide comprising EGVLYVGSK (SEQ ID NO: 7). The method may comprise detecting the a-synuclein peptide comprising EQVTNVGGAVVTGVTAVAQK (SEQ ID NO: 9) and the a/ [3-synuclein peptide comprising EGVLYVGSK (SEQ ID NO: 7). In a further embodiment, the method that discriminates PD from MSA comprises comparing an amount of each peptide of the at least one combination in the sample with a control. The control in such an embodiment may be a predetermined corresponding value obtained from a subject with MSA or a predetermined corresponding value obtained from a subject with PD. Likewise, the method can be used to discriminate PD from any one of the synucleinopathies or control.

[0043] In additional embodiments, the methods of distinguishing PD from other synucleinopathies may further comprise applying amounts of the at least one peptide to a calculation model adjusted for distinguishing PD from other type(s) of synucleinopathies. The synucleinopathy for which the calculation model is adjusted may be LBD, MSA, or any other synucleinopathy.

[0044] The method described above may be used to detect synuclein peptides from any subject that is suspected of having a neurodegenerative condition. For example, the subject may be a subject that was not previously diagnosed with any neurodegenerative condition. The subject may in another embodiment be a subject that was not previously diagnosed with any synucleinopathies. Further, the subject may be a subject that was not previously diagnosed with PD, MSA, or LBD. The subject to which the method described above can be used is not particularly limited but includes human subjects, non-human mammal subjects, as well as other vertebrates.

[0045] In one aspect, the method described herein may relates to a method of treating a subject with PD. In some embodiments, the method may include detecting or diagnosing PD as described above. In some embodiments, the treating may include administering a pharmaceutical composition to increase or substitute dopamine. Such a pharmaceutical composition may comprise at least one selected from the group consisting of dopamine promoter, antidepressant, cognition-enhancing medication or anti-tremor drugs. In some embodiments, the treating may include deep brain stimulation. In some embodiments, the treating may include implanting an electrical pulse generator.

EXAMPLES

[0046] Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited by these examples.

Example 1

[0047] Reagents and internal standards

[0048] Recombinant full-length a-synuclein and nitrogen-15 Uniform-labeled ("U-15N") recombinant full-length alpha synuclein protein came from the LGC (Teddington, UK). The U-15N [3 and y synuclein human recombinant proteins were purchased from Promise advanced proteomics (Grenoble, France). The beta and gamma human recombinant light form came from Anaspec (Fremont, California, USA) and Interchim (Montluçon, France) respectively. Eight protein standard concentration was determined for the a-synuclein by immuno-chemiluminescence with MesoScale Discovery (Rockville, Maryland, USA) system. Trypsin/Eys-C, rLys-C, glu-C and Lys-N with MS grade were purchased from Promega GmbH (Walldorf, Germany). Ammonium bicarbonate (ABC), 70 % perchloric acid, trifluoroacetic (TFA), and human serum albumin (HSA) were purchased from Sigma Aldrich (Saint Quentin Fallavier, France). RPW Cartridges tips were purchased from Agilent technologies (Santa Clara, California, USA). All liquid chromatography ("LC") solvents such as acetonitrile, water, formic acid were of LC-MS grade and purchased from Biosolve (Dieuze, France). AQUA (absolute quantitation) peptides for a-syn- 126/98, a- syn-112/98 and a-syn-41 with a 99% purity were purchased from Thermo Fisher scientific (Eincoln, Massachusetts, USA). Specific peptides, having a purity of 95%, of the alternative a-syn and its mutated form were also purchased from Thermo Fisher scientific (Eincoln, Massachusetts, USA). Goat serum (Capra hircus) was ordered from Clinisciences, (Nanterre, France). Immunoassay kit named U-PEEX Human a- Synuclein Kit was purchased from MesoScale Discovery.

[0049] Stock Solutions. Calibration Standards, and Quality Control ("OC") samples [0050] Proteins and peptides standards were dissolved in ammonium bicarbonate at 50 mM at a final concentration of 1 microgram/microliter. Aliquots were separated in low protein-binding tubes (1.5 or 0.5 mL) and stored at -20°C or -80°C.

[0051] Method development was made in a pool of sample of plasma on one level. The pools were made as follows: in a 50 ml falcon tube, patients' samples with AD suspicion were added, mixed and aliquoted in 1.5 ml Eppendorf LoBind tubes (150 microliter), then store at -80°C.

[0052] QC on three a-syn levels in plasma used for method validation was made by quantifying 42 plasma patients sample by immunoassay (MSD measurement). All these patients had different level of alpha synuclein and the 25% of most concentrated patients in a-syn was in the high-level pool, the 25% of the least concentrated patients were in the low-level pool, the rest of the patient samples were in the medium pool and was prepared as previously described.

[0053] For the clinical validation part, one level control QC was established for plasma, consisting of a pool of samples with a synuclein concentration determined beforehand with Meso Scale Discovery ("MSD") immunoassay, and considered as an intermediate mean value (52.16 ng/mL for plasma).

[0054] Calibration

[0055] For plasma, 9 points of calibration were prepared by diluting recombinant light a, [3, and y-synuclein standards into goat (Capra hircus) serum. Calibration ranges (detailed in Table 3) in plasma were as follows: [0-5571] ng/mE for a-synuclein, [0-410] ng/mE for [3-synuclein and [0-1401] ng/mL for y-synuclein. The N-terminal peptide concentration, a-synuclein isoform peptide concentration, and synuclein alternative protein heavy-labelled peptide concentration were as described in Table 4.

[0056]

[Table 3]

Table 3 : Synuclein concentrations of calibration points in plasma

[0057]

[Table 4]

Table 4: Heavy labelled peptides concentrations of calibration points in plasma.

[0058] Sample preparation

[0059] 95 microliter of plasma samples were thawed on ice for 1 hour, then 855 microliter of deionized water was added to the plasma samples. A 5.7 microliter at 10 ng/ microliter internal standard of recombinant labelled U-15N a, [3, and y-synuclein in 50 mM ammonium bicarbonate were added to samples and QCs. Samples were mixed and the proteins were precipitated by incubation on ice for 15 minutes with 142.5 microliter of 70% perchloric acid. After incubation, samples were spun for 15 min at 4 ° C, 16000 g and the supernatants were collected into new low-binding tube with the addition of 95 microliter of 1% trifluoroacetic acid (TFA). After a volume reduction by vacuum drying at room temperature (Speedvac, Labconco), the supernatants were cleaned up on RPW cartridges using the AssayMap Bravo (Agilent technologies) into a LoBind 96 deep well plate. The cartridges were washed with water at an aspiration rate of 10 microliter/min and then samples were loaded on the cartridges at a rate of 5 mi- croliter/min. Proteins were washed out on RP-W (reverse phase W) tips with 10% ACN and 0.1% formic acid at 10 microliter /min and eluted with 45% ACN and 0.1% formic acid at 5 microliter/min. Samples were evaporated dry at room temperature under vacuum for 1.5 hours using SpeedVac. The samples were reconstituted with 20 microliter of 50 mM ABC. Finally, 7 microliter at 1 microgram/microliter of trypsin/ Lys-C were added. Plasma samples were incubated for 4 hours at 37°C with gentle agitation (450 rpm). After incubation, 0.5 microliter of formic acid was added to the samples.

[0060] The entire workflow for the LC-MRM analysis of the a-synuclein peptides is illustrated in figure 1.

[0061] LC-MRM analysis

[0062] Samples were analyzed using a Shimadzu LC (Mikros) and a triple quadrupole - Shimadzu 8060 mass spectrometer (Duisburg, Germany) in a positive ionization mode. 15 microliter of a sample were injected on a ZORBAX SB-Aq (1 x 150 mm, 3,5 micrometer) column from Agilent technologies, at 35°C. The mobile phase A was composed of water with 0.1% of formic acid (FA) and the mobile phase B was composed of ACN with 0.1% of formic acid. The used gradient of solvent consists of a slow increase of the organic solvent (phase B) from 0% to 30% over 30 minutes. Total run time for LC-MRM analysis was 40 minutes at a flow rate of 50 microliter/min. LC gradients are described in table 5. The minimum dwell time used was 20 milliseconds ("msecs") (and range from 22 to 111 msecs) per peptide. The ion source parameters were 3 L/min for the nebulizing gas flow, 10 L/min for the heating gas flow, 300°C for the interface temperature, 250°C for the desolvation line temperature, 400°C for the heat block temperature and 10 L/min for the drying gas flow. These parameters were previously optimized for peptide analysis. Between three and six transitions were acquired for each peptide in biological samples. Peptide position, selection and transitions of the synucleins are described in table 6. The concentration of the a-syn and [3-syn peptides was determined by comparing unlabeled and labelled peptides with Multiple Reaction Monitoring (MRM) analysis.

[0063] [Table 5]

Table 5; LC gradient used.

[0064]

[Table 6-3]

[0067] Analytical validation

[0068] The intra and inter-precision assay was determined by analyzing one QC sample in triplicate of plasma samples at 3 different levels of a-syn (high at 230 ng/ml, medium at 87 ng/ml and low at 45 ng/ml) every day for 4 days. For CSF samples, it was determined by analyzing one QC in triplicate on one level every day for 4 days.

[0069] Sample's stability including the effect of thawing the sample on ice or at room temperature were tested as follows: one goat serum in triplicate were thaw on ice during 0, 2, 4 and 6h or at room temperature and then spiked with light and heavy recombinant standards of a-syn, [3-syn and y-syn (n=3). In addition, we also tested QC samples (one level CSF, and three level plasma, namely high, medium and low) after 0, 12, 24, 36 and 48 hours of storage into the autosampler at 4°C (n=3). The parallelism and the effect of dilution were achieved by spiking a-syn, [3-syn and y-syn to a high concentration (5 ng/ml for a-syn and y-syn and 10 ng/ml for [3-syn) in goat serum and then serially diluting by factors of 2, 3 and 4 with either ABC 50 mM or with normal goat serum already spiked with the same heavy concentration, respectively (n=3).

[0070] Accuracy and linearity were assessed during the intra and inter assay precision using three duplicative calibration standards on each of the 4 days.

[0071] The lower limit of quantification ("LLOQ") was determined as the lowest concentration with a variation coefficient superior to 20% (n=3) and a signal on noise (S/N) at least superior at 3. For this purpose, 4 different concentrations (6,45; 12,90; 19,35 and 32,25 ng/ml) for a-syn and y-syn peptides, which are close to the LLOQ of these peptides, were spiked into goat serum. The LLOQ are detailed in the table XO for a-syn peptides. For [3-syn peptides, 3 concentrations: 80,7; 96,9 and 113,1 ng/ml were spiked into goat serum. The same strategies were applied to AQUA peptides comprising N-terminal a/[3-syn peptide, alternative splicing and alternatives peptides with 3 concentrations each: 0.155; 0.306 and 0.613 ng/ml (N-ter); 0.0134; 0.0674 and 0.134 ng/ml (a-syn-41); 0.02; 0.101 and 0.202 ng/ml (a-syn- 112/98); 0.019; 0.093 and 0.186 ng/ml (a-syn- 126/98); and 0.008; 0.015 and 0.03 ng/ml (a-syn-alt), respectively.

[0072] Matrix effect and recovery were determined by comparing the MS signal between spiked normal goat serum at the beginning of the experiment and normal goat serum spiked after digestion at the same concentration and in a spiked solvent after digestion at the same concentration (n=3).

[0073] We analyzed blank samples with the same method as our samples after the highest concentration of the calibration standards to check carry-over effect (n=4). All these parameters are presented in tables 7 and 8 in the results section.

[0074]

[Table 7]

[0075] [Table 8-1]

[0076] [Table 8-2]

[0077] Immunoassay

[0078] Plasma samples were analysed by immuno-chemiluminescence assay with

MesoScale Discovery ("MSD"). Plasma samples were diluted 200 times for the determination of the total a-synuclein concentration. All measurements were performed according to the manufacturer’s instructions. Antibodies (AB) is directed against a C- terminal portion of a-syn (residues 110-125) for capture rabbit monoclonal antibody and between residues 15-125 for monoclonal mouse antibodies.

[0079] Patients and biofluid collection

[0080] All participants provided written consent to be enrolled in the study and were approved by the Montpellier University Hospital's regional Ethics Committee. General characteristics of the 143 patients are presented in Table 9. The mean age of the participants was 71 (+/-8.2) years, and 65 % of the participants were men. Disease groups were divided in three groups: PD (n=82), LBD (n=32) and MSA (n=8). Participants of the control group (n=21) suffered from non-neurodegenerative diseases as various neurologic diseases including neuropathic changes (33.3%), vascular changes (29.3%), immunologic changes (20.8%) and hydrocephalus (23.1%). Exclusion criteria for the control group was an abnormal Alzheimer biomarker profile (i.e. low amyloid beta ratio (Ab 40/42) and increased Tau/pTau protein levels). PD patients were diagnosed with the condition on average 11 (+/- 7.4) years before analysis, of which 41.9% presented cognitive impairment, 50.8% presented anosmia and 69.7% presented non motor signs. Mean Hoehn and Yahr and UPDRS (Unified Parkin's Disease Rating Scale) II and III scores were respectively 2.4 (+/- 0.7), 19.7 (+/- 11.2) and 36 (+/- 18.9), corresponding to a mild motor impairment. Plasma samples collected intravenously were stored at the Montpellier Neurobank (#DC-2008-417 of the certified NFS 96-900 CHU resource center BB-0033-00031). Authorization to handle personal data was granted by the French Data Protection Authority (CNIL). After reception, 0.5 mL of plasma were aliquoted in 1.5 mL polypropylene and LoBind tubes and stored at -80°C until further analysis.

[0081]

[Table 9]

[0082] Data retreatment

[0083] Statistical analysis was performed with MedCalc (19.0.3. version) and the normality of the data set were checked by Shapiro-Wilk test, result significance was tested by Mann- Whitney Wilcoxon method, and clinical performances were acquired using ROC curves (sensitivity corresponding to true positive results in function of 100- specificity that represent the false positives results). A statistical model that defines results was investigated by logistic regression with backward and enter methods (due to the small size of variables and large number of samples). For selection, we used a p-value <0.05, a minimal number of variables and the same coefficient given by the logistic regression methods. The coefficient, shown below, is applied to the quantification of alpha and beta syn peptides (a-syn [EQVTNVGGAVVTGVTAVAQK (SEQ ID NO: 9)]*-0.011268 + a/[3-syn [EGVLYVGSK (SEQ ID NO: 7)] * 0.0096545).

[0084] Extraction of chromatograms and area of synucleins peptides for quantification were obtained using Skyline Software (20.1.0 version) and LabSolutions Insight Browser. The LC-MRM analyses were launched with LabSolutions software (5.99 version). MS parameters such as collision energy and applied voltage in the quadrupoles were optimized using the "optimization for method" from LabSolutions software, those parameters and the peptides comprised in the method are described in the Table 5.

[0085] Results [0086] Method development

[0087] For a-synuclein, we selected all proteotypic peptides, except the C-terminal peptide that covers about 70% of the protein sequence. All peptides and transitions were selected based on sensitivity and selectivity observed in the biological sample compared to the spiked internal labelled standards. Since the C-terminal peptide contains a long residue chain (40 amino acids long) and is composed of many acidic residues, its MS analysis sensitivity is low. In biological fluids such as CSF and plasma, this C-terminal peptide was consistently under the LLOQ. The same methodology was applied for [3-syn and y-syn. All the synuclein species and sequences monitored in the method are displayed in table 7.

[0088] Analytical method validation

[0089] Both [3-syn and y-syn were consistently below LOQ into plasma QC at all three levels (high, medium and low of a-syn) and in patient samples. Similar results were obtained for the alternative splicing isoforms and alternative a-syn peptides. Only a- syn and a/[3-syn common peptides were detected and quantified in all QC samples and patient samples. Two peptides having sequences (ac)MDVFMK (SEQ ID NO: 1) and EGVVHGVATVAEK (SEQ ID NO: 8) were excluded from the analytical validation (and clinical) due to their instability and non-linear response in calibration by MS.

[0090] Intra-assay precision results for a-syn peptides were between 5-13% for low-level QC samples (at 46.11 ng/ml for a-syn peptides in mean and 195.95 ng/ml for a/[3-syn peptides), between 3-8% for medium-level QC samples (at 74.70 ng/ml for a-syn peptides in mean and 270.82 ng/ml for a/[3-syn peptides), and 3-5% for high-level QC samples (at 140.24 ng/ml for a-syn peptides in mean and 427.09 ng/ml for a/[3-syn peptides). For a/[3-syn peptides in low-level QC samples the intra assay precision were between 7-8%, 4-5% for medium QC samples and 2-6% in high level QC samples.

[0091] Inter-assay precision was comprised in the range of 12-19% for a-synuclein peptides in low level QC samples, except for TVEGAGSIAAATGFVK (SEQ ID NO: 10) peptides that were at 22% in low QC samples. In medium level QC samples, the coefficient of variation (CV) was 13-15%, and 10-13% in high QC samples for a-syn peptides. Inter-assay precision for common a/[3-syn peptides was 15-17% in low level, 13-16% for medium level, and 12-14% for high level QC samples.

[0092] The LOQ and linearity ranges for a-syn peptides were between 6.45 and 19.35 ng/ml and 0 to 5571 ng/ml, respectively. The LLOQs were similar with a-syn peptides and linearity range was from 0 to 5981 ng/ml for a/[3-syn common peptides.

[0093] For [3-syn peptides the LOQ was 113.1 ng/ml, and more than 32.25 ng/ml for [3/y-syn QGVTEAAEK peptide and ranged from 6.45 to 32.25 ng/ml for y-syn peptides. Linearity ranges from 0 and 410 ng/ml for [3-syn peptides, between 0 and 1811.5 ng/ml for common [3/y-syn and 0 and 1401.2 ng/ml for y-syn. [0094] The LLOQ for AQUA peptides was more than 0.613 ng/ml for N-terminal peptide, more than 0.134 ng/ml for a-syn-41 peptide, 0.101 ng/ml for a-syn- 112/98 peptide, 0.093 ng/ml for a-syn- 126/98 peptide and 0.03 ng/ml for a-syn-alt peptide. Mutated a- syn-alt peptide was not detected.

[0095] No peptides were retrieved after highly concentrated analyses of the synuclein peptides into our blank and carry over were below 1% except for a-syn EQVTNVG- GAVVTGVTAVAQK (SEQ ID NO: 9) that was at 1.18%. All these parameters are demonstrated in table 7 and 8.

[0096] For sample stability, the effect of dilution accuracy percentage ranged from 98 to 113% for a-syn peptides, except for TVEGAGSIAAATGFVK (SEQ ID NO: 10) that was stable only up to a 2-fold dilution (due to accuracy >120 %), and 87-117% for a/ [3-syn peptides, except for EGVLYVGSK (SEQ ID NO: 7) that was stable only to a 3-fold dilution.

[0097] Parallelism accuracy percentage ranged from 86 to 100 % for a-syn peptides, except for TVEGAGSIAAATGFVK (SEQ ID NO: 10) that was stable only up to a 2-fold dilution and EQVTNVGGAVVTGVTAVAQK stable on 3-fold dilution (due to accuracy >120 %). For a/[3-syn common peptides the accuracy was between 89-100 %.

[0098] Percentage accuracy of samples thawed on ice was 88-103% for a-syn peptides and 95-100% for a/[3-syn peptides after six hours of thawing. Samples thawed at room temperature were stable only until 4 hours for all synucleins peptides, and the accuracies ranged between 99-106% for a-syn peptides and 100-106% for a/[3-syn peptides.

[0099] For the stability in the autosampler at 4 °C, the accuracy percentages were between 95-121% for a-syn peptides, 93-103% for a/[3-syn peptides at the low level, 80-111% for a-syn peptides, 96-105% for a/[3-syn peptides at the medium level, except for TVEGAGSIAAATGFVK (SEQ ID NO: 10) peptide (<80%). For high level QC samples, the stability accuracy was between 97-115% for a-syn peptides and 96-108% for a/[3-syn peptides.

[0100] Matrix effect and recovery were determined at the same time (methodology are explained in the materials and methods section) on all peptide matrix effect were between -51% and 9%, meaning that there is a loss of signal of maximum 51% and a maximum gain of 9% due to interferences. And there is a maximum loss of recovery of 93% (between -93 to -41 % for all peptides). These parameters are described in details in Table 8.

[0101] Levels of a-syn and a/[3-syn peptides in our three level QCs followed Westgard rules (Westgard et al, 1981, Clin chem).

[0102] Clinical validation

[0103] In total 143 plasma patients were quantified, (n=82) for the PD group, (n=8) for the MSA group, (n=32) for the LBD group and (n=21) for the control group, all were acquired by MS and immunoassay. Details of the patients that participated in the study are presented in the table 9.

[0104] MS quantitation

[0105] During clinical validation only the peptides from a-syn and common a/[3-syn were detected into plasma patient samples. [3 and y-synucleins were below LOQ into patient samples. Similar results were obtained for the alternative splicing isoforms and alternative a-syn peptides.

[0106] Area under the ROC curve (AUC), sensitivity and specificity, in addition to p value to distinguish between pathological groups, were assessed to describe the clinical performance of the developed MS method. The clinical performance results for a-syn peptides in plasma patients samples were summarized in the table 10.

[0107] [Table 10]

[0108] Specifically, a-syn TVEGAGSIAAATGFVK (SEQ ID NO: 10) peptide gave no significant differences between groups (lowest p value =0.2134). a-syn EQVTNVG- GAVVTGVTAVAQK (SEQ ID NO: 9) and a/[3-syn EGVVAAAEK (SEQ ID NO: 5) peptides were able to discriminate the PD group from the control group (p value = 0.0205 and 0.0277, respectively) with sensitivity and specificity of 0.87 and 0.67, respectively. Their areas under the ROC curve (AUC) were 0.664 and 0.656, respectively. Two peptides, a/[3-syn EGVLYVGSK (SEQ ID NO: 7) and a-syn QGVAEAAGK (SEQ ID NO: 6), were able to discriminate the PD group from control and LBD patients. The p values were 0.0001 and 0.0014 for a/[3-syn EGVLYVGSK (SEQ ID NO: 7) for distinguishing from control and LBD, respectively, and 0.0014 and 0.0331 for a-syn QGVAEAAGK (SEQ ID NO: 6) for distinguishing from control and LBD, respectively. Sensitivity, specificity and AUC, evaluated for the PD patients when compared to control patients, were 0.84, 0.76 and 0.787, respectively, for a/ [3-syn EGVLYVGSK (SEQ ID NO: 7). These values have been determined for the discrimination of the PD group from the LBD group for the same peptide at 0.7, 0.69 and 0.694, respectively, a-syn QGVAEAAGK (SEQ ID NO: 6) sensitivity, specificity and AUC were 0.83, 0.76 and 0.726 for distinguishing PD patients from control patients. Only the MSA group was not distinguished from PD patients.

[0109] Data comparison graphs of groups disease and control patients were illustrated in the figure 2 for a/[3-syn EGVLYVGSK (SEQ ID NO: 7) peptide. The best clinical performance for distinguishing PD and control patients were obtained with a/[3-syn EGVLYVGSK (SEQ ID NO: 7) peptide. Respectively, the sensitivity and specificity were 0.84 and 0.76 for PD vs control. The clinical performance results for a/[3-syn EGVLYVGSK (SEQ ID NO: 7) peptide are illustrated via ROC curve with figure 3.

[0110] The combination of these two peptides enables a good discrimination of the PD group from the MSA patients (p value = 0.0021). This combination and its coefficient sign is similar to the Ab 40/42 ratio, so a ratio of a-syn peptides was tested and results as comparison graphs between groups are described in figure 5.

[0111] The peptide combination has a slightly lower p value for discriminating PD vs MSA than the ratio but the ratio is more easily applicable because, unlike when a logistic regression method is used, there is no coefficient that needs to be applied.

[0112] The best clinical performance for distinguishing PD and DLB groups was obtained from combinations of peptides. Indeed, sensitivity and specificity values of 0.7 and 0.68 respectively were obtained. The ROC curve describing the clinical performance of the peptide combination in PD vs LBD is demonstrated in figure 6.

[0113] The best clinical performance for distinguishing PD and MSA group were obtained with the peptide combination. Sensitivity, specificity and AUC were 0.84, 0.85 and 0.831, respectively for the peptide combination in order to discriminate PD patients from MSA. The ROC curve describing the clinical performance of the peptide combination in PD vs LBD is demonstrated in figure 7.

[0114] Immunoassay quantitation

[0115] The same plasma cohort has also been quantified by sensitive immunochemiluminescence assay. The data comparison graph between diseases is presented in figure 8. No significant difference in a-syn level quantified by immunochemiluminescence assay were observed. A slightly lower a-syn level was observed in the control group compared to the PD group. [0116] Correlation

[0117] The same plasma cohort has also been quantified by sensitive immunochemiluminescence assay. The data comparison graph between disease is presented in figure 8. No significant difference in a-syn level quantified by immune- chemiluminescence assay were observed. A slightly lower a-syn level was observed in the control group compared to the PD group.

[0118] a-syn QGVAEAAGK (SEQ ID NO: 6) and a/[3-syn EGVLYVGSK (SEQ ID NO: 7) were observed to be highly correlated with r= 0.827 in patients' plasma samples, a/ [3-syn EGVLYVGSK (SEQ ID NO: 7) peptide were moderately correlated with peptide combination and ratio (r= 0.568 and r= 0.66 respectively). No correlation was observed (r<0.277) between peptides and immunoassay quantitation. The sex, age and date of sampling did not correlate with a-syn peptides, combinations including a-syn peptides, or ratio (the Pearson coefficient was r<-0.124 in mean). The a/[3-syn EGVLYVGSK (SEQ ID NO: 7) peptide concentration was correlated with the Ab 40 concentration in CSF (r= 0.765) and inversely correlated with the Ab 42/40 ratio (r=-0.74). Also, a-syn QGVAEAAGK (SEQ ID NO: 6) peptide was inversely and slightly correlated with the Ab 42/40 ratio (r=-0.52). The ratio of the peptides in plasma were almost correlated (r=0,444) with a ratio of Ab 42/40 in CSF. A-syn peptides did not correlate with any UPDRS that monitor PD disease severity (r<-0.206).

[0119] The results of the LC-MRM method for quantitation in patient samples are interesting in terms of diagnosis or developing treatment due to the different levels of peptides observed in plasma.

[0120] Discussion

[0121] During method development and validation, the lack of a cysteine residue in synuclein sequences allows omitting denaturation and alkylation steps that are usually performed on proteins.

[0122] Alternative splicing isoforms of [3-syn, y-syn, a-syn and alternative a-syn peptides were consistently below LOQ in plasma QCs and patient samples, due their very low concentrations in the biological fluids (believed to be lower than the pg/ml range).

[0123] At the RNA level, alternative splicing isoforms have been retrieved at different levels of expression in different synucleinopathies. The aim was to target these a-syn species to have the same insights at a protein level since such differences in protein levels have not been described in literature at our knowledge.

[0124] As a proof of concept, two proteotypic peptides from the alternative a-syn was monitored in an attempt to detect these particular species in plasma QCs or patient sample.

[0125] During MS clinical validation, we observed a tendency for all a-syn peptides, and in a significant way for all except a-syn TVEGAGSIAAATGFVK (SEQ ID NO: 10) peptide, to be higher in PD patients’ plasma relative to the control. Taking into consideration the gut-brain axis hypothesis for PD, this means that a-syn species may originate from peripheral enteric plexus or erythrocyte in early stages of PD, which may not be the case for other synucleinopathies, especially MSA (Chang et al, 2020 (Frontiers in neurology)). All the synuclein peptides were correlated to each other (r min<0.727) but none correlated to disease severity (UPDRS II, III and V, r<-0,206). It could be explained by the low range of the different UPDRS values, as PD patients had only light or moderate evolution (see patient biofluid collection section).

[0126] The detected peptides from the same patients were quantified by sensitive immunoassay, no significant difference was observed between diseases groups. The main difference between these two-proteomics methods is the use of antibodies by immunoassay. In this case, the antibodies used targets the C-terminal part of the proteins that underwent an important number of Post-Translational Modifications and truncations (described in previous studies, Pons et al, 2022, Frontiers in aging neuroscience). These have an important impact in the total quantification of the soluble forms of a-syn. Also, immunoassay technique is not able to quantify proteoforms or truncations segment simultaneously that MS is capable of.

[0127] The literature underlines the fact that depending on a-syn localization in cells, for example oligodendrocyte for MSA and dopaminergic neurons for PD/DLB (Mehra et al, 2019; Mou et al., and Porro et al.), the a-syn strains/conformations (content of B sheet structure conformations in the total a-syn conformations) yield distinct results between different synucleinopathies. It has been proven 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 pathology, different a-synuclein species would be expressed in the insoluble fraction (LB or aggregate form) and in the soluble fraction (monomeric, dimeric, oligomeric). Here, with the LC-MRM method, we analyzed the soluble fraction of a-syn in the plasma of the participating patients.

[0128] The discrimination of the PD group from all other synucleinopathies and control was aimed. For this purpose, a peptide combination and a ratio of a-syn peptides was investigated. The peptide combinations and ratios were based on the same principle of the amyloid beta ratio (Ab 40/42). In fact, the ratio Ab 40/ Ab 42 is obtained by dividing the concentration of a peptide that have less propensity to aggregate divided by the concentration of a longer peptide that has more chance to aggregate. With respect to AD, the ratio of the Ab 40/ Ab 42 is more robust than the two separated peptides and enhanced clinical performance. (Hansson et al, 2019, Alzheimer's Research & Therapy).

[0129] The peptides in the peptide combinations involving a-syn do not originate from the same part of the protein. Indeed, a/[3-syn EGVLYVGSK (SEQ ID NO: 7) peptide (position 35-43 on sequence), is part of the N-terminal region.

[0130] McGlinchey et al, 2021 has described that 36-40 residues were important in the replication of full-length structure and for the formation of different fibril structure. This means that the N-terminal portion can modulate the mechanism of amyloid formation and the molecular interactions of fibril assembly. This author showed that truncation of the C-terminus resulted in a modest conformational change compared to N-terminal truncation. Usually a deletion of the C-terminal or/and N-terminal portion(s) results in more interactions of the hydrophobic NAC part with other proteins or other a-syn fragments/species (Sorrentino et al, 2020 (JBC)).

[0131] For the a-syn EQVTNVGGAVVTGVTAVAQK (SEQ ID NO: 9) peptide (position 61-80 of the full sequence), is retrieved into the NAC part that constitute the fibril core of the protein and is responsible of the aggregation process of a-syn. (Sorrentino et al, 2020 (JBC and (McGlinchey et al, 2021)).

[0132] As was done for the Ab 40/42 ratio for AD, we established a peptide combination and/or a ratio of peptides of alpha syn to enhanced clinical performance for PD (to distinguish from the other synucleinopathies in particular).

[0133] Currently, it is difficult for neurologists to differentiate between synucleinopathies (especially MSA and PD) due to overlapping cardinal symptoms and it is even more true at a prodromal stage. With this combination or ratio, distinction of these diseases with biomarkers becomes possible with good sensitivity and specificity (similar to the one obtained by Ab40/42). (Hansson et al, 2019, Alzheimer's Research & Therapy).

Claims

Claims

[Claim 1] A method of detecting a combination of synuclein peptides in a subject, comprising detecting, in a sample of 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 TVEG- AGSIAAATGFVK (SEQ ID NO: 10).

[Claim 2] The method according to claim 1, wherein the at least one combination is

(i) an a-synuclein peptide comprising QGVAEAAGK (SEQ ID NO: 6), an a-synuclein peptide comprising EQVTNVGGAVVTGVTAVAQK (SEQ ID NO: 9), an a/[3- synuclein peptide comprising EGVVAAAEK (SEQ ID NO: 5), and an a/[3- synuclein peptide comprising EGVLYVGSK (SEQ ID NO: 7);

(ii) the a-synuclein peptide comprising QGVAEAAGK (SEQ ID NO: 6) and the a/[3- synuclein peptide comprising EGVLYVGSK (SEQ ID NO: 7); or

(iii) the a-synuclein peptide comprising EQVTNVGGAVVTGVTAVAQK (SEQ ID NO: 9) and the a/[3- synuclein peptide comprising EGVLYVGSK (SEQ ID NO: 7).

[Claim 3] The method according to any one of the preceding claims, wherein said at least one combination comprises (i) the a-synuclein peptide comprising QGVAEAAGK (SEQ ID NO: 6), the a-synuclein peptide comprising EQVTNVGGAVVTGVTAVAQK (SEQ ID NO: 9), the a/ [3- synuclein peptide comprising EGVVAAAEK (SEQ ID NO: 5), and the a/[3- synuclein peptide comprising EGVLYVGSK (SEQ ID NO: 7).

[Claim 4] The method according to any one of the preceding claims, wherein said at least one combination comprises (ii) the a-synuclein peptide comprising QGVAEAAGK (SEQ ID NO: 6) and the a/[3- synuclein peptide comprising EGVLYVGSK (SEQ ID NO: 7).

[Claim 5] The method according to any one of the preceding claims, wherein said at least one combination comprises (iii) the a-synuclein peptide comprising EQVTNVGGAVVTGVTAVAQK (SEQ ID NO: 9) and the a/[3- synuclein peptide comprising EGVLYVGSK (SEQ ID NO: 7).

[Claim 6] The method according to any one of the preceding claims, wherein peptides of said at least one combination each have a length of 20 amino acids or less.

[Claim 7] The method according to any one of the preceding claims, wherein said detecting comprises quantifying peptides of the at least one combination.

[Claim 8] A method of detecting Parkinson disease (PD) in a subject, comprising detecting the at least one combination according to the method of any one of the preceding claims.

[Claim 9] The method according to claim 8, further comprising comparing an amount of each peptide of the at least one combination in the sample with a control.

[Claim 10] The method according to claim 9, wherein the control is a predetermined corresponding value obtained from a subject without PD.

[Claim 11] The method according to claim 9 or 10, wherein the control is a predetermined corresponding value obtained from a subject with PD.

[Claim 12] The method according to any one of claims 8-11, wherein the method further discriminates PD from Lewy Body Dementia (LBD), and the at least one combination comprises (ii) the a-synuclein peptides comprising QGVAEAAGK (SEQ ID NO: 6) and the a/[3- synuclein peptide comprising EGVLYVGSK (SEQ ID NO: 7).

[Claim 13] The method according to claim 12, further comprising comparing an amount of each peptide of the at least one combination in the sample with a control.

[Claim 14] The method according to claim 13, wherein the control is a predetermined corresponding value obtained from a subject with LBD.

[Claim 15] The method according to claim 13 or 14, wherein the control is a predetermined corresponding value obtained from a subject with PD.

[Claim 16] The method according to any one of claims 9-15, wherein the method further discriminates PD from multiple system atrophy (MSA), and the at least one combination comprises (iii) the a-synuclein peptide comprising EQVTNVGGAVVTGVTAVAQK (SEQ ID NO: 9) and the a/[3- synuclein peptide comprising EGVLYVGSK (SEQ ID NO: 7).

[Claim 17] The method according to claim 16, further comprising comparing a ratio of amount of the a-synuclein peptides comprising EQVTNVGGAVVTGVTAVAQK (SEQ ID NO: 9) and the a/[3- synuclein peptide comprising EGVLYVGSK (SEQ ID NO: 7) in the sample with a control.

[Claim 18] The method according to claim 17, wherein the control is a prede- termined corresponding value obtained from a subject with MSA.

[Claim 19] The method according to claim 17 or 18, wherein the control is a predetermined corresponding value obtained from a subject with PD.

[Claim 20] The method according to any one of claims 8-19, further comprising applying an amount of a peptide of the at least one combination to a calculation model adjusted for distinguishing PD from other type(s) of synucleinopathies.

[Claim 21] The method according to claim 20, wherein said other type(s) of synucleinopathies includes MSA, and the calculation model is adjusted for distinguishing PD from MSA.

[Claim 22] The method according to any one of the preceding claims, wherein the subject is not previously diagnosed with PD.

[Claim 23] The method according to any one of the preceding claims, wherein the detecting is performed by a mass spectrometer.

[Claim 24] The method according to any one of the preceding claims, wherein the detecting excludes immunoassay.

[Claim 25] The method according to any one of the preceding claims, wherein the subject is human.

[Claim 26] The method according to any one of the preceding claims, wherein the sample is blood or plasma.

[Claim 27] The method according to any one of the preceding claims, wherein the sample excludes cerebrospinal fluid.

[Claim 28] A method of treating a subject with PD, comprising detecting PD according to the method of any one of claims 8-27, and treating the subject.

[Claim 29] A method of diagnosing Parkinson disease (PD) in a subject, comprising quantifying at least one peptide selected from the group consisting of an a-synuclein peptide comprising QGVAEAAGK (SEQ ID NO: 6), an a-synuclein peptide comprising EQVTNVGGAVVT- GVTAVAQK (SEQ ID NO: 9), an a/[3- synuclein peptide comprising EGVVAAAEK (SEQ ID NO: 5), and an a/[3- synuclein peptide comprising EGVLYVGSK (SEQ ID NO: 7).

[Claim 30] The method according to claim 29, wherein said at least one peptide comprises the a-synuclein peptide.

[Claim 31] The method according to claim 29 or 30, wherein said at least one peptide comprises the a/[>- synuclein peptide.

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

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

[Claim 34] The method according to any one of claims 29-33, wherein said at least one peptide comprises at least one a-synuclein peptide and at least one a/[3- synuclein peptide.

[Claim 35] The method according to any one of claims 29-34, wherein said at least one peptide includes the a-synuclein peptide comprising QGVAEAAGK (SEQ ID NO: 6).

[Claim 36] The method according to any one of claims 29-35, wherein said at least one peptide includes the a-synuclein peptide comprising EQVTNVG- GAVVTGVTAVAQK (SEQ ID NO: 9).

[Claim 37] The method according to any any one of claims 29-36, wherein said at least one peptide includes the a/[>- synuclein peptide comprising EGVVAAAEK (SEQ ID NO: 5).

[Claim 38] The method according to any any one of claims 29-37, wherein said at least one peptide includes the a/[>- synuclein peptide comprising EGVLYVGSK (SEQ ID NO: 7).

[Claim 39] The method according to any one of claims 29-38, wherein said 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).

[Claim 40] The method according to any one of claims 29-39, wherein said detecting comprises quantifying the at least one peptide.

[Claim 41] The method according to any one of claims 29-40, further comprising comparing an amount of each of the at least one peptide in the sample with a control.

[Claim 42] The method according to claim 41, wherein the control is a predetermined corresponding value obtained from a subject without PD.

[Claim 43] The method according to claim 41, wherein the control is a predetermined corresponding value obtained from a subject with PD.

[Claim 44] The method according to any one of claims 29-43, wherein the method distinguishes PD from other types of synucleinopathies.

[Claim 45] The method according to any one of claims 29-44, further comprising applying an amount of each of said at least one peptide to a calculation model adjusted for distinguishing Parkinson disease and another type of synucleinopathies.

[Claim 46] The method according to claim 44 or 45, wherein said other type(s) of synucleinopathies includes multiple system atrophy (MSA).

[Claim 47] The method according to claim 46, wherein the calculation model is adjusted for distinguishing Parkinson disease and multiple system atrophy.

[Claim 48] The method according to any one of claims 29-47, wherein the method further discriminates PD from LBD, and the at least one peptide comprises the a-synuclein peptides comprising QGVAEAAGK (SEQ ID NO: 6) and/or the a/[3- synuclein peptide EGVLYVGSK (SEQ ID NO: 7).

[Claim 49] The method according to claim 48, further comprising comparing an amount of each of the at least one peptide in the sample with a control.

[Claim 50] The method according to claim 49, wherein the control is a predetermined corresponding value obtained from a subject with LBD.

[Claim 51] The method according to claim 49, wherein the control is a predetermined corresponding value obtained from a subject with PD.

[Claim 52] The method according to any one of claims 29-51, wherein the method further discriminates PD from multiple system atrophy (MSA), and the at least one peptide comprises the a-synuclein peptides comprising EQVTNVGGAVVTGVTAVAQK (SEQ ID NO: 9) and the a/[3- synuclein peptide EGVLYVGSK (SEQ ID NO: 7).

[Claim 53] The method according to claim 52, further comprising comparing a ratio of amount of the a-synuclein peptides comprising EQVTNVGGAVVTGVTAVAQK (SEQ ID NO: 9) and the a/[3- synuclein peptide EGVLYVGSK (SEQ ID NO: 7) in the sample with a control.

[Claim 54] The method according to claim 53, wherein the control is a predetermined corresponding value obtained from a subject with MSA.

[Claim 55] The method according to claim 53, wherein the control is a predetermined corresponding value obtained from a subject with PD.

[Claim 56] The method according to any one of claims 29-55, wherein the detecting is performed by a mass spectrometer.

[Claim 57] The method according to any one of claims 29-56, wherein the detecting excludes immunoassay.

[Claim 58] The method according to any one of claims 29-57, wherein the subject is human.

[Claim 59] The method according to any one of claims 29-58, wherein the sample is blood or plasma.

[Claim 60] The method according to any one of claims 29-59, wherein the sample excludes cerebrospinal fluid.

[Claim 61] A method of treating a subject with PD, comprising diagnosing PD according to the method of any one of claims 29-60, and treating the subject diagnosed with the PD.